Novel yeast display libraries and uses thereof

Abstract

The technology described herein provides a novel method for discovering, optimizing, diversifying, and characterizing binding proteins (e.g. antibodies) that bind to protein targets of interest, especially membrane proteins.

Description

Attorney Docket No. 701586-000163WOPTNOVEL YEAST DISPLAY LIBRARIES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application 63 / 778,581 filed on March 27, 2025, the content of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 19, 2026, is named 701586-000163WOPT_SL.xml and is 208,896 bytes in size.TECHNICAL FIELD

[0003] The technology described herein relates to a novel method for discovering, optimizing, diversifying, and characterizing binding proteins (e.g. antibodies) that bind to protein targets of interest, such as membrane proteins.BACKGROUND

[0004] A cell membrane contains thousands of different proteins. These proteins can sense and respond to the environment outside the cell through a variety of binding interactions, often through protein-protein interactions known as membrane protein-protein interactions (MPPIs). These interactions allow for membrane proteins to act as a system for information flow and decision making across the cell membrane. Because of their significant role in cellular function, these membrane proteins are important targets in drug development. Specifically, identifying binding proteins that interact with membrane proteins can advance drug development. Therefore, discovering, optimizing, diversifying and characterizing these binding proteins is a needed step in such drug development. However, understanding membrane protein interactions in a live cell environment is challenging due to the complexity of the cell membrane.SUMMARY

[0005] Prior methods for discovering, optimizing, diversifying, and characterizing binding proteins generally require biochemical purification of the target protein. This can be failure-prone, slow, and expensive, especially for biochemically challenging proteins. Genetic systems for assaying MPPIs within cells have the potential to streamline this process. Such a genetic system allows for expression of the proteins of interest in live cells. Existing genetic systems use a split-ubiquitin architecture and are primarily used in yeast cells. When the components of the target membrane protein and the binding protein outside of the cell membrane interact, their intracellular components also undergo an interaction. This intracellular interaction leads to expression of a reporter gene, such as a fluorescent 14939-1760-0399 2Attorney Docket No. 701586-000163WOPTprotein, which allows for the binding event to be detected and quantified. However, many examples show that this split-ubiquitin architecture is unusable for most applications due to severe defects, including extremely high rate of false positives, poor dynamic range, and limited tunability.

[0006] The inventors have now developed synthetic proteins and systems that permit improved identification and characterization of binding proteins, e.g., membrane protein binders. The compositions and systems described herein are the first to use an architecture based on proteases, rather than ubiquitin. These protease-based architectures overcome previous limitations and permit many new applications. Critically, two features — expression level control and the ability to perform negative selection — dramatically improve the performance of the system as compared to prior art technologies.

[0007] Disclosed herein are several examples of results and performance that would not be feasible with existing MPPI assays: 1) Discovery of novel antibodies for challenging, high-value human membrane protein drug targets, 2) Optimization of antibodies for affinity, specificity, and other developability properties, 3) Diversification of antibodies to find variants with new functional properties, and 4) High-throughput characterization of protein-protein interactions.

[0008] In one aspect of any of the embodiments is a set of proteins comprising: a) a target protein comprising: i) at least one extracellular domain and / or extracellular loop; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain; and b) a binding protein or binding dimer comprising: i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) at least one intracellular protease domain.

[0009] In some embodiments of any of the aspects, the set further comprises at least one off-target protein. An off-target protein can comprise i) at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

[0010] In some embodiments of any of the aspects, described herein is a set of proteins comprising: a) a target protein comprising: i) at least one extracellular domain and / or extracellular loop; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain; iv) an intracellular first portion of a split protease, located between the transmembrane domain and the intracellular transcription factor domain; and v) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain;24939-1760-0399 2Attorney Docket No. 701586-000163WQPTand b) a binding protein or binding dimer comprising: i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) an intracellular second portion of the split protease.

[0011] In some embodiments of any of the aspects is an expression system comprising: a) one or more nucleic acid molecule encoding a set of proteins of any one of the preceding aspects; and b) a nucleic acid molecule comprising: i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control sequence modulated by the intracellular transcription factor domain of the off-target protein; and ii) operably linked to a reporter gene.

[0012] In some embodiments of any of the aspects is an expression system comprising: a) one or more nucleic acid molecule encoding a set of proteins of any one of the preceding aspects; and b) a nucleic acid molecule comprising: i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control sequence modulated by the intracellular transcription factor domain of the off-target protein; and ii) operably linked to a reporter gene.

[0013] In one aspect of any of the embodiments, described herein is a set of proteins comprising:a) a target protein comprising:i) at least one extracellular domain and / or extracellular loop;ii) at least one transmembrane domain;iii) at least one intracellular transcription factor domain; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain;andb) a binding protein or binding dimer comprising:i) at least one extracellular binding domain;ii) at least one transmembrane domain; andiii) at least one intracellular protease domain.

[0014] In some embodiments of any of the aspects, the set of proteins further comprises:c) at least one off-target protein comprising:i) at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein;ii) at least one transmembrane domain;34939-1760-0399 2Attorney Docket No. 701586-000163WQPTiii) at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein; andiv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

[0015] In one aspect of any of the embodiments, described herein is set of proteins comprising:a) a target protein comprising:i) at least one extracellular domain and / or extracellular loop;ii) at least one transmembrane domain;iii) at least one intracellular transcription factor domain;iv) an intracellular first portion of a split protease, located between the transmembrane domain and the intracellular transcription factor domain; andv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain; andb) a binding protein or binding dimer comprising:i) at least one extracellular binding domain;ii) at least one transmembrane domain; andiii) an intracellular second portion of the split protease.

[0016] In some embodiments of any of the aspects, the first portion of the split protease is a N-terminal fragment of the split protease and the second portion of the split protease is a C-terminal fragment of the split protease. In some embodiments of any of the aspects, the first portion of the split protease is a C-terminal fragment of the split protease and the second portion of the split protease is a N-terminal fragment of the split protease.

[0017] In some embodiments of any of the aspects, the set of proteins further comprises:c) at least one off-target protein comprising:iv) at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein;v) at least one transmembrane domain;vi) at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein;44939-1760-0399 2Attorney Docket No. 701586-000163WQPTvii) an intracellular first portion of a split protease, located between the transmembrane domain and the intracellular transcription factor domain; andviii) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

[0018] In some embodiments of any of the aspects, the extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of the target protein are: G-protein coupled receptor (GPCR) sequences; receptor tyrosine kinases sequences; toll-like receptors sequences; T-cell receptors sequences; B-cell receptors sequences; transporters sequences; solute carriers sequences; proton pumps sequences; ion channels sequences; porins sequences; aquaporins sequences; viroprotein sequences; integrins sequences; cadherins sequences; 7TM protein sequences; nuclear receptor sequences; gap junction protein sequences; connexin sequences; selectin sequences; spectrin sequences; N-CAM sequences; claudin sequences; occludin sequences; Fc receptor sequences; tetraspanin sequences; transferrin receptor sequences; carrier protein sequences; or channelrhodopsin sequences.

[0019] In some embodiments of any of the aspects, the transcription factor domain is a polypeptide sequence comprising a DNA binding domain and a transcriptional activation domain.

[0020] In some embodiments of any of the aspects, the protease or split protease binds to and cleaves the intracellular protease cleavage target domain. In some embodiments of any of the aspects, the protease or split protease is selected from the group consisting of: TEV protease and 3C protease. In some embodiments of any of the aspects, the intracellular first portion of a split protease of an off-target protein is not identical to the intracellular first portion of a split protease of the target protein. In some embodiments of any of the aspects, the intracellular first portion of a split protease of an off-target protein and the intracellular first portion of a split protease of the target protein are variants of the same split protease. In some embodiments of any of the aspects, the protease cleavage site is selected from one of SEQ ID NOs: 1-27. In some embodiments of any of the aspects, the protease is TEV and the protease cleavage site is selected from one of SEQ ID NOs: 1-27.

[0021] In some embodiments of any of the aspects, the extracellular binding domain is an antibody or antibody reagent. In some embodiments of any of the aspects, the at least one transmembrane domain of the binding protein or binding dimer is a single-pass transmembrane domain.

[0022] In some embodiments of any of the aspects, the binding protein or binding dimer further comprises a linker between the at least one extracellular binding domain and the at least one transmembrane domain. In some embodiments of any of the aspects, the linker comprises 2-60 amino acids. In some embodiments of any of the aspects, the linker comprises 20-40 amino acids.

[0023] In some embodiments of any of the aspects, the set of proteins further comprises a binding dimer wherein the binding dimer comprises:54939-1760-0399 2Attorney Docket No. 701586-000163WOPTc) a first partner protein comprising:ix) at least one extracellular binding domain; andx) a first dimerization domain; andd) a second partner protein comprising:xi) a second dimerization domain that binds to the first dimerization domain; xii) at least one transmembrane domain; andxiii) one of:1) at least one intracellular protease domain that recognizes and cleaves the intracellular protease cleavage target domain; or2) an intracellular second portion of the split protease.

[0024] In one aspect of any of the embodiments, described herein is an expression system comprising:a) a set of proteins described herein; andb) a nucleic acid molecule comprising:i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control sequence modulated by the intracellular transcription factor domain of the off-target protein, andii) operably linked to a reporter gene.

[0025] In one aspect of any of the embodiments, described herein is an expression system comprising:a) one or more nucleic acid molecule encoding a set of proteins described herein; and b) a nucleic acid molecule comprising:i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control sequence modulated by the intracellular transcription factor domain of the off-target protein; andii) operably linked to a reporter gene .

[0026] In some embodiments of any of the aspects, the one or more nucleic acid molecule encoding a set of proteins of any one of the preceding claims further comprise a constitutive promoter operably linked to a sequence encoding the target protein, a constitutive promoter operably linked to a sequence encoding the binding protein, a constitutive promoter operably linked to a sequence encoding the first partner protein, a constitutive promoter operably linked to a sequence encoding the second partner protein, and / or a constitutive promoter operably linked to a sequence encoding the off-target protein.

[0027] In some embodiments of any of the aspects, the one or more nucleic acid molecule encoding a set of proteins of any one of the preceding claims further comprise an inducible promoter operably linked to a sequence encoding the target protein, an inducible promoter operably linked to a sequence encoding the binding protein, an inducible promoter operably linked to a sequence encoding the first64939-1760-0399 2Attorney Docket No. 701586-000163WOPTpartner protein, an inducible promoter operably linked to a sequence encoding the second partner protein, and / or an inducible promoter operably linked to a sequence encoding the off-target protein.

[0028] In one aspect of any of the embodiments, described herein is a cell comprising the set of proteins or expression system described herein. In some embodiments of any of the aspects, the cell is a yeast cell. In some embodiments of any of the aspects, the reporter gene is a fluorescent protein or yeast survival protein.

[0029] In one aspect of any of the embodiments, described herein is a library comprising a plurality of cells described herein.

[0030] In one aspect of any of the embodiments, described herein is a method comprising detecting a signal of a reporter gene of i) an expression system described herein or ii) a cell described herein.

[0031] In some embodiments of any of the aspects, a plurality of different binding proteins or a plurality of different first partner proteins is expressed in a plurality of cells. In some embodiments of any of the aspects, detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates the extracellular binding domain is specific for binding to the target protein. In some embodiments of any of the aspects, detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the off-target protein indicates the extracellular binding domain is not specific for binding to the target protein. In some embodiments of any of the aspects, the extracellular binding domain of the binding protein or binding dimer is an antibody or antibody reagent, the target protein comprises at least one extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of a therapeutic target protein, and the detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates the extracellular binding domain is specific for binding to the therapeutic target protein. In some embodiments of any of the aspects, the extracellular binding domain of the binding protein or binding dimer is an antibody or antibody reagent, the target protein comprises at least one extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of a therapeutic target protein, and the strength of the detected signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates a binding affinity of the extracellular binding domain for the target protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1. Schematic of one embodiment of the technology disclosed herein: a genetic system in yeast co-expressing binding protein(s) and target protein(s) at the cellular membrane, and then produces a signal output if a binding interaction occurs.74939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0033] FIG. 2. Schematic of one embodiment of the MMPI assay based on proteases as described herein. Split-ubiquitin MPPI assay as disclosed in prior art is shown on the left. Novel MMPI assays disclosed herein, involving protease recruitment and split-protease are shown on the right. TF = transcription factor. Upon cleavage, the free TF localizes to the nucleus and activates reporter genes such as GFP or yeast survival proteins.

[0034] FIG. 3. A schematic of an embodiment of the split-protease MMPI assay version 2. Splitprotease MMPI assay version 2 incorporates two critical features: 1) control of expression and 2) selection against off-target.

[0035] FIG. 4. Description of an embodiment of the protease-recruitment MPPI assay.

[0036] FIG. 5A. Description of an embodiment of the split-protease MPPI assay (vl). FIG. 5B. Description of an embodiment of the split-protease MPPI assay (v2).

[0037] FIG. 6A. Schematic of one embodiment of the protease-recruitment gene circuit. Ste2 is a yeast GPCRthat specifically recognizes the alpha-factor peptide (and does not recognize the apelin peptide). WSC3 and SS01 are Type I and Type II transmembrane domains, respectively, that were used to anchor the peptides to the yeast membrane. TEVp is a protease that recognizes and cleaves a cleavage sequence (CS). Here, CS was mutated at a single residue for optimization of the signal output. LexA-VP16 is a synthetic transcription factor that, when released from Ste2 by TEVp, can localize to the nucleus and activate expression of GFP. FIG. 6B. GFP output with alpha-factor and apelin displayed using the WSC3 and SS01 anchoring domains.

[0038] FIG. 7A. Schematic of an embodiment of the split-protease gene circuit. N-TEVp and C-TEVp are N-terminal and C- terminal portions, respectively, of a split TEV protease that can reconstitute and then recognize and cleave a cleavage sequence (CS). Here, CS was mutated at a single residue when testing with SS01, and C-TEVp was mutated at a single residue when testing with WSC3, both for optimization of signal output. LexA-VP16 is a synthetic transcription factor that, when released from Ste2 by reconstituted TEVp, can localize to the nucleus and activate expression of GFP. FIG. 7B. GFP output with alpha-factor and apelin displayed using the WSC3 and SS01 anchoring domains.

[0039] FIG. 8A. Schematic of an embodiment of the split-protease gene circuit used to test nanobodies that bind the human APJ GPCR. FIG. 8B. GFP output with positive control nanobodies that bind APJ (JN241 , JN241.9) negative control nanobodies that do not bind APJ (BC2, Pep), and a negative control without any nanobody (-). The affinity for JN241 is 83 pM. FIG. 8C. Schematic of an embodiment of the split-protease gene circuit used to test nanobodies that bind the HCMV US28 GPCR. FIG. 8D. GFP output with positive control nanobodies that bind US28 (VUN100, US28-2, US28-3, US28-4) and negative control nanobodies that do not bind US28 (Pep, BC2, JN241).Affinities for US28- binding nanobodies are shown.84939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0040] FIG. 9A. Schematic of an embodiment of the split-protease gene circuit with expression control. The target protein is the BILF1 GPCR fused N-terminally to the BC2 peptide. Expression of the target is under control of a beta-estradiol-inducible promoter. Positive and negative control nanobodies targeting the BC2 peptide are fused to the SPYCATCHER™-protein and is tested with a series of constitutive promoters. The SPYTAG™ protein is anchored to the membrane with a WSC3 transmembrane domain, and expression of this is kept constant and dose-limiting with the weak RAD27 promoter. Transcription factor output drives expression of GFP and the HIS3 selectable marker. FIG. 9B. Dose-response of GFP output vs. beta-estradiol for a series of nanobody display constructs that vary promoter level (RPL18B>RET2>RNR2>REV1) for a BC2 binding (BC2) and non-binding (JN241) nanobody. Inset shows yeast growth for a subset of constructs on media selecting for HIS3 expression. FIG. 9C. Characterization of US28 -binding (blue) and non-binding (yellow) nanobodies in the original split-protease format (without expression control) and the improved SPYCATCHER™-SPYTAG™ format (with expression control).

[0041] FIG. 10A. Schematic of an embodiment of the split-protease gene circuit with expression control and negative selection. The target protein is the BILF1 GPCR fused N-terminally to the BC2 peptide. The transcription factor fused to the target controls expression of GFP and the HIS3 selectable marker. The off-target protein is the US28 GPCR. The transcription factor fused to US28 controls expression of RFP and the URA3 marker, which can be negatively selected against. The C-terminal protease fragments are the same for the two targets and can both reconstitute with the N-terminal protease fragment bound to the nanobody anchor. FIG. 10B. Growth of yeast expressing the gene circuit with nanobodies that either bind the target (BC2 nanobody) or the off-target (US28 nanobody). Cells were grown on media that does not select for reporter output, only selects for HIS3 expression, or selects for HIS3 expression and against URA3 expression. Improvement in background growth is boxed.

[0042] FIG. 11A Schematic of an embodiment of the v2 split-protease gene circuit used to test nanobodies that bind targets of interest. FIG. 11B. Growth of yeast expressing the gene circuit with the AT1R GPCR as the target and nanobodies that either bind AT1R (ATI 18i4, ATI 18) or do not. Cells were grown on media that does not select for reporter output or on media that selects for HIS3 expression and against URA3 expression. The target is a human GPCR. FIG. 11C. Growth of yeast expressing the gene circuit with the APJ GPCR as the target and nanobodies that either bind APJ (JN241) or do not. Cells were grown on media that does not select for reporter output or on media that selects for HIS3 expression and against URA3 expression. The target is a human GPCR. FIG. 11D.Growth of yeast expressing the gene circuit with the BC2 peptide as the target fused to a GPCR, and nanobodies that either bind BC2 or do not. Cells were grown on media that does not select for reporter output or on media that selects for HIS3 expression and against URA3 expression. The target is a soluble BC2 peptide tethered to the C-terminal protease fragment using a GPCR.94939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0043] FIG. 12A. Schematic of an embodiment of the split-protease gene circuit used to characterize the relationship between affinity and assay output. The target is the yeast STE2 GPCR and the binding proteins are various mutants of the receptor’s peptide ligand, alpha-factor. STE2 is under control of a beta-estradiol inducible promoter. FIG. 12B. Mutants of the alpha-factor peptide tested and their reported affinities. FIG. 12B discloses SEQ ID NOS 28-37, respectively, in order of appearance. FIG.12C. GFP out for each of the alpha-factor mutants vs. their reported affinities. The dashed line corresponds to a non-binding protein (APLN nanobody). FIG. 12D. Growth of yeast expressing a subset of the alpha-factor variants, the APLN nanobody, or no binding protein. Yeast were grown in non-selective media (SDC) or media that selects for HIS3 expression (SD- His) with various levels of STE2 induction via beta-estradiol (EST). Growth was assessed by measuring OD600 after 15 hrs growth in liquid media at 30 °C.

[0044] FIG. 13A. Schematic of an embodiment of the v2 split-protease gene circuit used to test generalizability across multiple targets of interest. Since binding proteins are not available for many high-value drug targets, the BC2 peptide was fused N-terminally to each target, and the expression of the target was verified using a BC2 -binding nanobody. FIG. 13B. Growth of yeast expressing the gene circuit with various membrane protein targets in the presence of either a BC2-binding nanobody or a non-binding nanobody. Cells were grown on media that does not select for reporter output (without selection) or on media that selects for HIS3 expression and against URA3 expression (with selection). SSTR5, APJ and AT1R are human GPCRs. BILF1 is a viral GPCR. LMP2B is a viral multipass membrane protein. SLC7A11 is a human solute transporter.

[0045] FIG. 14A. Candidate nanobody binders discovered using the exemplary v2 split-protease MPPI assay for multiple membrane protein drug targets. A naive, 10E9-member nanobody library was transformed into yeast encoding the MPPI circuit and subject to selection for binding to the target of interest. Candidates were harvested and verified in yeast by re-testing in the v2 assay using either the target (x-axis) or an off-target receptor (y-axis) and measuring fluorescence output. Potential hits are those with strong on-target and weak off-target binding. FIG. 14B. Verification of two BILFl-binding candidates isolated from discovery campaigns. Nanobodies were purified as Fc-fiisions and used to stain HEK cells expressing either BILF1-GFP or SSTR5-GFP. X-axis represents receptor quantity (GFP), and y-axis represents binding signal. FIG. 14C is similar to FIG. 14B but for nanobodies discovered against SSTR5. FIG. 14D is similar to FIG. 14B but for nanobodies discovered against APJ.

[0046] FIG. 15A. Affinity maturation using the exemplary v2 split-protease MPPI assay of a candidate nanobody binder discovered for the BILF 1 GPCR. A scanning mutagenesis library of the candidate was transformed into yeast encoding the MPPI gene circuit and subject to a pooled growth selection. Growth rates for the variants were measured by tracking enrichment of each sequence over time via sequencing. Growth selections were performed in duplicates as shown. Sequences with high104939-1760-0399 2Attorney Docket No. 701586-000163WOPTenrichment are colored. FIG. 15B. Verification of individual affinity matured variants in the v2 MPPI assay. Nbl98 is a naive BILF1 binder initially discovered using the v2 MPPI assay and then affinity matured. Select matured variants with strong growth rates were individually transformed into yeast encoding the MPPI gene circuit and subject to growth selection. Yeast were grown on media that selects for HIS3 expression (CSM-His + 3AT) across a range of 3AT concentrations. All conditions also select against URA3 expression (not labeled). BC2 nanobody is a 1.4 nM binder against the BC2 peptide that was fused N-terminally to BILF1. FIG. 15C. Verification of two affinity matured variants of Nbl98 isolated from affinity maturation campaigns. Nanobodies were purified as Fc-fiisions and used to stain HEK cells expressing BILF1. X-axis represents nanobody concentration and y-axis represents binding signal. FIG. 15D. Affinity matured variants display improvements in developability properties such as protein yield, precipitation, and specificity. Select variants were purified as Fc-fusions and characterized. Protein yield was measured using the standard bicinchoninic assay (BCA). Precipitation was assessed visually. Affinity and specificity were assessed by staining HEK cells expressing BILF1 GFP or an off-target GPCR fused to GFP, respectively. X-axis represents receptor quantity (GFP signal), and y-axis represents binding signal.

[0047] FIG. 16A. Library design for diversification of ATI 18i4, a published nanobody that binds to the AT1R GPCR and acts as an antagonist. In this design, CDR3 was randomized and lengthened in order to discover variants with new functional properties. FIG. 16B. Verification of newly discovered variants in the exemplary v2 MPPI assay. The libraries were transformed into yeast encoding the MPPI gene circuit and subject to selection. From each library, 12 variants that enriched were characterized by re-testing in the MPPI assay, alongside ATI 18 (parent to AT118i4), AT118i4 and a non-binding nanobody. Yeast were grown on non-selective media (SDC) or selective media lacking histidine across a range of estradiol, 3-AT, and 5-FOA concentrations (shown as Est_3AT_5FOA). FIG. 16C. Verification of four new ATIR-binding candidates. Nanobodies were purified as Fc-fiisions and used to stain HEK cells expressing AT1R-GFP. X-axis represents receptor quantity (GFP), and y-axis represents binding signal. FIG. 16D.AlphaFold predicted structures of ten of the new variants that were confirmed to bind to AT1R on HEK cells. For comparison, ATI 18i4 and the AT1R ligand, Angll, are shown. FIG. 16E. Functional characterization of seven of the newly discovered AT1R binders. Nanobodies were purified as an Fc-fiision and incubated with 100 nM Angll in a standard beta-arrestin signaling assay for ATI R.

[0048] FIG. 17. High throughput characterization of protein-protein interactions. High-throughput affinity characterization of nanobodies that bind the AT1R GPCR. 16 nanobodies that bind to AT1R were transformed into yeast encoding the v2 MPPI gene circuit and characterized via growth selection. For verification, the nanobodies were also purified as Fc-fiisions and used to stain HEK cells expressing AT1R-GFP. The x-axis represents AT1R abundance in terms of GFP fluorescence. The y-axis represents staining signal. For each nanobody’s staining curve, the corresponding yeast signal is shown using color coding.114939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0049] FIG. 18 depicts a comparison of an embodiment of the circuit output (GFP) for the split-ubiquitin MPPI system, the protease recruitment MPPI system, and the split-protease recruitment MPPI system for both the binder and the non-binder.

[0050] FIG. 19A: GFP output of yeast expressing one embodiment of the v2 split-protease MPPI circuit with various membrane protein targets, including GPCRs and solute transporters, in the presence of either cognate or non-cognate nanobody binders. Output normalized to the minimum and maximum for each target. The circuit used in this experiment comprises a split TEV protease with wild-type N-tev fragment and L190K C-tev fragment; WSC3 domain for anchoring the nanobodies; and LexA-VP16 TF.

[0051] FIG. 19B: GFP output of yeast expressing one embodiment of the v2 split-protease MPPI circuit with various membrane protein targets, including GPCRs and multipass enzymes, in the presence of either cognate or non-cognate scFV or peptide binders. Output normalized to the minimum and maximum for each target. The circuit used in this experiment comprises a split TEV protease with wild-type N-tev fragment and L190K C-tev fragment; WSC3 domain for anchoring the nanobodies; and LexA-VP16 TF.

[0052] FIG. 19C: GFP output of yeast expressing one embodiment of the v2 split-protease MPPI circuit with the CXCR4 GPCR, in the presence of either cognate or non-cognate chemokines or miniprotein binders. The cognate minibinder CXCR4 has been described in the art in, for example, Muratspahic et al., bioRxiv 2025.03.23.644666, the contents of which are hereby incorporated in its entirety. The non-cognate minibinders have been described in the art in, for example, Cao et al., Nature 605 (2022) 551-560 and Watson et al., Nature 620 (2023) 1089-1100, the contents of each of which are hereby incorporated in their entireties. Output normalized to the minimum and maximum for each target. The circuit used in this experiment comprises a split TEV protease with wild-type N-tev fragment and L190K C-tev fragment; WSC3 domain for anchoring the nanobodies; and LexA-VP16 TF.DETAILED DESCRIPTION

[0053] Described herein are compositions and methods that can be used to identify and / or optimize binding molecules (e.g., drugs) that target membrane or membrane-displayed proteins. The compositions and methods described herein are the first to use an architecture based on proteases. Protease-based architectures overcome previous limitations and permit many new applications. For example, the compositions and methods disclosed herein permit discovery of novel antibodies for challenging, high-value human membrane protein drug targets, allow for optimization of antibodies for affinity, specificity, and other developability properties, permit diversification of antibodies to find variants with new functional properties, and can be used for high-throughput characterization of124939-1760-0399 2Attorney Docket No. 701586-000163WQPTprotein-protein interactions. The methods and compositions described herein can be used to identify variants from a library of protein binders (e.g. antibodies) that can bind to human therapeutic target proteins, especially membrane proteins, can be used to improve the affinity, specificity or developability of a protein binder (e.g. antibody), can be used to generate diverse variants of a protein binder (e.g. antibody) that are still capable of binding to its target, and can be used to quantitatively assess the affinity of an interaction between two proteins, including an antibody (or any other type of binding protein) and its target, as well as other uses.

[0054] In one aspect of any of the embodiments, described herein is a set of proteins comprising: a) a target protein comprising: i) at least one extracellular domain and / or extracellular loop; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain; and b) a binding protein or binding dimer comprising: i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) at least one intracellular protease domain.

[0055] As used herein, “set” refers to a group of proteins of at least two proteins, i.e., at least two separate and distinguishable macromolecules, each comprising an amino acid chain that is distinguishable from the other members of the set and which is not bound to other members of the set via peptide bonds. The members of a set are considered separate proteins even if bound to each other by non-peptide bonds such as hydrogen bonds. In some embodiments of any of the aspects, the set comprises two proteins. In some embodiments of any of the aspects, the set consists of two proteins. In some embodiments of any of the aspects, the set comprises more than two proteins.

[0056] As used herein, “target protein” refers to the protein in a set of proteins which undergoes a binding interaction with the binding protein (or binding dimer) if the binding protein (or binding dimer) is in proximity to the target protein and comprises a sequence that is recognizable by (or which recognizes) the target protein.

[0057] As used herein, “binding” refers to a interaction between macromolecules (e.g. the target protein and the binding protein). In some cases binding will be sequence-specific. Depending on the amino acid sequence a protein may bind to or recognize more than one target sequence, although typically one sequence will be bound in preference to any other recognized sequences, depending on the relative specificity of the individual interactions. In some embodiments of any of the aspects, the binding is a non-covalent binding. Binding can comprise ionic bonding, hydrogen bonds, ionic bonds, van der Waals interactions, and / or London dispersion forces. In some embodiments, specific binding does not refer to covalent bonding. In some embodiments, binding does not refer to a peptide bond.

[0058] A binding event involving a protein may be accompanied by a conformational change in the protein, which may or may not result in an alteration to the protein’s function. In some embodiments of any of the aspects, the binding interaction produces a signal. In some embodiments of any of the134939-1760-0399 2Attorney Docket No. 701586-000163WQPTaspects, the binding interaction results in cleavage of the target protein at an intracellular protease cleavage target domain.

[0059] In some embodiments of any of the aspects, the target protein is not a naturally occurring protein. In some embodiments of any of the aspects, the target protein is not a protein found in nature. In some embodiments of any of the aspects, the combination of the target protein’s at least one extracellular domain and / or extracellular loop, at least one transmembrane domain, at least one intracellular transcription factor domain, and at least one intracellular protease cleavage target domain are a combination not found in a single naturally occurring protein.

[0060] In some embodiments of any of the aspects, the target protein comprises i) at least one extracellular domain and / or extracellular loop; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain. In some embodiments of any of the aspects, the target protein consists essentially of i) at least one extracellular domain and / or extracellular loop; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain. In some embodiments of any of the aspects, the target protein consists of i) at least one extracellular domain and / or extracellular loop; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

[0061] In some embodiments of any of the aspects, the target protein comprises i) an extracellular domain and / or extracellular loop; ii) a transmembrane domain; iii) an intracellular transcription factor domain; and iv) an intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain. In some embodiments of any of the aspects, the target protein consists essentially of i) an extracellular domain and / or extracellular loop; ii) a transmembrane domain; iii) an intracellular transcription factor domain; and iv) an intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain. In some embodiments of any of the aspects, the target protein consists of i) an extracellular domain and / or extracellular loop; ii) a transmembrane domain; iii) an intracellular transcription factor domain; and iv) an intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

[0062] As used herein, “extracellular domain” refers to the portion of the target protein which extends out from a cellular membrane into the cytoplasm when the target protein is expressed in a cell. In some embodiments of any of the aspects, the extracellular domain terminates at the N-terminus of the target protein. In some embodiments of any of the aspects, the extracellular domain terminates at the C-terminus of the target protein. Alternatively, the extracellular domain is located144939-1760-0399 2Attorney Docket No. 701586-000163WOPTbetween two transmembrane domains or two transmembrane passes and thereby forms an extracellular loop. In some embodiments of any of the aspects, both the N-terminus and C-terminus of the target protein are intracellular, such that the extracellular domain forms an extracellular loop. In some embodiments of any of the aspects, there are at least two transmembrane passes in a target protein, and the extracellular domain is located between the two transmembrane domains, forming an extracellular loop. In some embodiments of any of the aspects, the extracellular domain or extracellular loop is naturally occurring sequence. In some embodiments of any of the aspects, the extracellular domain or extracellular loop is a fusion polypeptide sequence, e.g., comprising sequences from at least two different sources. In some embodiments of any of the aspects, the extracellular domain or extracellular loop is a display polypeptide sequence. In some embodiments of any of the aspects, the extracellular domain or extracellular loop is not a naturally occurring polypeptide sequence. In some embodiments of any of the aspects, the extracellular domain or extracellular loop is not a polypeptide sequence found in nature.

[0063] Suitable extracellular domains include, for example, extracellular domains of proteins which are known to be involved in pathological processes. Such proteins are known in the art and one of ordinary skill can readily identify such an extracellular domain for a disease of interest. Suitable Extracellular domains sequences and extracellular loop sequences include but are not limited to: G-protein coupled receptor (GPCR) sequences; receptor tyrosine kinases sequences; toll-like receptors sequences; T-cell receptors sequences; B-cell receptors sequences; transporters sequences; solute carriers sequences; proton pumps sequences; ion channels sequences; porins sequences; aquaporins sequences; viroprotein sequences; integrins sequences; cadherins sequences; 7TM protein sequences; nuclear receptor sequences; gap junction protein sequences; connexin sequences; selectin sequences; spectrin sequences; N-CAM sequences; claudin sequences; occludin sequences; Fc receptor sequences; tetraspanin sequences; transferrin receptor sequences; carrier protein sequences; or channelrhodopsin sequences. Information on the structure, sequence, and function of such proteins is available in the art, e.g., Gurdap Biophys J. 2022 Oct 18; 121 (20):3826-3836, and Rosenbaum Nature.2009 May 21;459(7245):356-63, each of which is incorporated by reference herein in their entireties.

[0064] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a G-protein coupled receptor (GPCR) sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a G-protein coupled receptor (GPCR) sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a G-protein coupled receptor (GPCR) sequence.

[0065] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a receptor tyrosine kinase sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a receptor tyrosine kinase sequence. In some154939-1760-0399 2Attorney Docket No. 701586-000163WQPTembodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a receptor tyrosine kinase sequence.

[0066] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a toll-like receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of atoll-like receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a toll-like receptor sequence.

[0067] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a T-cell receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a T-cell receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a T-cell receptor sequence.

[0068] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a B-cell receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a B-cell receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a B-cell receptor sequence.

[0069] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a transporter sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a transporter sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a transporter sequence.

[0070] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a solute carrier sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a solute carrier sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a solute carrier sequence.

[0071] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a proton pump sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a proton pump sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a proton pump sequence.

[0072] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises an ion channel sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of an ion channel sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of an ion channel sequence.

[0073] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a porin sequence. In some embodiments of any of the aspects, an extracellular domain or164939-1760-0399 2Attorney Docket No. 701586-000163WQPTextracellular loop consists of a porin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a porin sequence.

[0074] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises an aquaporin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of an aquaporin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of an aquaporin sequence.

[0075] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a viroprotein sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a viroprotein sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a viroprotein sequence.

[0076] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises an integrin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of an integrin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of an integrin sequence.

[0077] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a cadherin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a cadherin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a cadherin sequence.

[0078] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a 7TM protein sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a 7TM protein sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a 7TM protein sequence.

[0079] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a nuclear receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a nuclear receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a nuclear receptor sequence.

[0080] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a gap junction protein sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a gap junction protein sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a gap junction protein sequence.

[0081] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a connexin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a connexin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a connexin sequence.174939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0082] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a selectin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a selectin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a selectin sequence.

[0083] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a spectrin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a spectrin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a spectrin sequence.

[0084] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises an N-CAM sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of an N-CAM sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of an N-CAM sequence.

[0085] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a claudin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a claudin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a claudin sequence.

[0086] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises an occludin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of an occludin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of an occludin sequence.

[0087] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises an Fc receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of an Fc receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of an Fc receptor sequence.

[0088] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a tetraspanin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a tetraspanin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a tetraspanin sequence.

[0089] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a transferrin receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a transferrin receptor sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a transferrin receptor sequence.

[0090] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a carrier protein sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a carrier protein sequence. In some embodiments of any of184939-1760-0399 2Attorney Docket No. 701586-000163WQPTthe aspects, an extracellular domain or extracellular loop consists essentially of a carrier protein sequence.

[0091] In some embodiments of any of the aspects, an extracellular domain or extracellular loop comprises a channelrhodopsin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists of a channelrhodopsin sequence. In some embodiments of any of the aspects, an extracellular domain or extracellular loop consists essentially of a channelrhodopsin sequence.

[0092] As used herein, “transmembrane domain” refers to a membrane-spanning protein domain. The sequences and structures of transmembrane domains are known in the art. In some embodiments of any of the aspects, the transmembrane domain of the membrane-spanning protein is a single pass transmembrane domain. In some embodiments of any of the aspects, the transmembrane domain of the membrane-spanning protein is a multi-pass transmembrane domain. Transmembrane domains include but are not limited to: G-protein coupled receptor (GPCR) sequences; receptor tyrosine kinases sequences; toll-like receptors sequences; T-cell receptors sequences; B-cell receptors sequences; transporters sequences; solute carriers sequences; proton pumps sequences; ion channels sequences; porins sequences; aquaporins sequences; viroprotein sequences; integrins sequences; cadherins sequences; 7TM protein sequences; nuclear receptor sequences; gap junction protein sequences; connexin sequences; selectin sequences; spectrin sequences; N-CAM sequences; claudin sequences; occludin sequences; Fc receptor sequences; tetraspanin sequences; transferrin receptor sequences; carrier protein sequences; or channelrhodopsin sequences. Information on the structure, sequence, and function of such transmembrane domains are available in the art, e.g., Avner Fink Biochimica et Biophysica Acta (BBA) - Biomembranes. Volume 1818, Issue 4, 2012, pp 974-983, which is incorporated by reference herein in its entirety.

[0093] In some embodiments of any of the aspects, a transmembrane domain comprises a G-protein coupled receptor (GPCR) sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a G-protein coupled receptor (GPCR) sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a G-protein coupled receptor (GPCR) sequence.

[0094] In some embodiments of any of the aspects, a transmembrane domain comprises a receptor tyrosine kinase sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a receptor tyrosine kinase sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a receptor tyrosine kinase sequence.

[0095] In some embodiments of any of the aspects, a transmembrane domain comprises a toll-like receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a toll -like receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a toll-like receptor sequence.194939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0096] In some embodiments of any of the aspects, a transmembrane domain comprises a T-cell receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a T-cell receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a T-cell receptor sequence.

[0097] In some embodiments of any of the aspects, a transmembrane domain comprises a B-cell receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a B-cell receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a B-cell receptor sequence.

[0098] In some embodiments of any of the aspects, a transmembrane domain comprises a transporter sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a transporter sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a transporter sequence.

[0099] In some embodiments of any of the aspects, a transmembrane domain comprises a solute carrier sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a solute carrier sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a solute carrier sequence.

[0100] In some embodiments of any of the aspects, a transmembrane domain comprises a proton pump sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a proton pump sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a proton pump sequence.

[0101] In some embodiments of any of the aspects, a transmembrane domain comprises an ion channel sequence. In some embodiments of any of the aspects, a transmembrane domain consists of an ion channel sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of an ion channel sequence.

[0102] In some embodiments of any of the aspects, a transmembrane domain comprises a porin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a porin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a porin sequence.

[0103] In some embodiments of any of the aspects, a transmembrane domain comprises an aquaporin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of an aquaporin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of an aquaporin sequence.

[0104] In some embodiments of any of the aspects, a transmembrane domain comprises a viroprotein sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a viroprotein sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a viroprotein sequence.204939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0105] In some embodiments of any of the aspects, a transmembrane domain comprises an integrin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of an integrin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of an integrin sequence.

[0106] In some embodiments of any of the aspects, a transmembrane domain comprises a cadherin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a cadherin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a cadherin sequence.

[0107] In some embodiments of any of the aspects, a transmembrane domain comprises a 7TM protein sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a 7TM protein sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a 7TM protein sequence.

[0108] In some embodiments of any of the aspects, a transmembrane domain comprises a nuclear receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a nuclear receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a nuclear receptor sequence.

[0109] In some embodiments of any of the aspects, a transmembrane domain comprises a gap junction protein sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a gap junction protein sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a gap junction protein sequence.

[0110] In some embodiments of any of the aspects, a transmembrane domain comprises a connexin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a connexin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a connexin sequence.

[0111] In some embodiments of any of the aspects, a transmembrane domain comprises a selectin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a selectin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a selectin sequence.

[0112] In some embodiments of any of the aspects, a transmembrane domain comprises a spectrin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a spectrin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a spectrin sequence.

[0113] In some embodiments of any of the aspects, a transmembrane domain comprises an N-CAM sequence. In some embodiments of any of the aspects, a transmembrane domain consists of an N-CAM sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of an N-CAM sequence.214939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0114] In some embodiments of any of the aspects, a transmembrane domain comprises a claudin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a claudin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a claudin sequence.

[0115] In some embodiments of any of the aspects, a transmembrane domain comprises an occludin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of an occludin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of an occludin sequence.

[0116] In some embodiments of any of the aspects, a transmembrane domain comprises an Fc receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists of an Fc receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of an Fc receptor sequence.

[0117] In some embodiments of any of the aspects, a transmembrane domain comprises a tetraspanin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a tetraspanin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a tetraspanin sequence.

[0118] In some embodiments of any of the aspects, a transmembrane domain comprises a transferrin receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a transferrin receptor sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a transferrin receptor sequence.

[0119] In some embodiments of any of the aspects, a transmembrane domain comprises a carrier protein sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a carrier protein sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a carrier protein sequence.

[0120] In some embodiments of any of the aspects, a transmembrane domain comprises a channelrhodopsin sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a channelrhodopsin sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a channelrhodopsin sequence.

[0121] In some embodiments of any of the aspects, a transmembrane domain comprises a WSC3 transmembrane domain sequence. In some embodiments of any of the aspects, a transmembrane domain consists of aWSC3 transmembrane domain sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of aWSC3 transmembrane domain sequence.

[0122] In some embodiments of any of the aspects, a transmembrane domain comprises a SS01 transmembrane domain sequence. In some embodiments of any of the aspects, a transmembrane domain consists of a SS01 transmembrane domain sequence. In some embodiments of any of the aspects, a transmembrane domain consists essentially of a SS01 transmembrane domain sequence.224939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0123] In some embodiments of any of the aspects, the extracellular domain or extracellular loop and the transmembrane domain are found in the same protein in nature. In some embodiments of any of the aspects, the target protein further comprises intracellular sequence which is found in the same protein in nature as the extracellular domain or extracellular loop. In some embodiments of any of the aspects, the target protein further comprises intracellular sequence which is found in the same protein in nature as the extracellular domain or extracellular loop and the transmembrane domain.

[0124] As used herein, “transcription factor domain” refers to the portion of the target protein which can interact with a DNA regulatory element to modulate expression of a reporter gene, e.g., when it is cleaved from the target protein. In some embodiments of any of the aspects, a cleaved transcription factor domain increases expression of a reporter gene when cleaved from the target protein. In some embodiments of any of the aspects, the transcription factor domain is located intracellularly when the target protein is expressed in a cell. In some embodiments of any of the aspects, the transcription factor domain is intracellular. Structures, sequences, and functions of transcription factors are known in the art and further information can be found, e.g., in Latchman 1997, The International Journal of Biochemistry & Cell Biology, 29(12), 1305-1312, and Yusuf 2012, Genome Biology 13:R24; each of which is incorporated by reference herein in its entirety.

[0125] In some embodiments of any of the aspects, the transcription factor domain comprises a DNA binding domain and a transcriptional activation domain. In some embodiments of any of the aspects, the transcription factor domain is a synthetic transcription factor domain comprising a DNA binding domain and transcriptional activation domain not found together in a naturally occurring protein. Structures, sequences, and functions of DNA binding domains and transcriptional activation domains, along with their cognate expression control sequences, are known in the art and further information can be found, e.g., in Harrison 1991, Nature 353(6346) 715, and Triezenberg 1995, Current Opinion in Genetics & Development, 5(2) 190-196; each of which is incorporated by reference herein in its entirety. Suitable domains and cognate expression control sequences are also described in databases such as the Registry of Standard Biological Parts, e.g., parts.igem.org / Protein_domains / DNA_binding; which is incorporated by reference in its entirety herein.

[0126] Suitable DNA binding domains include but are not limited to the DNA binding domains of: Gal4 DBD, LexA, Zif268, TetR, synthetic DNA-binding zinc finger protein domain (ZF), ZF 43-8, and dCas9. Suitable transcriptional activation domains include but are not limited to the transcriptional activation domains of VP 16, VP64, Gal4 AD, Msn2, and rTA.

[0127] In some embodiments of any of the aspects, a DNA binding domain is a DNA binding domain of Gal4 DBD. In some embodiments of any of the aspects, a DNA binding domain comprises a DNA binding domain of Gal4 DBD. In some embodiments of any of the aspects, a DNA binding234939-1760-0399 2Attorney Docket No. 701586-000163WQPTdomain consists of a DNA binding domain of Gal4 DBD. In some embodiments of any of the aspects, a DNA binding domain consists essentially of a DNA binding domain of Gal4 DBD.

[0128] In some embodiments of any of the aspects, a DNA binding domain is a DNA binding domain of LexA. In some embodiments of any of the aspects, a DNA binding domain comprises a DNA binding domain of LexA. In some embodiments of any of the aspects, a DNA binding domain consists of a DNA binding domain of LexA. In some embodiments of any of the aspects, a DNA binding domain consists essentially of a DNA binding domain of LexA.

[0129] In some embodiments of any of the aspects, a DNA binding domain is a DNA binding domain of Zif268. In some embodiments of any of the aspects, a DNA binding domain comprises a DNA binding domain of Zif268. In some embodiments of any of the aspects, a DNA binding domain consists of a DNA binding domain of Zif268. In some embodiments of any of the aspects, a DNA binding domain consists essentially of a DNA binding domain ofZif268.

[0130] In some embodiments of any of the aspects, a DNA binding domain is a DNA binding domain of TetR. In some embodiments of any of the aspects, a DNA binding domain comprises a DNA binding domain of TetR. In some embodiments of any of the aspects, a DNA binding domain consists of a DNA binding domain of TetR. In some embodiments of any of the aspects, a DNA binding domain consists essentially of a DNA binding domain of TetR.

[0131] In some embodiments of any of the aspects, a DNA binding domain is a DNA binding domain of synthetic DNA-binding zinc finger protein domain (ZF). In some embodiments of any of the aspects, a DNA binding domain comprises a DNA binding domain of synthetic DNA-binding zinc finger protein domain (ZF). In some embodiments of any of the aspects, a DNA binding domain consists of a DNA binding domain of synthetic DNA-binding zinc finger protein domain (ZF). In some embodiments of any of the aspects, a DNA binding domain consists essentially of a DNA binding domain of synthetic DNA-binding zinc finger protein domain (ZF).

[0132] In some embodiments of any of the aspects, a DNA binding domain is a DNA binding domain of ZF 43-8. In some embodiments of any of the aspects, a DNA binding domain comprises a DNA binding domain of ZF 43-8. In some embodiments of any of the aspects, a DNA binding domain consists of a DNA binding domain of ZF 43-8. In some embodiments of any of the aspects, a DNA binding domain consists essentially of a DNA binding domain ofZF 43-8.

[0133] In some embodiments of any of the aspects, a DNA binding domain is a DNA binding domain of dCas9. In some embodiments of any of the aspects, a DNA binding domain comprises a DNA binding domain of dCas9. In some embodiments of any of the aspects, a DNA binding domain consists of a DNA binding domain of dCas9. In some embodiments of any of the aspects, a DNA binding domain consists essentially of a DNA binding domain of dCas9.

[0134] In some embodiments of any of the aspects, a transcriptional activation domain is VP 16. In some embodiments of any of the aspects, a transcriptional activation domain comprises a244939-1760-0399 2Attorney Docket No. 701586-000163WQPTtranscriptional activation domain of VP 16. In some embodiments of any of the aspects, a transcriptional activation domain consists of a transcriptional activation domain of VP 16. In some embodiments of any of the aspects, the DNA binding domain consists essentially of VP 16.

[0135] In some embodiments of any of the aspects, a transcriptional activation domain is VP64. In some embodiments of any of the aspects, a transcriptional activation domain comprises a transcriptional activation domain ofVP64. In some embodiments of any of the aspects, a transcriptional activation domain consists of a transcriptional activation domain ofVP64. In some embodiments of any of the aspects, the DNA binding domain consists essentially of VP64.

[0136] In some embodiments of any of the aspects, a transcriptional activation domain is Gal4 AD. In some embodiments of any of the aspects, a transcriptional activation domain comprises a transcriptional activation domain of Gal4 AD. In some embodiments of any of the aspects, a transcriptional activation domain consists of a transcriptional activation domain of Gal4 AD. In some embodiments of any of the aspects, a transcriptional activation domain consists essentially of a transcriptional activation domain of Gal4 AD.

[0137] In some embodiments of any of the aspects, a transcriptional activation domain is Msn2. In some embodiments of any of the aspects, a transcriptional activation domain comprises a transcriptional activation domain of Msn2. In some embodiments of any of the aspects, a transcriptional activation domain consists of a transcriptional activation domain of Msn2. In some embodiments of any of the aspects, a transcriptional activation domain consists essentially of a transcriptional activation domain of Msn2.

[0138] In some embodiments of any of the aspects, a transcriptional activation domain is rTA. In some embodiments of any of the aspects, a transcriptional activation domain comprises a transcriptional activation domain of rTA. In some embodiments of any of the aspects, a transcriptional activation domain consists of a transcriptional activation domain of rTA. In some embodiments of any of the aspects, a transcriptional activation domain consists essentially of a transcriptional activation domain of rTA.

[0139] As used herein, “protease domain” refers to a portion of the binding protein which can catalyze proteolysis of the protease cleavage target domain of the target protein when the a) extracellular domain of the target protein and b) extracellular binding domain of the binding protein or binding dimer are bound to each other. In some embodiments of any of the aspects, the protease domain is located intracellularly when the set is expressed in a cell. In some embodiments of any of the aspects, the protease domain is intracellular.

[0140] As used herein, “protease cleavage target domain” refers to a portion of the target protein which undergoes proteolysis by the protease domain of the binding protein when the a) extracellular domain of the target protein and b) extracellular binding domain of the binding protein or binding dimer are bound to each other. The protease cleavage target domain may also be referred to as the254939-1760-0399 2Attorney Docket No. 701586-000163WQPTprotease cleavage site interchangeably herein. In one embodiment of any of the aspects, the at least one protease cleavage target domain is located between the transmembrane domain and the intracellular transcription factor domain of the target protein.

[0141] In some embodiments of any of the aspects, the at least one intracellular protease domain recognizes and cleaves the intracellular protease cleavage target domain, e.g, when the a) extracellular domain of the target protein and b) extracellular binding domain of the binding protein or binding dimer are bound to each other. In some embodiments of any of the aspects, the at least one intracellular protease domain specifically recognizes and cleaves the intracellular protease cleavage target domain, e.g., when the a) extracellular domain of the target protein and b) extracellular binding domain of the binding protein or binding dimer are bound to each other.

[0142] Further information on proteases and protease cleavage target domains is provided elsewhere herein.

[0143] As used herein, “binding protein” refers to a transmembrane protein which is capable of undergoing a binding interaction with the target protein or off-target protein if the binding protein or binding dimer is in proximity to the target protein and comprises a sequence that is recognizable by (or recognizes) the target protein. In some embodiments of any of the aspects, the binding protein comprises i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) at least one intracellular protease domain.

[0144] In some embodiments of any of the aspects, a binding protein comprises i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) at least one intracellular protease domain. In some embodiments of any of the aspects, a binding protein consists essentially of i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) at least one intracellular protease domain. In some embodiments of any of the aspects, a binding protein consists of i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) at least one intracellular protease domain.

[0145] In some embodiments of any of the aspects, a binding protein comprises i) an extracellular binding domain; ii) a transmembrane domain; and iii) an intracellular protease domain. In some embodiments of any of the aspects, a binding protein consists essentially of i) an extracellular binding domain; ii) a transmembrane domain; and iii) an intracellular protease domain. In some embodiments of any of the aspects, a binding protein consists of i) an extracellular binding domain; ii) a transmembrane domain; and iii) an intracellular protease domain.

[0146] Alternatively, the elements of a “binding protein” may be present as “binding dimer”, e.g., consist of a dimerizing protein pair, with the pair collectively comprising i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) at least one intracellular protease domain.264939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0147] When a target protein and a binding protein are in physical proximity, the extracellular domain of the target protein and the extracellular binding domain of the binding protein will be able to interact. If the two domains are capable of binding, e.g., specific binding, under the environmental conditions, the domains will bind to each other. This will retain the target protein and binding protein in physical proximity and the intracellular domains of the two proteins will be able to interact and result in protease cleavage as described elsewhere herein. If the extracellular domain of the target protein and the extracellular binding domain of the binding protein are not capable of binding, e.g., specific binding, under the environmental conditions, the domains will not bind to each other. This will permit the target protein and binding protein to move away from each other, and the intracellular domains of the two proteins will not interact and protease cleavage will not occur.

[0148] In some embodiments of any of the aspects, the binding protein is not a naturally occurring protein. In some embodiments of any of the aspects, the binding protein is not a protein found in nature. In some embodiments of any of the aspects, the combination of the binding protein’s binding domain, transmembrane domain, and protease domain are a combination not found in a single naturally occurring protein.

[0149] As used herein, “binding domain” refers to the portion of the binding protein or binding dimer which can interact with the extracellular domain or extracellular loop of the target protein if the binding protein or binding dimer is in proximity to the target protein and comprises a sequence that is recognizable by (or recognizes) the target protein. The binding domain can optionally include a native intracellular domain. In some embodiments of any of the aspects, the binding domain is not a naturally occurring polypeptide sequence. In some embodiments of any of the aspects, the binding domain is not a polypeptide sequence found in nature. In some embodiments of any of the aspects, the binding domain does not comprise a naturally occurring polypeptide sequence. In some embodiments of any of the aspects, the binding domain does not comprise a polypeptide sequence found in nature. In some embodiments of any of the aspects, the binding domain sequence is not a naturally occurring sequence. In some embodiments of any of the aspects, the binding domain sequence is not a sequence found in nature.

[0150] In some embodiments of any of the aspects, the binding domain can be an antibody or antibody reagent. In some embodiments of any of the aspects, the binding domain comprises an antibody or antibody reagent. In some embodiments of any of the aspects, the binding domain consists essentially of an antibody or antibody reagent. In some embodiments of any of the aspects, the binding domain consists of an antibody or antibody reagent.

[0151] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms274939-1760-0399 2Attorney Docket No. 701586-000163WOPTincluding full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a nanobody, a functionally active epitope -binding portion thereof, and / or bifunctional hybrid antibodies.

[0152] Each heavy chain is composed of a variable region of said heavy chain (abbreviated here as HCVR or VH) and a constant region of said heavy chain. The heavy chain constant region consists of three domains CHI, CH2 and CH3. Each light chain is composed of a variable region of said light chain (abbreviated here as LCVR or VL) and a constant region of said light chain. The light chain constant region consists of a CL domain. The VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR). Each VH and VL region thus consists of three CDRs and four FRs which are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.

[0153] As used herein, the term “antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments as well as complete antibodies.

[0154] An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.

[0155] In some embodiments, the antibody is a fully human antibody. In some embodiments, the antibody is a humanized antibody or antibody reagent. In some embodiments, the antibody is a fully humanized antibody or antibody reagent. In some embodiments, the antibody is a chimeric antibody or antibody reagent. In some embodiments, the antibody is a recombinant polypeptide.

[0156] Further information on binding domains is provided elsewhere herein.284939-1760-0399 2Attorney Docket No. 701586-000163WOPT

[0157] In some embodiments of any of the aspects, the set further comprises at least one off-target protein. In some embodiments of any of the aspects, the off-target protein comprises i) at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

[0158] As used herein, “off-target protein” refers to a protein other than the target protein of interest. If the binding protein or binding dimer is in proximity to the off-target protein and comprises a sequence that is recognizable by (or recognizes) the off-target protein, a binding interaction can occur. A binding interaction between a binding protein or binding dimer and an off-taiget protein would indicate that the binding protein or binding dimer is non-specific to the target protein.Therefore, the inclusion of off-target proteins in the system provides a negative selection control. In some embodiments of any of the aspects, the off-target protein is not a naturally occurring protein. In some embodiments of any of the aspects, the off-target protein is not a protein found in nature. In some embodiments of any of the aspects, the combination of the off-target protein’s at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein, at least one transmembrane domain, at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein, and at least one intracellular protease cleavage target domain are a combination not found in a single naturally occurring protein. For off-target proteins, the protease of the binding protein is able to interact with both the target protein’s protease cleavage site and the off-target protein’s protease cleavage site if the protease domain and the protease cleavage site are in close proximity (e.g., if the target protein and binding protein, or the off-target protein and binding protein are bound to each other), however, the protease cleavage site of the off-target protein need not be identical to the protease cleavage site of the target protein. In some embodiments of any of the aspects, the protease cleavage site of the off-target protein and the protease cleavage site of the target protein are identical.

[0159] As used herein, “not identical” means that less than 100% of the identity by sequence (e.g., amino acid sequence, nucleotide sequence) is the same. For not identical off-target proteins or elements thereof, not identical can mean a variant of the target protein (or target protein’s element), a wild-type version of the target protein (or target protein’s element), a disease variant of a wild type target protein (or target protein elements), a family member of the target protein (or target protein’s element), or a different protein entirely, allowing screening for different off-target effects. For not identical transcription factors, not identical can mean the transcription factor is able to bind to294939-1760-0399 2Attorney Docket No. 701586-000163WOPTdifferent DNA, and activate a different gene, allowing for different readouts depending on whether a binding protein binds a target protein, off-target protein, or both.

[0160] In some embodiments of any of the aspects, the extracellular domain or extracellular loop and the transmembrane domain are found in the same protein in nature. In some embodiments of any of the aspects, the off-target protein further comprises intracellular sequence which is found in the same protein in nature as the extracellular domain or extracellular loop. In some embodiments of any of the aspects, the off-target protein further comprises intracellular sequence which is found in the same protein in nature as the extracellular domain or extracellular loop and the transmembrane domain.

[0161] Where a target protein or off-target protein comprises further elements than the protease cleavage target and the transcription factor domain in the intracellular portion of the protein, the additional elements can be located ii) between the cleavage target domain and the transcription factor domain, ii) closer in the amino acid chain to the transmembrane domain than both the cleavage target domain and the transcription factor domain and / or iii) further from the transmembrane domain than both the cleavage target domain and the transcription factor domain.

[0162] In some embodiments of any of the aspects, the extracellular binding domain of the binding protein (or binding dimer) can specifically bind the extracellular domain (or extracellular loop) of the target protein. Upon binding of the binding protein and target protein, the intracellular protease domain of the binding protein (or binding dimer) is brought into contact with the intracellular protease cleavage target domain of the target protein and cleaves the cleavage target sequence. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene.

[0163] In some embodiments of any of the aspects, the extracellular binding domain of the binding protein (or binding dimer) can specifically bind the extracellular domain (or extracellular loop) of the off-target protein. Upon binding of the binding protein and off-target protein, the intracellular protease domain of the binding protein (or binding dimer) is brought into contact with the intracellular protease cleavage target domain of the off-target protein and cleaves the cleavage off-target sequence. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene. Expression of the off-target reporter gene provides an indication that the binding protein or binding dimer is non-specific to the target protein.

[0164] The sets described herein can also utilize split protease technology, e.g., where two different proteins each comprise a portion of a protease, wherein when the two different proteins are in close proximity, the portions of the protease will complement each other and collectively exhibit protease activity. In other words, the split protease reconstitutes when the binding protein or binding dimer comprising a first portion of a split protease comes into proximity with a target protein or off-target304939-1760-0399 2Attorney Docket No. 701586-000163WQPTprotein comprising a second portion of the split protease. Such split protease technology is known in the art, e.g., Dolberg, Nat Chem Biol 17, 531-539 (2021), and J. Bae, ChemBioChem 2024, 25, e202400123, each of which is incorporated by reference herein in their entireties.

[0165] Accordingly, in some embodiments of any of the aspects, described herein is a set of proteins comprising: a) a target protein comprising: i) at least one extracellular domain and / or extracellular loop; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain; iv) an intracellular first portion of a split protease, located between the transmembrane domain and the intracellular transcription factor domain; and v) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain; and b) a binding protein or binding dimer comprising: i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) an intracellular second portion of the split protease.

[0166] In one aspect of any of the embodiments, the first portion of the split protease can be located between the protease cleavage target domain and the transcription factor domain. In one aspect of any of the embodiments, the first portion of the split protease can be located closer in the amino acid chain of the protein to the transmembrane domain than both the protease cleavage target domain and the transcription factor domain.

[0167] In some embodiments of any of the aspects, the first portion of the split protease is a N-terminal fragment of the split protease, and the second portion of the split protease is a C-terminal fragment of the split protease. In one aspect of any of the embodiments, the first portion of the split protease is a C-terminal fragment of the split protease, and the second portion of the split protease is a N-terminal fragment of the split protease.

[0168] In some embodiments of any of the aspects, the first portion of the split protease comprises a N-terminal fragment of the split protease and the second portion of the split protease comprises a C-terminal fragment of the split protease. In one aspect of any of the embodiments, the first portion of the split protease comprises a C-terminal fragment of the split protease and the second portion of the split protease comprises a N-terminal fragment of the split protease.

[0169] In some embodiments of any of the aspects, the first portion of the split protease consists of a N-terminal fragment of the split protease and the second portion of the split protease consists of a C-terminal fragment of the split protease. In one aspect of any of the embodiments, the first portion of the split protease consists of a C-terminal fragment of the split protease and the second portion of the split protease consists of a N-terminal fragment of the split protease.

[0170] In some embodiments of any of the aspects, the first portion of the split protease consists essentially of a N-terminal fragment of the split protease and the second portion of the split protease consists essentially of a C-terminal fragment of the split protease. In one aspect of any of the embodiments, the first portion of the split protease consists essentially of a C-terminal fragment of the314939-1760-0399 2Attorney Docket No. 701586-000163WQPTsplit protease and the second portion of the split protease consists essentially of a N-terminal fragment of the split protease.

[0171] In some embodiments of any of the aspects, the set of proteins further comprises: a) at least one off-target protein comprising: i) at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein; iv) an intracellular first portion of a split protease, located between the transmembrane domain and the intracellular transcription factor domain; and v) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

[0172] In some embodiments of any of the aspects, the protease or split protease binds to and cleaves the intracellular protease cleavage target domain if the binding protein (or binding dimer) is bound to the target protein (or off-target protein) via the extracellular binding domain and the extracellular domain (or loop) In some embodiments of any of the aspects, the protease or split protease is selected from the group consisting of: Tobacco Etch Virus (TEV) protease and 3C-protease.

[0173] Suitable TEV protease variants include but are not limited to wild type H75E N-terminal split TEV protease, H75S N-terminal split TEV protease, H75T N-terminal split TEV protease, L190K C-terminal split TEV protease, PE10 C-terminal split TEV protease, PH21 C-terminal split TEV protease. In some aspects of any of the embodiments, suitable TEV proteases include mutants of wild type H75E N-terminal split TEV protease, H75S N-terminal split TEV protease, H75T N-terminal split TEV protease, L190K C-terminal split TEV protease, PE 10 C-terminal split TEV protease, PH21 C-terminal split TEV protease. In some aspects of any of the embodiments, suitable TEV proteases include combinations of mutants of wild type H75E N-terminal split TEV protease, H75S N-terminal split TEV protease, H75T N-terminal split TEV protease, L190K C-terminal split TEV protease, PE 10 C-terminal split TEV protease, PH21 C-terminal split TEV protease.

[0174] In some embodiments of any of the aspects, a TEV protease is a wild type H75E N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a wild type H75E N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a wild type H75E N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a wild type H75E N-terminal split TEV protease.

[0175] In some embodiments of any of the aspects, a TEV protease is a wild type H75S N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a wild type H75S N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a wild type H75S N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a wild type H75S N-terminal split TEV protease.324939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0176] In some embodiments of any of the aspects, a TEV protease is a wild type H75T N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a wild type H75T N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a wild type H75T N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a wild type H75T N-terminal split TEV protease.

[0177] In some embodiments of any of the aspects, a TEV protease is a wild type L190K C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a wild type L190K C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a wild type L190K C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a wild type L190K C-terminal split TEV protease.

[0178] In some embodiments of any of the aspects, a TEV protease is a wild type PE 10 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a wild type PE 10 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a wild type PE 10 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a wild type PE10 C-terminal split TEV protease.

[0179] In some embodiments of any of the aspects, a TEV protease is a wild type PH21 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a wild type PH21 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a wild type PH21 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a wild type PH21 C-terminal split TEV protease.

[0180] In some embodiments of any of the aspects, a TEV protease is a mutant of H75E N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a mutant of H75E N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a mutant of H75E N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a mutant of H75E N-terminal split TEV protease.

[0181] In some embodiments of any of the aspects, a TEV protease is a mutant of H75S N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a mutant of H75S N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a mutant of H75S N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a mutant of H75S N-terminal split TEV protease.

[0182] In some embodiments of any of the aspects, a TEV protease is a mutant of H75T N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a mutant of H75T N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a mutant of H75T N-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a mutant of H75T N-terminal split TEV protease.334939-1760-0399 2Attorney Docket No. 701586-000163WOPT

[0183] In some embodiments of any of the aspects, a TEV protease is a mutant of L190K C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a mutant of L190K C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a mutant of L190K C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a mutant of LI 901< C-terminal split TEV protease.

[0184] In some embodiments of any of the aspects, a TEV protease is a mutant of PE 10 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a mutant of PE 10 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a mutant of PE 10 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a mutant of PE10 C-terminal split TEV protease.

[0185] In some embodiments of any of the aspects, a TEV protease is a mutant of PH21 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease comprises a mutant of PH21 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists of a mutant of PH21 C-terminal split TEV protease. In some embodiments of any of the aspects, a TEV protease consists essentially of a mutant of PH21 C-terminal split TEV protease.

[0186] Split protease variants, including TEV variants, variants are known in the art and are discussed in, for example, L. Yi, Proc. Natl. Acad. Sci. U.S.A. 110 (18) 7229-7234, (2013); Carl A. Denard, ACS Synthetic Biology 2021 10 (1), 63-71; J. Bae, ChemBioChem 2024, 25, e202400123; Dolberg, T.B., Nat Chem Biol 17, 531-539 (2021); Hailey I EdelsteinSynthetic Biology, Volume 5, Issue 1, 2020; Rachel B Kapust, Biochemical and Biophysical Research Communications, Volume 294, Issue 5, 2002, p 949-955; Mateo I. Sanchez and Alice Y. Ting. Nature Methods 17, 177-174 (2020); and Michael S. Packer, Nature Communications 8, 956 (2017), each of which is incorporated by reference herein in their entireties.

[0187] In some embodiments of any of the aspects, the intracellular first portion of a split protease of an off-target protein is not identical to the intracellular first portion of a split protease of the target protein. In some embodiments of any of the aspects, the intracellular first portion of a split protease of an off-target protein and the intracellular first portion of a split protease of the target protein are variants of the same split protease. In some embodiments of any of the aspects, the intracellular first portion of a split protease of an off-target protein is identical to the intracellular first portion of a split protease of the target protein.

[0188] In some embodiments of any of the aspects, the extracellular binding domain of the binding protein (or binding dimer) can specifically bind the extracellular domain (or extracellular loop) of the target protein. Upon binding of the binding protein and target protein, the intracellular split protease domain (C-terminus) of the binding protein (or binding dimer) is brought into contact with the intracellular split protease of the target protein (N-terminus). The protease reconstitutes and cleaves344939-1760-0399 2Attorney Docket No. 701586-000163WOPTthe protease cleavage site. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene.

[0189] In some embodiments of any of the aspects, the extracellular binding domain of the binding protein (or binding dimer) can specifically bind the extracellular domain (or extracellular loop) of the target protein. Upon binding of the binding protein and target protein, the intracellular split protease domain (N-terminus) of the binding protein (or binding dimer) is brought into contact with the intracellular split protease of the target protein (C-terminus). The protease reconstitutes and cleaves the protease cleavage site. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene.

[0190] In some embodiments of any of the aspects, the extracellular binding domain of the binding protein (or binding dimer) can specifically bind the extracellular domain (or extracellular loop) of the off-target protein. Upon binding of the binding protein and off-target protein, the intracellular split protease domain (C-terminus) of the binding protein (or binding dimer) is brought into contact with the intracellular split protease of the off-target protein (N-terminus). The protease reconstitutes and cleaves the protease cleavage site. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene. Expression of the off-target reporter gene provides indication that the binding protein or binding dimer is non-specific to the target protein.

[0191] In some embodiments of any of the aspects, the extracellular binding domain of the binding protein (or binding dimer) can specifically bind the extracellular domain (or extracellular loop) of the off-target protein. Upon binding of the binding protein and off-target protein, the intracellular split protease domain (N-terminus) of the binding protein (or binding dimer) is brought into contact with the intracellular split protease of the off-target protein (C-terminus). The protease reconstitutes and cleaves the protease cleavage site. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene. Expression of the off-target reporter gene provides indication that the binding protein or binding dimer is non-specific to the target protein.

[0192] Cognate protease cleavage target domains / sites for a given protease are well known in the art, e.g., are described in the references provided herein. As an illustrative example, suitable protease cleavage target sites for TEV protease are provided in Table 1. In some embodiments of any of the aspects, the protease cleavage site has the sequence of one of SEQ ID NOs: 1-27:Table 1: Examples of suitable protease cleavage target sites for TEV protease.SEQ ID NO: SEQUENCE354939-1760-0399 2Attorney Docket No. 701586-000163WQPTSEQ ID NO 1 ENLYFQMSEQ ID NO 2 ENLYFQLSEQ ID NO 3 ENLYFQESEQ ID NO 4 ENLYFQISEQ ID NO 5 ENLYFQVSEQ ID NO 6 ENLYFQPSEQ ID NO 7 ENLYFQKSEQ ID NO 8 ENLYFQRSEQ ID NO 9 ENLYFQNSEQ ID NO 10 ENLYFQYSEQ ID NO 11 ENLYFQKSEQ ID NO 12 ENLYFQDSEQ ID NO 13 ENLYFQQSEQ ID NO 14 ENLYFQFSEQ ID NO 15 ENLYFQTSEQ ID NO 16 ENLYFQWSEQ ID NO 17 MGSENLYFQMSEQ ID NO 18 MGSENLYFQASEQ ID NO 19 MGSENLYFQLSEQ ID NO 20 AGSENLYFQMSEQ ID NO 21 AGSENLYFQASEQ ID NO 22 AGSENLYFQLSEQ ID NO 23 YGSENLYFQMSEQ ID NO 24 YGSENLYFQASEQ ID NO 25 YGSENLYFQLSEQ ID NO 26 ENLYFEMSEQ ID NO 27 ENLYFHM

[0193] In some embodiments of any of the aspects, the protease is TEV and the protease cleavage site has the sequence of one of SEQ ID NOs: 1-27.

[0194] In some embodiments of any of the aspects, the extracellular binding domain is an antibody or antibody reagent. Suitable extracellular binding domains include, but are not limited to: antibodies, antibody reagents, antibody-based scaffold, full length antibodies, Fab, F(ab’)2, scFy, single-domain antibodies, BiTEs, diabodies, triabodies, tetrabodies, bivalent antibodies, bispecific antibodies, bivalent and bispecific antibodies, DARPins, affibodies, adnectins, anticalins, avimers, knottins, centyrins, scaffoldins, three-finger toxins, minibinders, peptide-based scaffolds, peptides, bicyclic peptides, macrocylic peptides, protein domains, engineered proteins, Fibmectin type III domains, tetratricopeptide repeats, SH3 domains, PDZ domains, chemokines, cytokines, and computationally designed binders.

[0195] As used herein, the terms “minibinder” and “miniprotein binder” are used interchangeably and generally refer to a protein that is less than 100 amino acids in length. In some embodiments of any of the aspects, the minibinder or miniprotein binder is between about 37 and 65 amino acids in364939-1760-0399 2Attorney Docket No. 701586-000163WQPTlength. In some embodiments of any of the aspects, the minibinder or miniprotein binder is less than 40 amino acids in length. In some embodiments of any of the aspects, the minibinder or miniprotein binder is computationally designed. In some embodiments of any of the aspects, the minibinder or miniprotein binder can bind to a target molecule. Miniprotein binders are discussed in the art in, for example, Weinberg et al. (2024) eLife 13:RP96154, the contents of which are incorporated herein in its entirety. Exemplary miniprotein binders are described in, for example, Muratspahic et al., bioRxiv 2025.03.23.644666, Cao et al., Nature 605 (2022) 551-560 and Watson et al., Nature 620 (2023) 1089-1100, the contents of each of which are hereby incorporated in their entireties.

[0196] In some embodiments of any of the aspects, the extracellular binding domain is an antibody. In some embodiments of any of the aspects, the extracellular binding domain is an antibody-based scaffold. In some embodiments of any of the aspects, the extracellular binding domain is a full-length antibody. In some embodiments of any of the aspects, the extracellular binding domain is a Fab. In some embodiments of any of the aspects, the extracellular binding domain is a F(ab')2. In some embodiments of any of the aspects, the extracellular binding domain is a scFv. In some embodiments of any of the aspects, the extracellular binding domain is a single-domain antibody. In some embodiments of any of the aspects, the extracellular binding domain is a BiTEs. In some embodiments of any of the aspects, the extracellular binding domain is a diabody. In some embodiments of any of the aspects, the extracellular binding domain is a triabody. In some embodiments of any of the aspects, the extracellular binding domain is a tetrabody. In some embodiments of any of the aspects, the extracellular binding domain is a bivalent antibody. In some embodiments of any of the aspects, the extracellular binding domain is a bispecific antibody. In some embodiments of any of the aspects, the extracellular binding domain is a bivalent and bispecific antibody. In some embodiments of any of the aspects, the extracellular binding domain is a DARPin. In some embodiments of any of the aspects, the extracellular binding domain is an aflfibody. In some embodiments of any of the aspects, the extracellular binding domain is an adnectin. In some embodiments of any of the aspects, the extracellular binding domain is an anticalin. In some embodiments of any of the aspects, the extracellular binding domain is an avimer. In some embodiments of any of the aspects, the extracellular binding domain is a knottin. In some embodiments of any of the aspects, the extracellular binding domain is a centyrin. In some embodiments of any of the aspects, the extracellular binding domain is a scaffoldin. In some embodiments of any of the aspects, the extracellular binding domain is a three-finger toxin. In some embodiments of any of the aspects, the extracellular binding domain is a minibinder. In some embodiments of any of the aspects, the extracellular binding domain is a peptide-based scaffold. In some embodiments of any of the aspects, the extracellular binding domain is a peptide. In some embodiments of any of the aspects, the extracellular binding domain is a bicyclic peptide. In some embodiments of any of the aspects, the extracellular binding domain is a macrocyclic peptide. In some embodiments of any of the aspects, the extracellular binding domain is a protein domain. In some 374939-1760-0399 2Attorney Docket No. 701586-000163WQPTembodiments of any of the aspects, the extracellular binding domain is an engineered protein. In some embodiments of any of the aspects, the extracellular binding domain is a Fibronectin type III domain. In some embodiments of any of the aspects, the extracellular binding domain is a tetratricopeptide repeat. In some embodiments of any of the aspects, the extracellular binding domain is a SH3 domain. In some embodiments of any of the aspects, the extracellular binding domain is a PDZ domain. In some embodiments of any of the aspects, the extracellular binding domain is a chemokine. In some embodiments of any of the aspects, the extracellular binding domain is a cytokine. In some embodiments of any of the aspects, the extracellular binding domain is a computationally designed binder.

[0197] In some embodiments of any of the aspects, the extracellular binding domain comprises an antibody. In some embodiments of any of the aspects, the extracellular binding domain comprises an antibody-based scaffold. In some embodiments of any of the aspects, the extracellular binding domain comprises a full-length antibody. In some embodiments of any of the aspects, the extracellular binding domain comprises a Fab. In some embodiments of any of the aspects, the extracellular binding domain comprises a F(ab')2. In some embodiments of any of the aspects, the extracellular binding domain comprises a scFv. In some embodiments of any of the aspects, the extracellular binding domain comprises a single-domain antibody. In some embodiments of any of the aspects, the extracellular binding domain comprises a BiTEs. In some embodiments of any of the aspects, the extracellular binding domain comprises a diabody. In some embodiments of any of the aspects, the extracellular binding domain comprises a triabody. In some embodiments of any of the aspects, the extracellular binding domain comprises a tetrabody. In some embodiments of any of the aspects, the extracellular binding domain comprises a bivalent antibody. In some embodiments of any of the aspects, the extracellular binding domain comprises a bispecific antibody. In some embodiments of any of the aspects, the extracellular binding domain comprises a bivalent and bispecific antibody. In some embodiments of any of the aspects, the extracellular binding domain comprises a DARPin. In some embodiments of any of the aspects, the extracellular binding domain comprises an affibody. In some embodiments of any of the aspects, the extracellular binding domain comprises an adnectin. In some embodiments of any of the aspects, the extracellular binding domain comprises an anticalin. In some embodiments of any of the aspects, the extracellular binding domain comprises an avimer. In some embodiments of any of the aspects, the extracellular binding domain comprises a knottin. In some embodiments of any of the aspects, the extracellular binding domain comprises a centyrin. In some embodiments of any of the aspects, the extracellular binding domain comprises a scaffoldin. In some embodiments of any of the aspects, the extracellular binding domain comprises a three-finger toxin. In some embodiments of any of the aspects, the extracellular binding domain comprises a minibinder. In some embodiments of any of the aspects, the extracellular binding domain comprises a peptide-based scaffold. In some embodiments of any of the aspects, the extracellular binding domain comprises a peptide. In some embodiments of any of the aspects, the extracellular binding domain 384939-1760-0399 2Attorney Docket No. 701586-000163WQPTcomprises a bicyclic peptide. In some embodiments of any of the aspects, the extracellular binding domain comprises a macrocyclic peptide. In some embodiments of any of the aspects, the extracellular binding domain comprises a protein domain. In some embodiments of any of the aspects, the extracellular binding domain comprises an engineered protein. In some embodiments of any of the aspects, the extracellular binding domain comprises a Fibronectin type III domain. In some embodiments of any of the aspects, the extracellular binding domain comprises a tetratricopeptide repeat. In some embodiments of any of the aspects, the extracellular binding domain comprises a SH3 domain. In some embodiments of any of the aspects, the extracellular binding domain comprises a PDZ domain. In some embodiments of any of the aspects, the extracellular binding domain comprises a chemokine. In some embodiments of any of the aspects, the extracellular binding domain comprises a cytokine. In some embodiments of any of the aspects, the extracellular binding domain comprises a computationally designed binder.

[0198] In some embodiments of any of the aspects, the extracellular binding domain consists of an antibody. In some embodiments of any of the aspects, the extracellular binding domain consists of an antibody-based scaffold. In some embodiments of any of the aspects, the extracellular binding domain consists of a full-length antibody. In some embodiments of any of the aspects, the extracellular binding domain consists of a Fab. In some embodiments of any of the aspects, the extracellular binding domain consists of a F(ab')2. In some embodiments of any of the aspects, the extracellular binding domain consists of a scFv. In some embodiments of any of the aspects, the extracellular binding domain consists of a single-domain antibody. In some embodiments of any of the aspects, the extracellular binding domain consists of a BiTEs. In some embodiments of any of the aspects, the extracellular binding domain consists of a diabody. In some embodiments of any of the aspects, the extracellular binding domain consists of a triabody. In some embodiments of any of the aspects, the extracellular binding domain consists of a tetrabody. In some embodiments of any of the aspects, the extracellular binding domain consists of a bivalent antibody. In some embodiments of any of the aspects, the extracellular binding domain consists of a bispecific antibody. In some embodiments of any of the aspects, the extracellular binding domain consists of a bivalent and bispecific antibody. In some embodiments of any of the aspects, the extracellular binding domain consists of a DARPin. In some embodiments of any of the aspects, the extracellular binding domain consists of an affibody. In some embodiments of any of the aspects, the extracellular binding domain consists of an adnectin. In some embodiments of any of the aspects, the extracellular binding domain consists of an anticalin. In some embodiments of any of the aspects, the extracellular binding domain consists of an avimer. In some embodiments of any of the aspects, the extracellular binding domain consists of a knottin. In some embodiments of any of the aspects, the extracellular binding domain consists of a centyrin. In some embodiments of any of the aspects, the extracellular binding domain consists of a scaffoldin. In some embodiments of any of the aspects, the extracellular binding domain consists of a three-finger toxin. In some embodiments of any of the aspects, the extracellular binding domain consists of a 394939-1760-0399 2Attorney Docket No. 701586-000163WQPTminibinder. In some embodiments of any of the aspects, the extracellular binding domain consists of a peptide-based scaffold. In some embodiments of any of the aspects, the extracellular binding domain consists of a peptide. In some embodiments of any of the aspects, the extracellular binding domain consists of a bicyclic peptide. In some embodiments of any of the aspects, the extracellular binding domain consists of a macrocyclic peptide. In some embodiments of any of the aspects, the extracellular binding domain consists of a protein domain. In some embodiments of any of the aspects, the extracellular binding domain consists of an engineered protein. In some embodiments of any of the aspects, the extracellular binding domain consists of a Fibronectin type III domain. In some embodiments of any of the aspects, the extracellular binding domain consists of a tetratricopeptide repeat. In some embodiments of any of the aspects, the extracellular binding domain consists of a SH3 domain. In some embodiments of any of the aspects, the extracellular binding domain consists of a PDZ domain. In some embodiments of any of the aspects, the extracellular binding domain consists of a chemokine. In some embodiments of any of the aspects, the extracellular binding domain consists of a cytokine. In some embodiments of any of the aspects, the extracellular binding domain consists of a computationally designed binder.

[0199] In some embodiments of any of the aspects, the extracellular binding domain consists essentially of an antibody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of an antibody-based scaffold. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a full-length antibody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a Fab. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a F(ab')2. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a scFv. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a single-domain antibody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a BiTEs. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a diabody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a triabody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a tetrabody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a bivalent antibody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a bispecific antibody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a bivalent and bispecific antibody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a DARPin. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of an affibody. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of an adnectin. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of an anticalin. In some embodiments of any of the 404939-1760-0399 2Attorney Docket No. 701586-000163WQPTaspects, the extracellular binding domain consists essentially of an avimer. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a knottin. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a centyrin. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a scaffoldin. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a three -finger toxin. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a minibinder. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a peptide-based scaffold. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a peptide. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a bicyclic peptide. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a macrocyclic peptide. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a protein domain. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of an engineered protein. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a Fibronectin type III domain. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a tetratricopeptide repeat. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a SH3 domain. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a PDZ domain. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a chemokine. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a cytokine. In some embodiments of any of the aspects, the extracellular binding domain consists essentially of a computationally designed binder.

[0200] In some embodiments of any of the aspects, the at least one transmembrane domain of the binding protein or binding dimer is a single-pass transmembrane domain. In some embodiments of any of the aspects, the at least one transmembrane domain of the binding protein or binding dimer is a multi-pass transmembrane domain. In some embodiments of any of the aspects, the binding protein or binding dimer further comprises a linker between the at least one extracellular binding domain and the at least one transmembrane domain.

[0201] As used herein, linker is defined as a sequence of amino acids within a protein that connects distinct protein domains. In some embodiments of any of the aspects, the linker comprises one or more alanine, glycine, and / or serine residues. In some embodiments of any of the aspects, the linker comprises one or more glycine and / or serine residues. In some embodiments of any of the aspects, the linker is a flexible linker.

[0202] In some embodiments of any of the aspects, the linker comprises 2-60 amino acids. In some embodiments of any of the aspects, the linker comprises 20-40 amino acids.414939-1760-0399 2Attorney Docket No. 701586-000163WOPT

[0203] In some embodiments of any of the aspects, the set of proteins comprises a binding dimer wherein the binding dimer comprises: a) a first partner protein comprising: i) at least one extracellular binding domain; and ii) a first dimerization domain; and b) a second partner protein comprising: i) a second dimerization domain that binds to the first dimerization domain; ii) at least one transmembrane domain; and iii) one of: 1) at least one intracellular protease domain that recognizes and cleaves the intracellular protease cleavage target domain; or 2) an intracellular second portion of the split protease. As used herein, partner protein is defined as one of the two proteins that form a binding dimer, which collectively comprises at least one extracellular domain, at least one transmembrane domain, and at least one intracellular proteinase domain (or second portion of a split protease). As used herein, dimerization domain is defined as the region of the first partner protein or second partner protein or which is able to interact and bind with a cognate dimerization domain of the other of the second partner protein or first partner protein.

[0204] Suitable dimerization domains are known in the art. Exemplary dimerization domains are provided herein. In some embodiments of any of the aspects, the first dimerization domain and the second dimerization domain are, in either order, SPYTAG™-SPYCATCHER™. In some embodiments of any of the aspects, the first dimerization domain and the second dimerization domain are, in either order, ABI1-PYL1 In some embodiments of any of the aspects, the first dimerization domain and the second dimerization domain are, in either order, aeyCFaN-CfaC. In some aspects of any of the embodiments, the first dimerization domain and the second dimerization domain are, in either order, GID1-GAI.

[0205] In some embodiments of any of the aspects, the extracellular binding domain of the binding dimer can specifically bind the extracellular domain (or extracellular loop) of the target protein. Upon binding of the binding dimer and target protein, the intracellular protease domain of the binding dimer is brought into contact with the intracellular protease cleavage target domain of the target protein and cleaves the cleavage target sequence. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene.

[0206] In some embodiments of any of the aspects, the extracellular binding domain of the binding dimer can specifically bind the extracellular domain (or extracellular loop) of the off-target protein. Upon binding of the binding dimer and off-target protein, the intracellular protease domain of the binding dimer is brought into contact with the intracellular protease cleavage target domain of the off-target protein and cleaves the cleavage off-target sequence. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene. Expression of the off-target reporter gene provides indication that the binding dimer is non-specific to the target protein.424939-1760-0399 2Attorney Docket No. 701586-000163WOPT

[0207] In some embodiments of any of the aspects, the extracellular binding domain of the binding dimer can specifically bind the extracellular domain (or extracellular loop) of the target protein. Upon binding of the binding dimer and target protein, the intracellular split protease domain (C-terminus) of the binding dimer is brought into contact with the intracellular split protease of the target protein (N-terminus). The protease reconstitutes and cleaves the protease cleavage site. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene.

[0208] In some embodiments of any of the aspects, the extracellular binding domain of the binding dimer can specifically bind the extracellular domain (or extracellular loop) of the target protein. Upon binding of the binding dimer and target protein, the intracellular split protease domain (N-terminus) of the binding dimer is brought into close contact with the intracellular split protease of the target protein (C-terminus). The protease reconstitutes and cleaves the protease cleavage site. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene.

[0209] In some embodiments of any of the aspects, the extracellular binding domain of the binding dimer can specifically bind the extracellular domain (or extracellular loop) of the off-target protein. Upon binding of the binding dimer and off-target protein, the intracellular split protease domain (C-terminus) of the binding dimer is brought into close contact with the intracellular split protease of the off-target protein (N-terminus). The protease reconstitutes and cleaves the protease cleavage site. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene. Expression of the off-target reporter gene provides indication that the binding dimer is non-specific to the target protein.

[0210] In some embodiments of any of the aspects, the extracellular binding domain of the binding dimer can specifically bind the extracellular domain (or extracellular loop) of the off-target protein. Upon binding of the binding dimer and off-target protein, the intracellular split protease domain (N-terminus) of the binding dimer is brought into contact with the intracellular split protease of the off-target protein (C-terminus). The protease reconstitutes and cleaves the protease cleavage site. This cleavage releases the intracellular transcription factor domain, which can then translocate to the nucleus, bind a DNA sequence, and activate transcription of a reporter gene. Expression of the off-target reporter gene provides indication that the binding dimer is non-specific to the target protein.

[0211] In some embodiments of any of the aspects described herein is an expression system comprising: a) a set of proteins as described herein; and b) a nucleic acid molecule comprising: i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control sequence modulated by the intracellular transcription factor domain of the off-target protein, and ii) operably linked to a reporter gene.434939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0212] As used herein, an “expression control sequence” is defined as a segment of a nucleic acid molecule which is specifically recognized by a DNA binding protein. Expression control sequences are known in the art and include promoters, enhancers, and the like. As discussed elsewhere herein, cognate expression control sequences for transcription factor domains and / or DNA binding domains are known in the art.

[0213] As used herein, reporter gene is defined as a nucleotide sequence which encodes an expression product that is, produces, or generates a detectable signal. Numerous reporter gene systems are known in the art and include, for example, alkaline phosphatase Berger, J., et al (1988) Gene 66 1-10; Kain, S.R. (1997) Methods. Mol. Biol. 63 49-60), 13 -galactosidase (See, U.S. Patent No. 5,070,012, issued Dec 3, 1991 to Nolan et al., and Bronstein, I., et al., (1989) J. Chemilum. Biolum. 499-111), chloramphenicol acetyltransferase (See Gorman et al., Mol Cell Biol. (1982) 2 1044-51), 13 -glucuronidase, peroxidase, 13-lactamase (U.S. Patent Nos. 5,741,657 and 5,955,604), catalytic antibodies, luciferases (U.S. Patents 5,221,623; 5,683,888; 5,674,713; 5,650,289; 5,843,746) and naturally fluorescent proteins (Tsien, R.Y. (1998) Annu. Rev. Biochem.67509-44).

[0214] Detectable signals, methods of detecting them are well known in the art. In some embodiments of any of the aspects, detectable signals can include signals that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable signals used in the methods described herein can be primary signals (where the signal comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary signal (where the reporter gene expression product binds to another moiety to produce a detectable signal). Detectable signals can include, but are not limited to bioluminescent compounds, chromophores, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes. In some embodiments of any of the aspects, the detectable signal is a fluorescent compound. In some embodiments of any of the aspects, the detectable signal is a chemiluminescent compound. In some embodiments of any of the aspects, the detectable signal is an enzymatic signal, e.g., an enzyme that can produce a chemiluminescent signal, a color signal, or a fluorescent signal.

[0215] A reporter gene encoding any fluorescent protein can be applicable in the technology described herein. The fluorescent protein can include, but is not limited to, for example, GFP, mCherry, Venus, and Cerulean. Examples of genes encoding fluorescent proteins that can be used in accordance with the compositions and methods described herein include, without limitation, those proteins provided in U.S. Patent Application No. 2012 / 0003630 (see Table 59), incorporated herein by reference in its entirety.444939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0216] Similarly, a reporter gene encoding any enzyme can be applicable as well. Enzymes that produce colored substrates ("colorimetric enzymes") can also be used for visualization and / or quantification. Enzymatic products can be quantified using spectrophotometers or other instruments that can take absorbance measurements including plate readers. Examples of genes encoding colorimetric enzymes that can be used in accordance with the compositions and methods described herein include, without limitation, lacZ alpha fragment, lacZ (encoding betagalactosidase, full-length), and xylE. An enzyme (e.g., glucose oxidase) can also change the conductivity of a reaction volume, permitting an electrical or electronic readout (Malitesta et al., Anal Chem 1990, 62, 2735-2740). In yet another example, an enzyme can separate a fluorescence resonance energy transfer (FRET) or quenching pair to induce a change in fluorescence.

[0217] A reporter gene encoding any antigen for which a specific antibody is available or can be made can also be applicable.

[0218] For non-limiting examples of reporter genes, see Reporter Genes: A Practical Guide, D. Anson (Ed.), 2007, Humana Press, the contents of which are incorporated by reference for examples on reporter genes.

[0219] A reporter gene encoding luciferases can also be used in the technology described herein. Luciferases produce luminescence, which can be readily quantified using a plate reader or luminescence counter. Examples of genes encoding luciferases for that can be used in accordance with the compositions and methods described herein include, without limitation, Rluc and firefly luciferase (from Photinus pyralis).

[0220] In some embodiments of any of the aspects, the detectable signal can be a cell phenotype, e.g., survival or growth under the selected culture conditions.

[0221] In some embodiments of any of the aspects described herein is an expression system comprising: a) one or more nucleic acid molecules encoding a set of proteins described herein; and b) a nucleic acid molecule comprising: i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control sequence modulated by the intracellular transcription factor domain of the off-target protein; and ii) operably linked to a reporter gene.

[0222] As used herein, a promoter is defined as a region of DNA upstream of a gene where proteins bind to initiate transcription of the gene. A constitutive promoter is active under all circumstances, whereas an inducible promoter must be activated.

[0223] In some embodiments of any of the aspects, the one or more nucleic acid molecule encoding a set of proteins further comprise a constitutive promoter operably linked to a sequence encoding the target protein, a constitutive promoter operably linked to a sequence encoding the binding protein, a constitutive promoter operably linked to a sequence encoding the first partner protein, a constitutive454939-1760-0399 2Attorney Docket No. 701586-000163WQPTpromoter operably linked to a sequence encoding the second partner protein, and / or a constitutive promoter operably linked to a sequence encoding the off-target protein.

[0224] In some embodiments of any of the aspects, the one or more nucleic acid molecule encoding a set of proteins further comprise an inducible promoter operably linked to a sequence encoding the target protein, an inducible promoter operably linked to a sequence encoding the binding protein, an inducible promoter operably linked to a sequence encoding the first partner protein, an inducible promoter operably linked to a sequence encoding the second partner protein, and / or an inducible promoter operably linked to a sequence encoding the off-target protein.

[0225] In some aspects of any of the embodiments, described herein is a cell comprising the set of proteins or expression system of any one of the aspects described herein. In some embodiments of any of the aspects, the cell is a yeast cell. In some embodiments of any of the aspects, the cell is a yeast cell selected from the group of species consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces unisporus, Saccharomyces servazzii (also referred to as Kazachstania servazzii), Saccharomyces kluyveri (also referred to as Lachancea kluyveri), Kluyveromyces marxianus, Pichia angusta (also referred to as Hansenula polymorpha or Ogataea polymorpha), Deharyomyces hansenii (also referred to as Candida famata), Candida tropicalis, and Yarrowia lipolytica. In some embodiments of any of the aspects, the yeast cell is of the species .S', cerevisiae.

[0226] In some embodiments of any of the aspects, the reporter gene is a fluorescent protein or yeast survival protein. In some embodiments of any of the aspects, the reporter gene is a fluorescent protein, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), TagBFP, Cerulean, Monomerig EGFP, Venus, Citrine, mCherry, mKate2, PA-GFP, or PA-mCherry. Fluorescent proteins are known in the art and are described in, for example, Snapp., Trends Cell Biol. 2009 Oct 8; 19(11): 649-655.

[0227] A yeast survival protein can convey a survival phenotype under culture with an antibiotic or provide the ability to metabolize or catabolize an essential nutrient (e.g., amino acid), or can be a gene necessary for cell survival and growth under any conditions. Some suitable yeast survival proteins include, but are not limited to, HIS3, URA3, LEU2, TRP1, LYS2, MET15, HIS6, ADE1, ADE2, KanamycinR, NourseothricinR, HygromycinR, and ZeocinR. Suitable yeast survival proteins, and appropriate culture conditions for detecting their expression are known in the art, e.g., Solis-Escalante, FEMS Yeast Research, 2013 13(1): 126-139 which is incorporated by reference herein in its entirety.

[0228] In some embodiments of any of the aspects, the reporter gene is HIS3. In some embodiments of any of the aspects, the reporter gene comprises HIS3. In some embodiments of any of the aspects, the reporter gene consists of HIS3. In some embodiments of any of the aspects, the reporter gene consists essentially of HIS3.464939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0229] In some embodiments of any of the aspects, the reporter gene is URA3. In some embodiments of any of the aspects, the reporter gene comprises URA3. In some embodiments of any of the aspects, the reporter gene consists of URA3. In some embodiments of any of the aspects, the reporter gene consists essentially of URA3.

[0230] In some embodiments of any of the aspects, the reporter gene is LEU2. In some embodiments of any of the aspects, the reporter gene comprises LEU2. In some embodiments of any of the aspects, the reporter gene consists of LEU2. In some embodiments of any of the aspects, the reporter gene consists essentially of LEU2.

[0231] In some embodiments of any of the aspects, the reporter gene is TRP 1. In some embodiments of any of the aspects, the reporter gene comprises TRP1. In some embodiments of any of the aspects, the reporter gene consists of TRP 1. In some embodiments of any of the aspects, the reporter gene consists essentially of TRP 1.

[0232] In some embodiments of any of the aspects, the reporter gene is LYS2. In some embodiments of any of the aspects, the reporter gene comprises LYS2. In some embodiments of any of the aspects, the reporter gene consists of LYS2. In some embodiments of any of the aspects, the reporter gene consists essentially ofLYS2.

[0233] In some embodiments of any of the aspects, the reporter gene is MET15. In some embodiments of any of the aspects, the reporter gene comprises MET15. In some embodiments of any of the aspects, the reporter gene consists of MET15. In some embodiments of any of the aspects, the reporter gene consists essentially ofMET15.

[0234] In some embodiments of any of the aspects, the reporter gene is HIS6. In some embodiments of any of the aspects, the reporter gene comprises HIS6. In some embodiments of any of the aspects, the reporter gene consists of HIS6. In some embodiments of any of the aspects, the reporter gene consists essentially of HIS6.

[0235] In some embodiments of any of the aspects, the reporter gene is ADE1. In some embodiments of any of the aspects, the reporter gene comprises ADE1. In some embodiments of any of the aspects, the reporter gene consists of ADE 1. In some embodiments of any of the aspects, the reporter gene consists essentially of ADE 1.

[0236] In some embodiments of any of the aspects, the reporter gene is ADE2. In some embodiments of any of the aspects, the reporter gene comprises ADE2. In some embodiments of any of the aspects, the reporter gene consists of ADE2. In some embodiments of any of the aspects, the reporter gene consists essentially of ADE2.

[0237] In some embodiments of any of the aspects, the reporter gene is KanamycinR. In some embodiments of any of the aspects, the reporter gene comprises KanamycinR. In some embodiments of any of the aspects, the reporter gene consists of KanamycinR. In some embodiments of any of the aspects, the reporter gene consists essentially of KanamycinR.474939-1760-0399 2Attorney Docket No. 701586-000163WOPT

[0238] In some embodiments of any of the aspects, the reporter gene is NourseothricinR. In some embodiments of any of the aspects, the reporter gene comprises NourseothricinR. In some embodiments of any of the aspects, the reporter gene consists of NourseothricinR. In some embodiments of any of the aspects, the reporter gene consists essentially of NourseothricinR.

[0239] In some embodiments of any of the aspects, the reporter gene is HygromycinR. In some embodiments of any of the aspects, the reporter gene comprises HygromycinR. In some embodiments of any of the aspects, the reporter gene consists of HygromycinR. In some embodiments of any of the aspects, the reporter gene consists essentially of HygromycinR.

[0240] In some embodiments of any of the aspects, the reporter gene is ZeocinR. In some embodiments of any of the aspects, the reporter gene comprises ZeocinR. In some embodiments of any of the aspects, the reporter gene consists of ZeocinR. In some embodiments of any of the aspects, the reporter gene consists essentially of ZeocinR.

[0241] In some aspects of any of the embodiments, described herein is a library comprising a plurality of cells of any one of the aspects described herein.

[0242] As used herein, library is defined as a plurality of cells which are not all identical, wherein the cells are not identical at least in that the cell comprises distinct binding proteins, distinct target proteins, and / or distinct off-target proteins. In some embodiments of any of the aspects, the library comprises a plurality of cells, each of the cells comprising distinct binding proteins. In some embodiments of any of the aspects, the library comprises a plurality of cells, each of the cells comprising distinct binding proteins, wherein the binding proteins are distinct because of their different binding domains. A library of cells having different binding proteins permits high-throughput screening of potential or candidate binding domains.

[0243] In some aspects of any of the embodiments, described herein is a method comprising detecting a signal of a reporter gene of i) an expression system of as described herein or ii) a cell as described herein.

[0244] In some embodiments of any of the aspects, a plurality of different binding proteins or a plurality of different first partner proteins is expressed in a plurality of cells. In some embodiments of any of the aspects, a plurality of different binding proteins is expressed in a plurality of cells. In some embodiments of any of the aspects, a plurality of different first partner proteins is expressed in a plurality of cells.

[0245] In some embodiments of any of the aspects, detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates the extracellular binding domain of the binding protein binds to the extracellular domain (or extracellular loop) of the target protein.

[0246] In some embodiments of any of the aspects, detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of484939-1760-0399 2Attorney Docket No. 701586-000163WOPTthe off-target protein indicates the extracellular binding domain of the binding protein is not specific for binding to the extracellular domain (or extracellular loop) of the target protein.

[0247] In some embodiments of any of the aspects, detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein and no detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the off-target protein indicates the extracellular binding domain of the binding protein specifically binds to the extracellular domain (or extracellular loop) of the target protein.

[0248] In some embodiments of any of the aspects, the extracellular binding domain of the binding protein or binding dimer is an antibody or antibody reagent, the target protein comprises at least one extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of a therapeutic target protein, and the detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates the extracellular binding domain of the binding protein is binds to the therapeutic target protein.

[0249] In some embodiments of any of the aspects, the extracellular binding domain of the binding protein or binding dimer is an antibody or antibody reagent, the target protein comprises at least one extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of a therapeutic target protein, and the strength of the detected signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates a binding affinity of the extracellular binding domain of the binding protein for the therapeutic target protein.

[0250] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising”). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of’ the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.494939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0251] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0252] Other aspects and embodiments of the invention provide the aspects and embodiments described herein with the term "comprising" replaced by the term "consisting of." Other aspects and embodiments of the invention provide the aspects and embodiments described herein with the term "comprising" replaced by the term "consisting essentially of."

[0253] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level.

[0254] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or signal, an “increase” is a statistically significant increase in such level.

[0255] As used herein, the terms “protein" and “polypeptide" are used interchangeably to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of 504939-1760-0399 2Attorney Docket No. 701586-000163WOPTamino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0256] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0257] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested to confirm that a desired activity.

[0258] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; lie into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu,514939-1760-0399 2Attorney Docket No. 701586-000163WQPTinto Tyr or into lie; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into lie or into Leu.

[0259] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” or a “functional portion” is a fragment, portion, or segment of a polypeptide which retains at least 50% of the wild-type reference polypeptide’s activity. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0260] In some embodiments, the polypeptide described herein can be a variant of a polypeptide sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a protein or fragment thereof that retains activity of the native or reference polypeptide. A wide variety of, for example, PCR-based, site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan to generate and test artificial variants.

[0261] A variant amino acid or DNA sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).

[0262] A variant amino acid sequence can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to a native or reference sequence. Accordingly, the percentage of “sequence similarity” is the percentage of amino acids which is either identical or conservatively changed; e.g., “sequence similarity” = (% sequence identity) +(% conservative changes). It should be understood that a sequence that has a specified percent similarity to a reference sequence necessarily encompasses a sequence with the same specified percent identity to that reference sequence. The skilled person will be aware of various computer programs, using different mathematical algorithms, that are available to determine the identity or similarity between two sequences. For instance, use can be made of a computer program employing the Needleman and Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego U.S.A.); the algorithm of E. Meyers and W. Miller (Meyers et al.524939-1760-0399 2Attorney Docket No. 701586-000163WQPT(1989)) which has been incorporated into the ALIGN program (version 2.0); or more preferably the BLAST (Basic Local Alignment Tool using default parameters); see e.g., US Patent 10,023,890, the content of which is incorporated by reference herein in its entirety.

[0263] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. A wide variety of, site-specific mutagenesis approaches, e.g., Kunkel’s method, cassette mutagenesis, PCR site-directed mutagenesis (e.g., traditional PCR, primer extension, or inverse PCR), whole plasmid mutagenesis, in vivo site-directed mutagenesis, CRISPR / Cas-guided mutagenesis, are known in the art and can be applied by the ordinarily skilled artisan to introduce mutations into specific nucleic acid loci. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Braman, Jeff, ed. (2002) In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol. 182 (2nd ed.); Khudyakov and Fields (2002), Artificial DNA: Methods and Applications, CRC Press; Hsu et al. (2014), Cell 157 (6): 1262-78; Cerchione et al. (2020) PLOS ONE 15 (4): e0231716; and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0264] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double -stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single -stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0265] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and534939-1760-0399 2Attorney Docket No. 701586-000163WOPTprocessing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0266] In some embodiments, the expression of a target(s) or gene / polypeptide described herein is / are tissue-specific. In some embodiments, the expression of a target(s) or gene / polypeptide described herein is / are global. In some embodiments, the expression of a target(s) or gene / polypeptide described herein is systemic.

[0267] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g.5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0268] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0269] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0270] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one reporter gene or signal. As used herein, the term "detecting" or “measuring” refers to observing a signal from, e.g. a probe, label, target molecule, or reporter gene expression product to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.544939-1760-0399 2Attorney Docket No. 701586-000163WOPT

[0271] Accordingly, as used herein, "selectively binds" or “specifically binds” or “specific binding” in reference to the interaction of an antibody, or antibody fragment thereof, or a binding protein described herein, means that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope or target) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody. Generally, specific binding is preferably achieved with a dissociation constant (Kd) of 1 nM or lower, 100 pM or lower; or 10 pM or lower. In certain embodiments, a binding protein or antibody or antigen-binding fragment thereof that specifically binds to an antigen binds to that antigen with a KDgreater than 106M, 107M, 108M, IO'9M, 1010M, 1011M, 1012M, 1013M, 1014M. In other embodiments, a binding protein or antibody or antigen binding fragment thereof that specifically binds to an antigen binds to that antigen with a KDbetween IO-6and 10-7M, IO-6and I0-8M, IO-6and 109M, 10'6and 1010M, IO6and 10nM, IO6and 1012M, IO6and 1013M, IO6and 1014M, IO9and 1010M, IO9and I011M, IO9and 1012M, IO9and 1013M, IO9and 1014M. In some embodiments, a binding protein or antibody or antigen-binding fragment thereof binds to an epitope, with a KDIO5M (10000 nM) or less, e.g., IO6M, 107M, 108M, IO9M, 1010M, 1011M, 1012M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent. The person of ordinary skill in the art can determine appropriate conditions under which the polypeptide agents described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay. In certain embodiments, a binding protein or antibody or antigen-binding fragment thereof is said to “specifically bind” an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. Binding proteins, antibodies or antigen-binding fragments that bind to the same or similar epitopes will likely cross-compete (one prevents the binding or modulating effect of the other). Cross-competition, however, can occur even without epitope overlap, e.g., if epitopes are adjacent in three-dimensional space and / or due to steric hindrance.

[0272] Specific binding can comprise ionic bonding, hydrogen bonds, ionic bonds, van der Waals interactions, and / or London dispersion forces. In some embodiments, specific binding does not refer to covalent bonding. In some embodiments, specific binding does not refer to a peptide bond. In some embodiments, specific binding does not refer to a phosphodiester bond.

[0273] As used herein, “binding” refers to interaction between macromolecules (e.g. the target protein and the binding protein). In some cases binding will be sequence-specific. It will be appreciated, however, that not all components of a binding interaction need be sequence-specific. “Affinity” refers to the strength of binding, such that increased binding affinity correlates with a lower Kd value. It will be appreciated, however, that depending on the amino acid sequence it may bind to554939-1760-0399 2Attorney Docket No. 701586-000163WOPTor recognize more than one target sequence, although typically one sequence will be bound in preference to any other recognized sequences, depending on the relative specificity of the individual interactions.

[0274] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0275] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0276] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0277] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0278] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0279] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0280] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0281] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely fortheir disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the564939-1760-0399 2Attorney Docket No. 701586-000163WQPTinformation available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0282] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0283] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0284] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in cell biology, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia ofMolecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and574939-1760-0399 2Attorney Docket No. 701586-000163WOPTCurrent Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0285] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.

[0286] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0287] Embodiments of various aspects described herein can be defined as in any of the following numbered embodiments:

[0288] Embodiment 1: A set of proteins comprising: a) a target protein comprising: i) at least one extracellular domain and / or extracellular loop; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain; and b) a binding protein or binding dimer comprising: i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) at least one intracellular protease domain.

[0289] Embodiment 2: The set of proteins of any one of the preceding Embodiments, further comprising: c) at least one off-target protein comprising: i) at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.584939-1760-0399 2Attorney Docket No. 701586-000163WQPT

[0290] Embodiment 3: A set of proteins comprising: a) a target protein comprising: i) at least one extracellular domain and / or extracellular loop; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain; iv) an intracellular first portion of a split protease, located between the transmembrane domain and the intracellular transcription factor domain; and v) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain; and b) a binding protein or binding dimer comprising: i) at least one extracellular binding domain; ii) at least one transmembrane domain; and iii) an intracellular second portion of the split protease.

[0291] Embodiment 4: The set of proteins of Embodiment 3, wherein the first portion of the split protease is a N-terminal fragment of the split protease and the second portion of the split protease is a C-terminal fragment of the split protease.

[0292] Embodiment 5: The set of proteins of Embodiment 3, wherein the first portion of the split protease is a C-terminal fragment of the split protease and the second portion of the split protease is a N-terminal fragment of the split protease.

[0293] Embodiment 6: The set of proteins of any one of Embodiments 3-4, further comprising: c) at least one off-target protein comprising: i) at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein; ii) at least one transmembrane domain; iii) at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein; iv) an intracellular first portion of a split protease, located between the transmembrane domain and the intracellular transcription factor domain; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

[0294] Embodiment 7: The set of proteins of any one of the preceding Embodiments, wherein the extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of the target protein are: G-protein coupled receptor (GPCR) sequences; receptor tyrosine kinases sequences; tolllike receptors sequences; T-cell receptors sequences; B-cell receptors sequences; transporters sequences; solute carriers sequences; proton pumps sequences; ion channels sequences; porins sequences; aquaporins sequences; viroprotein sequences; integrins sequences; cadherins sequences; 7TM protein sequences; nuclear receptor sequences; gap junction protein sequences; connexin sequences; selectin sequences; spectrin sequences; N-CAM sequences; claudin sequences; occludin sequences; Fc receptor sequences; tetraspanin sequences; transferrin receptor sequences; carrier protein sequences; or channelrhodopsin sequences.

[0295] Embodiment 8: The set of proteins of any one of the preceding Embodiments, wherein the transcription factor domain is a polypeptide sequence comprising a DNA binding domain and a transcriptional activation domain.594939-1760-0399 2Attorney Docket No. 701586-000163WOPT

[0296] Embodiment 9: The set of proteins of any one of the preceding Embodiments, wherein the protease or split protease binds to and cleaves the intracellular protease cleavage target domain.

[0297] Embodiment 10: The set or proteins of any one of the preceding Embodiments, wherein the protease or split protease is selected from the group consisting of TEV protease and 3C protease.

[0298] Embodiment 11 : The set of proteins of any one of the preceding Embodiments, wherein the intracellular first portion of a split protease of an off-target protein is not identical to the intracellular first portion of a split protease of the target protein.

[0299] Embodiment 12: The set of proteins of any one of the preceding Embodiments, wherein the intracellular first portion of a split protease of an off-target protein and the intracellular first portion of a split protease of the target protein are variants of the same split protease.

[0300] Embodiment 13: The set of proteins of any one of the preceding Embodiments, wherein the protease cleavage site is selected from one of SEQ ID NOs: 1-27.

[0301] Embodiment 14: The set of proteins of any one of the preceding Embodiments, wherein the protease is TEV and the protease cleavage site is selected from one of SEQ ID NOs: 1-27.

[0302] Embodiment 15: The set of proteins of any one of the preceding Embodiments, wherein the extracellular binding domain is an antibody or antibody reagent.

[0303] Embodiment 16: The set of proteins of any one of the preceding Embodiments, wherein the at least one transmembrane domain of the binding protein or binding dimer is a single-pass transmembrane domain.

[0304] Embodiment 17: The set of proteins of any one of the preceding Embodiments, wherein the binding protein or binding dimer further comprises a linker between the at least one extracellular binding domain and the at least one transmembrane domain.

[0305] Embodiment 18: The set of proteins of Embodiment 14, wherein the linker comprises 2-60 amino acids.

[0306] Embodiment 19: The set of proteins of Embodiment 14, wherein the linker comprises 20-40 amino acids.

[0307] Embodiment 20: The set of proteins of any one of the preceding Embodiments, comprising a binding dimer wherein the binding dimer comprises: a) a first partner protein comprising: i) at least one extracellular binding domain; and ii) a first dimerization domain; and b) a second partner protein comprising: i) a second dimerization domain that binds to the first dimerization domain; ii) at least one transmembrane domain; and iii) one of: 1) at least one intracellular protease domain that recognizes and cleaves the intracellular protease cleavage target domain; or 2) an intracellular second portion of the split protease.

[0308] Embodiment 21: An expression system comprising: a) a set of proteins of any one of the preceding Embodiments; and a) a nucleic acid molecule comprising: i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control604939-1760-0399 2Attorney Docket No. 701586-000163WOPTsequence modulated by the intracellular transcription factor domain of the off-target protein, and ii) operably linked to a reporter gene.

[0309] Embodiment 22: An expression system comprising: a) one or more nucleic acid molecule encoding a set of proteins of any one of the preceding Embodiments; and b) a nucleic acid molecule comprising: i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control sequence modulated by the intracellular transcription factor domain of the off-target protein; and ii) operably linked to a reporter gene.

[0310] Embodiment 23 : The expression system of Embodiment 22, wherein the one or more nucleic acid molecule encoding a set of proteins of any one of the preceding Embodiments further comprise a constitutive promoter operably linked to a sequence encoding the target protein, a constitutive promoter operably linked to a sequence encoding the binding protein, a constitutive promoter operably linked to a sequence encoding the first partner protein, a constitutive promoter operably linked to a sequence encoding the second partner protein, and / or a constitutive promoter operably linked to a sequence encoding the off-target protein.

[0311] Embodiment 24: The expression system of Embodiment 19, wherein the one or more nucleic acid molecule encoding a set of proteins of any one of the preceding Embodiments further comprise an inducible promoter operably linked to a sequence encoding the target protein, an inducible promoter operably linked to a sequence encoding the binding protein, an inducible promoter operably linked to a sequence encoding the first partner protein, an inducible promoter operably linked to a sequence encoding the second partner protein, and / or an inducible promoter operably linked to a sequence encoding the off-target protein.

[0312] Embodiment 25: A cell comprising the set of proteins or expression system of any one of the preceding Embodiments.

[0313] Embodiment 26: The cell of Embodiment 25, wherein the cell is a yeast cell.

[0314] Embodiment 27: The expression system or cell of any one of the preceding Embodiments, wherein the reporter gene is a fluorescent protein or yeast survival protein.

[0315] Embodiment 28: A library comprising a plurality of cells of any one of Embodiments 25-27.

[0316] Embodiment 29: A method comprising detecting a signal of a reporter gene of i) an expression system of any one of the preceding Embodiments or ii) a cell of any one of the preceding Embodiments.

[0317] The method of Embodiment 29, wherein a plurality of different binding proteins or a plurality of different first partner proteins of any one of the preceding Embodiments is expressed in a plurality of cells.

[0318] Embodiment 30: The method of any one of the preceding Embodiments, wherein detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates the extracellular binding domain is specific for binding to the target protein.614939-1760-0399 2Attorney Docket No. 701586-000163WOPT

[0319] Embodiment 31 : The method of any one of the preceding Embodiments, wherein detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the off-target protein indicates the extracellular binding domain is not specific for binding to the target protein.

[0320] Embodiment 32: The method of any one of the preceding Embodiments, wherein the extracellular binding domain of the binding protein or binding dimer is an antibody or antibody reagent, the target protein comprises at least one extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of a therapeutic target protein, and the detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates the extracellular binding domain is specific for binding to the therapeutic target protein.

[0321] Embodiment 33: The method of any one of the preceding Embodiments, wherein the extracellular binding domain of the binding protein or binding dimer is an antibody or antibody reagent, the target protein comprises at least one extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of a therapeutic target protein, and the strength of the detected signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates a binding affinity of the extracellular binding domain for the target protein.

[0322] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.EXAMPLES

[0323] Example 1: Method for discovering, optimizing, diversifying, and characterizing proteins that bind to target proteins.

[0324] Described herein is a novel method for discovering, optimizing, diversifying, and characterizing binding proteins (e.g. antibodies) that bind to protein targets of interest, especially membrane proteins. One embodiment of the technology disclosed herein is a genetic system in yeast that co-expresses binding protein(s) and target protein(s) at the cell membrane and then produces a signal output if a binding interaction occurs (FIG. 1). Non-limiting use cases of the technology disclosed herein include: 1) Biologies discovery: the systems described herein can be used to identify variants from a library of protein binders (e.g. antibodies) that can bind to human therapeutic target proteins, especially membrane proteins, 2) Biologies optimization: the systems described herein can be used to improve the affinity, specificity, or developability of a protein binder (e.g. antibody), 3) Biologies diversification: the systems described herein can be used to generate diverse variants of a protein binder (e.g. antibody) that are still capable of binding to its target, and 4) Characterization of protein-protein interactions: the systems described herein can be used to quantitatively assess the624939-1760-0399 2Attorney Docket No. 701586-000163WOPTaffinity of an interaction between two proteins, including an antibody (or any other type of binding protein) and its target.

[0325] Prior methods for discovering, optimizing, diversifying, and characterizing binding proteins generally require biochemical purification of the target protein. This can be failure-prone, idiosyncratic, low-throughput, slow and expensive, especially for biochemically challenging proteins such as multipass membrane proteins (e.g. GPCRs, solute transporters, ion channels). A few genetic systems have been devised for assaying membrane protein-protein interactions (MPPIs) within cells (e.g. E. coli, yeast, or mammalian cells). These systems use the split-ubiquitin architecture and are primarily in yeast. However, many examples show that this architecture is unusable for most applications due to severe defects, including extremely high rate of false positives, poor dynamic range, and limited tunability.

[0326] In one embodiment of any of the aspects, disclosed herein includes an MPPI assay (in yeast) which is the first to use an architecture based on proteases, rather than ubiquitin (FIG. 2). Provided herein is evidence that protease-based architectures overcome the limitations of the prior art and permit many new applications. Critically, two new features — expression level control and negative selection — dramatically improve the performance of the system.

[0327] The utility of the aspects disclosed herein are illustrated in several examples that would not be feasible with existing MPPI assays: 1) Discovery of novel antibodies for challenging, high-value human membrane protein drug targets, 2) Optimization of antibodies for affinity, specificity, and other developability properties, 3) Diversification of antibodies to find variants with new functional properties, and 4) High-throughput characterization of protein-protein interactions.Technical Description

[0328] A schematic of an exemplary split-protease MPPI assay version 2 is shown in FIG. 3. The key genetic components for an exemplary protease-recruitment MPPI assay are noted in FIG. 4. In both of these architectures, the genetic components are modular and tunable. Depending on the usecase or target protein, the assay can be customized by choosing from a number of promoter levels, binding proteins, proteases with different kinetics, synthetic transcription factors of various strengths, reporter genes for fluorescence or cell viability, among other potential factors. Likewise, the key genetic components for the exemplary version 1 split-protease MPPI assay are noted in FIG. 5A, and the key genetic components for the exemplary version 2 split-protease MPPI assay are noted in FIG.5BResults

[0329] FIGs. 6A and 6B demonstrate the protease -recruitment MPPI assay with a yeast receptorligand pair. FIG. 6A is a schematic of the exemplary protease-recruitment gene circuit. Ste2 is a yeast GPCRthat specifically recognizes the alpha-factor peptide (and does not recognize the apelin peptide). WSC3 and SS01 are Type I and Type II transmembrane domains, respectively, that were634939-1760-0399 2Attorney Docket No. 701586-000163WQPTused to anchor the peptides to the yeast membrane. TEVp is a protease that recognizes and cleaves a cleavage sequence (CS). Here, CS was mutated at a single residue for optimization of the signal output. LexA-VP16 is a synthetic transcription factor that, when released from Ste2 by TEVp, can localize to the nucleus and activate expression of GFP. FIG. 6B demonstrates the GFP output with alpha-factor and apelin displayed using the WSC3 and SSO1 anchoring domains.

[0330] FIGs. 7A and 7B demonstrate an exemplary split-protease MPPI assay (vl) with a yeast receptor-ligand pair. FIG. 7A is a schematic of the exemplary split-protease gene circuit. N-TEVp and C-TEVp are N-terminal and C- terminal portions, respectively, of a split TEV protease that can reconstitute and then recognize and cleave a cleavage sequence (CS). Here, CS was mutated at a single residue when testing with SSO1, and C-TEVp was mutated at a single residue when testing with WSC3, both for optimization of signal output. LexA-VP16 is a synthetic transcription factor that, when released from Ste2 by reconstituted TEVp, can localize to the nucleus and activate expression of GFP. FIG. 7B shows the GFP output with alpha-factor and apelin displayed using the WSC3 and SS01 anchoring domains.

[0331] FIGs. 8A-8D demonstrate the exemplary split-protease MPPI assay (vl) with antibodies that bind human & viral membrane protein drug targets. FIG. 8A is a schematic of the exemplary splitprotease gene circuit used to test nanobodies that bind the human APJ GPCR. FIG. 8B shows the GFP output with positive control nanobodies that bind APJ (JN241 , JN241.9) negative control nanobodies that do not bind APJ (BC2, Pep), and a negative control without any nanobody (-). The affinity for JN241 is 83 pM. FIG. 8C is a Schematic of the exemplary split-protease gene circuit used to test nanobodies that bind the HCMV US28 GPCR. FIG. 8D shows the GFP output with positive control nanobodies that bind US28 (VUN100, US28-2, US28-3, US28-4) and negative control nanobodies that do not bind US28 (Pep, BC2, JN241). Affinities for US28- binding nanobodies are shown.

[0332] FIGs. 9A-9C demonstrate expression control for the exemplary split-protease MPPI assay.FIG. 9A is a schematic of the exemplary split-protease gene circuit with expression control. The target protein is the BILF1 GPCR fused N-terminally to the BC2 peptide. Expression of the target is under control of a beta-estradiol-inducible promoter. Positive and negative control nanobodies targeting the BC2 peptide are fused to the SPYCATHCER™ protein and is tested with a series of constitutive promoters. The SPYTAG™ protein is anchored to the membrane with a WSC3 transmembrane domain, and expression of this is kept constant and dose-limiting with the weak RAD27 promoter. Transcription factor output drives expression of GFP and the HIS3 selectable marker. FIG. 9B shows the dose-response of GFP output vs. beta-estradiol for a series of nanobody display constructs that vary promoter level (RPL18B>RET2>RNR2>REV1) for a BC2 binding (BC2) and non-binding (JN241) nanobody. Inset shows yeast growth for a subset of constructs on media selecting for HIS3 expression. FIG. 9C shows characterization of US28-binding (blue) and non-644939-1760-0399 2Attorney Docket No. 701586-000163WOPTbinding (yellow) nanobodies in the original split-protease format (without expression control) and the improved SPYCATCHER™-SPYTAG™ format (with expression control).

[0333] FIGs. 10A-10B demonstrate negative selection (along with expression control) for the exemplary split-protease MPPI assay (v2). FIG. 10A is a schematic of the exemplary split-protease gene circuit with expression control and negative selection. The target protein is the BILF1 GPCR fused N-terminally to the BC2 peptide. The transcription factor fused to the target controls expression of GFP and the HIS3 selectable marker. The off-target protein is the US28 GPCR. The transcription factor fused to US28 controls expression of RFP and the URA3 marker, which can be negatively selected against. The C- terminal protease fragments are the same for the two targets and can both reconstitute with the N-terminal protease fragment bound to the nanobody anchor. FIG. 10B shows growth of yeast expressing the gene circuit with nanobodies that either bind the target (BC2 nanobody) or the off-target (US28 nanobody). Cells were grown on media that does not select for reporter output, only selects for HIS3 expression, or selects for HIS3 expression and against URA3 expression. Improvement in background growth is boxed.

[0334] FIGs. 11A-11D demonstrate the exemplary v2 split-protease MPPI assay with antibodies that bind human membrane protein & soluble drug targets. FIG. 11A is a schematic of the exemplary v2 split-protease gene circuit used to test nanobodies that bind targets of interest. In FIG. 11B and FIG. 11C, the target is a human GPCR. In FIG. 11D, the target is a soluble BC2 peptide tethered to the C-terminal protease fragment using a GPCR. FIG. 11B shows growth of yeast expressing the gene circuit with the AT 1 R GPCR as the target and nanobodies that either bind AT 1 R (AT 118i4, AT 118) or do not. Cells were grown on media that does not select for reporter output or on media that selects for HIS3 expression and against URA3 expression. FIG. 11C shows growth of yeast expressing the gene circuit with the APJ GPCR as the target and nanobodies that either bind APJ (JN241) or do not. Cells were grown on media that does not select for reporter output or on media that selects for HIS3 expression and against URA3 expression. FIG. 11D shows growth of yeast expressing the gene circuit with the BC2 peptide as the target fused to a GPCR, and nanobodies that either bind BC2 or do not. Cells were grown on media that does not select for reporter output or on media that selects for HIS3 expression and against URA3 expression.

[0335] FIGs. 12A-12D demonstrate a quantitative characterization of the exemplary split-protease MPPI assay. FIG. 12A is a schematic of the exemplary split-protease gene circuit used to characterize the relationship between affinity and assay output. The target is the yeast STE2 GPCR and the binding proteins are various mutants of the receptor’s peptide ligand, alpha-factor. STE2 is under control of a beta-estradiol inducible promoter. FIG. 12B shows mutants of the alpha-factor peptide tested and their reported affinities. FIG. 12C shows GFP out for each of the alpha-factor mutants vs. their reported affinities. The dashed line corresponds to a non-binding protein (APLN nanobody). FIG. 12D shows growth of yeast expressing a subset of the alpha-factor variants, the APLN nanobody, or654939-1760-0399 2Attorney Docket No. 701586-000163WOPTno binding protein. Yeast were grown in non-selective media (SDC) or media that selects for HIS3 expression (SD- His) with various levels of STE2 induction via beta-estradiol (EST). Growth was assessed by measuring OD600 after 15 hrs growth in liquid media at 30 °C.

[0336] FIGs. 13A-13B demonstrate the generalizability of the exemplary v2 split-protease MPPI assay for challenging, high-value drug targets. FIG. 13A is a schematic of the exemplary v2 splitprotease gene circuit used to test generalizability across multiple targets of interest. Since binding proteins are not available for many high-value drug targets, the BC2 peptide was fused N-terminally to each target, and the expression of the target was verified using a BC2-binding nanobody. FIG. 13B shows growth of yeast expressing the gene circuit with various membrane protein targets in the presence of either a BC2-binding nanobody or a non-binding nanobody. Cells were grown on media that does not select for reporter output (without selection) or on media that selects for HIS3 expression and against URA3 expression (with selection). SSTR5, APJ and AT1R are human GPCRs. BILF1 is a viral GPCR. LMP2B is a viral multipass membrane protein. SLC7A11 is a human solute transporter.

[0337] FIGs. 14A-14D demonstrate the discovery of novel antibodies for challenging, high-value multipass membrane protein drug targets. FIG. 14A shows candidate nanobody binders discovered using the v2 split-protease MPPI assay for multiple membrane protein drug targets. A naive, 10E9-member nanobody library was transformed into yeast encoding the MPPI circuit and subject to selection for binding to the target of interest. Candidates were harvested and verified in yeast by retesting in the v2 assay using either the target (x-axis) or an off-target receptor (y-axis) and measuring fluorescence output. Potential hits are those with strong on-target and weak off-target binding. FIG.14B shows the verification of two BILFl-binding candidates isolated from discovery campaigns. Nanobodies were purified as Fc-fiisions and used to stain HEK cells expressing either BILF1-GFP or SSTR5-GFP. X-axis represents receptor quantity (GFP), and y-axis represents binding signal. FIG.14C is similar to FIG. 14B but for nanobodies discovered against SSTR . FIG. 14D is similar to FIG. 14B but for nanobodies discovered against APJ.

[0338] FIGs. 15A-15D demonstrate optimization of novel antibodies for a challenging, high-value multipass membrane protein drug target. FIG. 15A shows affinity maturation using the v2 splitprotease MPPI assay of a candidate nanobody binder discovered for the BILF1 GPCR. A scanning mutagenesis library of the candidate was transformed into yeast encoding the MPPI gene circuit and subject to a pooled growth selection. Growth rates for the variants were measured by tracking enrichment of each sequence overtime via sequencing. Growth selections were performed in duplicates as shown. Sequences with high enrichment are colored. FIG. 15B shows the verification of individual affinity matured variants in the v2 MPPI assay. Nbl98 is a naive BILF1 binder initially discovered using the v2 MPPI assay and then affinity matured. Select matured variants with strong growth rates were individually transformed into yeast encoding the MPPI gene circuit and subject to664939-1760-0399 2Attorney Docket No. 701586-000163WOPTgrowth selection. Yeast were grown on media that selects for HIS3 expression (CSM-His + 3AT) across a range of 3AT concentrations. All conditions also select against URA3 expression (not labeled). BC2 nanobody is a 1.4 nM binder against the BC2 peptide that was fused N-terminally to BILF1. FIG. 15C shows the verification of two affinity matured variants ofNbl98 isolated from affinity maturation campaigns. Nanobodies were purified as Fc-fusions and used to stain HEK cells expressing BILF1. X-axis represents nanobody concentration and y-axis represents binding signal. FIG. 15D shows the affinity matured variants display improvements in developability properties such as protein yield, precipitation, and specificity. Select variants were purified as Fc-fusions and characterized. Protein yield was measured using the standard bicinchoninic assay (BCA).Precipitation was assessed visually. Affinity and specificity were assessed by staining HEK cells expressing BILF1 GFP or an off-target GPCR fused to GFP, respectively. X-axis represents receptor quantity (GFP signal), and y-axis represents binding signal.

[0339] FIGs. 16A-16E demonstrate diversification of an antibody to create variants with new functional properties. FIG. 16A shows a library design for diversification of ATI 18i4, a published nanobody that binds to the AT1R GPCR and acts as an antagonist. In this design, CDR3 was randomized and lengthened in order to discover variants with new functional properties. FIG. 16B shows the verification of newly discovered variants in the exemplary v2 MPPI assay. The libraries were transformed into yeast encoding the MPPI gene circuit and subject to selection. From each library, 12 variants that enriched were characterized by re-testing in the MPPI assay, alongside ATI 18 (parent to ATI 18i4), ATI 18i4 and a non-binding nanobody. Yeast were grown on non-selective media (SDC) or selective media lacking histidine across a range of estradiol, 3-AT, and 5-FOA concentrations (shown as Est_3AT_5FOA). FIG. 16C shows verification of four new ATI R-binding candidates. Nanobodies were purified as Fc-fusions and used to stain HEK cells expressing AT1R-GFP. X-axis represents receptor quantity (GFP), and y-axis represents binding signal. FIG. 16D shows AlphaFold predicted structures of ten of the new variants that were confirmed to bind to AT1R on HEK cells. For comparison, AT118i4 and the AT1R ligand, Angll, are shown. FIG. 16E shows the functional characterization of seven of the newly discovered AT1R binders. Nanobodies were purified as an Fc-fusion and incubated with 100 nM Angll in a standard beta-arrestin signaling assay for AT1R.

[0340] FIG. 17 demonstrates high throughput characterization of protein-protein interactions. High-throughput affinity characterization of nanobodies that bind the AT1R GPCR. 16 nanobodies that bind to AT1R were transformed into yeast encoding the exemplary v2 MPPI gene circuit and characterized via growth selection. For verification, the nanobodies were also purified as Fc-fiisions and used to stain HEK cells expressing AT1R-GFP. The x-axis represents AT1R abundance in terms of GFP fluorescence. The y-axis represents staining signal. For each nanobody’s staining curve, the corresponding yeast signal is shown using color coding.674939-1760-0399 2Attorney Docket No. 701586-000163WOPT

[0341] FIG. 18 depicts a comparison of the circuit output (GFP) for the split-ubiquitin MPPI system, the protease recruitment MPPI system, and the split-protease recruitment MPPI system for both the binder and the non-binder.

[0342] FIG. 19A depicts GFP output of yeast expressing the v2 split-protease MPPI circuit with various membrane protein targets, including GPCRs and solute transporters, in the presence of either cognate or non-cognate nanobody binders. Output normalized to the minimum and maximum for each target. FIG. 19B depicts GFP output of yeast expressing the v2 split-protease MPPI circuit with various membrane protein targets, including GPCRs and multipass enzymes, in the presence of either cognate or non-cognate scFV or peptide binders. Output normalized to the minimum and maximum for each target. FIG. 19C depicts GFP output of yeast expressing the v2 split-protease MPPI circuit with the CXCR4 GPCR, in the presence of either cognate or non-cognate chemokines or miniprotein binders. Output normalized to the minimum and maximum for each target.Table 2. Exemplary Sequences.Name SEQ ID NO3C-protease 38ABI1 39ADE1 40ADE2 41aeyCFaN 42Apelin peptide 43APLNR 44ATI 18 45AT118i4 46AT1R 47BC2 48BILF1 49BLF198 50BLFI98.III 51BLF198.313 52CCL5 53CfaC 54CX3CL1 55684939-1760-0399 2Atorney Docket No. 701586-000163WQPTCXCL12 56 CXCR4 57 E2A 58 GAI 59 Gal4 DBD 60 Gal4 AD 61 GFP 62 GID1 63 GR28-10 64 GR28-2 65 GR28-25 66 GR28-30 67 GR28-31 68 GR28-36 69 GR28-7 70 H75E N-terminal split TEV protease 71 H75S N-terminal split TEV protease 72 H75T N-terminal split TEV protease 73 HIS3 74 HIS6 75 HygromycinR 76 IL-7mb 77 JN241 78 JN241.9 79 KanamycinR 81 L190K C-terminal split TEV 81 proteaseLEU2 82 LexA 83 LexA-VP16 84 LMP1 85694939-1760-0399 2Atorney Docket No. 701586-000163WQPTLMP2B 86 LYS2 87 MET 15 88 Msn2 89 Nb352 90 Nb421 91 Nb437 92 Nb630 93 NourseothricinR 94 N-TEVp 95 PDZ 96 PE 10 C-terminal split TEV protease 97 Pep 98 PH21 C-terminal split TEV protease 99 PYL1 100 RAD27 101 RET2 102 REV1 103 RFP 104 RNR2 105 RPL18B 106 rTA 107 SH3 108 SLC7A11 109 SS01 110 SSTR5 111 Ste2 112 TetR 113 TRP1 114 URA3 115 US28 116704939-1760-0399 2Atorney Docket No. 701586-000163WQPTUS28-2 117 US28-3 118 US28-4 119 VP16 120 VP64 121 VUN100 122 WSC3 123 ZeocinR 124 Zif268 125 WSC3 126 SLG1 127 SKG1 128 Ste2-TM1 129 SS01 130 SKN1 131 TRE1 132 ALG5 133 YEH2 134 TEV 135 Ntev(WT) 136 Ntev(H75E) 137 Ntev(H75S) 138 Ntev(H75T) 139 Ctev(WT) 140 Ctev(L190K) 141 Ctev(PElO) 142 Ctev(PH21) 143 Ctev(PE10, L190K) 144 Ctev(PH21, L190K) 145 CS(WT) 1 CS(L) 2714939-1760-0399 2Atorney Docket No. 701586-000163WQPTCS(E) 3 CS(I) 4 CS(V) 5 CS(P) 6 CS(K) 7 CS(R) 8 CS(N) 9 CS(Y) 10 CS(K) 11 CS(D) 12 CS(Q) 13 CS(F) 14 CS(T) 15 CS(W) 16 AIP(M)-CS(WT) 17 AIP(M)-CS(A) 18 AIP(M)-CS(L) 19 AIP(A)-CS(WT) 20 AIP(A)-CS(A) 21 AIP(A)-CS(L) 22 AIP(Y)-CS(WT) 23 AIP(Y)-CS(A) 24 AIP(Y)-CS(L) 25 CS(Q>E) 26 CS(Q>H) 27 SpyTag 146 SpyCatcher 147 ABI1 148 PYL1 149 aey-CfaN 150 CfaC 151724939-1760-0399 2Attorney Docket No. 701586-000163WQPTGID1 152GAI 153LexA-VP16 154VP16-Zif268 155TetR-Gal4AD 156VP16-43-8 157(Lex0)x6 158pminLEU2 159pminRNR2 160p(TetO)x7-ZRTlm 161HIS3 162URA3 163sfGFP 164m Scarlett 165HISA 166sfGFP-E2A-HIS3 167URA3 -E2 A-m Scarlett 168

[0343] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely fortheir disclosure prior to the fding date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.734939-1760-0399 2

Claims

Attorney Docket No. 701586-000163WOPTCLAIMSWe claim:

1. A set of proteins comprising:a) a target protein comprising:i) at least one extracellular domain and / or extracellular loop;ii) at least one transmembrane domain;iii) at least one intracellular transcription factor domain; and iv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain;andb) a binding protein or binding dimer comprising:i) at least one extracellular binding domain;ii) at least one transmembrane domain; andiii) at least one intracellular protease domain.

2. The set of proteins of any one of the preceding claims, further comprising:c) at least one off-target protein comprising:i) at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein;ii) at least one transmembrane domain;iii) at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein; andiv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

3. A set of proteins comprising:a) a target protein comprising:i) at least one extracellular domain and / or extracellular loop;ii) at least one transmembrane domain;iii) at least one intracellular transcription factor domain;iv) an intracellular first portion of a split protease, located between the transmembrane domain and the intracellular transcription factor domain; and744939-1760-0399 2Attorney Docket No. 701586-000163WOPTv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain; andb) a binding protein or binding dimer comprising:i) at least one extracellular binding domain;ii) at least one transmembrane domain; andiii) an intracellular second portion of the split protease.

4. The set of proteins of claim 3, wherein the first portion of the split protease is a N-terminal fragment of the split protease and the second portion of the split protease is a C-terminal fragment of the split protease.

5. The set of proteins of claim 3, wherein the first portion of the split protease is a C-terminal fragment of the split protease and the second portion of the split protease is a N-terminal fragment of the split protease.

6. The set of proteins of any one of claims 3-4, further comprising:c) at least one off-target protein comprising:i) at least one extracellular domain and / or extracellular loop which is not identical to the at least one extracellular domain and / or extracellular loop of the target protein;ii) at least one transmembrane domain;iii) at least one intracellular transcription factor domain which is not identical to the at least one intracellular transcription factor domain of the target protein;iv) an intracellular first portion of a split protease, located between the transmembrane domain and the intracellular transcription factor domain; andv) at least one intracellular protease cleavage target domain located between the transmembrane domain and the intracellular transcription factor domain.

7. The set of proteins of any one of the preceding claims, wherein the extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of the target protein are:G-protein coupled receptor (GPCR) sequences; receptor tyrosine kinases sequences; toll-like receptors sequences; T-cell receptors sequences; B-cell receptors sequences; transporters sequences; solute carriers sequences; proton pumps sequences; ion 754939-1760-0399 2Attorney Docket No. 701586-000163WOPTchannels sequences; porins sequences; aquaporins sequences; viroprotein sequences; integrins sequences; cadherins sequences; 7TM protein sequences; nuclear receptor sequences; gap junction protein sequences; connexin sequences; selectin sequences; spectrin sequences; N-CAM sequences; claudin sequences; occludin sequences; Fc receptor sequences; tetraspanin sequences; transferrin receptor sequences; carrier protein sequences; or channelrhodopsin sequences.

8. The set of proteins of any one of the preceding claims, wherein the transcription factor domain is a polypeptide sequence comprising a DNA binding domain and a transcriptional activation domain.

9. The set of proteins of any one of the preceding claims, wherein the protease or split protease binds to and cleaves the intracellular protease cleavage target domain.

10. The set or proteins of any one of the preceding claims, wherein the protease or split protease is selected from the group consisting of:TEV protease and 3C protease.

11. The set of proteins of any one of the preceding claims, wherein the intracellular first portion of a split protease of an off-target protein is not identical to the intracellular first portion of a split protease of the target protein.

12. The set of proteins of any one of the preceding claims, wherein the intracellular first portion of a split protease of an off-target protein and the intracellular first portion of a split protease of the target protein are variants of the same split protease.

13. The set of proteins of any one of the preceding claims, wherein the protease cleavage site is selected from one of SEQ ID NOs: 1-27.

14. The set of proteins of any one of the preceding claims, wherein the protease is TEV and the protease cleavage site is selected from one of SEQ ID NOs: 1-27.

15. The set of proteins of any one of the preceding claims, wherein the extracellular binding domain is an antibody or antibody reagent.764939-1760-0399 2Attorney Docket No. 701586-000163WQPT16. The set of proteins of any one of the preceding claims, wherein the at least one transmembrane domain of the binding protein or binding dimer is a single-pass transmembrane domain.

17. The set of proteins of any one of the preceding claims, wherein the binding protein or binding dimer further comprises a linker between the at least one extracellular binding domain and the at least one transmembrane domain.

18. The set of proteins of claim 14, wherein the linker comprises 2-60 amino acids.

19. The set of proteins of claim 14, wherein the linker comprises 20-40 amino acids.

20. The set of proteins of any one of the preceding claims, comprising a binding dimer wherein the binding dimer comprises:a) a first partner protein comprising:i) at least one extracellular binding domain; andii) a first dimerization domain; andb) a second partner protein comprising:i) a second dimerization domain that binds to the first dimerization domain; ii) at least one transmembrane domain; andiii) one of:1) at least one intracellular protease domain that recognizes and cleaves the intracellular protease cleavage target domain; or2) an intracellular second portion of the split protease.

21. An expression system comprising:a) a set of proteins of any one of the preceding claims; andb) a nucleic acid molecule comprising:i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control sequence modulated by the intracellular transcription factor domain of the off-target protein, andii) operably linked to a reporter gene.

22. An expression system comprising:a) one or more nucleic acid molecule encoding a set of proteins of any one of the preceding claims; and774939-1760-0399 2Attorney Docket No. 701586-000163WQPTb) a nucleic acid molecule comprising:i) an expression control sequence modulated by the intracellular transcription factor domain of the target protein or an expression control sequence modulated by the intracellular transcription factor domain of the off-target protein; andii) operably linked to a reporter gene.

23. The expression system of claim 22, wherein the one or more nucleic acid molecule encoding a set of proteins of any one of the preceding claims further comprise a constitutive promoter operably linked to a sequence encoding the target protein, a constitutive promoter operably linked to a sequence encoding the binding protein, a constitutive promoter operably linked to a sequence encoding the first partner protein, a constitutive promoter operably linked to a sequence encoding the second partner protein, and / or a constitutive promoter operably linked to a sequence encoding the off-target protein.

24. The expression system of claim 22, wherein the one or more nucleic acid molecule encoding a set of proteins of any one of the preceding claims further comprise an inducible promoter operably linked to a sequence encoding the target protein, an inducible promoter operably linked to a sequence encoding the binding protein, an inducible promoter operably linked to a sequence encoding the first partner protein, an inducible promoter operably linked to a sequence encoding the second partner protein, and / or an inducible promoter operably linked to a sequence encoding the off-target protein.

25. A cell comprising the set of proteins or expression system of any one of the preceding claims.

26. The cell of claim 25, wherein the cell is a yeast cell.

27. The expression system or cell of any one of the preceding claims, wherein the reporter gene is a fluorescent protein or yeast survival protein.

28. A library comprising a plurality of cells of any one of claims 25-27.

29. A method comprising detecting a signal of a reporter gene of i) an expression system of any one of the preceding claims or ii) a cell of any one of the preceding claims.784939-1760-0399 2Attorney Docket No. 701586-000163WOPT30. The method of claim 29, wherein a plurality of different binding proteins or a plurality of different first partner proteins of any one of the preceding claims is expressed in a plurality of cells.

31. The method of any one of the preceding claims, wherein detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates the extracellular binding domain is specific for binding to the target protein.

32. The method of any one of the preceding claims, wherein detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the off-target protein indicates the extracellular binding domain is not specific for binding to the target protein.

33. The method of any one of the preceding claims, wherein the extracellular binding domain of the binding protein or binding dimer is an antibody or antibody reagent, the target protein comprises at least one extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of a therapeutic target protein, and the detection of a signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates the extracellular binding domain is specific for binding to the therapeutic target protein.

34. The method of any one of the preceding claims, wherein the extracellular binding domain of the binding protein or binding dimer is an antibody or antibody reagent, the target protein comprises at least one extracellular domain and / or extracellular loop, and / or at least one transmembrane domain of a therapeutic target protein, and the strength of the detected signal of a reporter gene operably linked to the expression control sequence modulated by the intracellular transcription factor domain of the target protein indicates a binding affinity of the extracellular binding domain for the target protein.794939-1760-0399 2

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