RU2802770C2 - Yeast protein display in the periplasmatic space - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: compositions and methods of displaying antibodies in the periplasmic space of yeast cells are described. Besides, the invention relates to high throughput screening of antibody libraries using periplasmic display in yeast cells. Antibodies are associated with a membrane-spanning transmembrane domain, a cell membrane-associated protein domain that resides on the outer surface of the yeast cell membrane, a protein that binds to the inner surface of the yeast cell wall, or a periplasmic protein to display antibodies in the yeast periplasmic space.

EFFECT: possibility to co-express a target protein of interest in yeast, so that it is localized on the plasma membrane or periplasmic space and is available for binding by exposed antibodies.

117 cl, 7 dwg, 2 tbl, 8 ex

Description

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to US Provisional Application No. 62/597388, filed December 11, 2017, the contents of which are incorporated herein by reference in their entirety.

STATEMENT REGARDING GOVERNMENT SPONSORED RESEARCH

[0002] The present invention was made with the support of the Government under Grant No. 1747391 issued by the National Science Foundation. The government has certain rights to an invention.

TECHNICAL FIELD

[0003] The invention relates to cell display and methods for high-throughput screening of protein libraries. In particular, the invention relates to methods for displaying proteins in the yeast periplasmic space and the use of such methods for screening protein libraries for specific binding or functional characteristics.

BACKGROUND OF THE ART

[0004] Molecular display technology has proven to be invaluable for the discovery, production and optimization of proteins and peptides for a variety of biotechnological and biomedical applications. Various approaches including phage display (Smith (1985) Science 228:1315-1317), mRNA display (Wilson et al. (2001) Proc. Natl. Acad. Sci. USA 98:3750-3755) and DNA display (Yonezawa et al. (2003) Nucleic Acids Res. 31:e118), ribosomal display (Hanes & Pluckthun (1997) Proc. Natl. Acad. Sci. USA 94:4937-4942), eukaryotic virus display (Bupp & Roth (2002) ) Mol. Ther. 5:329-335; Muller et al. (2003) Nat. Biotechnol. 21:1040-1046), bacterial display (Lu et al. (1995) Biotechnology13:366-372) and yeast display (Boder & Wittrup (1997) Nat. Biotechnol. 15:553-557) were developed to screen combinatorial libraries of recombinant proteins for desired characteristics. Such display technologies are widely used in protein engineering to identify proteins with increased stability and desired binding affinities and enzymatic activities, and they have proven useful in a variety of applications including targeting, affinity maturation, engineering of therapeutic proteins and antibodies, biofuel production, adsorption of pollutants environment, epitope mapping and protein-protein interaction studies.

[0005] In particular, yeast display is used for a wide variety of prokaryotic and eukaryotic proteins (Cherf et al. (2015) Methods Mol. Biol. 1319:155-175). Expression in yeast cells provides the advantage of allowing eukaryotic proteins to be properly folded and glycosylated. In conventional yeast display, recombinant proteins are displayed on the surface of yeast cells by fusion with a cell wall protein. Although Saccharomyces cerevisiae is the most commonly used species for cell surface display, other yeast species, including Pichia, Candida, and Yarrowia, have proven useful for some applications (Tanaka et al. (2012) Appl. Microbiol. Biotechnol. 95(3):577 -591, Buerth et al (2016) Appl Microbiol Biotechnol 100(16):6981-6990, Madzak (2015) Appl Microbiol Biotechnol 99(11):4559-4577).

[0006] There remains a need for improved methods that expose proteins more efficiently, particularly for high-throughput screening of protein-protein interactions with membrane proteins.

SUMMARY OF THE INVENTION

[0007] The present invention relates to high-throughput screening of protein libraries for specific binding or functional characteristics by displaying proteins in the periplasmic space of yeast cells.

[0008] In one aspect, the invention relates to a yeast periplasmic display library containing a plurality of yeast host cells, wherein each yeast cell contains: a) a variant protein for display in the periplasmic space of the yeast host cell, wherein the variant displayed protein is different in each yeast cell. host cell, such that multiple yeast host cells display multiple protein variants; b) a periplasmic anchor protein, wherein the periplasmic anchor protein is associated with a variant protein such that the variant protein is exposed in the periplasmic space; and c) a target membrane protein of interest, wherein the membrane protein of interest is located on the plasma membrane of the host yeast cell and is accessible to the protein variant exposed in the periplasmic space of the host yeast cell. The yeast host cell can be haploid or diploid.

[0009] In certain embodiments, the variant protein and the periplasmic anchor protein are covalently linked together in a fusion protein. In other embodiments, the variant protein and the periplasmic anchor protein are non-covalently linked together through molecular binding interactions in a complex. In other embodiments, the variant protein and the periplasmic anchor protein are linked via a non-peptide bond in a complex. In some embodiments, the non-peptide bond is a disulfide bond.

[0010] In certain embodiments, the periplasmic anchor protein comprises a signal sequence that causes transport of the fusion protein into the periplasm of the host yeast cell, the plasma membrane, or the cell wall such that the fusion protein is exposed in the periplasm. An exemplary signal sequence that may be used is the prepro-alpha factor signal sequence.

[0011] In certain embodiments, the periplasmic anchor protein comprises a membrane spanning transmembrane domain that exposes the fusion protein into the periplasm.

[0012] In certain embodiments, the periplasmic anchor protein comprises a cell membrane-associated protein domain that is localized on the outer surface of the cell membrane such that the exposed variant of the protein protrudes into the periplasm. In certain embodiments, the cell membrane-associated protein domain is a plasma membrane-anchoring glycosylphosphatidylinositol (GPI) domain. For example, the plasma membrane-anchoring GPI domain may be a plasma membrane-anchoring yapsin GPI domain, such as, but not limited to, a YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7 plasma membrane-anchoring GPI domain. .

[0013] In certain embodiments, a periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the exposed variant of the protein protrudes into the periplasm.

[0014] In certain embodiments, the periplasmic anchor protein contains a signal sequence that causes transport of the fusion protein into the periplasm of the host yeast cell, and the periplasmic anchor protein is large enough to ensure that the fusion protein remains in the periplasm.

[0015] In certain embodiments, the anchor protein is a component of a periplasmic protein complex that is large enough that formation of the complex in the periplasm results in retention of the fusion protein in the periplasm.

[0016] In another embodiment, the fusion protein further comprises a tag.

[0017] In certain embodiments, protein variants are antibodies, antibody mimetics, aptamers, antigens, enzymes, receptors, hormones, substrates, agonists, antagonists, or ligands.

[0018] In certain embodiments, the protein variants are antibodies selected from the group consisting of monoclonal antibodies, chimeric antibodies, nanoantibodies, recombinant antibody fragments, Fab fragments, Fab' fragments, F(ab') ₂ fragments, F _v -fragments and scFv-fragments.

[0019] In certain embodiments, each yeast host cell in the yeast periplasmic display library further contains a target protein of interest that is expressed in a location accessible to the exposed protein variant (e.g., close enough for the exposed protein variant to bind to the representing protein variant). interest in the target protein). For example, the target protein of interest may be located on the plasma membrane or periplasm of a yeast host cell. The protein of interest may be, for example, a membrane protein, receptor, ion channel or transporter. In one embodiment, the target protein of interest is a G protein coupled receptor (GPCR).

[0020] In certain embodiments, each yeast host cell further comprises a reporter system for detecting the response of a target protein of interest to a protein-protein interaction with an exposed protein variant. In certain embodiments, the exposed protein variant is an antagonist of a target protein of interest, and the response is a decrease in the activity of the target protein of interest upon binding of the antagonist to the target protein of interest, wherein the reporter system detects a decrease in the activity of the target protein of interest upon binding an antagonist with a target protein of interest. In other embodiments, the exposed protein variant is an agonist of a target protein of interest and the response is an increase in the activity of the target protein of interest upon binding of the agonist to the target protein of interest, wherein the reporter system allows detection of an increase in the activity of the target protein of interest upon binding agonist with a target protein of interest.

[0021] In certain embodiments, activation of a target protein of interest increases the growth of host yeast cells. In this case, the yeast periplasmic display library can be screened for an agonist of the target protein of interest by culturing at least a subset of yeast host cells of the yeast periplasmic display library in a medium wherein growth of the yeast host cells in the medium indicates that the protein variant , exposed in a yeast host cell, is an agonist of the target protein of interest.

[0022] In other embodiments, activation of a target protein of interest reduces the growth of host yeast cells. In this case, the yeast periplasmic display library can be screened for an antagonist of the target protein of interest by culturing at least a subset of yeast host cells of the yeast periplasmic display library in a medium wherein growth of the yeast host cells in the medium indicates that the protein variant , exposed in a yeast host cell, is an antagonist of the target protein of interest.

[0023] In another embodiment, the invention relates to a yeast periplasmic display library containing a plurality of yeast host cells, wherein each yeast host cell contains: a) a fusion protein comprising a periplasmic anchor protein fused to an antibody for yeast periplasmic display a host cell, wherein the antibody displayed is different in each host yeast cell such that a plurality of host yeast cells display a plurality of antibodies; and b) a target membrane protein of interest, wherein the membrane protein of interest is located on the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell.

[0024] The target membrane protein of interest may be, for example, a receptor, an ion channel, and a transporter. In some embodiments, the target membrane protein of interest contains a mutation that increases or decreases its activity.

[0025] Antibodies that can be displayed with a target membrane protein of interest may include, but are not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, nanoantibodies, recombinant antibody fragments, Fab fragments, Fab' fragments, F( ab') ₂ fragments, F _v fragments and scFv fragments.

[0026] In certain embodiments, the yeast periplasmic display library further comprises a reporter system comprising a reporter gene operably linked to an inducible promoter that is activated when the target membrane protein of interest is activated to allow detection of an increase or decrease in the activity of the membrane protein of interest - target when the antibody binds to a target membrane protein of interest. For example, the reporter gene may be a nutritional marker (e.g., HIS3, HIS7, ARG6, LEU2, URA3, and TRP1), an antibiotic resistance marker (e.g., conferring resistance to an antibiotic such as geneticin (e.g., aphA1), zeocin (e.g., ble), hygromycin B, nourseothricin or bialaphos), fluorescent marker (eg green fluorescent protein, red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein and orange fluorescent protein), bioluminescent marker (eg luciferase or aequorin) , or a counterselection marker (e.g., CAN1, URA3, MET15, TRP1, and TK). In certain embodiments, the reporter gene is a selectable marker such that an increase in the activity of a target membrane protein of interest upon binding of an antibody to the target membrane protein of interest is detected by the growth of host yeast cells on positive selection media. In other embodiments, the reporter gene is a counterselection marker such that a decrease in the activity of the target membrane protein of interest upon binding of the antibody to the target membrane protein of interest is detected by the growth of host yeast cells on the medium containing the counterselection agent.

[0027] In certain embodiments, the target membrane protein of interest is a G protein-coupled receptor (GPCR), e.g., an exogenous GPCR, such as a mammalian GPCR (e.g., from a human or non-human primate, rodent, laboratory animal, livestock). In certain embodiments, the mammalian GPCR is a human GPCR selected from the group consisting of CXCR4, CXCR5, SSTR2, MOR, AVPR2, FPR2/ALX, ADORA2A, CHRM3, CGRP2, CCR2, CCR4, CCR5, CHRM4, PAC1, b2AR, CXCR2 , CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB1, CB2, A3AR and AT1R.

[0028] In certain embodiments, the yeast periplasmic display library further comprises an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered Gα subunit is capable of activating a detectable pheromone response in a host yeast cell.

[0029] In certain embodiments, the engineered G(subunit) is a chimeric G protein alpha (Gα) subunit containing the N-terminal domain of a yeast G(subunit) and the C-terminal domain of an exogenous Gα subunit. For example, a yeast G(subunit) may belong to the family G proteins Gαi, Gαq, Gαs, or Gαo. In a chimeric G(subunit), at least five C-terminal residues of the yeast G(subunit) can be replaced by the corresponding C-terminal residues of the mammalian G(subunit, such that the chimeric G(subunit is capable of activation by Mammalian GPCR In some embodiments, at least 20 C-terminal residues of the yeast G subunit are replaced with corresponding C-terminal residues of the mammalian G subunit such that the chimeric G subunit is capable of being activated by a mammalian GPCR. In another embodiment, the chimeric G subunit contains at least 41 N-terminal residues of the yeast Gα subunit.

[0030] Exemplary mammalian G subunits include G alpha-S, G alpha-I, G alpha-O, G alpha-T, G alpha-Z, G alpha-Q, G alpha-11, G alpha-12, G alpha-13 and transducin.

[0031] In some embodiments, the target GPCR of interest has constitutive ligand-independent activity. In other embodiments, a ligand is added to activate a GPCR target of interest.

[0032] In certain embodiments, the yeast host cell is a haploid or diploid yeast host cell. In certain embodiments, the host yeast cell is a Δfar1, Δsst2, Δste14, Δste3, or Δmat strain. The Δmat strain may contain, for example, a deleted or inactivated MAT locus (or a deleted or inactivated MATa locus.

[0033] In another embodiment, the yeast host cell further comprises a modified CLN3 protein containing a C-terminal truncation that increases the CLN3 content of the yeast host cell compared to wild-type CLN3 protein. For example, a modified CLN3 protein may retain at least the N-terminal amino acids 1-387 or 1-408 of the wild-type CLN3 protein, or any number of N-terminal amino acids in these ranges, such as 1-388, 1-389, 1-390 , 1-391, 1-392, 1-393, 1-394, 1-395, 1-396, 1-397, 1-398, 1-399, 1-400, 1-401, 1-402, 1 -403, 1-404, 1-405, 1-406, 1-407, or 1-408, where the C-terminal truncation contains a deletion of all or some of the remaining residues of the wild-type CLN3 protein.

[0034] In another embodiment, the yeast host cell is a GPCR antagonist antibody selection strain FAR1.

[0035] In another embodiment, the yeast host cell is a Δfar1 strain containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selecting GPCR agonist antibodies.

[0036] In another aspect, the invention relates to a yeast periplasmic display library containing a plurality of yeast host cells, wherein each yeast host cell contains: a) an antibody for display in the periplasmic space of the yeast host cell, wherein the displayed antibody is different in each yeast host cell such that a plurality of host yeast cells display a plurality of antibodies, wherein the antibody is linked to a signal sequence that allows transport into the periplasm of the host yeast cell, the plasma membrane or cell wall, such that the antibody is displayed in the periplasmic space of the yeast host cell ; and b) a target membrane protein of interest, wherein the membrane protein of interest is located on the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell.

[0037] In another aspect, the invention relates to a method for producing the yeast periplasmic display library described herein, the method comprising: a) providing a plurality of recombinant polynucleotides encoding fusion proteins, wherein each recombinant polynucleotide encodes a different fusion protein comprising a periplasmic anchor protein, fused to a different antibody, for display; b) transfecting a plurality of yeast host cells with a plurality of recombinant polynucleotides encoding fusion proteins; c) transfecting a plurality of yeast host cells with a recombinant polynucleotide encoding a target membrane protein of interest; and d) culturing a plurality of yeast host cells under conditions that allow expression of the fusion proteins and the target membrane protein of interest, wherein each yeast host cell displays a different antibody in the periplasmic space and the target membrane protein of interest is localized to the plasma membrane ( i.e., where it is available for binding by the exposed antibody). In certain embodiments, expression vectors provide recombinant polynucleotides encoding fusion proteins or a recombinant polynucleotide encoding a target membrane protein of interest. In other embodiments, recombinant polynucleotides encoding fusion proteins or a target membrane protein of interest are inserted into the genome of a host yeast cell at the target locus.

[0038] In another aspect, the invention relates to a method for producing a yeast periplasmic display library, the method comprising: a) providing a plurality of recombinant antibody-encoding polynucleotides for display in the periplasmic space of a host yeast cell, wherein the displayed antibody is different in each yeast cell; host, such that a plurality of host yeast cells exhibit a plurality of antibodies; b) providing a second recombinant polynucleotide encoding a periplasmic anchor protein, wherein the periplasmic anchor protein is linked to an antibody such that the antibody is exposed in the periplasmic space; c) transfecting a plurality of host yeast cells with a first plurality of recombinant polynucleotides and a second recombinant polynucleotide; d) transfecting a plurality of yeast host cells with a recombinant polynucleotide encoding a target membrane protein of interest; and e) culturing a plurality of yeast host cells under conditions that allow expression of antibodies, a periplasmic anchor protein, and a target membrane protein of interest, wherein each yeast host cell displays a different antibody in the periplasmic space and the target membrane protein of interest is localized to the plasma membrane to obtain a yeast periplasmic display library.

[0039] Expression of the fusion proteins and the target membrane protein of interest typically depends on the presence of a promoter, which may be included in a vector or in a chromosomal locus into which the recombinant polynucleotides are inserted. The promoter may be a constitutive or inducible promoter. In certain embodiments, each recombinant polynucleotide contains a promoter operably linked to a polynucleotide encoding a fusion protein or target membrane protein of interest. The recombinant polynucleotide may be provided in a vector containing a promoter. In other embodiments, the chromosomal target locus comprises a promoter that becomes operably linked to a polynucleotide encoding a fusion protein or target membrane protein of interest that is inserted into the chromosomal target locus.

[0040] In another embodiment, the method further includes introducing into a plurality of yeast host cells a recombinant polynucleotide encoding an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in the host yeast cell.

[0041] In another embodiment, the invention provides a periplasmic targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a cloning site suitable for inserting in frame a polynucleotide encoding a protein variant after a polynucleotide encoding a signal peptide; c) a polynucleotide encoding a plasma membrane-anchoring glycophosphatidylinositol (GPI) domain, positioned such that the vector is capable of producing a fusion protein comprising a signal peptide and a variant protein fused to the plasma membrane-anchoring GPI domain; and d) a promoter operably linked to the fusion protein coding sequences. In one embodiment, the signal peptide comprises a prepro-alpha factor signal sequence. In another embodiment, the cloning site contains one or more restriction sites. In certain embodiments, the plasma membrane-anchoring GPI domain is a plasma membrane-anchoring yapsin GPI domain, such as, but not limited to, a plasma membrane-anchoring yapsin GPI domain YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7. In another embodiment, the periplasmic-targeting expression vector further comprises a polynucleotide encoding a linker, wherein the polynucleotide encoding the linker is located between the cloning site and the polynucleotide encoding a plasma membrane anchoring GPI domain. The linker may further comprise a label. In another embodiment, the periplasmic-targeting expression vector further comprises a selectable marker.

[0042] In another aspect, the invention relates to a method for producing the yeast periplasmic display library described herein, the method comprising: a) providing a plurality of recombinant polynucleotides encoding antibody variants, wherein each recombinant polynucleotide encodes a different antibody variant; b) transfecting a plurality of yeast host cells with the periplasmic targeting expression vector described herein, c) transfecting a plurality of yeast host cells with a plurality of recombinant polynucleotides encoding antibody variants, wherein in each yeast host cell, a recombinant polynucleotide encoding an antibody variant, inserted into the cloning site of a periplasmic-targeting expression vector by homologous recombination to allow expression of a fusion protein containing a periplasmic anchor protein fused to a variant antibody for display; c) transfecting a plurality of yeast host cells with a recombinant polynucleotide encoding a target membrane protein of interest; and d) culturing a plurality of yeast host cells under conditions that allow expression of the fusion proteins and the target membrane protein of interest, wherein each yeast host cell displays a different antibody in the periplasmic space and the target membrane protein of interest is localized to the plasma membrane (i.e. .e., where it is available for binding by the exposed antibody). In another embodiment, the method further comprises introducing into a plurality of host yeast cells a recombinant polynucleotide encoding an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in the host yeast cell.

[0043] In another aspect, the invention provides a method of screening a yeast periplasmic display library containing a reporter system as described herein for an antibody that modulates the activity of a target membrane protein of interest, the method comprising culturing at least a subset of yeast cells -hosts of the yeast periplasmic display library described herein in a selective environment; and detecting reporter gene expression, wherein increased expression of the reporter gene indicates that the antibody increases the activity of the target membrane protein of interest, and decreased expression of the reporter gene indicates that the antibody reduces the activity of the target membrane protein of interest.

[0044] Exemplary reporter genes include a nutritional marker (e.g., HIS3, HIS7, ARG6, LEU2, URA3, and TRP1), an antibiotic resistance marker (e.g., communicates resistance to an antibiotic such as geneticin (aphA1), zeocin (ble), hygromycin B, nourseothricin and bialaphos), a fluorescent marker (eg, green fluorescent protein, red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and orange fluorescent protein), a bioluminescent marker (eg, luciferase or aequorin), and a counterselection marker ( for example CAN1, URA3, MET15, TRP1 and TK).

[0045] In another embodiment, the method further includes positive selection for expression of a nutritional marker, wherein growth of the host yeast cells in a selective nutrient-deficient environment indicates that the target membrane protein of interest is activated.

[0046] In another embodiment, the method further includes positive selection for expression of an antibiotic resistance marker, wherein growth of the host yeast cells in the selective medium containing the antibiotic indicates that the target membrane protein of interest is activated.

[0047] In another embodiment, the method further includes positive selection for expression of a fluorescent marker, wherein detection of fluorescence emitted by the host yeast cells indicates that the target membrane protein of interest is activated.

[0048] In another embodiment, the method further includes positive selection for expression of a bioluminescent marker, wherein detection of bioluminescence emitted by the host yeast cells indicates that the target membrane protein of interest is activated.

[0049] In another embodiment, the method further includes negative selection against a counterselection marker, wherein the reduction in activity of the target membrane protein of interest upon binding of the exposed antibody to the target membrane protein of interest is determined by the growth of host yeast cells in a medium containing the agent, which allows selection against cells expressing a counterselection marker.

[0050] In another embodiment, the invention provides a method of screening a yeast periplasmic display library for an antibody that modulates the activity of a GPCR target of interest, the method comprising culturing at least a subset of yeast host cells of the yeast periplasmic display library in an environment where detection activation or inhibition of the pheromone response in at least one yeast host cell compared to a control yeast host cell that does not have antibody exposed in the periplasmic space, indicates that the exposed antibody in the at least one yeast host cell binds and modulates GPCR activity. In some embodiments, the method further includes contacting the human GPCR with a ligand. In other embodiments, the GPCR has constitutive ligand-independent activity.

[0051] In certain embodiments, the yeast host cell contains an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in the yeast host cell. In certain embodiments, the engineered G subunit is a chimeric a G protein alpha (Gα) subunit containing the N-terminal domain of the yeast G subunit and the C-terminal domain of the exogenous Gα subunit. For example, the yeast G subunit may belong to the Gαi, Gαq, Gαs, or Gαo family of G proteins. In a chimeric the G subunit, at least five C-terminal residues of the yeast G subunit may be replaced by the corresponding C-terminal residues of the mammalian G subunit, such that the chimeric G subunit is capable of being activated by a mammalian GPCR. In some embodiments, at least 20 C- terminal residues of the yeast G subunit are replaced by the corresponding C-terminal residues of the mammalian G subunit, so that the chimeric G subunit is capable of activation by the mammalian GPCR. In another embodiment, the chimeric G(subunit) contains at least 41 N-terminal residues of the yeast Gα subunit. Exemplary mammalian G(subunits) include G alpha-S, G alpha-I, G alpha-O, G alpha-T, G alpha- Z, G alpha-Q, G alpha-11, G alpha-12, G alpha-13 and transducin.

[0052] In certain embodiments, the host yeast cell is a FAR1 strain, wherein inhibition of the pheromone response by an antibody acting as an antagonist that binds and inhibits GPCRs in the host yeast cell results in cell cycle arrest and growth of the yeast cell. owner. In other embodiments, the yeast host cell is a Δfar1 strain containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene, wherein activation of the pheromone response by an antibody acting as an agonist that binds and activates a GPCR in the yeast host cell results in to increase reporter gene expression.

[0053] In another aspect, the invention relates to a yeast host cell comprising: a) an antibody for display in the periplasmic space of the yeast host cell, b) a periplasmic anchor protein, wherein the periplasmic anchor protein is linked to the antibody such that the antibody is displayed in the periplasmic space ; and c) a target membrane protein of interest, wherein the membrane protein of interest is located on the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell.

[0054] In another aspect, the invention provides an antibody coupled to a periplasmic anchor protein. In some embodiments, in which the antibody is produced in a host yeast cell, the antibody is localized in the periplasmic space of the host yeast cell. In some embodiments, the antibody and the periplasmic anchor protein are non-covalently linked to each other through molecular binding interactions in the complex or are linked by a covalent non-peptide bond in the complex. In some embodiments, the non-peptide bond is a disulfide bond. In some embodiments, the antibody and the periplasmic anchor protein are covalently linked together in a fusion protein.

[0055] In another aspect, the invention provides a method of localizing an antibody to the periplasmic space of a yeast host cell, comprising binding the antibody to a periplasmic anchor protein such that the antibody is localized to the periplasmic space. In some embodiments, the antibody and the periplasmic anchor protein are non-covalently linked together through molecular binding interactions in a complex or linked through a covalent non-peptide bond in a complex. In some embodiments, the non-peptide bond is a disulfide bond. In some embodiments, the antibody and the periplasmic anchor protein are covalently linked together in a fusion protein.

[0056] In another aspect, the invention provides a kit containing the yeast periplasmic display library described herein and instructions for screening multiple protein variants for their ability to bind and/or modulate the activity of a target protein of interest.

[0057] These and other embodiments of the present invention will become readily apparent to those skilled in the art with reference to the present description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figures 1A-1D present a new method for displaying yeast cells to screen for antibodies that modulate GPCR function. Figure 1A presents a unique combination of 1) functional coupling of human-yeast GPCRs with 2) secretion of an affinity molecule and 3) localization of the affinity molecule together in a highly productive, highly modifiable yeast cell platform. A functional, properly folded GPCR provides ScFvs (which can be easily converted into IgG antibodies) or nanoantibodies that are more likely to function as therapeutics in the context of the human body. FIG. 1B illustrates the use of an "antagonist selection strain" to search for antagonists. FIG. 1C illustrates the use of an "agonist selection strain" to search for agonists. By changing the logic of reporters and selectable markers coupled to the output of the pheromone response system, the platform can be used to select agonists or antagonists. Figure 1D shows that direct functional screening provides therapeutic antibody candidates that would normally be missed by traditional screening, which may provide novel binding modes and functional modulation of GPCR targets. Because of the ease of genetic engineering techniques in yeast, it is possible to adjust both antibody expression levels and GPCR expression levels, and tailor selective and screenable reporters to be highly sensitive. Both of these allow one to find low-affinity but functional candidates that can be easily subjected to affinity maturation later.

[0059] Figure 2 illustrates a method for reducing background/false positives in a "halo assay". 10 ⁷ cells of the parental strain (left) and the current platform strain (NIY326, right) were plated on agar medium. A disk of filter paper was placed on the dish and 3 μl of 1 mM alpha factor was applied as a spot. A zone of no growth in response to the ligand (the desired phenotypic response) was observed in both cases, but suppressor mutants emerged in the parental strain (left) and grew into colonies in the presence of the pheromone (halo colonies). In the platform strain NI326 (right), the background rate was reduced to ~10 ^-7 , as demonstrated by the clear halo zone and the absence of background suppressor mutations that could act as false positives for antagonist selection.

[0060] Figure 3 illustrates the structure and concept of a targeting vector for an affinity molecule. We cloned an affinity molecule downstream of the secretion signal and upstream of the linker and extracellular membrane-anchoring GPI domain. When expressed in cells, the protein is secreted into the extracellular space and the GPI domain is then processed, leaving a GPI domain that binds to the membrane, which tethers affinity molecules to that cell and leaves them free to interact with the GPCR target on its extracellular surface.

[0061] Figures 4A and 4B provide confirmation of expression of the affinity molecule/targeting vector. Figure 4A shows that if the expressed anti-GFP nanoantibody is properly folded and localized, GFP applied outside the cell (after cell wall cleavage) should label the cell membrane. Figure 4B shows images of yeast expressing an anti-GFP nanoantibody using our targeting vector after cell wall digestion and application of purified GFP protein, which indicate GFP binding to the membrane. No fluorescence was observed in control cells (data not shown). Left, light field; right, GFP channel.

[0062] Figures 5A and 5B provide confirmation of plasmid dependence of alpha factor resistant clones. FIG. 5A shows a diagram of the strategy. Figure 5B shows an example of a "candidate" clone that exhibited alpha-factor resistant growth when tested by halo assay and then exhibited no resistance after plasmid knockout.

[0063] Figure 6 is a process flow diagram.

[0064] Figure 7 shows the effect on the growth rate of yeast cells through activation of the cannabinoid receptor type 2 (CB2 receptor) using VHH domain agonists exposed in the periplasmic space in various ways.

DETAILED DESCRIPTION OF THE INVENTION

[0065] Many therapeutic targets for diseases such as cancer and inflammation involve cell membrane-associated proteins. However, many cell membrane-associated proteins that have the greatest therapeutic potential for high-impact diseases are difficult to convert into drugs. Although small molecule compounds that affect the function of these proteins are easy to find, they are often nonspecific. In contrast to small molecules, antibodies and related affinity molecules (eg, nanoantibodies, and ScFvs and Fabs) are an attractive class of therapeutics due to their potentially higher specificity, functional diversity, and pharmacological properties. In addition, antibodies may better interact with extracellular domains and loops that can modulate the structure (and thus function) of cell membrane-associated proteins such as GPCRs in more complex ways than small molecule compounds. However, to date, there is no approved antibody therapeutic against GPCR in the United States, and there is only one in the world, in Japan.

[0066] Modern yeast and phage display technologies identify antibodies that bind strongly, but often do not interfere with the function of cell membrane-associated proteins such as GPCRs. The antigens used are often fragments that do not represent a functional protein available to the antibody in vivo, or are heterogeneously structured full-length protein preparations. This technology also misses a huge proportion of the overall functional diversity, since most antibodies are never subjected to functional analysis. A high-throughput platform is needed for the direct selection of antibodies that modulate the function of cell membrane-associated proteins such as GPCRs.

[0067] It is much less straightforward to develop antibodies that alter the function of cell membrane-associated proteins such as GPCRs (Jo 2015, Hutchings 2010). This is primarily a consequence of the following problems with many existing solutions: 1) there are no usable antigens. Antigens derived from extracellular peptides or fragments may be well suited for the development of antibodies for Western blotting, but do not structurally reflect therapeutic targets. In addition, uniformly functionally folded full-length protein in lipids or detergents may be difficult to obtain in sufficient quantities for immunization, phage display, or yeast display. 2) Antibodies selected for their high affinity are largely nonfunctional; they bind to regions in the protein that do not affect function. 3) Technologies lose much of the antibody diversity and thus the functionality of the selected antibodies. By first selecting antibodies that bind strongly and excluding others, much of the functional diversity is lost. Mammalian cell systems have been designed to functionally screen subsets of antibody candidates in an autocrine manner (Zhang 2014), which partially address problem 2, but due to transformation efficiency (~10 ⁴ ) and limited ability to construct selectable/screenable outputs, they are limited to screening small subsets of candidates .

[0068] The present inventors' innovation involves combining cell membrane-bound protein coupling with yeast pheromone response and expression of affinity molecules having cis-action in the same cell in a high-throughput platform. This allows direct and high-throughput functional selection of affinity molecules in the yeast periplasmic space. Antibodies and related affinity molecules are large molecules with complex folding patterns that must be maintained to maintain binding activity. Although in unstructured form short peptides may be able to localize to the yeast periplasmic space, it was not previously known that antibodies and related affinity molecules could be exposed to the yeast periplasmic space and retain binding activity.

[0069] To practice the present invention, unless otherwise indicated, conventional methods of pharmacology, chemistry, biochemistry, recombinant DNA and immunology methods related to the art can be used. Such methods are fully explained in the literature. See, for example, High Throughput Screening: Methods and Protocols (Methods in Molecular Biology, WP Janzen ed., Humana Press, ^3rd edition, 2016); G Protein-Coupled Receptors: Structure, Signaling, and Physiology (S. Siehler and G. Milligan eds., Cambridge University Press, 2010); Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell eds., Blackwell Scientific Publications); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual ( ^3rd Edition, 2001); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0070] All publications, patents and patent applications cited herein, both above and below, are incorporated herein by reference in their entirety.

I. DEFINITIONS

[0071] In describing the present invention, the following terms are used and are intended to be defined as defined below.

[0072] It should be noted that, as used in the present description and in the accompanying claims, the singular form includes the plural unless the context clearly indicates otherwise. Thus, for example, reference to “cell” includes a mixture of two or more cells, etc.

[0073] The term "about", particularly with respect to a given amount, covers variations of plus or minus five percent.

[0074] The term "about", particularly with respect to a given amount, covers and describes the given amount itself.

[0075] The terms "polypeptide" and "protein" refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, the definition includes peptides, oligopeptides, dimers, multimers, etc. The definition covers both full-length proteins and their fragments. The terms also include post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, hydroxylation, and the like. Moreover, for purposes of the present invention, “polypeptide” refers to a protein that includes modifications, such as deletions, insertions, and substitutions in the native sequence . These modifications may be intentional, such as through site-directed mutagenesis, or may be accidental, such as through mutations in the hosts that produce the proteins or errors during PCR amplification.

[0076] The term "antibody" includes monoclonal antibodies, as well as hybrid antibodies, modified antibodies, chimeric antibodies and humanized antibodies. The term "antibody" includes: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and US patent No. 4816567); F(ab') ₂ - and F(ab) fragments; F _v molecules (non-covalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single chain Fv molecules (scFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanoantibodies or single domain antibodies (sdAbs) (see, for example, Wang et al. (2016) Int J Nanomedicine 11:3287-3303, Vincke et al. (2012) Methods Mol Biol 911:15-26; dimeric and trimeric fragment designs antibodies; mini-antibodies (see, for example, Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, for example, Riechmann et al. al (1988) Nature 332:323-327; Verhoeyan et al (1988) Science 239:1534-1536; and UK Patent Publication No. GB 2276169 published September 21, 1994); and any functional moieties derived from such molecules , where such fragments retain the specific binding properties of the original antibody molecule.

[0077] The expression "specifically (or selectively) binds" with respect to the binding of an antibody to an antigen (eg, a GPCR) refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biological agents. Thus, under the intended conditions of the immunoassay, specific antibodies bind to a particular antigen at a level at least twice the background level, and essentially do not bind at a significant level to other antigens present in the sample. Specific binding to an antigen under such conditions may require an antibody that is selected for its specificity for a particular antigen. For example, antibodies raised against an antigen from a particular species, such as rat, mouse or human, can be selected to produce only those antibodies that are specifically immunoreactive against the antigen and not other proteins, excluding polymorphic variants and alleles. This selection can be accomplished by eliminating antibodies that cross-react with molecules from another species. Various immunoassay formats can be used to select antibodies that are specifically immunoreactive for a particular antigen. For example, ELISA is commonly used to select antibodies that are specifically immunoreactive for a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity) . Typically, the specific or selective response is at least twice the background signal or noise and, more specifically, more than 10 to 100 times the background.

[0078] A protein is considered to “interact” with another protein if it binds to it specifically (eg, in a lock-and-key manner), nonspecifically, or some combination of specific and nonspecific binding. A first protein "preferentially interacts" with a second protein if it binds (nonspecifically and/or specifically) to the second protein with greater affinity and/or greater specificity than other proteins. The term "affinity" refers to the strength of binding, and it can be expressed quantitatively as a dissociation constant (K _d ). It should be understood that specific binding does not necessarily require interactions between specific amino acid residues and/or motifs of each protein. For example, in certain embodiments, the first protein may preferentially interact with a second protein but may nonetheless be able to bind other polypeptides at a weak but detectable level (e.g., 10% or less of the binding shown for the protein of interest). polypeptide). Typically, weak binding or background binding is readily distinguishable from preferential interaction with the compound or polypeptide of interest, for example, using appropriate controls.

[0079] As used herein, the term “binding pair” refers to first and second molecules that specifically bind to each other. “Specific binding” of the first member of a binding pair to the second member of a binding pair is indicated by binding of the first member to the second member, or vice versa, with higher affinity and specificity than to other components in the sample. The binding between members of the binding pair is generally non-covalent. Examples include antigen-antibody, receptor-hormone, receptor-ligand, receptor-agonist, and receptor-antagonist binding pairs.

[0080] As used herein, the term “ligand” refers to a molecule that binds to another molecule, for example, an antigen binding to an antibody, a hormone, an agonist or antagonist binding to a receptor, a neurotransmitter binding to an ion channel, or substrate, inhibitor or allosteric effector binding to an enzyme, and includes natural and synthetic biomolecules such as proteins, polypeptides, peptides, nucleic acid molecules, carbohydrates, sugars, lipids, lipoproteins, low molecular weight compounds, natural and synthetic organic and inorganic materials, synthetic polymers, aptamers, etc.

[0081] The term “polynucleotide”, as known in the art, generally refers to a nucleic acid molecule. "Polynucleotide" may include both double-stranded and single-stranded sequences, and refers to, but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA from viral, prokaryotic or eukaryotic mRNA, RNA genomic sequences, and DNA from viruses (e.g., RNA and DNA viruses and retroviruses), prokaryotic DNA or eukaryotic (eg mammalian) DNA, and especially synthetic DNA sequences. The term also covers sequences that include any known analogues of DNA and RNA bases, and includes modifications such as deletions, insertions and substitutions (generally conservative in nature) in the native sequence. These modifications may be intentional, such as through site-directed mutagenesis, or may be random, such as through mutations in hosts involving polynucleotides encoding display polypeptide variants. Modifications to polynucleotides can have any number of effects, including, for example, facilitating expression of the polypeptide product in the host cell.

[0082] The polynucleotide may encode a biologically active protein or polypeptide. Depending on the nature of the polypeptide the polynucleotide encodes, the polynucleotide may comprise only 10 nucleotides, for example where the polynucleotide encodes an antigen or an epitope. Typically, the polynucleotide encodes peptides of at least 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 30 or even more amino acids.

[0083] The terms “variant,” “analogue,” and “mutein” refer to biologically active derivatives of the reference molecule that retain the desired activity (eg, effective polypeptide display) as described herein. In general, the terms "variant" and "analogue" refer to compounds having a native polypeptide sequence and structure with one or more insertions, substitutions and/or deletions of amino acids relative to the native molecule, provided that the modifications do not interfere with biological activity, and which are " "substantially homologous" to the reference molecule, as defined below. Typically, the amino acid sequences of such analogues have a high degree of sequence homology with the reference sequence, for example, amino acid sequence homology of greater than 50%, typically greater than 60%-70%, even more particularly 80%-85% or greater, e.g. at least 90%-95% or more when the two sequences are aligned. Often, analogues include the same number of amino acids, but include substitutions as explained herein. The term "mutein" further includes polypeptides having one or more amino acid-like molecules, including, but not limited to, compounds containing only amino and/or imino molecules, polypeptides containing one or more amino acid analogues (including, e.g., unnatural amino acids, etc.), polypeptides with substituted bonds, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules, etc. P. The term also includes molecules containing one or more N-substituted glycine residues ("peptoids") and other synthetic amino acids or peptides. (See, for example, US Patent Nos. 5,831,005; 5,877,278 and 5,977,301; Nguyen et al., Chem. Biol. (2000) 7:463-473; and Simon et al., Proc. Natl. Acad. Sci. USA (1992 ) 89:9367-9371 for the description of peptoids). Methods for preparing polypeptide and mutein analogues are known in the art and are further described below.

[0084] Analogues typically include substitutions that are conservative in nature, i.e. substitutions that occur in a family of amino acids that are similar in their side chains. In particular, amino acids are generally divided into four families: (1) acidic - aspartate and glutamate; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar ones - glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonable to assume that a single substitution of isoleucine or valine for leucine, glutamate for aspartate, or serine for threonine, or a similarly conservative amino acid substitution for a structurally similar amino acid, would not have a significant effect on biological activity. For example, a polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer in the range of 5-25, as long as the desired function of the molecule remains intact. One skilled in the art can readily determine regions of a molecule of interest that may be susceptible to variation according to Hopp/Woods and Kyte-Doolittle plots well known in the art.

[0085] “Recombinant,” as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin that, due to its origin or manipulation, is not associated with all or part of the polynucleotide with which it is associated in nature. The term "recombinant" as used in relation to a protein, polypeptide or peptide means a polypeptide produced by expression of a recombinant polynucleotide. Typically, the gene of interest is cloned and then expressed in transformed organisms, as further described below. The host organism expresses a foreign gene to produce a protein under expression conditions.

[0086] A "polynucleotide coding sequence" or a sequence that "codes" for a single polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when under the control of appropriate regulatory authorities. sequences (or "controls"). The boundaries of the coding sequence are defined by a start codon at the 5'(amino) end and a translation stop codon at the 3'(carboxy) end. The transcription termination sequence may be located 3' to the coding sequence. Exemplary "control elements" include, but are not limited to, transcriptional regulators such as promoters, transcriptional enhancer elements, transcription termination signals, and polyadenylation sequences; and translation regulators, such as sequences for optimizing translation initiation, for example, Shine-Dalgarno sequences (ribosome binding site), Kozak sequences (i.e. sequences for optimizing translation, located, for example, on the 5' side of the coding sequence), leader sequences (heterologous or native), translation initiation codon (eg, ATG) and translation termination sequences. Promoters may include inducible promoters (where expression of a polynucleotide sequence operably linked to a promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to a promoter is induced by an analyte, cofactor, regulatory protein, etc.) and constitutive promoters.

[0087] "Operably associated" refers to an arrangement of elements where the components so described are organized to perform their normal function. Thus, a given promoter, operably linked to the coding sequence, is capable of driving expression of the coding sequence when the proper enzymes are present. The promoter does not need to be adjacent to the coding sequence so long as it functions to drive its expression. Thus, for example, intervening untranslated but transcribed sequences may be present between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

[0088] By "fragment" is meant a molecule consisting of only a portion of the intact full-length sequence and structure. The fragment may include a C-terminal deletion and an N-terminal deletion, and/or an internal deletion in the peptide. The active fragments of a particular protein or peptide typically comprise at least about 5-10 consecutive amino acid residues of a full-length molecule, preferably at least about 15-25 consecutive amino acid residues of a full-length molecule, and most preferably at least about 20-50 or more consecutive amino acid residues of the full-length molecule, or any integer from 5 amino acids to the full-length sequence, provided that the fragment in question retains biological activity.

[0089] “Substantially purified” generally refers to the isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance constitutes a major percentage of the sample in which it is found. Typically, the substantially purified component of the sample comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Methods for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and density sedimentation.

[0090] By “isolated” we mean, when referring to a polypeptide, that the molecule is separated and distinct from the whole organism in which it naturally occurs, or is present substantially in the absence of other biological macromolecules of the same type. The term "isolated" with respect to a polynucleotide is a nucleic acid molecule lacking, in whole or in part, the sequences normally associated with it in nature; or a sequence as it exists in nature, but having heterologous sequences associated with it; or a molecule separated from a chromosome.

[0091] "Homology" refers to the percentage identity between two polynucleotide or two polypeptide molecules. Two nucleic acid sequences or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80%-85%, preferably at least at least about 90%, and most preferably at least about 95%-98% sequence identity over a certain molecular length. As used herein, essentially homologous also refers to sequences exhibiting complete identity to the specified sequence.

[0092] Generally, “identity” refers to an exact nucleotide-nucleotide or amino acid-amino acid match between two polynucleotide or polypeptide sequences, respectively. Percent identity can be determined by directly comparing the sequence information between two molecules (the reference sequence and a sequence with unknown % identity to the reference sequence) by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. Well-available computer programs such as ALIGN, Dayhoff, M.O., Atlas of Protein Sequence and Structure M.O. can be used to facilitate analysis. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, DC, which uses the local homology algorithm of Smith and Waterman, Advances in Appl. Math. 2:482-489, 1981, for peptide analysis. Programs for determining nucleotide sequence identities are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.), such as the BESTFIT, FASTA, and GAP programs, which are also based on the Smith and Waterman algorithm. These programs can be easily used with the manufacturer's recommended default settings and are described in the Wisconsin Sequence Analysis Package referenced above. For example, the percent identity of a particular nucleotide sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default scoring table and a deletion penalty of six nucleotide positions.

[0093] Another method of establishing percent identity in the context of the present invention is the use of the MPSRCH software package, copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). This software package uses the Smith-Waterman algorithm, which uses the default parameters for the scoresheet (for example, a skip penalty of 12, a continuation penalty of one, and a skip penalty of six). From the obtained data, the value "Match" reflects "sequence identity". Other suitable programs for calculating percent identity or similarity between sequences are well known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=no; chain=obi; threshold=60; wait=10; matrix=BLOSUM62; descriptions=50 sequences; sort by=highest exponent; databases=non-redundant GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Detailed descriptions of these programs are freely available.

[0094] Alternatively, homology can be determined by hybridizing the polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-strand specific nuclease(s), and determining the size of the digested fragments. DNA sequences that are substantially homologous can be identified in an experiment using Southern hybridization, for example, under stringent conditions, as determined for that particular system. Determining suitable hybridization conditions is within the ability of one skilled in the art. See, for example, Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

[0095] "Expression cassette" or "expression construct" refers to a system that is capable of directing the expression of sequence(s) or gene(s) of interest. The expression cassette typically includes control elements as described above, such as a promoter that is operably linked to (ie, directs transcription) the sequence(s) or genome(s) of interest, and also often includes a polyadenylation sequence. In certain embodiments of the invention, the expression cassette described herein may be contained in a plasmid or viral vector construct. In addition to the expression cassette components, the construct may also include one or more selectable markers, a signal that allows the construct to exist as single-stranded DNA (eg, M13 origin of replication), at least one multiple cloning site, and an origin of replication (eg, autonomously replicating sequence in yeast).

[0096] The term "transfection" is used to refer to the uptake of foreign DNA into a cell. A cell is "transfected" when exogenous DNA is introduced into the cell membrane. A number of transfection methods are well known in the art. See, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such methods can be used to introduce one or more exogenous portions of DNA into suitable host cells. The term refers to both stable and transient capture of genetic material, and includes capture of peptide- or antibody-associated DNA.

[0097] A “vector” is capable of transporting nucleic acid sequences into target cells (eg, viral vectors, non-viral vectors, particulate carriers, and liposomes). In general, “vector construct,” “expression vector,” and “gene transfer vector” mean any nucleic acid construct that is capable of directing the expression of a nucleic acid of interest and that can transfer nucleic acid sequences into target cells. Thus, the term includes cloning and expression vehicles as well as plasmid and viral vectors.

[0098] The term “transformation” refers to the introduction of an exogenous polynucleotide into a host cell, regardless of the method used for insertion. For example, direct capture, transduction or f-crossing are included. The exogenous polynucleotide may remain as a non-integrated vector, such as a plasmid, or alternatively it may be incorporated into the host genome.

[0099] "Recombinant host cells", "host cells", "cells", "cell lines", "cell cultures" and other such terms designating microorganisms or eukaryotic cell lines cultured as single cells refer to cells , which can be or are used as recipients for the recombinant vector or other transferred DNA, and include the original progeny of the original cell that is transfected.

[0100] A "coding sequence" or sequence that "encodes" a particular polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence may be defined by a start codon at the 5'(amino) end and a translation stop codon at the 3'(carboxy) end. The coding sequence may include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence may be located 3' to the coding sequence.

[0101] Typical "control elements" include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), translation initiation optimization sequences (located 5 '-sides of the coding sequence), and translation termination sequences.

[0102] The terms "label" and "detectable label" refer to a molecule capable of detecting, including, but not limited to, radioactive isotopes, stable (non-radioactive) heavy isotopes, fluorophores, chemiluminescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, ligands (eg biotin or haptens), etc. The term "fluorophore" refers to a substance or part thereof that is capable of fluorescence in a detectable range. Specific examples of tracers that can be used within the scope of the invention include, but are not limited to, radioactive tracers (for example, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P), stable (non-radioactive) heavy isotopes (for example, ¹³ C or ¹⁵ N), phycoerythrin, fluorescein, 7-nitrobenzo-2-oxa-1,3-diazole (NBD), YPet, CyPet, Cascade blue, allophycocyanin, Alexa dyes (e.g. Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 594, Alexa 647, Alexa 660, Alexa 680 and Alexa 750), Atto dyes (for example, Atto 488, Atto 532, Atto 550, Atto 565, Atto 590, Atto 610, Atto 620, Atto 635, Atto 647, Atto 655 and Atto 680), cyanine dyes (e.g. Cy3, Cy5 and Cy7), TYE 563, TYE 665, TYE 705, TEX 615, JOE, TET, HEX, TAMRA, ROX, rhodamine, dansyl , umbelliferone, Texas red, luminol, akradimium esters, biotin or other streptavidin-binding proteins, magnetic beads, electron-dense reagents, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, mApple, mCherry, rsCherry, rsCherryRev, firefly luciferase, Renilla luciferase, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase , alkaline phosphatase, chloramphenicol acetyltransferase and urease. Enzyme tags are used with their own substrate. As with many standard techniques associated with the practice of the invention, those skilled in the art will be aware of additional labels that can be used.

II. METHODS FOR IMPLEMENTING THE INVENTION

[0103] Before describing the present invention in detail, it should be understood that the present invention is not limited to specific compositions or process parameters, as these may of course vary. It should also be understood that the terminology provided in the present description is provided only to describe specific embodiments of the invention and is not limiting.

[0104] Although a number of methods and materials similar or equivalent to the methods and materials described herein may be used in the practice of the present invention, preferred materials and methods are described herein.

[0105] The present invention is based on the development of methods for displaying recombinant proteins in the periplasmic space of yeast cells. Specifically, the recombinant proteins are associated with a cell membrane-spanning transmembrane domain, a cell membrane-bound protein domain that is found on the outer surface of the yeast cell membrane, a protein that binds to the inner surface of the yeast cell wall, or a periplasmic protein for displaying proteins in the periplasmic space yeast. Recombinant proteins can also be targeted to the periplasm by binding the recombinant protein to a secretory signal. In addition, a target protein of interest can be coexpressed in yeast such that it is localized to the plasma membrane or periplasmic space and is accessible to exposed proteins. In specific embodiments, the inventors used their yeast periplasmic display method to screen for antibodies that bind and modulate human GPCR function (see examples). Antibodies exposed in the periplasmic space of the yeast cell are close enough to bind GPCRs expressed on the cell membrane. We have further developed a method for high-throughput screening of GPCRs for antagonists and agonists using periplasmic display by coupling human GPCRs to the yeast pheromone response cascade.

[0106] To further understand the invention, a more detailed discussion regarding yeast periplasmic display and methods for using it for high-throughput screening of protein libraries is provided below.

A. Periplasmic display of recombinant proteins in yeast

[0107] In one aspect, the invention relates to the display of a protein in the periplasmic space of a yeast host cell. In some embodiments, the yeast host cell comprises a yeast periplasmic display protein, a periplasmic anchor protein associated with a periplasmic display protein, and a target membrane protein of interest located in the yeast host cell periplasmic space. In some embodiments, a host yeast cell can be used to determine whether a protein to be displayed in the periplasmic space specifically binds to or affects the function of a target membrane protein of interest. A protein to be displayed in the periplasmic space of a host yeast cell can be produced by linking the recombinant protein to a periplasmic anchor protein, which localizes the recombinant protein to the periplasmic space of the yeast cell. The bond may be covalent or non-covalent. For example, the recombinant protein may be covalently linked to a periplasmic anchor protein in a fusion protein. Alternatively, the recombinant protein variant may form a complex with a periplasmic anchor protein, wherein the recombinant protein and the periplasmic anchor protein are non-covalently linked through molecular binding interactions in the complex. Alternatively, the recombinant protein variant and the periplasmic anchor protein are linked covalently through a non-peptide bond in the complex. In some embodiments, the non-peptide bond is a disulfide bond. A protein to be displayed in the periplasmic space of a yeast host cell can also be produced by coupling the protein to be displayed to a secretory signal. In some embodiments, the host yeast cell genus is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the host yeast cell genus is Saccharomyces. In some embodiments, the host yeast cell species is Saccharomyces cerevisiae.

[0108] In another aspect, the invention provides an antibody coupled to a periplasmic anchor protein. In some embodiments, the periplasmic anchor protein-linked antibody further comprises an additional modification, portion, or interacting protein. In some embodiments, the additional modification is a post-translational modification. In some embodiments, the portion is an affinity tag, an epitope, a label, or the like. In some embodiments, the antibody is localized in the periplasmic space of the host yeast cell. In some embodiments, when an antibody is produced in or introduced into a host yeast cell, the antibody is localized to the periplasmic space of the host yeast cell. In some embodiments, the antibody is coupled to a periplasmic anchor protein such that the antibody is localized to the periplasmic space. The association of the antibody with the periplasmic anchor protein may be covalent or non-covalent. For example, the antibody may be covalently linked to a periplasmic anchor protein in a fusion protein. Alternatively, the antibody may form a complex with a periplasmic anchor protein, where the antibody and the periplasmic anchor protein are non-covalently linked through molecular binding interactions in the complex. Alternatively, the antibody and periplasmic anchor protein are linked covalently through a non-peptide bond in the complex. In some embodiments, the non-peptide bond is a disulfide bond. Yeast host cells containing the antibody are also provided as described herein. In some embodiments, the host yeast cell genus is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the host yeast cell genus is Saccharomyces. In some embodiments, the host yeast cell species is Saccharomyces cerevisiae.

[0109] In another aspect, the invention provides methods for localizing an antibody to the periplasmic space of a yeast host cell, comprising linking the antibody to a periplasmic anchor protein such that the antibody is localized to the periplasmic space. In some embodiments, the antibody is associated with a periplasmic anchor protein such that the antibody is localized in the periplasmic space. The association of the antibody with the periplasmic anchor protein may be covalent or non-covalent. For example, the antibody may be covalently linked to a periplasmic anchor protein in a fusion protein. Alternatively, the antibody may form a complex with a periplasmic anchor protein, where the antibody and the periplasmic anchor protein are non-covalently linked through molecular binding interactions in the complex. Alternatively, the antibody and periplasmic anchor protein are linked covalently through a non-peptide bond in the complex. In some embodiments, the non-peptide bond is a disulfide bond. In some embodiments, the host yeast cell genus is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the host yeast cell genus is Saccharomyces. In some embodiments, the host yeast cell species is Saccharomyces cerevisiae.

[0110] In another aspect, the invention relates to methods for high-throughput screening of protein libraries for specific binding or functional characteristics by displaying proteins in the yeast periplasmic space. A yeast periplasmic display library can be prepared by linking recombinant proteins to a periplasmic anchor protein, which localizes the recombinant proteins to the periplasmic space of the yeast cell. The bond may be covalent or non-covalent. For example, the recombinant protein may be covalently linked to a periplasmic anchor protein in a fusion protein. Alternatively, the recombinant protein variant may form a complex with a periplasmic anchor protein, where the recombinant protein and the periplasmic anchor protein are non-covalently linked through molecular binding interactions in the complex. Alternatively, the recombinant protein variant and the periplasmic anchor protein are linked covalently through a non-peptide bond in the complex. In some embodiments, the non-peptide bond is a disulfide bond. A yeast periplasmic display library can also be generated by coupling recombinant proteins to secretory signals. In some embodiments, the host yeast cell genus is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia. In some embodiments, the host yeast cell genus is Saccharomyces. In some embodiments, the host yeast cell species is Saccharomyces cerevisiae.

[0111] Localization in the periplasmic space can be achieved in various ways. In certain embodiments, the recombinant protein is targeted to the periplasm by binding the recombinant protein to a secretory signal, which results in secretion of the recombinant protein into the extracellular space. In certain embodiments, the periplasmic anchor protein comprises a signal sequence that causes the periplasmic anchor protein to be transported into the periplasm of the host yeast cell, to the plasma membrane, or to the cell wall, such that the bound recombinant is exposed in the periplasm. For example, the periplasmic anchor protein may contain a signal sequence that directs the transport of the periplasmic anchor protein and associated recombinant protein into the periplasm of the host yeast cell. Preferably, the periplasmic anchor protein is large enough such that the periplasmic anchor protein and associated recombinant protein are retained in the periplasm. Alternatively, the periplasmic anchor protein may be a component of a periplasmic protein complex that is large enough that complex formation in the periplasm results in retention in the periplasm. In other embodiments, the periplasmic anchor protein comprises a membrane-spanning transmembrane domain or membrane-associated protein domain that is localized to the outer surface of the cell membrane such that the bound recombinant protein protrudes into the periplasm. For example, the glycosylphosphatidylinositol (GPI) anchoring domain, which is localized on the plasma membrane, can be used for this purpose. In one embodiment, the plasma membrane-anchoring GPI domain is a plasma membrane-anchoring yapsin GPI domain, such as, but not limited to, a plasma membrane-anchoring yapsin GPI domain YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7. In another embodiment, a periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the exposed variant of the protein protrudes into the periplasm. In certain embodiments, the periplasmic anchor protein and the recombinant protein are covalently linked in a fusion protein, and the periplasmic anchor protein comprises a signal sequence that directs transport of the fusion protein into the periplasm of the yeast host cell, to the plasma membrane, or to the cell wall, such that the fusion protein exposed in the periplasm. For example, the periplasmic anchor protein may contain a signal sequence that directs transport of the fusion protein into the periplasm of the host yeast cell. Preferably, the periplasmic anchor protein is large enough that the fusion protein is retained in the periplasm. Alternatively, the periplasmic anchor protein may be a component of a periplasmic protein complex that is large enough that formation of the complex in the periplasm results in retention of the fusion protein in the periplasm. In other embodiments, the periplasmic anchor protein comprises a membrane-spanning transmembrane domain or membrane-associated protein domain that is localized to the outer surface of the cell membrane such that the fusion protein protrudes into the periplasm. For example, the glycosylphosphatidylinositol (GPI) anchoring domain, which is localized on the plasma membrane, can be used for this purpose. In one embodiment, the plasma membrane-anchoring GPI domain is a plasma membrane-anchoring yapsin GPI domain, such as, but not limited to, a plasma membrane-anchoring yapsin GPI domain YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7. In another embodiment, a periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the exposed variant of the protein protrudes into the periplasm. In certain embodiments, the periplasmic anchor protein is a fragment of a full-length protein that retains the ability to localize to the periplasm.

[0112] Any type of protein can be exposed in the periplasmic space of a yeast cell, including, but not limited to, antibodies, antibody mimetics, aptamers, antigens, enzymes, receptors, transporters, ion channels, hormones, substrates, agonists, antagonists, or ligands. The yeast periplasmic display library presents a variety of such proteins that can be screened for folding and/or biological activity in the presence of a target molecule of interest. When located extracellularly, the target molecule must be able to penetrate the yeast cell wall to reach exposed proteins for screening. In certain embodiments, the target protein of interest is coexpressed with the exposed protein variant in the yeast cell at a position accessible to the exposed protein variant (e.g., close enough that the exposed protein variant binds and/or modulates the activity of the target protein of interest). For example, the target protein of interest may be located on the plasma membrane of the host yeast cell or in the periplasm near the site where the protein variant is exposed. In particular, this method is applicable to receptors, ion channels, transporters and other membrane proteins that are localized on the plasma membrane. Thus, yeast periplasmic display can be used to present proteins to a target membrane protein in an environment essentially similar to the native environment.

[0113] Any polypeptides included in the fusion construct, including the periplasmic anchor protein and the exposed variant protein, may be linked directly to each other by peptide bonds or may be separated by intervening amino acid sequences or linkers. Linker amino acid sequences are typically short, such as 20 amino acids or less (i.e., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 , 5, 4, 3, 2 or 1). Examples include short peptide sequences that facilitate cloning, polyglycine linkers (Gly _n , where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), histidine tags (His _n , where n=3 , 4, 5, 6, 7, 8, 9, 10 or more), linkers consisting of glycine and serine residues, where n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more), GSAT, SEG and Z-EGFR linkers. Linkers may include restriction sites that facilitate cloning and manipulation. Other suitable linker amino acid sequences are known to those skilled in the art (see, for example, Argos (1990) J. Mol. Biol. 211(4):943-958; Crasto et al. (2000) Protein Eng. 13:309-312 ; George et al. (2002) Protein Eng. 15:871-879; Arai et al. (2001) Protein Eng. 14:529-532; and Registry of Standard Biological Parts (partsregistry.org/Protein_domains/Linker).

[0114] Optionally, a label may be included in the fused structures. Tags that can be used to practice the invention include, but are not limited to, a His tag, a Strep tag, a TAP tag, an S tag, an SBP tag, an Arg tag, a calmodulin-binding peptide tag, a cellulose binding domain tag, DsbA tag, c-myc tag, glutathione S-transferase tag, FLAG tag, HAT tag, maltose binding protein tag, NusA tag and thioredoxin tag.

B. Polynucleotides encoding periplasm-anchored protein variants and target proteins and library construction

[0115] Polynucleotides encoding periplasm-anchored protein variants and target proteins of interest can be produced by a number of methods, all of which are well known in the art. For example, polynucleotides can be produced using recombinant methods well known in the art. One skilled in the art can readily determine nucleotide sequences that encode the desired proteins using standard methodology and the concepts described herein.

[0116] Oligonucleotide probes can be designed based on known gene sequences and can be used to probe genomic or cDNA libraries. Polynucleotides with the desired sequences can then be further isolated using standard methods and, for example, restriction enzymes can be used to truncate the gene at the desired regions of the full-length sequence. Likewise, polynucleotides with sequences of interest can be isolated directly from cells and tissues containing them using known methods such as phenol extraction, and the sequence can be further manipulated to produce desired protein variants. See, for example, Sambrook et al., supra, for a description of methods used to obtain and isolate DNA.

[0117] Sequences encoding variant proteins can also be obtained synthetically, for example, based on known sequences. The nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. The complete sequence is typically assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, for example, Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. 259:6311; Stemmer et al. (1995) Gene 164:49–53.

[0118] Recombinant methods are readily used to clone sequences encoding protein variants (eg, antibodies) useful for the claimed invention, which can then be mutagenized in vitro by replacing the appropriate base pair(s) to produce a codon for the desired amino acid. Such a change may involve just one base pair, representing a change in one amino acid, or it may involve a change in several base pairs. Alternatively, mutations can be made using a mismatch primer that hybridizes to the original nucleotide sequence (typically a cDNA corresponding to the RNA sequence) at a temperature below the melting temperature of the mismatch duplex. Primer specificity can be ensured by keeping the primer length and base composition within relatively narrow limits and by positioning the mutant base centrally. See, for example, Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is achieved using DNA polymers, the product is cloned and clones containing mutant DNA obtained by segregation of the extended primer strand are selected. Selection can be carried out using a mutant primer as a hybridization probe. This method is also applicable to introducing multiple point mutations. See, for example, Dalbie-McFarland et al. Proc. Natl. Acad. Sci. USA (1982) 79:6409.

[0119] The diversity of the display library depends on the mutagenesis method used. To quickly obtain large numbers of mutations, cassette mutagenesis can be used by inserting mutagenic cassettes into nucleic acid (see, for example, Worrall (1994) Methods Mol. Biol. 30:199-210, Kegler-Ebo et al. (1994) Nucleic Acids Research 22(9):1593-1599). In addition, random mutagenesis can also be used to obtain a large number of variants for a display library. Suitable random mutagenesis methods include, but are not limited to, reduced fidelity PCR, reduced fidelity rolling ring PCR, chemical mutagenesis, mutagenesis in a mutator strain deficient in DNA repair cascades, insertional mutagenesis using a transposon system, or DNA shuffling ( see, for example, McCullum et al. (2010) Methods Mol. Biol. 634:103-109, Fujii et al. (2014) Methods Mol. Biol. 1179:23-29, Muteeb (2010) Methods Mol. Biol. 634:411-419, Bose (2016) Methods Mol Biol 1373:111-115, Labrou (2010) Curr Protein Pept Sci 11(1):91-100 Wilson et al (2011) Methods Mol Biol 765:359-371). Such methods can be used to efficiently produce a large number of variants with modifications of the original nucleic acid molecule. In some embodiments, a DNA library containing at least 10 ⁶ , preferably at least 10 ⁸ , and more preferably at least 10 ¹⁰ unique sequence variants is constructed using methods known in the art.

[0120] In some embodiments, antibody libraries for yeast periplasmic display are constructed by cloning natural antibodies from B lymphocytes obtained from blood donors. Nucleic acids encoding antibody light and heavy chains or fragments thereof containing complementarity-determining variable domain regions (eg, Fab) can be amplified by PCR and cloned into vectors. ScFv antibodies can be produced by cloning into a vector that connects the light and heavy chains via a linker in a single open reading frame. A blood donor can be of any type. In some embodiments, human blood donors are used to generate the human antibody library. In other embodiments, camelid blood donors are used to generate the camelid antibody library. Camel antibodies can come from, for example, dromedaries, Bactrian camels, llamas or alpacas. These camelids produce a unique type of antibody that lacks a light chain. These heavy chain antibodies (HCAbs) or variable domain fragments thereof (eg, single domain antibodies or nanoantibodies) can be used to construct an antibody library (see, for example, Vincke et al. (2012) Methods Mol. Biol. 911:15-26 , Krah et al (2016) Immunopharmacol. Immunotoxicol. 38(1):21-8; incorporated herein by reference).

[0121] Once coding sequences for protein variants (eg, antibodies) have been isolated and/or synthesized, they can be cloned into any suitable vector or replicon for expression in yeast. A "vector" is a composition that can be used to deliver a nucleic acid of interest into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The expression construct can be produced by replication in a living cell, or it can be produced synthetically. For purposes of this application, the terms "expression construct", "expression vector" and "vector" are used interchangeably to demonstrate the application of the invention in a general illustrative sense, and they are not intended to limit the invention.

[0122] In certain embodiments, the nucleic acid encoding the polynucleotide of interest is under the transcriptional control of a promoter. "Promoter" refers to a DNA sequence recognized by a cell's synthetic machinery or introduced by a synthetic machinery required to initiate specific transcription of a gene. The term "promoter" is used herein to refer to a group of transcriptional control modules that cluster around the initiation site for RNA polymerase I, II or III. Enhancer elements can be used in conjunction with a promoter to increase the level of expression of the constructs. In certain embodiments, the expression vector contains a promoter "operably linked" to a polynucleotide encoding a fusion protein or target protein of interest. The expression "operably linked" or "under transcriptional control" as used herein means that the promoter is in the correct position and orientation relative to the polynucleotide to control the initiation of transcription by the RNA polymerase and the expression of the fusion protein or target protein of interest.

[0123] Typically, transcription terminator/polyadenylation signals are also present in the expression construct. Examples of such sequences include, but are not limited to, sequences derived from SV40, as described in Sambrook et al., supra, as well as the bovine growth hormone terminator sequence (see, for example, US Pat. No. 5,122,458). In addition, 5'UTR sequences can be placed adjacent to the coding sequence to enhance its expression. Such sequences may include UTRs containing an internal ribosome entry site (IRES).

[0124] Enabling IRES allows translation of one or more open reading frames from the vector. The IRES element recruits the eukaryotic ribosomal translation initiation complex and stimulates translation initiation. See, for example, Kaufman et al., Nuc. Acids Res. (1991) 19:4485–4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (1997 22 150-161). Many IRES sequences are known, and these include sequences derived from a wide variety of viruses, such as the leader sequences of picornaviruses, such as the UTR of encephalomyocarditis virus (EMCV) (Jang et al. J. Virol. (1989) 63:1651-1660), the leader polio sequence, hepatitis A virus leader sequence, hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25):15125-15130), IRES element from virus foot and mouth disease (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(5):3389-3397), etc. Also useful within the scope of the present invention are various non-viral IRES sequences, including, but not limited to, IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al., Mol. Cell Endocrinol. (2003) 212 :51-61), fibroblast growth factor IRES (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105(12):4733-4738, Stein et al. (1998) Mol Cell Biol 18(6):3112-3119, Bert et al (2006) RNA 12(6):1074-1083), and the insulin-like growth factor 2 IRES (Pedersen et al (2002) Biochem J. 363(Pt 1):37-44). An IRES sequence may be included in a vector, for example, to express a fusion protein comprising a periplasmic anchor protein fused to a variant display protein in combination with a target protein of interest from an expression cassette.

[0125] Alternatively, a polynucleotide encoding a T2A viral peptide can be used to enable the production of multiple protein products from a single vector. 2A linker peptides are inserted between coding sequences in a multicistronic construct. Peptide 2A, which is self-cleaving, allows the production of coexpressed proteins from a multicistronic construct in equimolar quantities. 2A peptides from various viruses can be used, including, but not limited to, 2A peptides derived from foot-and-mouth disease virus, equine rhinitis virus A, Thosea asigna virus, and porcine teschovirus 1. See, for example, Kim et al. (2011) PLoS One 6(4):e18556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10):625–629, Furler et al. (2001) Gene Ther. 8(11):864-873; incorporated herein by reference in their entirety.

[0126] In certain embodiments, the expression construct comprises a plasmid useful for transforming a yeast cell. Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and nutritional selection markers (e.g. HIS3, URA3, LYS2, LEU2, TRP1, MET15, ura4+, leu1+, ade6+), antibiotic selection markers (e.g. aphA1 or ble), fluorescent markers (eg, mCherry, green fluorescent protein), bioluminescent markers (eg, luciferase), or other markers for selection of transformed yeast cells. The yeast plasmid may further contain components to facilitate transfer between a bacterial host (eg, E. coli) and yeast cells. A number of different types of yeast plasmids are available, including yeast insertion plasmids (YIp), which lack an ORI and integrate into host chromosomes via homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromere plasmids (YCp), which are low copy vectors containing part of the ARS and part of the centromere sequence (CEN); and yeast episomal plasmids (YEp), which are high-copy number plasmids containing a 2-micron ring fragment (a naturally occurring yeast plasmid) that allows stable production of 50 or more copies per cell.

[0127] It may also be desirable to include regulatory sequences that allow the expression of protein sequences to be regulated relative to the growth of the host cell. Such regulatory sequences are known to those skilled in the art, and examples include sequences that cause gene expression to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. For example, a pheromone-inducible promoter, such as the PRM1 or FUS2 promoter, can be used to achieve transcription dependent on the activation of the pheromone signaling cascade. Control sequences and other regulatory sequences can be ligated to the coding sequence before insertion into the vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains control sequences and an appropriate restriction site.

[0128] In some cases, it may be necessary to modify the coding sequence so that it is associated with the control sequences in the proper orientation; those. to maintain proper reading frame. Mutants or can be generated by deleting part of the protein coding sequence, by inserting a sequence, and/or by replacing one or more nucleotides in the sequence. Methods for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, for example, Sambrook et al., supra; DNA Cloning, Vols. I and II, above; Nucleic Acid Hybridization, supra.

[0129] In one embodiment, recombinant polynucleotides encoding protein variants are cloned into a periplasmic-targeting expression vector containing: a) a polynucleotide encoding a signal peptide; b) a cloning site suitable for insertion in frame of a polynucleotide encoding a protein variant after a polynucleotide encoding a signal peptide; c) a polynucleotide encoding a plasma membrane-anchoring glycophosphatidylinositol (GPI) domain, positioned such that the vector is capable of producing a fusion protein comprising a signal peptide and a variant protein fused to the plasma membrane-anchoring GPI domain; and d) a promoter operably linked to the fusion protein coding sequences. In certain embodiments, the plasma membrane-anchoring GPI domain is a plasma membrane-anchoring yapsin GPI domain, such as, but not limited to, a plasma membrane-anchoring yapsin GPI domain YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7. The periplasmic-targeting expression vector may further comprise a polynucleotide encoding a linker located between the cloning site and a polynucleotide encoding a plasma membrane anchoring GPI domain. The cloning site may contain one or more restriction sites. The periplasmic-targeting expression vector may further contain a selectable marker.

[0130] In some embodiments, an affinity tag, epitope, label, or the like is added to the protein variant to enable determination of the overall level of display in yeast cells. As used herein, the term “affinity tag” refers to a biomolecule, such as a polypeptide segment, that can be associated with a second biomolecule to provide purification or detection of the second biomolecule or to provide sites for attachment of the second biomolecule to a substrate. Examples of affinity tags include poly-histidine stretch, protein A (Nilsson et al. (1985) EMBO J. 4:1075; Nilsson et al. (1991) Methods Enzymol. 198:3, glutathione S transferase (Smith and Johnson (1988) Gene 67:31), Glu-Glu affinity tag (Grussenmeyer et al., (1985) PNAS USA 82:7952), substance P, FLAG peptide (Hopp et al. (1988) Biotechnology 6:1204), streptavidin-binding peptide , or other antigenic epitope or binding domain, etc. (Ford et al. (1991) Protein Expression and Purification 2:950), all of which are incorporated herein by reference. As used herein, "labeling " is a molecule or atom that can be conjugated to a biomolecule to make a biomolecule or a form of a biomolecule, such as a conjugate, detectable or quantifiable. Examples of labels include fluorescent agents, bioluminescent proteins, photoactive agents, radioisotopes, paramagnetic ions, chelators and etc.

[0131] An expression vector is used to transform a suitable yeast host cell. A number of yeast hosts are known in the art, including, but not limited to, Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces boulardii, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces chevalieri, Saccharomyces dairenensis, Sac charomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguus , Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencero rum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, Saccharomyces zonatus, Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida auris, Candida blattae, Candida bromeliacearum, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydali, Candida do sseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, Candida kefyr, Candida keroseneae, Candida krusei, Candida lusitaniae, Candida lyxosophila, Cand ida maltosa, Candida marina , Candida membranifaciens, Candida mogii, Candida oleophila, Candida oregonensis, Candida parapsilosis, Candida quercitrusa, Candida rugosa, Candida sake, Candida shehatea, Candida temnochilae, Candida tenuis, Candida theae, Candida tolerans, Candida tropicalis, Candida tsuchiyae, Candida sin olaborantium, Candida sojae, Candida subhashii, Candida viswanathii, Candida utilis, Candida ubatubensis, Candida zemplinina, Pichia farinosa, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pich ia methanolica, Pichia subpelliculosa, Kluyveromyces aestuarii, Kluyveromyces africanus, Kluyveromyces bacillisporus, Kluyveromyces blattae, Kluyveromyces dobzhanskii, Kluyveromyces hubeiensis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Kluyveromyces nonfer mentans, Kluyveromyces piceae, Kluyveromyces sinensis, Kluyveromyces thermotolerans, Kluyveromyces waltii, Kluyveromyces wickerhamii, Kluyveromyces yarrowii, Yarrowia bubula , Yarrowia deformans, Yarrowia lipolytica, Yarrowia porcina and Yarrowia yakushimensis, which can be used in the expression constructs of the present invention. In some embodiments, the yeast is a Saccharomyces species. In some embodiments, the yeast is Saccharomyces cerevisiae.

[0132] To enable expression of sense or antisense gene constructs, the expression construct must be delivered into the yeast cell. This delivery can be accomplished in vitro using laboratory yeast cell transformation techniques well known in the art, such as spheroplast transformation, alkaline ion treatment (e.g., Cs ⁺ or Li ⁺ ), electroporation, inter-kingdom conjugation, electroporation, and biolistic and glass-mediated granule methods (see, for example, Kawai et al. (2010) Bioeng. Bugs. 1(6):395-403, Gietz et al. (1995) Yeast 11(4):355-360, Gietz et al. ( 2007) Nat Protoc 2(1):38-41, Hinnen et al (1978) Proc Natl Acad Sci USA 75:1929-1933, Avery et al (1995) Mol Med 1( 4):344-365, Ito et al (1983) J Bacteriol 153:163-168, Johnston et al (1988) Science 240:1538-1541, Dohmen et al (1991) Yeast 7(7) :691-246, Hayama et al (2002) J Biosci Bioeng 94(2):166-171, and Wang et al (2001) Crit Rev Biotechnol 21(3):177-218; incorporated herein by reference).

[0133] After delivery of the expression construct to the cell, the nucleic acid encoding the gene of interest can be placed and expressed at various sites. In certain embodiments, the nucleic acid encoding the gene can be stably integrated into the genome of a cell through homologous recombination. This insertion may occur at a similar position and orientation (gene replacement), within a gene (gene interruption), or at a random, nonspecific position (gene amplification). Insertion of a construct at a target locus that interrupts a gene may be acceptable as long as the gene interruption does not interfere with cell growth or screening of a yeast periplasmic display library (eg, does not disrupt the pheromone response if used in the screen). In other embodiments, the nucleic acid may be stably maintained in a cell as a distinct episomal DNA segment. Such nucleic acid segments or "episomes" encode sequences sufficient to allow maintenance and replication independent of or synchronous with the host cell cycle. How the expression construct is delivered into the cell and where the nucleic acid remains in the cell depends on the type of expression construct used.

[0134] In certain embodiments, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct can be accomplished by any of the methods mentioned above that physically or chemically permeabilize the cell membrane.

[0135] In another embodiment, the naked DNA expression construct can be transferred into cells via particle bombardment. This method is based on the ability to accelerate DNA-coated microprojectiles to high speeds, allowing them to pass through yeast cell walls and membranes and enter cells without killing them (Armaleo et al. (1990) Curr. Genet. 17(2):97- 103). Several devices have been developed to accelerate small particles. One such device relies on a high-voltage discharge to generate an electrical current, which in turn provides the driving force (Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572). Microprojectiles may consist of biologically inert substances such as tungsten or gold granules.

[0136] In some embodiments, a set of linear DNA molecules encoding protein variants is obtained. Instead of cloning linear DNA molecules into a vector before transformation, yeast cells are transformed with an empty vector along with a set of linear DNA molecules encoding protein variants, which are then incorporated into the vector in vivo, for example, through homologous recombination in host yeast cells.

C. Sets

[0137] The kit may include a yeast periplasmic display library as described herein, or means for producing a yeast periplasmic display library, such as suitable vectors for cloning nucleic acids encoding protein variants to produce protein variants associated with a periplasmic anchor protein, yeast cells, transfection agents, media suitable for growing yeast cells, positive and negative cell selection agents, and other reagents as necessary. The kit will typically include instructions (e.g., printed form, CD-ROM, DVD, Blu-ray, flash memory, digital download, etc.) for preparing and/or screening the yeast periplasmic display library, as described herein.

[0138] The kit may include a yeast periplasmic display library as described herein, or means for producing a yeast periplasmic display library, such as suitable vectors for cloning nucleic acids encoding protein variants to produce periplasmic anchor protein fusion constructs in yeast cells , transfection agents, media suitable for growing yeast cells, agents for positive or negative cell selection and other reagents as required. The kit will typically include instructions (e.g., printed form, CD-ROM, DVD, Blu-ray, flash memory, digital download, etc.) for preparing and/or screening the yeast periplasmic display library, as described herein.

[0139] In one embodiment, the kit contains a periplasmic targeting expression vector comprising: a) a polynucleotide encoding a signal peptide; b) a cloning site suitable for inserting in frame a polynucleotide encoding a protein variant (eg, an antibody) after a polynucleotide encoding a signal peptide; c) a polynucleotide encoding a plasma membrane-anchoring glycophosphatidylinositol (GPI) domain, arranged so that the vector is capable of producing a fusion protein comprising a signal peptide and a variant protein fused to a plasma membrane-anchoring GPI domain; and d) a promoter operably linked to the fusion protein coding sequences. In another embodiment, the signal peptide comprises a prepro-alpha factor signal sequence. In certain embodiments, the plasma membrane-anchoring GPI domain is a plasma membrane-anchoring yapsin GPI domain, such as, but not limited to, a plasma membrane-anchoring yapsin GPI domain YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7. In another embodiment, the periplasmic-targeting expression vector further comprises a polynucleotide encoding a linker, wherein the polynucleotide encoding the linker is located between the cloning site and the polynucleotide encoding a plasma membrane anchoring GPI domain. The linker may also contain a label. In another embodiment, the periplasmic-targeting expression vector further comprises a selectable marker. In another embodiment, the cloning site contains one or more restriction sites.

[0140] In another embodiment, the kit includes a yeast periplasmic display library containing a plurality of yeast host cells, wherein each yeast host cell contains: a) a fusion protein comprising a periplasmic anchor protein fused to a variant protein (e.g., antibody) for display in the periplasmic space of the yeast host cell, wherein the displayed protein variant is different in each host yeast cell such that a plurality of host yeast cells display a plurality of protein variants; b) a G protein coupled receptor (GPCR) target of interest, wherein the target GPCR of interest is located on the plasma membrane of the host yeast cell and is accessible to the protein variant exposed in the periplasmic space of the host yeast cell; and c) an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in a host yeast cell. In certain embodiments, the engineered G subunit is a chimeric G protein alpha (Gα) subunit, containing the N-terminal domain of the yeast G subunit and the C-terminal domain of the exogenous Gα subunit. For example, the yeast G subunit may belong to the Gαi, Gαq, Gαs, or Gαo family of G proteins. The chimeric G subunit has at least five C-terminal residues of the yeast G subunit may be replaced by the corresponding C-terminal residues of the mammalian G subunit such that the chimeric G subunit is capable of being activated by a mammalian GPCR. In some embodiments, at least 20 C-terminal residues of the yeast G subunit are replaced by the corresponding C- terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of activation by a mammalian GPCR. In another embodiment, the chimeric Gα subunit comprises at least 41 N-terminal residues of the yeast Gα subunit. In certain embodiments, the mammalian GPCR is a mouse GPCR. In certain embodiments, the mammalian GPCR is a human GPCR selected from the group consisting of CXCR4 , CXCR5, SSTR2, MOR, AVPR2, FPR2/ALX, ADORA2A, CHRM3, CGRP2, CCR2, CCR4, CCR5, CHRM4, PAC1, b2AR, CXCR2, CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB1, CB2, A3AR and AT1R.

[0141] In another embodiment, the kit includes a yeast periplasmic display library comprising: a plurality of yeast host cells, wherein each yeast host cell contains a fusion protein comprising a periplasmic anchor protein fused to a variant of the display protein, wherein the periplasmic anchor protein is sufficient large so that the fusion protein is retained in the periplasm, and the protein variant displayed is different in each yeast host cell such that multiple yeast host cells display multiple protein variants.

[0142] In another embodiment, the kit includes a yeast periplasmic display library comprising: a plurality of yeast host cells, wherein each yeast host cell contains a fusion protein comprising a display protein variant linked to a target membrane protein of interest, wherein the displayed variant protein is different in each yeast host cell such that a plurality of yeast host cells display a plurality of protein variants with the target membrane protein of interest.

D. Applications

[0143] The present invention can be generally used to screen for proteins that perform useful or desired functions, including binding, catalysis, assembly, transport, and the like. For example, yeast periplasmic display can be used to identify receptor agonists and antagonists, design therapeutic proteins and antibodies, identify protein-protein interactions, and map epitopes.

[0144] Yeast periplasmic display is particularly useful for screening a library of proteins for candidates that bind and/or modulate the function of a target protein that is a membrane protein, such as a receptor, ion channel or transporter. Localization of a protein to the membrane makes it accessible to exposed protein variants in the periplasmic space (eg, close enough for the exposed protein variant to bind to the target protein of interest).

[0145] In certain embodiments, activation of a target protein of interest enhances the growth of host yeast cells. In this case, the yeast periplasmic display library can be screened for an agonist of the target protein of interest by culturing at least a subset of yeast host cells of the yeast periplasmic display library in a medium where growth of the yeast host cell in the medium indicates that the protein variant , exposed in a yeast host cell, is an agonist of the target protein of interest.

[0146] In other embodiments, activation of a target protein of interest reduces the growth of host yeast cells. In this case, the yeast periplasmic display library can be screened for an antagonist of the target protein of interest by culturing at least a subset of yeast host cells of the yeast periplasmic display library in a medium wherein growth of the yeast host cell in the medium indicates that the protein variant , exposed in a yeast host cell, is an antagonist of the target protein of interest.

[0147] In certain embodiments, each yeast host cell further comprises a reporter system comprising a reporter gene operably linked to an inducible promoter that is activated when the target protein of interest is activated, allowing detection of an increase or decrease in the activity of the target protein of interest when binding the exposed protein variant to a target protein of interest. For example, the reporter gene may be a nutritional marker (e.g., HIS3, HIS7, ARG6, LEU2, URA3, and TRP1), an antibiotic resistance marker (e.g., communicates resistance to an antibiotic such as geneticin (e.g., aphA1), zeocin (e.g., ble), hygromycin B, nourseothricin or bialaphos), fluorescent marker (eg green fluorescent protein, red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein and orange fluorescent protein), bioluminescent marker (eg luciferase or aequorin) or a counterselection marker (eg, CAN1, URA3, MET15, TRP1, and TK).

[0148] For example, positive selection (ie, selection for activation of reporter gene expression) can be used to detect an increase in the activity of a target membrane protein of interest upon binding of an exposed protein variant to the target membrane protein of interest. Detection of nutritional marker expression can be carried out, for example, by the growth of host yeast cells in a selective nutrient-deficient medium. Detection of the expression of an antibiotic resistance marker can be carried out, for example, by the growth of host yeast cells in a selective medium containing the antibiotic. Detection of fluorescent marker expression can be achieved, for example, by the fluorescence emitted by the host yeast cells. Detection of bioluminescent marker expression can be carried out, for example, by bioluminescence emitted by host yeast cells.

[0149] Alternatively, counterselection (ie, growth-based selection for loss of reporter gene expression) can be used to detect a decrease in the activity of a target membrane protein of interest upon binding of an exposed protein variant to the target membrane protein of interest. The counterselection marker may kill cells by inducing apoptosis, convert a nontoxic drug into a toxic compound, or transport a toxic molecule into the cell. Counter-selection can be accomplished by culturing host yeast cells in a medium containing an agent that selectively kills cells expressing the counter-selection marker. Exemplary counterselection markers include CAN1 (canavanine counterselection), URA3 (5-fluoroorotic acid (5-FOA) counterselection), MET15 (methylmercury counterselection), TRP1 (5-fluoroanthranilic acid (5-FAA) counterselection), and TK thymidine kinase human herpes virus (counterselection with floxuridine (FUDR)).

[0150] In particular, a yeast periplasmic display library can be used to screen for antibodies that bind and modulate GPCR function. In some embodiments, the GPCR in the host yeast cell is replaced with a mammalian GPCR, such as a human GPCR. In some embodiments, the host yeast cell expresses a mammalian GPCR, such as a human GPCR, and an endogenous yeast GPCR. In some embodiments, antagonists and agonists are identified using a reporter system that couples the GPCR response to exposed antibody binding to yeast pheromone secretion levels (see Examples). For this purpose, a host yeast cell can be genetically modified to express an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in the yeast host cell. In certain embodiments, the engineered G subunit is a chimeric a G protein alpha (Gα) subunit containing the N-terminal domain of the yeast G subunit and the C-terminal domain of the exogenous Gα subunit. For example, the yeast G subunit may belong to the Gαi, Gαq, Gαs, or Gαo family of G proteins. In a chimeric subunit The G(at least five C-terminal residues of the yeast G(subunit) may be replaced by the corresponding C-terminal residues of the mammalian G(subunit) such that the chimeric G(subunit is capable of being activated by a mammalian GPCR. In some embodiments, the at least 20 C-terminal residues of the mammalian G(subunit) residues of the yeast G subunit are replaced by the corresponding C-terminal residues of the mammalian G subunit, such that the chimeric G subunit is capable of activation by the mammalian GPCR. In another embodiment, the chimeric G(subunit) contains at least 41 N-terminal residues of the yeast Gα subunit. Exemplary mammalian G(subunits) include G alpha-S, G alpha-I, G alpha-O, G alpha-T, G alpha- Z, G alpha-Q, G alpha-11, G alpha-12, G alpha-13 and transducin.

[0151] In particular, yeast strains Δfar1, Δsst2, Δste14, Δste3 or Δmat are useful for screening GPCR antagonists and agonists. The Δmat strain may contain, for example, a deleted or inactivated MAT locus (or a deleted or inactivated MATa locus. The host yeast cell may further contain a modified CLN3 protein containing a C-terminal truncation that increases the content of CLN3 in the yeast host cell by compared to wild-type CLN3 protein. For example, a modified CLN3 protein may retain at least N-terminal amino acids 1-387 or 1-408 of wild-type CLN3 protein, or any number of N-terminal amino acids in these ranges, such as 1-388, 1-389, 1-390, 1-391, 1-392, 1-393, 1-394, 1-395, 1-396, 1-397, 1-398, 1-399, 1-400, 1- 401, 1-402, 1-403, 1-404, 1-405, 1-406, 1-407, or 1-408, where the C-terminal truncation contains a deletion of all or some of the remaining residues of the wild-type CLN3 protein. Yeast cells -hosts used to generate the periplasmic display library can be haploid or diploid.Illustrative yeast strains for use in antagonist and agonist selection are described in Examples 2 and 5, respectively.

[0152] In certain embodiments, the host yeast cell is a FAR1 strain, wherein inhibition of the pheromone response by an antagonist antibody that binds and inhibits GPCRs in the host yeast cell results in cell cycle arrest and growth of the yeast cell. owner. In other embodiments, the host yeast cell is a Δfar1 strain comprising a pheromone-inducible PRM1 promoter operably linked to a reporter gene, wherein activation of the pheromone response by an agonist antibody that binds and activates a GPCR in the host yeast cell results in to increase reporter gene expression.

[0153] Any type of GPCR from any species can be screened using periplasmic display as described herein. In some embodiments, the target GPCR of interest is a mammalian GPCR (eg, from a human or non-human primate, rodent, laboratory animal, livestock). For example, a mammalian GPCR may be a human GPCR (e.g., CXCR4, CXCR5, SSTR2, MOR, AVPR2, FPR2/ALX, ADORA2A, CHRM3, CGRP2, CCR2, CCR4, CCR5, CHRM4, PAC1, b2AR, CXCR2, CYSLTR2, KSHV vGPCR , PKR1, PKR2, CB1, CB2, A3AR and AT1R). The GPCR target of interest may have constitutive ligand-independent activity. Alternatively, a ligand can be added to activate a GPCR target of interest during agonist or antagonist screening. In some embodiments, the yeast host cell expresses a target GPCR of interest, such as a human GPCR of interest, and an endogenous yeast GPCR.

[0154] In some embodiments, the protein variants are antibodies. Using the yeast periplasmic display methods described herein, any type of antibody can be screened, including monoclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al (1991) Nature 349:293-299; and US Patent No. 4816567); F(ab') ₂ - and F(ab) fragments; F _v molecules (non-covalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single chain Fv molecules (scFv) (see, for example, Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanoantibodies or single domain antibodies (sdAbs) (see, for example, Wang et al. (2016) Int J Nanomedicine 11:3287–3303, Vincke et al. (2012) Methods Mol Biol 911:15–26; dimer and trier fragment designs antibodies; mini-antibodies (see, for example, Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, for example, Riechmann et al. al (1988) Nature 332:323-327; Verhoeyan et al (1988) Science 239:1534-1536; and UK Patent Publication No. GB 2276169 published September 21, 1994); and any functional moieties derived from such molecules , where such fragments retain the specific binding properties of the original antibody molecule.

[0155] In other embodiments, the protein variants are aptamers. Aptamers can be isolated from a combinatorial library and improved through targeted mutation or repeated rounds of mutagenesis and selection. For descriptions of methods for preparing aptamers, see, for example, Aptamers: Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols (Methods in Molecular Biology, G. Mayer ed. , Humana Press, 2009), Aptamers Selected by Cell-SELEX for Theranostics (W. Tan, X. Fang eds., Springer, 2015), Cox et al. (2001) Bioorg. Med. Chem. 9(10):2525-2531; Cox et al. (2002) Nucleic Acids Res. 30(20): e108, Kenan et al. (1999) Methods Mol. Biol. 118:217-231; Platella et al. (2016) Biochim. Biophys. Acta Nov 16 pii: S0304-4165(16)30447-0, and Lyu et al. (2016) Theranostics 6(9):1440–1452; incorporated herein by reference in their entirety.

[0156] In other embodiments, the protein variants are antibody mimetics. Any type of antibody mimetics can be used, including, but not limited to, affiantibody molecules (Nygren (2008) FEBS J. 275 (11):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372 (1):172-185), affmers (Johnson et al. (2012) Anal. Chem. 84 (15):6553-6560), affins (Krehenbrink et al. (2008) J. Mol. Biol. 383 (5 ):1058-1068), alphabodies (Desmet et al. (2014) Nature Communications 5:5237), anticalins (Skerra (2008) FEBS J. 275 (11):2677-2683), avimers (Silverman et al. (2005) ) Nat. Biotechnol. 23 (12):1556-1561), darpins (Stumpp et al. (2008) Drug Discov. Today 13 (15-16):695-701), finomers (Grabulovski et al. (2007) J . Biol. Chem. 282(5):3196-3204) and monobodies (Koide et al. (2007) Methods Mol. Biol. 352:95-109).

[0157] In addition, directed evolution can be performed through multiple rounds of mutagenesis and yeast periplasmic display screening to enrich libraries for protein variants (eg, antibodies) that bind the target protein of interest with high affinity. Directed evolution, in particular, may be useful for improving the binding performance of candidates with desired functional activity but weak binding affinity.

III. Exemplary Embodiments

[0158] The embodiments described herein are as follows:

1. A yeast periplasmic display library containing a plurality of yeast host cells, where each yeast host cell contains:

a) an antibody for display in the periplasmic space of a yeast host cell, where the displayed antibody is different in each yeast host cell such that a plurality of yeast host cells display a plurality of antibodies;

b) periplasmic anchor protein, where the periplasmic anchor protein is associated with the antibody so that the antibody is exposed in the periplasmic space; And

c) a target membrane protein of interest, wherein the membrane protein of interest is located on the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell.

2. The yeast periplasmic display library according to embodiment 1, wherein the antibody and the periplasmic anchor protein are non-covalently linked to each other through molecular binding interactions in a complex or linked through a covalent non-peptide bond in a complex.

3. The yeast periplasmic display library according to embodiment 1, wherein the antibody and the periplasmic anchor protein are covalently linked together in a fusion protein.

4. The yeast periplasmic display library according to any one of embodiments 1-3, wherein the periplasmic anchor protein further comprises a signal sequence that directs transport of the periplasmic anchor protein into the periplasm of the yeast host cell, the plasma membrane, or the cell wall, such that the antibody is displayed in periplasm.

5. The yeast periplasmic display library according to any one of embodiments 1-3, wherein the periplasmic anchor protein comprises a membrane spanning transmembrane domain or membrane-associated protein domain that exposes the antibody to the periplasm.

6. The yeast periplasmic display library of Embodiment 5, wherein the cell membrane-associated protein domain is a plasma membrane-anchoring glycosylphosphatidylinositol (GPI) domain.

7. The yeast periplasmic display library according to embodiment 6, wherein the plasma membrane-anchoring GPI domain is a japasine plasma membrane-anchoring GPI domain.

8. The yeast periplasmic display library of Embodiment 7, wherein the plasma membrane-anchoring yapsin GPI domain is a plasma membrane-anchoring yapsin GPI domain YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7.

9. The yeast periplasmic display library of Embodiment 2, wherein the periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the displayed protein variant protrudes into the periplasm.

10. The yeast periplasmic display library of Embodiment 3, wherein the periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the displayed protein variant protrudes into the periplasm.

11. The yeast periplasmic display library of Embodiment 2, wherein the periplasmic anchor protein is large enough such that the periplasmic anchor protein and associated antibody are retained in the periplasm.

12. The yeast periplasmic display library of Embodiment 3, wherein the periplasmic anchor protein is large enough that the fusion protein is retained in the periplasm.

13. A yeast periplasmic display library containing a plurality of yeast host cells, wherein each yeast host cell contains:

a) an antibody for display in the periplasmic space of a yeast host cell, wherein the displayed antibody is different in each yeast host cell such that a plurality of yeast host cells display a plurality of antibodies, wherein the antibody is linked to a signal sequence that directs transport of the antibody into the yeast periplasm host cell, to the plasma membrane or cell wall, such that the antibody is exposed in the periplasmic space of the yeast host cell; And

b) a target membrane protein of interest, wherein the membrane protein of interest is located on the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell.

14. The yeast periplasmic display library according to any one of embodiments 1-13, further comprising a reporter system comprising a reporter gene operably linked to an inducible promoter that is activated when the target membrane protein of interest is activated, allowing detection of increases or decreases in the activity of the target of interest target membrane protein upon binding of the antibody to the target membrane protein of interest.

15. The yeast periplasmic display library of embodiment 14, wherein the reporter gene is a nutritional marker, an antibiotic resistance marker, a fluorescent marker, a bioluminescent marker, or a counterselection marker.

16. The yeast periplasmic display library according to embodiment 15, wherein the nutritional marker is selected from the group consisting of HIS3, HIS7, ARG6, LEU2, URA3 and TRP1.

17. The yeast periplasmic display library of embodiment 15, wherein the antibiotic resistance marker conveys resistance to an antibiotic selected from the group consisting of geneticin, zeocin, hygromycin B, nourseothricin, and bialaphos.

18. The yeast periplasmic display library of embodiment 15, wherein the fluorescent marker is selected from the group consisting of green fluorescent protein, red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and orange fluorescent protein.

19. The yeast periplasmic display library according to embodiment 15, wherein the bioluminescent marker is luciferase or aequorin.

20. The yeast periplasmic display library according to embodiment 15, wherein the counterselection marker is selected from the group consisting of CAN1, URA3, MET15, TRP1 and TK.

21. The yeast periplasmic display library of embodiment 14, wherein the reporter gene is a selectable marker such that said increase in activity of a target membrane protein of interest upon binding of an antibody to the target membrane protein of interest is detectable by growth of host yeast cells on the medium for positive selection.

22. The yeast periplasmic display library of embodiment 14, wherein the reporter gene is a counterselection marker such that said reduction in activity of a target membrane protein of interest upon binding of an antibody to the target membrane protein of interest is detectable by growth of host yeast cells on the medium for negative selection.

23. The yeast periplasmic display library according to any one of embodiments 1-22, wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel, and a transporter.

24. The yeast periplasmic display library of embodiment 23, wherein the receptor is a G protein coupled receptor (GPCR).

25. The yeast periplasmic display library of embodiment 24, wherein the GPCR is an exogenous GPCR.

26. The yeast periplasmic display library of embodiment 25, wherein the yeast host cell further comprises an endogenous GPCR.

27. The yeast periplasmic display library of embodiment 25 or 26, further comprising an engineered Gα subunit capable of being activated by an exogenous GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in a host yeast cell.

28. The yeast periplasmic display library according to embodiment 27, wherein the engineered G(subunit) is a chimeric G protein alpha (Gα) subunit containing the N-terminal domain of the yeast G(subunit) and the C-terminal domain of the exogenous Gα subunit.

29. A yeast periplasmic display library according to embodiment 28, wherein the yeast G(subunit belongs to the G(i, G(q, G(s, or G(o) family of G proteins.

30. The yeast periplasmic display library according to any one of embodiments 25-29, wherein the exogenous GPCR is a mammalian GPCR.

31. The yeast periplasmic display library of Embodiment 30, wherein the at least five C-terminal residues of the yeast G(subunit) are replaced with the corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

32. The yeast periplasmic display library of Embodiment 31, wherein at least 20 C-terminal residues of the yeast G(subunit) are replaced with corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

33. The yeast periplasmic display library of embodiment 31 or 32, wherein the mammalian G(subunit) is selected from the group consisting of G alpha-S, G alpha-I, G alpha-O, G alpha-T, G alpha-Z, G alpha-Q, G alpha-11, G alpha-12, G alpha-13 and transducin.

34. A yeast periplasmic display library according to any one of embodiments 28-33, wherein the chimeric G(subunit) contains at least 41 N-terminal residues of the yeast G(subunit).

35. The yeast periplasmic display library according to any one of embodiments 30-34, wherein the mammalian GPCR is a human GPCR.

36. The yeast periplasmic display library of embodiment 35, wherein the human GPCR is selected from the group consisting of CXCR4, CXCR5, SSTR2, MOR, AVPR2, FPR2/ALX, ADORA2A, CHRM3, CGRP2, CCR2, CCR4, CCR5, CHRM4, PAC1, b2AR, CXCR2, CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB1, CB2, A3AR and AT1R.

37. The yeast periplasmic display library according to any one of embodiments 24-36, wherein the GPCR target membrane protein of interest has constitutive ligand-independent activity.

38. The yeast periplasmic display library according to any one of embodiments 24-37, wherein the yeast host cell is a FAR1 strain for selecting antagonist antibodies of a GPCR target membrane protein of interest.

39. The yeast periplasmic display library of any one of embodiments 24-36, wherein the host yeast cell is a Δfar1 strain containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selecting agonist antibodies of a GPCR target membrane protein of interest.

40. The yeast periplasmic display library according to any one of embodiments 1-39, wherein the antibodies are selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, nanoantibodies, recombinant antibody fragments, Fab fragments, Fab' fragments, F(ab ') ₂ fragments, F _v fragments and scFv fragments.

41. The yeast periplasmic display library according to any one of embodiments 1-40, wherein the target membrane protein of interest contains a mutation that increases or decreases its activity.

42. The yeast periplasmic display library according to any one of embodiments 1-40, wherein the host yeast cell is a Δfar1, Δsst2, Δste14, Δste3, or Δmat strain.

43. The yeast periplasmic display library of embodiment 42, wherein the host yeast cell is a Δmat strain containing a deleted or inactivated MAT locus (or a deleted or inactivated MATa locus.

44. The yeast periplasmic display library according to any one of embodiments 1-43, wherein the yeast host cell further comprises a modified CLN3 protein containing a C-terminal truncation that increases the content of CLN3 in the yeast host cell compared to the wild-type CLN3 protein.

45. The yeast periplasmic display library of embodiment 44, wherein the modified CLN3 protein retains at least the N-terminal amino acids 1-387 of the wild-type CLN3 protein.

46. The yeast periplasmic display library of embodiment 44, wherein the modified CLN3 protein retains at least the N-terminal amino acids 1-408 of the wild-type CLN3 protein.

47. The yeast periplasmic display library according to any one of embodiments 1-46, wherein the yeast host cell is a haploid or diploid yeast host cell.

48. A method for producing a yeast periplasmic display library according to Embodiment 1, the method comprising:

a) providing a first plurality of recombinant antibody-encoding polynucleotides for display in the periplasmic space of a host yeast cell, wherein the displayed antibody is different in each yeast host cell such that a plurality of host yeast cells display a plurality of antibodies;

b) providing a second recombinant polynucleotide encoding a periplasmic anchor protein, wherein the periplasmic anchor protein is linked to an antibody such that the antibody is exposed in the periplasmic space;

c) transfecting a plurality of yeast host cells with a first plurality of recombinant polynucleotides and a second recombinant polynucleotide;

d) transfecting a plurality of yeast host cells with a recombinant polynucleotide encoding a target membrane protein of interest; And

e) culturing a plurality of yeast host cells under conditions that allow expression of antibodies, a periplasmic anchor protein, and a target membrane protein of interest, wherein each yeast host cell exposes a different antibody to the periplasmic space and the target membrane protein of interest is localized to the plasma membrane , obtaining the yeast periplasmic display library according to embodiment 1.

49. The method of embodiment 48, wherein the recombinant polynucleotides encoding antibodies or the recombinant polynucleotide encoding a periplasmic anchor protein or a target membrane protein of interest are provided via expression vectors.

50. The method of embodiment 48, wherein the recombinant polynucleotides encoding antibodies or the recombinant polynucleotide encoding a periplasmic anchor protein or a target membrane protein of interest is inserted into the genome of a host yeast cell at the target locus.

51. A method for producing a yeast periplasmic display library according to embodiment 3, the method comprising:

a) providing a plurality of recombinant polynucleotides encoding fusion proteins, wherein each recombinant polynucleotide encodes a different fusion protein comprising a periplasmic anchor protein linked to a different antibody for display;

b) transfecting a plurality of yeast host cells with a plurality of recombinant polynucleotides encoding fusion proteins;

c) transfecting host yeast cells with a recombinant polynucleotide encoding a target membrane protein of interest; And

d) culturing a plurality of yeast host cells under conditions that allow expression of the fusion proteins and the target membrane protein of interest, wherein each yeast host cell displays a different antibody in the periplasmic space and the target membrane protein of interest is localized to the plasma membrane, with by obtaining the yeast periplasmic display library according to embodiment 3.

52. The method of Embodiment 51, wherein the recombinant polynucleotides encoding the fusion proteins or the recombinant polynucleotide encoding the target membrane protein of interest are provided via expression vectors.

53. The method of embodiment 51, wherein the recombinant polynucleotides encoding the fusion proteins or target membrane protein of interest are inserted into the genome of the host yeast cell at the target locus.

54. The method of any one of embodiments 48-53, wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel, and a transporter.

55. The method of embodiment 54, wherein the receptor is a G protein coupled receptor (GPCR).

56. The method according to any one of embodiments 48-55, comprising introducing into a plurality of yeast host cells a recombinant polynucleotide encoding an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in the host yeast cell .

57. The method of Embodiment 56, wherein the engineered G(subunit) is a chimeric G protein alpha (Gα) subunit containing the N-terminal domain of a yeast G(subunit) and the C-terminal domain of an exogenous Gα subunit.

58. The method of Embodiment 57, wherein the yeast G(subunit belongs to the G(i, G(q, G(s, or G(o) family of G proteins.

59. The method according to embodiment 57 or 58, wherein the exogenous G subunit is a mammalian G subunit.

60. The method of Embodiment 59, wherein the at least five C-terminal residues of the yeast G(subunit) are replaced with corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

61. The method of Embodiment 60, wherein at least 20 C-terminal residues of the yeast G(subunit) are replaced with corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

62. The method of any one of embodiments 55-61, wherein the host yeast cell is a FAR1 strain for selecting antagonist antibodies of the GPCR target of interest.

63. The method according to any one of embodiments 55-61, wherein the yeast host cell is a Δfar1 strain containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selecting GPCR agonist antibodies.

64. A method of screening a yeast periplasmic display library according to embodiment 14 for an antibody that modulates the activity of a target membrane protein of interest, the method comprising culturing at least a subset of yeast host cells of the yeast periplasmic display library according to embodiment 14 in a selective medium; and detecting reporter gene expression, wherein increased expression of the reporter gene indicates that the antibody increases the activity of the target membrane protein of interest, and decreased expression of the reporter gene indicates that the antibody reduces the activity of the target membrane protein of interest.

65. The method of embodiment 64, wherein the reporter gene is a nutritional marker, an antibiotic resistance marker, a fluorescent marker, a bioluminescent marker, or a counterselection marker.

66. The method of embodiment 65, further comprising performing positive selection for expression of a nutritional marker, wherein growth of the host yeast cells in a selective nutrient-deficient environment indicates that the target membrane protein of interest is activated.

67. The method of embodiment 66, wherein the nutritional marker is HIS3, HIS7, ARG6, LEU2, URA3 and TRP1.

68. The method of embodiment 65, further comprising performing positive selection for expression of an antibiotic resistance marker, wherein growth of the host yeast cells in a selective medium containing the antibiotic indicates that the target membrane protein of interest is activated.

69. The method of embodiment 68, wherein the antibiotic resistance marker conveys resistance to an antibiotic selected from the group consisting of geneticin, zeocin, hygromycin B, nourseothricin, and bialaphos.

70. The method of embodiment 65, further comprising performing positive selection for expression of a fluorescent marker, wherein detecting fluorescence emitted by the host yeast cells indicates that the target membrane protein of interest is activated.

71. The method of embodiment 70, wherein the fluorescent marker is selected from the group consisting of green fluorescent protein, red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and orange fluorescent protein.

72. The method of embodiment 65 further comprising performing positive selection for expression of a bioluminescent marker, wherein detecting bioluminescence emitted by the host yeast cells indicates that the target membrane protein of interest is activated.

73. The method of embodiment 72, wherein the bioluminescent marker is luciferase or aequorin.

74. The method of embodiment 65, further comprising performing negative selection on expression of a counterselection marker, wherein the reduction in activity of the target membrane protein of interest upon binding of the exposed antibody to the target membrane protein of interest is detectable by growth of host yeast cells in the medium, containing an agent that selects against cells expressing a counterselection marker.

75. The method according to embodiment 74, wherein the counter-selection marker is selected from the group consisting of CAN1, URA3, MET15, TRP1 and TK.

76. A method of screening a yeast periplasmic display library according to embodiment 27 for an antibody that modulates the activity of a GPCR target of interest, the method comprising culturing at least a subset of yeast host cells of the yeast periplasmic display library according to embodiment 27 in a detection environment activation or inhibition of the pheromone response in at least one yeast host cell compared to a control yeast host cell that does not have antibody exposed in the periplasmic space, indicates that the exposed antibody in the at least one yeast host cell binds and modulates GPCR activity.

77. The method of embodiment 76, wherein the target GPCR of interest is a human GPCR.

78. The method of embodiment 77, further comprising contacting the human GPCR with a ligand.

79. The method of embodiment 78, wherein the GPCR has constitutive ligand-independent activity.

80. The method of any one of embodiments 76-79, wherein the host yeast cell is a FAR1 strain, wherein inhibition of the pheromone response by an antagonist antibody that binds and inhibits a GPCR in the host yeast cell results in cell cycle arrest. and growth of the yeast host cell.

81. The method according to any one of embodiments 76-79, wherein the yeast host cell is a Δfar1 strain containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene, wherein activation of the pheromone response by an antibody acting as an agonist that binds and activates A GPCR in a yeast host cell results in increased reporter gene expression.

82. The method of embodiment 73, wherein the reporter gene is a nutritional marker, an antibiotic resistance marker, a fluorescent marker, a bioluminescent marker, and a counterselection marker.

83. The method according to any one of embodiments 1-82, wherein the genus of yeast host cells is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces and Yarrowia.

84. The method of embodiment 83, wherein the host yeast cell genus is Saccharomyces.

85. The method of Embodiment 84, wherein the Saccharomyces species is Saccharomyces cerevisiae.

86. Yeast host cell containing:

a) antibody for display in the periplasmic space of the host yeast cell,

b) periplasmic anchor protein, where the periplasmic anchor protein is associated with the antibody so that the antibody is exposed in the periplasmic space; And

c) a target membrane protein of interest, wherein the membrane protein of interest is located on the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell.

87. The yeast host cell of embodiment 86, wherein the antibody and the periplasmic anchor protein are non-covalently linked together through molecular binding interactions in a complex or linked through a covalent non-peptide bond in a complex.

88. The yeast host cell of embodiment 86, wherein the antibody and the periplasmic anchor protein are covalently linked in a fusion protein.

89. The yeast host cell according to any one of embodiments 86-88, wherein the periplasmic anchor protein further comprises a signal sequence that causes the periplasmic anchor protein to be transported into the periplasm of the yeast host cell, to the plasma membrane, or to the cell wall, such that the antibody is exposed to periplasm.

90. The yeast host cell of any one of embodiments 86-88, wherein the periplasmic anchor protein comprises a membrane spanning transmembrane domain or membrane-associated protein domain that exposes the antibody to the periplasm.

91. The yeast host cell of any one of embodiments 86-88, wherein the periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the antibody protrudes into the periplasm.

92. The yeast host cell of any one of embodiments 86-88, wherein the periplasmic anchor protein is large enough such that the periplasmic anchor protein and associated antibody are retained in the periplasm.

93. The yeast host cell of any one of embodiments 86-92, wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel, and a transporter.

94. The yeast host cell of embodiment 93, wherein the receptor is a G protein coupled receptor (GPCR).

95. The yeast host cell of any one of embodiments 86-94, further comprising introducing into the yeast host cell a recombinant polynucleotide encoding an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in the yeast host cell.

96. The yeast host cell of Embodiment 95, wherein the engineered G subunit is a chimeric G protein alpha (Gα) subunit containing the N-terminal domain of a yeast G subunit and the C-terminal domain of an exogenous Gα subunit.

97. The yeast host cell of Embodiment 96, wherein the yeast G(subunit belongs to the G(i, G(q, G(s, or G(o) family of G proteins.

98. The yeast host cell of embodiment 96 or 97, wherein the exogenous G subunit is a mammalian G subunit.

99. The yeast host cell of Embodiment 98, wherein the at least five C-terminal residues of the yeast G(subunit) are replaced with the corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

100. The yeast host cell of Embodiment 99, wherein at least 20 C-terminal residues of the yeast G(subunit) are replaced with corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

101. The yeast host cell of any one of embodiments 86-100, wherein the yeast host cell is a FAR1 strain for selecting antagonist antibodies of the GPCR target of interest.

102. The yeast host cell of any one of embodiments 86-101, wherein the yeast host cell is a Δfar1 strain containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selecting GPCR agonist antibodies.

103. The yeast host cell of any one of embodiments 86-101, wherein the genus of the yeast host cell is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia.

104. The yeast host cell according to embodiment 103, wherein the genus of the yeast host cell is Saccharomyces.

105. The yeast host cell of embodiment 104, wherein the Saccharomyces is Saccharomyces cerevisiae.

106. Antibody bound to periplasmic anchor protein.

107. The antibody of embodiment 106, wherein the antibody is localized in the periplasmic space of the host yeast cell.

108. The antibody of embodiment 106, wherein when the antibody is produced in a host yeast cell, the antibody is localized in the periplasmic space of the host yeast cell.

109. The antibody of any one of embodiments 106-108, wherein the antibody and the periplasmic anchor protein are non-covalently linked together through molecular binding interactions in a complex or are linked through a covalent non-peptide bond in a complex.

110. The antibody of any one of embodiments 106-108, wherein the antibody and the periplasmic anchor protein are covalently linked together in a fusion protein.

111. The antibody of any one of embodiments 107-110, wherein the periplasmic anchor protein further comprises a signal sequence that causes the periplasmic anchor protein to be transported into the periplasm of the host yeast cell, to the plasma membrane, or to the cell wall, such that the antibody is exposed in the periplasm.

112. The antibody of any one of embodiments 107-110, wherein the periplasmic anchor protein comprises a membrane spanning transmembrane domain or membrane-associated protein domain that exposes the antibody to the periplasm.

113. The antibody of embodiment 112, wherein the membrane-associated protein domain is a plasma membrane anchoring glycosylphosphatidylinositol (GPI) domain.

114. The antibody of any one of embodiments 107-110, wherein the periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the antibody protrudes into the periplasm.

115. The antibody of any one of embodiments 107-110, wherein the periplasmic anchor protein is large enough such that the periplasmic anchor protein and associated antibody are retained in the periplasm.

116. The antibody of any one of embodiments 106-115, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a nanoantibody, a recombinant antibody fragment, a Fab fragment, a Fab' fragment, F(ab') ₂ -fragment, Fv _- fragment and scFv-fragment.

117. The antibody of any one of embodiments 107-116, wherein the host yeast cell genus is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia.

118. The antibody of embodiment 117, wherein the host yeast cell genus is Saccharomyces.

119. The antibody of embodiment 118, wherein the Saccharomyces species is Saccharomyces cerevisiae.

120. A yeast host cell comprising the antibody of any one of embodiments 106-119.

121. A method of localizing an antibody to the periplasmic space of a host yeast cell, comprising binding the antibody to a periplasmic anchor protein such that the antibody is localized to the periplasmic space.

122. The method of embodiment 121, wherein the antibody and the periplasmic anchor protein are non-covalently linked together through molecular binding interactions in a complex or linked through a covalent non-peptide bond in a complex.

123. The method of embodiment 122, wherein the antibody and the periplasmic anchor protein are covalently linked together in a fusion protein.

124. The method of any one of embodiments 121-123, wherein the periplasmic anchor protein further comprises a signal sequence that causes the periplasmic anchor protein to be transported into the periplasm of the host yeast cell, to the plasma membrane, or to the cell wall, such that the antibody is exposed in the periplasm.

125. The method of any one of embodiments 121-123, wherein the periplasmic anchor protein comprises a membrane spanning transmembrane domain or membrane-associated protein domain that exposes the antibody to the periplasm.

126. The method of embodiment 125, wherein the membrane-associated protein domain is a plasma membrane-anchoring glycosylphosphatidylinositol (GPI) domain.

127. The method of any one of embodiments 121-123, wherein the periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the antibody protrudes into the periplasm.

128. The method of any one of embodiments 120-123, wherein the periplasmic anchor protein is large enough such that the periplasmic anchor protein and associated antibody are retained in the periplasm.

129. The method according to any one of embodiments 121-128, wherein the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a nanoantibody, a recombinant antibody fragment, a Fab fragment, a Fab' fragment, F(ab') ₂ fragment, F _v fragment and scFv fragment.

130. The method of any one of embodiments 121-129, wherein the host yeast cell genus is selected from the group consisting of Saccharomyces, Candida, Pichia, Kluyveromyces, and Yarrowia.

131. The method of embodiment 130, wherein the host yeast cell genus is Saccharomyces.

132. The method of embodiment 131, wherein the Saccharomyces species is Saccharomyces cerevisiae.

133. A yeast periplasmic display library containing a plurality of yeast host cells, wherein each yeast host cell contains:

a) an antibody for display in the periplasmic space of a yeast host cell, where the displayed antibody is different in each yeast host cell such that a plurality of yeast host cells display a plurality of antibodies;

b) periplasmic anchor protein, wherein the periplasmic anchor protein is associated with the antibody such that the antibody is exposed in the periplasmic space; And

c) a target membrane protein of interest, wherein the membrane protein of interest is localized to the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell.

134. The yeast periplasmic display library of embodiment 133, wherein the antibody and the periplasmic anchor protein are covalently linked together in a fusion protein.

135. The yeast periplasmic display library of embodiment 133, wherein the antibody and the periplasmic anchor protein are non-covalently linked together through molecular binding interactions.

136. The yeast periplasmic display library of embodiment 133, further comprising a reporter system comprising a reporter gene operably linked to an inducible promoter that is activated when a target membrane protein of interest is activated, allowing detection of an increase or decrease in the activity of the target membrane protein of interest when the antibody binds to a target membrane protein of interest.

137. The yeast periplasmic display library of embodiment 136, wherein the reporter gene is a nutritional marker, an antibiotic resistance marker, a fluorescent marker, a bioluminescent marker, or a counterselection marker.

138. The yeast periplasmic display library of embodiment 137, wherein the nutritional marker is selected from the group consisting of HIS3, HIS7, ARG6, LEU2, URA3, and TRP1.

139. The yeast periplasmic display library of embodiment 137, wherein the antibiotic resistance marker conveys resistance to an antibiotic selected from the group consisting of geneticin, zeocin, hygromycin B, nourseothricin, and bialaphos.

140. The yeast periplasmic display library of embodiment 137, wherein the fluorescent marker is selected from the group consisting of green fluorescent protein, red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and orange fluorescent protein.

141. The yeast periplasmic display library of embodiment 137, wherein the bioluminescent marker is luciferase or aequorin.

142. The yeast periplasmic display library of embodiment 137, wherein the counterselection marker is selected from the group consisting of CAN1, URA3, MET15, TRP1, and TK.

143. The yeast periplasmic display library of Embodiment 136, wherein the reporter gene is a selectable marker such that an increase in the activity of the target membrane protein of interest upon binding of an antibody to the target membrane protein of interest is detectable by growth of the yeast host cells on media for positive selection.

144. The yeast periplasmic display library of embodiment 136, wherein the reporter gene is a counterselection marker such that said reduction in activity of a target membrane protein of interest upon binding of an antibody to the target membrane protein of interest is detectable by growth of host yeast cells on the medium for negative selection.

145. The yeast periplasmic display library of embodiment 133, wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel, and a transporter.

146. The yeast periplasmic display library of embodiment 145, wherein the receptor is a G protein coupled receptor (GPCR).

147. The yeast periplasmic display library of embodiment 146, wherein the GPCR is an exogenous GPCR.

148. The yeast periplasmic display library of embodiment 147, further comprising an engineered G subunit capable of being activated by an exogenous GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in a host yeast cell.

149. The yeast periplasmic display library of embodiment 148, wherein the engineered G(subunit) is a chimeric G protein alpha (Gα) subunit containing the N-terminal domain of the yeast G(subunit) and the C-terminal domain of the exogenous Gα subunit.

150. A yeast periplasmic display library according to embodiment 149, wherein the yeast G(subunit belongs to the G(i, G(q, G(s, or G(o) family of G proteins.

151. The yeast periplasmic display library of embodiment 149, wherein the exogenous GPCR is a mammalian GPCR.

152. The yeast periplasmic display library of embodiment 151, wherein the at least five C-terminal residues of the yeast G(subunit) are replaced with the corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

153. The yeast periplasmic display library of embodiment 152, wherein at least 20 C-terminal residues of the yeast G(subunit) are replaced with corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

154. The yeast periplasmic display library of embodiment 151, wherein the mammalian G(subunit) is selected from the group consisting of G alpha-S, G alpha-I, G alpha-O, G alpha-T, G alpha-Z, G alpha- Q, G alpha-11, G alpha-12, G alpha-13 and transducin.

155. A yeast periplasmic display library according to embodiment 149, wherein the chimeric G(subunit) contains at least 41 N-terminal residues of the yeast G(subunit.

156. The yeast periplasmic display library of embodiment 151, wherein the mammalian GPCR is a human GPCR.

157. The yeast periplasmic display library of embodiment 156, wherein the human GPCR is selected from the group consisting of CXCR4, b2AR, CXCR2, CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB2, A3AR, and AT1R.

158. The yeast periplasmic display library of embodiment 146, wherein the GPCR has constitutive ligand-independent activity.

159. The yeast periplasmic display library of embodiment 146, wherein the yeast host cell is a GPCR antagonist antibody selection strain FAR1.

160. The yeast periplasmic display library of embodiment 146, wherein the host yeast cell is a Δfar1 strain containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selecting GPCR agonist antibodies.

161. The yeast periplasmic display library of embodiment 133, wherein the antibodies are selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, nanoantibodies, recombinant antibody fragments, Fab fragments, Fab' fragments, F(ab') ₂ - fragments, _Fv fragments and scFv fragments.

162. The yeast periplasmic display library of embodiment 133, wherein the target membrane protein of interest contains a mutation that increases or decreases its activity.

163. The yeast periplasmic display library of embodiment 133, wherein the host yeast cell is a Δfar1, Δsst2, Δste14, Δste3, or Δmat strain.

164. A yeast periplasmic display library according to embodiment 163 wherein the Δmat strain contains a deleted or inactivated MAT locus (or a deleted or inactivated MATa locus.

165. The yeast periplasmic display library of embodiment 133, wherein the yeast host cell further comprises a modified CLN3 protein containing a C-terminal truncation that increases the CLN3 content of the yeast host cell compared to the wild-type CLN3 protein.

166. The yeast periplasmic display library of embodiment 165, wherein the modified CLN3 protein retains at least N-terminal amino acids 1-387 of the wild-type CLN3 protein.

167. The yeast periplasmic display library of embodiment 166, wherein the modified CLN3 protein retains at least the N-terminal amino acids 1-408 of the wild-type CLN3 protein.

168. The yeast periplasmic display library of embodiment 133, wherein the yeast host cell is a haploid or diploid yeast host cell.

169. The yeast periplasmic display library of embodiment 133, wherein the periplasmic anchor protein further comprises a signal sequence that causes the fusion protein to be transported into the periplasm of the host yeast cell to the plasma membrane or cell wall such that the fusion protein is displayed in the periplasm.

170. The yeast periplasmic display library of embodiment 133, wherein the periplasmic anchor protein comprises a membrane spanning transmembrane domain or membrane-bound protein domain that exposes the fusion protein to the periplasm.

171. The yeast periplasmic display library of embodiment 170, wherein the membrane-bound protein domain is a plasma membrane-anchoring glycosylphosphatidylinositol (GPI) domain.

172. The yeast periplasmic display library of embodiment 171, wherein the plasma membrane-anchoring GPI domain is a japsin plasma membrane-anchoring GPI domain.

173. The yeast periplasmic display library of embodiment 172, wherein the plasma membrane-anchoring yapsin GPI domain is a plasma membrane-anchoring yapsin GPI domain YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7.

174. The yeast periplasmic display library of embodiment 133, wherein the periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the exposed variant of the protein protrudes into the periplasm.

175. The yeast periplasmic display library of embodiment 133, wherein the periplasmic anchor protein is large enough to allow the fusion protein to be retained in the periplasm.

176. A method for producing a yeast periplasmic display library according to embodiment 134, the method comprising:

a) providing a plurality of recombinant polynucleotides encoding fusion proteins, wherein each recombinant polynucleotide encodes a different fusion protein comprising a periplasmic anchor protein linked to a different antibody for display;

b) transfecting a plurality of yeast host cells with a plurality of recombinant polynucleotides encoding fusion proteins;

c) transfecting a plurality of yeast host cells with a recombinant polynucleotide encoding a target membrane protein of interest; And

d) culturing a plurality of yeast host cells under conditions that allow expression of the fusion proteins and the target membrane protein of interest, wherein each yeast host cell exposes a different antibody to the periplasmic space and the target membrane protein of interest is localized to the plasma membrane, obtaining yeast periplasmic display libraries according to embodiment 134.

177. The method of embodiment 176, wherein the recombinant polynucleotides encoding the fusion proteins or the recombinant polynucleotide encoding a target membrane protein of interest is provided via expression vectors.

178. The method of embodiment 176, wherein recombinant polynucleotides encoding fusion proteins or a target membrane protein of interest are inserted into the genome of a host yeast cell at a predetermined locus.

179. The method of embodiment 176, wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel, and a transporter.

180. The method of embodiment 179, wherein the receptor is a G protein coupled receptor (GPCR).

181. The method of embodiment 180, further comprising introducing into a plurality of yeast host cells a recombinant polynucleotide encoding an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered G subunit is capable of activating a detectable pheromone response in the host yeast cell.

182. The method of Embodiment 181, wherein the engineered G subunit is a chimeric G protein alpha (Gα) subunit containing the N-terminal domain of a yeast G subunit and the C-terminal domain of an exogenous Gα subunit.

183. The method of embodiment 182, wherein the yeast G(subunit belongs to the G(i, G(q, G(s, or G(o) family of G proteins.

184. The method of embodiment 182, wherein the exogenous G(subunit) is a mammalian G(subunit.

185. The method of embodiment 184, wherein the at least five C-terminal residues of the yeast G(subunit) are replaced with corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

186. The method of embodiment 185, wherein at least 20 C-terminal residues of the yeast G(subunit) are replaced with corresponding C-terminal residues of the mammalian G(subunit), such that the chimeric G(subunit is capable of being activated by a mammalian GPCR.

187. The method of embodiment 181, wherein the host yeast cell is a FAR1 strain for selecting antagonist antibodies of the GPCR target of interest.

188. The method of embodiment 181, wherein the host yeast cell is a Δfar1 strain containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selecting GPCR agonist antibodies.

189. A method of screening a yeast periplasmic display library of embodiment 136 for an antibody that modulates the activity of a target membrane protein of interest, the method comprising culturing at least a subset of yeast host cells of the yeast periplasmic display library of embodiment 136 in a selective medium; and detecting reporter gene expression, wherein increased expression of the reporter gene indicates that the antibody increases the activity of the target membrane protein of interest, and decreased expression of the reporter gene indicates that the antibody reduces the activity of the target membrane protein of interest.

190. The method of embodiment 189, wherein the reporter gene is a nutritional marker, an antibiotic resistance marker, a fluorescent marker, a bioluminescent marker, and a counterselection marker.

191. The method of embodiment 190, further comprising performing positive selection for expression of a nutritional marker, wherein growth of the host yeast cells in a selective nutrient-deficient environment indicates that the target membrane protein of interest is activated.

192. The method of embodiment 191, wherein the nutritional marker is HIS3, HIS7, ARG6, LEU2, URA3, and TRP1.

193. The method of embodiment 190, further comprising performing positive selection for expression of an antibiotic resistance marker, wherein growth of the host yeast cells in a selective medium containing the antibiotic indicates that the target membrane protein of interest is activated.

194. The method of embodiment 193, wherein the antibiotic resistance marker conveys resistance to an antibiotic selected from the group consisting of geneticin, zeocin, hygromycin B, nourseothricin, and bialaphos.

195. The method of embodiment 190, further comprising performing positive selection for expression of a fluorescent marker, wherein detecting fluorescence emitted by the host yeast cells indicates that the target membrane protein of interest is activated.

196. The method of embodiment 195, wherein the fluorescent marker is selected from the group consisting of green fluorescent protein, red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and orange fluorescent protein.

197. The method of embodiment 190, further comprising performing positive selection for expression of a bioluminescent marker, wherein detecting bioluminescence emitted by the host yeast cells indicates that the target membrane protein of interest is activated.

198. The method of embodiment 197, wherein the bioluminescent marker is luciferase or aequorin.

199. The method of embodiment 190, further comprising performing negative selection on expression of a counterselection marker, wherein the reduction in activity of the target membrane protein of interest upon binding of the exposed antibody to the target membrane protein of interest is detectable by growth of host yeast cells in the medium, containing an agent that selects against cells expressing a counterselection marker.

200. The method of embodiment 199, wherein the counterselection marker is selected from the group consisting of CAN1, URA3, MET15, TRP1, and TK.

201. A method of screening a yeast periplasmic display library according to embodiment 148 for an antibody that modulates the activity of a GPCR target of interest, the method comprising culturing at least a subset of yeast host cells of the yeast periplasmic display library according to embodiment 148 in a detection environment activation or inhibition of the pheromone response in at least one yeast host cell compared to a control yeast host cell that does not have antibody exposed in the periplasmic space, indicates that the exposed antibody in the at least one yeast host cell binds and modulates GPCR activity.

202. The method of embodiment 201, further comprising contacting the human GPCR with a ligand.

203. The method of embodiment 201, wherein the GPCR has constitutive ligand-independent activity.

204. The method of Embodiment 201, wherein the host yeast cell is a FAR1 strain, wherein inhibition of the pheromone response by an antagonist antibody that binds and inhibits GPCRs in the yeast host cell results in cell cycle arrest and growth of the yeast host cells.

205. The method of Embodiment 201, wherein the yeast host cell is a Δfar1 strain containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene, wherein activating the pheromone response through an agonist antibody that binds and activates a GPCR in the yeast host cell, leads to increased expression of the reporter gene.

206. The method of embodiment 205, wherein the reporter gene is a nutritional marker, an antibiotic resistance marker, a fluorescent marker, a bioluminescent marker, or a counterselection marker.

IV. experimental part

[0159] The invention will become better understood with the help of the following examples of specific embodiments of the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Various modifications or changes may therefore occur to those skilled in the art and are within the spirit and scope of the present application and the scope of the appended claims.

[0160] Efforts have been made to ensure accuracy in terms of numbers used (eg, quantity, temperature, etc.), however some experimental error and variation is certainly acceptable.

Example 1

Yeast display for selection of antibodies that modulate GPCR function

Review

[0161] Many therapeutic targets for diseases such as cancer and inflammation involve G protein-coupled receptors (GPCRs). However, many of the GPCRs with the greatest therapeutic potential for high-impact diseases are difficult to develop into drugs. Although small molecule compounds that interfere with the function of these proteins are easy to find, they are often nonspecific due to the structural similarity between the ligand-binding pockets of GPCRs, potentially causing significant nonspecific side effects. In contrast to small molecules, antibodies and related affinity molecules (eg, nanoantibodies, and ScFvs and Fabs) are an attractive class of therapeutics due to their potentially higher specificity, functional diversity, and pharmacological properties. In addition, antibodies may better interact with extracellular domains and loops that can modulate the structure (and thus function) of GPCRs in more complex ways than small molecule compounds. However, to date, there is no approved antibody therapeutic against GPCR in the United States, and only one exists in the world, in Japan.

[0162] Current yeast and phage display technologies identify antibodies that bind strongly to, but often do not interfere with, GPCR function. The antigens used are often GPCR fragments that do not represent a functional protein available to the antibody in vivo, or are heterogeneously structured full-length protein preparations. This technology also misses a huge proportion of the overall functional diversity, since most antibodies are never subjected to functional analysis. A high-throughput platform is needed for the direct selection of antibodies that modulate GPCR function.

[0163] However, it is much less straightforward to develop antibodies that alter GPCR function (Jo 2015, Hutchings 2010). This is primarily a consequence of the following problems with many existing solutions: 1) there are no usable antigens. Antigens derived from extracellular peptides or GPCR fragments may be well suited for the development of antibodies for Western blotting, but do not structurally reflect therapeutic targets. In addition, uniformly functionally folded full-length protein in lipids or detergents may be difficult to obtain in sufficient quantities for immunization, phage display, or yeast display. 2) Antibodies selected for their high affinity are largely nonfunctional; they bind to regions in GPCRs that do not affect function. 3) Technologies lose much of the antibody diversity and thus the functionality of the selected antibodies. By first selecting antibodies that bind strongly and excluding others, much of the functional diversity is lost. Mammalian cell systems have been designed to functionally screen subsets of antibody candidates in an autocrine manner (Zhang 2014), which partially address problem 2, but due to transformation efficiency (~10 ⁴ ) and limited ability to construct selectable/screenable outputs, they are limited to screening small subsets of candidates .

[0164] The present inventors' innovation involves combining GPCR coupling with yeast pheromone response and expression of affinity molecules having cis-action in the same cell in a high-throughput platform. This allows direct and high-throughput functional selection of affinity molecules (Figure 1). To do this, the inventors combined two strategies that are optimal for yeast:

1. Functional expression of human GPCRs coupled with yeast pheromone response data

[0165] Yeast is the preferred system of choice for expressing full-length functional human GPCRs for in vitro biochemical and structural studies. Notably, yeast has also been successfully used to functionally couple human GPCRs to the yeast pheromone response cascade and, depending on the information obtained, screen/select small molecule peptide or protein ligands that functionally interact with GPCRs (King 1990, Brown 2000, Erlenbach 2001, Minic 2005, Dowell 2009, Dong 2010, Liu 2016). The most "universal" coupling method is to modify the yeast G(yeast) subunit, Gpa1, to bind to a human GPCR and transmit signals by grafting the 5 C-terminal residues of the corresponding human G(to replace the native 5 C-terminal amino acids in Gpa1 to produce a "Gα graft" (Conklin 1993, Brown 2000, Erlenbach 2001) Other methods include replacing Gpa1 entirely with the full-length corresponding human G(receptor) (King 1990), or constructing a more complex Gpa1/Gα chimera in which the various parts are combined into a hybrid human/yeast G( (Klein 1998, Price 1995).

2. Yeast display of affinity molecules - antibodies, nanoantibodies and ScFv

[0166] There is also fundamental knowledge about the expression and secretion of affinity molecules such as IgG, ScFv, Fab and nanoantibodies in yeast (Horwitz 1988, Hamilton 2006, Jeong 2011). Many antibodies have been developed as clinical candidates by yeast display companies such as Adimab. Many academic laboratories and companies have improved ways to express these molecules and “display” them in yeast by fusing affinity molecules with proteins that are covalently linked to the extracellular surface of the cell wall, such as Aga1 and Aga2 (Boder 1997, Bidlingmaier 2015). Libraries have been operationally developed for yeast to have 10 ⁹ complexity that are sufficient for routine binding studies and more than sufficient for the inventors' platform for functional selection (for the reasons described in FIG. 1).

[0167] In addition, the inventors have made efforts to bind the secreted affinity molecules not to the outside of the cell wall, as in the case of traditional yeast display, but to bind them to the extracellular surface of the cell membrane. Thus, the affinity molecule can functionally interact in cis with GPCRs in the membrane. The authors tested four different domains and determined that the YPS1 GPI-anchoring domain (Frieman 2003) was the best, based on membrane fluorescence from cells expressing GFP fusion constructs with the domains tested.

Example 2

Yeast strain with low background level

[0168] The inventors have created a strain of yeast that, when treated with a GPCR ligand, does not grow and is not prone to frequent mutations that can cause growth. The inventors' goal was background/false positive growth 10 ^-7 .

[0169] Haploid yeast mating type "a"("MATa" cells) undergo cell cycle arrest when the mating pheromone, alpha factor (α-factor), activates its recognition GPCR receptor Ste2. This growth arrest phenotype can be used to select Ste2 antagonists. Unfortunately, spontaneous "pheromone-resistant mutants" are appearing at a tremendous rate. The background or "false positive" level in the parental haploid strain of the inventors is ~10 ^-4 , i.e. ~100 colonies grow when 10 ⁶ cells are plated on plates with α -factor. To reduce background levels, we constructed a pheromone-responsive diploid master strain (Herskowitz 1989). False-positives caused by loss-of-function mutations are much less common in diploids. However, normal diploid yeast contains both MATa and MATalpha genes; a/alpha cells do not express Ste2 and do not respond to pheromones. We constructed a diploid organism that behaves like a MATa haploid by deleting the entire MATalpha locus from its genome. The MATa/Δmat-alpha diploid had ~100-fold lower background levels .

[0170] Virtually none of the "false positive" colonies in the diploid strain responded to pheromones at all. Presumably, they contained gain-of-function mutations that inactivated the signaling cascade. Thus, the inventors have developed a selectable marker that is active only in cells with a functioning pheromone response cascade. This marker depends on a basal level signaling cascade for its expression—this signaling does not require a pheromone or pheromone receptor and, instead, depends on the stochastic “baseline” activation of the G protein recognition protein (Hagen 1991; Oehlen 1995). This low basal signaling is insufficient to trigger cell cycle arrest; this response requires activation of the GPCR Ste2 via α-factor. Some constitutively expressed yeast genes depend on the basal activity of the pheromone response cascade to be expressed. The inventors constructed their selectable marker by inserting the promoter of one such gene, MFA1, which directs the expression of HIS3, a gene required for cells to synthesize their own histidine (Daniel 2006). The P(MFA1)-HIS3 construct allows cells to grow in a medium lacking histidine (H- medium) only if they can transmit the signal from G proteins further to the pheromone response transcription factor Ste12. A constructed diploid strain (NIY326) containing P(MFA1)-HIS3 and inoculated in H- medium with α-factor had a background level of 10 ^-7 .

Table 1. Summary of strain development to reduce background levels. The background level represents colonies formed on 10 ⁷ cells sown on agar containing pheromones

Strain Background level Parental haploid 10 ^-4 Engineered diploid 10 ^-6 Constructed diploid+cascade function confirmation cassette 10 ^-7

Example 3

Construction of a periplasm-localized nanoantibody library

[0171] We constructed a set of periplasmic-targeting expression vectors allowing expression of a chimera with an N-terminal secretion signal, MFalpha PrePro (Brake 1984), followed by an 18 amino acid linker containing a single FLAG epitope (DYKDDDDK) and terminating with a glycophosphatidylinositol anchor ( GPI) domain of YPS1. The PrePro signal directs the protein to translocate into the endoplasmic reticulum and secrete and is later cleaved off, while processing of the GPI domain of YPS1 in the ER provides an N-terminal GPI anchor that keeps the chimera bound to the plasma membrane (Frieman 2003). Restriction sites immediately following the MFalphaPrePro coding sequence allow cloning of affinity molecules (for example, to create a nanoantibody cDNA library). The vectors contain the selectable marker URA3, which allows positive selection in uracil-deficient environments as well as counter-selection (see below).

[0172] The inventors confirmed that these expression vectors localized the nanoantibody to the extracellular surface of the membrane by cloning the anti-GFP nanoantibody (Kirchhoffer 2009). When we digested the cell walls of these cells and applied GFP extracellularly, they observed green fluorescence coinciding with their cell membranes (Figure 4), confirming the periplasmic localization of the anti-GFP antibody.

[0173] Next, the inventors constructed a library of nanoantibodies. The authors used as a source a library of nanoantibodies with a clone titer of 10 ⁶ , previously cloned into an E. coli vector (Salema 2013). We amplified the nanoantibody coding sequences by PCR and cloned them into a plasma membrane targeting vector via yeast homologous recombination using the low background platform strain NIY326 (van Leeuwen 2015). The inventors obtained ~1×10 ⁶ independent colonies on agarose medium deficient in uracil and histidine (U-/H-). We scraped colonies using cryogenic storage medium and stored the suspension in aliquots at -80°C (Library 001).

Example 4

Selection of nanoantibodies that act as autocrine antagonists of Ste2

A. Selection of pheromone-resistant clones from a nanoantibody library:

[0174] We selected pheromone-resistant clones from library 001 described in Example 3. We seeded 10 ⁸ cells onto several U-/H- α-factor plates and incubated them for 5 days. They analyzed a random sample of 90 colonies from the approximately 300 that grew. We next determined whether their ability to grow on α-factor was plasmid dependent. They selected clones that had spontaneously lost the plasmid by plating them on medium containing 5-FOA, which is toxic to cells expressing URA3. We then tested clones that grew before 5-FOA selection but did not grow after it. They performed halo assays and found that 12 of the 90 clones initially isolated lost their ability to grow on agarose medium containing α-factor after selection with 5-FOA in the halo assays (Figure 5).

B. Specificity and site of action testing:

[0175] To test whether candidate nanoantibody affinity molecules are specific (ie, require the target receptor Ste2 to block the pheromone response), we express the candidates in the MATalpha strain. MATalpha cells express the a-factor receptor Ste3 and do not express Ste2. Ste3 is a GPCR only distantly related to Ste2, and its ligand, the pheromone a-factor, is a glycosylated peptide completely different from the α-factor. For both of them, the MATa and MATalpha pheromone signaling cascades downstream of the receptor are identical. Both MATa and MATalpha cells arrest their cell cycle and activate pheromone-inducible genes in response to the pheromone they recognize. Our MATa and MATalpha test cells contain a pheromone-inducible transcriptional reporter, P(PRM1)-YFP, which can be used to test cascade function in the absence and presence of candidate antagonists. Thus, we evaluate specificity by comparing the effect of candidate antagonists by determining cell cycle arrest and YFP induction in MATa and MATalpha cells exposed to their recognized pheromones. The inventors select candidates if they act as antagonists only in the MATa (Ste2) strain.

C. Site of Action Assessment Using Purified Nanoantibody Extracellularly

[0176] Although unlikely, GPCR antagonism could result from intracellular binding and disruption of plasma membrane localization of Ste2. Since any “binding” antibody could potentially act in this way, we are testing the ability of candidates to modulate GPCR function when added exogenously.

[0177] We express and purify nanoantibody proteins, add them to cells in the presence of a pheromone, and use pheromone growth and reporter induction assays to determine their effect on Ste2 (and Ste3) function. We express the candidates in bacteria with a C-terminal 6xHis tag using the pET28b vector and BL21(DE3) cells, and purify them using non-denaturing 6His affinity purification (Bornhorst 2000). Due to their small size (15 kDa), nanoantibodies are able to diffuse through the yeast cell wall (Ries 2012). In addition to this functional assay, we labeled recombinant nanoantibodies using a fluorescent dye (Alexa 488, compatible with GFP wavelengths; kit #A20181, Thermo-Fisher Scientific) and tested their ability to stain the plasma membrane of Ste2-expressing cells but not control-expressing cells. Ste3 cells. Immunofluorescence experiments also allow comparison of staining of fixed cells with or without their cell walls (which can be readily removed by treatment with the enzyme lyticase), and thus confirm that nanoantibodies can effectively diffuse through the cell wall. Finally, immunofluorescence is used to confirm that candidate antagonists directly bind the receptor rather than the ligand to exert their effects.

Example 5

Strains for agonist selection

[0178] The inventors construct strains to select agonists that require Ste2 stimulation for growth. In a version of our platform strain lacking the P(MFA1)-HIS3 marker, they switch off the pheromone-induced cell cycle arrest function by deleting both genomic copies of FAR1 using a CRISPR-Cas9 approach (Horwitz 2015). Next, we replace the PRM1 open reading frame in one of the PRM1 alleles of this diploid strain with the HIS3 ORF, generating the P(PRM1)-HIS3 selectable marker. We incorporated P(PRM1)-HIS3 into other strains and observed that there was no significant leakage, i.e. these cells grew on H- plates only in the presence of α-factor. We created MATa and MAT versions of this strain for specificity tests. As expected, the false positive colony rate of the P(PRM1)-HIS3 strains is significantly lower than for the antagonist selection strain because mutations that involve the pheromone cascade are more rarer than those that turn it off (Brown 2000).In the case where the second observe a large number of weakly "growing colonies" on H- plates when plating cells expressing a library of nanoantibodies, the inventors use the minimum empirically determined concentration of the His3 inhibitor 3-aminotriazole , which blocks their growth due to leakage of HIS3 expression (a standard strategy for HIS3 selection applications in yeast).

[0179] Similar to antagonist screening, we select clones expressing candidate Ste2 agonist nanoantibodies based on growth on H- plates. We demonstrate whether growth is plasmid/expression dependent and confirm specificity by testing the best results in a Ste3 agonist selection strain (using a P(PRM1)-HIS3 and P(PRM1)-YFP reporter). Also, as described above, the inventors produce nanoantibodies in bacteria and add them directly to cells to test their effectiveness.

Example 6

Coupling of human GPCRs with the yeast pheromone cascade

A. Gpa1 graft panel

[0180] A widely used method for coupling a human GPCR to the yeast pheromone cascade is to modify the yeast G(yeast, Gpa1) so that its 5 C-terminal amino acids are replaced with the 5 C-terminal amino acids of a human G(, producing a “GPA1 graft” (Brown 2000, Erlenbach 2001) For each receptor, the appropriate G(is often found empirically (Dowell 2009). In most cases, pairing is achieved using grafts for Gαi, Gαq, Gαs or Gαo (Dong 2010). Using the CRISPR approach (Horwitz 2015), the authors The inventions create a panel of diploid GPA1 graft strains that are agonist and antagonist for these 4 Gα graft strains.

B. Testing of Human GPCRs in the Inventors' System

[0181] In most cases, human GPCRs are best expressed in yeast with a genomic construct controlled by a moderate promoter like P(ACT1) (Shiroishi 2012, Schutz 2016). Often, well-expressed GPCRs are chimeras with an N-terminal cleavable secretory signal (typically MFalpha PrePro) followed by a FLAG epitope tag for immunodetection and sometimes a C-terminal GFP. The inventors have constructed vectors with these features, and they can be easily modified depending on the case. We clone the receptors shown in Table 2 into these vectors and test their expression level and plasma membrane localization by fluorescence microscopy and/or immunofluorescence with anti-FLAG antibody.

[0182] The inventors transform strains with grafts with constructs expressing GPCRs. Each GPCR is tested for its coupling strength to the pheromone response using the P(PRM1)-HIS3 and P(PRM1)-YFP reporters in an agonist selection strain.

Table 2. Therapeutic human GPCR targets that are candidates for pairing and antagonist/agonist discovery

Target Indication Co-author Type of selection Ligand/
constitutive mutant Yeast Nb/ScFv CXCR4 Malignant tumor (11, 40), inflammation (72) Handel (29), Gutkind (76) Antagonist cytokine SDF-1, small molecule agonist (79) and constitutive mutant (79) (73) (36, 40) b2AR asthma (48), COPD (2) Kobilka (66) Agonist N/A (agonist selection) (17) (45, 54) CXCR2 Immuno-oncology \(75, 37) Handel (18), Gutkind (24) Antagonist cytokine IL-8, constitutive mutant (53) (53) CYSLTR2 Oncology (70) Gutkind (35) Antagonist N/A (constitutively active) (70) KSHV vGPCR Oncology (9) Gutkind (47) Antagonist N/A (constitutively active) (3) PKR1 Oncology/
Angiogenesis (33) Ferrara (63) Antagonist EG-VEGF, BV8, peptide (50, 10) PKR2 Oncology /
Angiogenesis (33) Ferrara Antagonist EG-VEGF, BV8 (50) CB2 Immunosuppression (57), RA (25), IBD/Crohn's disease (44) Agonist N/A (agonist selection) A3AR RA (22), asthma (58), psoriasis (78) Agonist N/A (agonist selection) AT1R Heart disease (68), diabetes (68) Antagonist Angiotensin II, low molecular weight (27) (51)

Example 7

Screening for antibodies that modulate GPCR function

A. Carrying out the antibody selection process with selected GPCRs

[0183] The inventors follow a similar approach to their prior work with Ste2, except for the following. We use a diversified human ScFv library (assured diversity 10 ⁸ -10 ⁹ , Oak Biosciences). We clone this library into the same affinity vector as above expressing ScFv on the plasma membrane as GPI-anchored YPS1 chimeras. For antagonist selection, signal transduction from coupled receptors is not expected to be strong enough to trigger cell cycle arrest. Thus, we use the counterselection marker P(FUS2)-CAN1 (Erlenbach 2001). P(FUS2) is a pheromone-inducible promoter such as P(PRM1). CAN1 encodes a plasma membrane transporter for arginine as well as the toxic arginine analogue canavanine. Cells containing P(FUS2)-CAN1 cannot grow on canavanine plates if they contain an activated human GPCR coupled to the pheromone response. This phenotype allows for the selection of human GPCR antagonist antibodies on canavanine plates. To select agonists, the inventors use the P(PRM1)-HIS3 marker as described above. After candidate selection as described above, plasmid-free derivatives of these clones are isolated to test the plasmid dependence of their pheromone blocking phenotypes.

B. Testing the specificity and site of action of candidate antibodies

1) Testing on other GPCRs

[0184] The inventors confirm the specificity of the plasmid-dependent candidates by transforming the plasmids into strains expressing other receptors. In this case, the authors test them on strains MATa(Ste2) and MAT((Ste3), as well as strains expressing at least 2 other human GPCRs coupled to the pheromone response through the GPA1 graft (i.e., to test whether that the agonist or antagonist does not act on other GPCRs).

2) Testing using purified nanoantibody protein extracellularly

[0185] While nanoantibodies (15 kDa) can diffuse through the yeast cell wall (Ries 2012), larger ScFvs (27 kDa) can be significantly restricted. We perform immunofluorescence with labeled ScFv candidates in yeast with and without cell wall cleavage.

Example 8

Testing the effect of presentation of the CB2 agonist VHH domain on growth rate

[0186] To determine the effect of agonist presentation on the growth rate of yeast cells, we constructed yeast strains expressing the human GPCR protein, human cannabinoid receptor type 2 (CB2 receptor), and transformed them with either an empty plasmid (no VHH) or plasmids expressing the agonist , in which the single-domain VHH antibody Ab101 is presented in different ways. We tested four different VHH agonist expression plasmids in which the VHH domain is presented by 1) a short linker connected to the Yps1 plasma membrane anchor, 2) a long linker connected to the Yps1 plasma membrane anchor, 3) an N-terminal fusion construct, coupled to the soluble periplasmic enzyme Suc2, and 4) direct secretion of unlabeled VHH into the periplasm. Two to five independent clones were grown to saturation in the absence of selection. Saturated cells were used to start cultures with technical duplicates in a 96-well microtiter plate for automated absorbance measurement (OD630) in a plate reader. Two compositions of the culture medium were used. The first composition of the culture medium does not require expression of the pheromone response reporter (no selection). The second culture medium composition was the same as the first, but lacked one amino acid that can only be produced by expression of a pheromone response reporter (selection). Absorbance measurements were taken every five minutes for 48 hours. For each technical replicate, a computer calculation of the maximum growth rate was performed from the original absorption data. All obtained data were plotted on a graph, and the median growth rate is indicated by a horizontal line (Fig. 7). The figures below the graph depict each presentation method (Fig. 7).

[0187] Each of the four VHH agonist-expressing plasmids increased growth rate in a second formulation of culture medium compared to cells transformed with the empty plasmid (no VHH). This demonstrates that different modes of agonist presentation in the periplasm can be used to activate the CB2 receptor protein GPCR. In particular, we demonstrated that the CB2 receptor can be activated by VHH domain antibodies that are covalently linked to a plasma membrane anchor protein, either through a long or short linker. We also demonstrated that functional agonist antibodies with a VHH domain can be localized to the periplasm by fusing the antibody to a periplasmic protein, Suc2, that is large enough that the fusion protein is retained in the periplasm. Suc2 forms oligomers containing multiple Suc2 proteins linked through noncovalent interactions, and this multimerization is required for Suc2 retention in the periplasm. Thus, these conditions also demonstrate retention in the periplasm partly through noncovalent interactions between the antibody and the anchor protein. Finally, this experiment demonstrates that direct secretion of unlabeled VHH domain antibodies into the periplasm can activate the CB2 receptor.

[0188] Although preferred embodiments of the subject invention have been described in detail, it will be understood that obvious changes can be made without departing from the spirit and scope of the invention as described herein.

Claims (134)

1. A yeast periplasmic display library for selecting an antibody from an antibody library for specific binding or functional characteristics, comprising a plurality of yeast host cells, wherein each yeast host cell contains: a) an antibody for display in the periplasmic space of a yeast host cell, where the displayed antibody is different in each yeast host cell such that a plurality of yeast host cells display a plurality of antibodies; b) periplasmic anchor protein, where the periplasmic anchor protein is associated with the antibody so that the antibody is exposed in the periplasmic space; And c) a target membrane protein of interest, wherein the membrane protein of interest is localized to the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell. 2. The yeast periplasmic display library according to claim 1, wherein the antibody and the periplasmic anchor protein are non-covalently linked together through molecular binding interactions in a complex or linked through a covalent non-peptide bond in a complex. 3. The yeast periplasmic display library according to claim 1, wherein the antibody and the periplasmic anchor protein are covalently linked together in a fusion protein. 4. The yeast periplasmic display library of claim 1, wherein the periplasmic anchor protein further comprises a signal sequence that causes the periplasmic anchor protein to be transported into the periplasm of the host yeast cell, to the plasma membrane, or to the cell wall, such that the antibody is displayed in the periplasm. 5. The yeast periplasmic display library of claim 1, wherein the periplasmic anchor protein comprises a membrane-spanning transmembrane domain or membrane-associated protein domain that exposes the antibody to the periplasm. 6. The yeast periplasmic display library of claim 5, wherein the membrane-bound protein domain is a plasma membrane-anchoring glycosylphosphatidylinositol (GPI) domain. 7. The yeast periplasmic display library according to claim 6, wherein the plasma membrane-anchoring GPI domain is a japsin plasma membrane-anchoring GPI domain. 8. The yeast periplasmic display library of claim 7, wherein the plasma membrane-anchoring yapsin GPI domain is a plasma membrane-anchoring yapsin GPI domain YPS1, YPS2, YPS3, YPS4, YPS5, YPS6, or YPS7. 9. The yeast periplasmic display library according to claim 2, wherein the periplasmic anchor protein is a protein that binds to the inner surface of the cell wall such that the antibody projects into the periplasm. 10. The yeast periplasmic display library of claim 3, wherein the periplasmic anchor protein is an inner cell wall binding protein that exposes the fusion protein to the periplasm. 11. The yeast periplasmic display library of claim 2, wherein the periplasmic anchor protein is large enough such that the periplasmic anchor protein and associated antibody are retained in the periplasm. 12. The yeast periplasmic display library of claim 3, wherein the periplasmic anchor protein is large enough such that the fusion protein is retained in the periplasm. 13. A yeast periplasmic display library for selecting an antibody from an antibody library for specific binding or functional characteristics, comprising a plurality of yeast host cells, wherein each yeast host cell contains: a) an antibody for display in the periplasmic space of a yeast host cell, wherein the displayed antibody is different in each yeast host cell such that a plurality of yeast host cells display a plurality of antibodies, wherein the antibody is linked to a signal sequence that enables transport of the antibody into the yeast periplasm host cell, to the plasma membrane or to the cell wall, such that the antibody is exposed in the periplasmic space of the yeast host cell; And b) a target membrane protein of interest, wherein the membrane protein of interest is localized to the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell. 14. The yeast periplasmic display library of claim 1, wherein each yeast host cell further comprises a reporter system comprising a reporter gene operably linked to an inducible promoter that is activated when the target membrane protein of interest is activated, allowing detection of increases or decreases in activity the target membrane protein of interest upon binding of the antibody to the target membrane protein of interest. 15. The yeast periplasmic display library of claim 14, wherein the reporter gene is a nutritional marker, an antibiotic resistance marker, a fluorescent marker, a bioluminescent marker, or a counterselection marker. 16. The yeast periplasmic display library according to claim 15, wherein the nutritional marker is selected from the group consisting of HIS3, HIS7, ARG6, LEU2, URA3 and TRP1. 17. The yeast periplasmic display library of claim 15, wherein the antibiotic resistance marker indicates resistance to an antibiotic selected from the group consisting of geneticin, zeocin, hygromycin B, nourseothricin, and bialaphos. 18. The yeast periplasmic display library of claim 15, wherein the fluorescent marker is selected from the group consisting of green fluorescent protein, red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, and orange fluorescent protein. 19. The yeast periplasmic display library according to claim 15, where the bioluminescent marker is luciferase or aequorin. 20. The yeast periplasmic display library according to claim 15, wherein the counterselection marker is selected from the group consisting of CAN1, URA3, MET15, TRP1 and TK. 21. The yeast periplasmic display library of claim 14, wherein the reporter gene is a selectable marker such that an increase in the activity of the target membrane protein of interest upon binding of the antibody to the target membrane protein of interest is detectable by growth of the yeast host cells on media for positive selection. 22. The yeast periplasmic display library of claim 14, wherein the reporter gene is a counterselection marker such that said reduction in the activity of the target membrane protein of interest upon binding of the antibody to the target membrane protein of interest is detectable by growth of the host yeast cells on the medium for negative selection. 23. The yeast periplasmic display library according to any one of claims. 1-22, wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel and a transporter. 24. The yeast periplasmic display library of claim 23, wherein the receptor is a G protein-coupled receptor (GPCR). 25. The yeast periplasmic display library according to claim 24, wherein the GPCR is an exogenous GPCR. 26. The yeast periplasmic display library of claim 25, wherein the yeast host cells further contain an endogenous GPCR. 27. The yeast periplasmic display library of claim 25, wherein each yeast host cell further comprises an engineered Gα subunit capable of being activated by an exogenous GPCR, wherein the activated engineered Gα subunit is capable of activating a detectable pheromone response in the yeast host cell. 28. The yeast periplasmic display library of claim 27, wherein the engineered Gα subunit is a chimeric G protein alpha (Gα) subunit containing the N-terminal domain of the yeast Gα subunit and the C-terminal domain of the exogenous Gα subunit. 29. The yeast periplasmic display library according to claim 28, wherein the yeast Gα subunit belongs to the Gα i, Gα q, Gα s, or Gα o family of G proteins. 30. The yeast periplasmic display library according to claim 25, wherein the exogenous GPCR is a mammalian GPCR. 31. The yeast periplasmic display library of claim 30, wherein at least five C-terminal residues of the yeast Gα subunit are replaced with corresponding C-terminal residues of a mammalian Gα subunit such that the chimeric Gα subunit is capable of being activated by a mammalian GPCR. 32. The yeast periplasmic display library of claim 31, wherein at least 20 C-terminal residues of the yeast Gα subunit are replaced with corresponding C-terminal residues of the mammalian Gα subunit such that the chimeric Gα subunit is capable of being activated by a mammalian GPCR. 33. The yeast periplasmic display library of claim 31, wherein the mammalian Gα subunit is selected from the group consisting of G alpha-S, G alpha-I, G alpha-O, G alpha-T, G alpha-Z, G alpha-Q , G alpha-11, G alpha-12, G alpha-13 and transducin. 34. The yeast periplasmic display library of claim 28, wherein the chimeric Gα subunit contains at least 41 N-terminal residues of the yeast Gα subunit. 35. The yeast periplasmic display library of claim 30, wherein the mammalian GPCR is a human GPCR. 36. The yeast periplasmic display library according to claim 35, where the human GPCR is selected from the group consisting of CXCR4, CXCR5, SSTR2, MOR, AVPR2, FPR2/ALX, ADORA2A, CHRM3, CGRP2, CCR2, CCR4, CCR5, CHRM4, PAC1, b2AR, CXCR2, CYSLTR2, KSHV vGPCR, PKR1, PKR2, CB1, CB2, A3AR and AT1R. 37. The yeast periplasmic display library of claim 24, wherein the target membrane protein GPCR of interest has constitutive ligand-independent activity. 38. The yeast periplasmic display library of claim 24, wherein the host yeast cell is a FAR1 strain containing both genomic copies of FAR1, for selecting antagonist antibodies of the target membrane protein GPCR of interest. 39. The yeast periplasmic display library of claim 24, wherein the host yeast cell is a Δfar1 strain lacking both genomic copies of FAR1, containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene for selection of agonist antibodies of the membrane protein of interest GPCR that is the target. 40. The yeast periplasmic display library according to any one of claims. 1-22, where the antibodies are selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, nanoantibodies, recombinant antibody fragments, Fab fragments, Fab' fragments, F(ab') ₂ fragments, F _v fragments and scFv fragments. 41. The yeast periplasmic display library according to any one of claims. 1-22, wherein the target membrane protein of interest contains a mutation that increases or decreases its activity. 42. The yeast periplasmic display library according to any one of claims. 1-22, wherein the host yeast cell is a Δfar1 strain lacking both genomic copies of FAR1, Δsst2 containing an inactivated SST2 gene, Δste14 lacking both genomic copies of STE14, Δste3 lacking both genomic copies of STE3, or Δmat containing a deleted or inactivated MATα locus or a deleted or inactivated MATa locus. 43. The yeast periplasmic display library of claim 42, wherein the host yeast cell is a Δmat strain containing a deleted or inactivated MATα locus or a deleted or inactivated MATa locus. 44. The yeast periplasmic display library according to any one of claims. 1-22, wherein the yeast host cell further contains a modified CLN3 protein containing a C-terminal truncation that increases the CLN3 content of the yeast host cell compared to wild-type CLN3 protein. 45. The yeast periplasmic display library of claim 44, wherein the modified CLN3 protein retains at least the N-terminal amino acids 1-387 of the wild-type CLN3 protein. 46. The yeast periplasmic display library of claim 44, wherein the modified CLN3 protein retains at least the N-terminal amino acids 1-408 of the wild-type CLN3 protein. 47. The yeast periplasmic display library according to any one of claims. 1-22, wherein the host yeast cell is a haploid or diploid yeast host cell. 48. A method for obtaining a yeast periplasmic display library according to claim 1, wherein the method includes: a) providing a first plurality of recombinant polynucleotides encoding antibodies for display in the periplasmic space of a yeast host cell, wherein the displayed antibody is different in each yeast host cell such that a plurality of yeast host cells display a plurality of antibodies; b) providing a second recombinant polynucleotide encoding a periplasmic anchor protein; c) transfecting a plurality of host yeast cells with a first plurality of recombinant polynucleotides and a second recombinant polynucleotide; d) transfecting a plurality of yeast host cells with a recombinant polynucleotide encoding a target membrane protein of interest; And e) culturing a plurality of yeast host cells under conditions that allow expression of antibodies, a periplasmic anchor protein, and a target membrane protein of interest, wherein the periplasmic anchor protein is associated with the antibody such that the antibody is exposed in the periplasmic space where each yeast host cell exhibits The different antibody to the periplasmic space and the target membrane protein of interest are localized to the plasma membrane, yielding the yeast periplasmic display library of claim 1. 49. The method of claim 48, wherein the recombinant polynucleotides encoding antibodies or the recombinant polynucleotide encoding a periplasmic anchor protein or a target membrane protein of interest are provided via expression vectors. 50. The method of claim 48, wherein the recombinant polynucleotides encoding antibodies or the recombinant polynucleotide encoding a periplasmic anchor protein or a target membrane protein of interest is inserted into the genome of the host yeast cell at the target locus. 51. A method for obtaining a yeast periplasmic display library according to claim 3, wherein the method includes: a) providing a plurality of recombinant polynucleotides encoding fusion proteins, wherein each recombinant polynucleotide encodes a different fusion protein comprising a periplasmic anchor protein linked to a different antibody for display; b) transfecting a plurality of yeast host cells with a plurality of recombinant polynucleotides encoding fusion proteins; c) transfecting a plurality of yeast host cells with a recombinant polynucleotide encoding a target membrane protein of interest; And d) culturing a plurality of yeast host cells under conditions that allow expression of the fusion proteins and the target membrane protein of interest, wherein each yeast host cell exposes a different antibody to the periplasmic space and the target membrane protein of interest is localized to the plasma membrane, obtaining yeast periplasmic display libraries according to claim 3. 52. The method of claim 51, wherein the recombinant polynucleotides encoding the fusion proteins or the recombinant polynucleotide encoding the target membrane protein of interest are provided via expression vectors. 53. The method of claim 51, wherein the recombinant polynucleotides encoding the fusion proteins or target membrane protein of interest are inserted into the genome of the host yeast cell at a predetermined locus. 54. Method according to any one of paragraphs. 48-53, wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel and a transporter. 55. The method of claim 54, wherein the receptor is a G protein-coupled receptor (GPCR). 56. Method according to any one of paragraphs. 48-53, further comprising introducing into a plurality of yeast host cells a recombinant polynucleotide encoding an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered Gα subunit is capable of activating a detectable pheromone response in the yeast host cell. 57. The method of claim 56, wherein the engineered Gα subunit is a chimeric G protein alpha (Gα) subunit containing the N-terminal domain of a yeast Gα subunit and the C-terminal domain of an exogenous Gα subunit. 58. The method according to claim 57, wherein the yeast Gα subunit belongs to the Gα i, Gα q, Gα s or Gα o family of G proteins. 59. The method of claim 57, wherein the exogenous Gα subunit is a mammalian Gα subunit. 60. The method of claim 59, wherein at least five C-terminal residues of the yeast Gα subunit are replaced with corresponding C-terminal residues of the mammalian Gα subunit such that the chimeric Gα subunit is capable of being activated by a mammalian GPCR. 61. The method of claim 60, wherein at least 20 C-terminal residues of the yeast Gα subunit are replaced with corresponding C-terminal residues of the mammalian Gα subunit such that the chimeric Gα subunit is capable of being activated by a mammalian GPCR. 62. The method of claim 55, wherein the host yeast cell is a FAR1 strain containing both genomic copies of FAR1 for selecting antagonist antibodies of a GPCR target of interest. 63. The method of claim 55, wherein the host yeast cell is a Δfar1 strain lacking both genomic copies of FAR1 containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene for the selection of GPCR agonist antibodies. 64. The method of screening the yeast periplasmic display library of claim 14 for an antibody that modulates the activity of a target membrane protein of interest, the method comprising culturing at least a subset of yeast host cells of the yeast periplasmic display library of claim 14 in a selective medium; and detecting reporter gene expression, wherein increased expression of the reporter gene indicates that the antibody increases the activity of the target membrane protein of interest, and decreased expression of the reporter gene indicates that the antibody reduces the activity of the target membrane protein of interest. 65. The method of claim 64, wherein the reporter gene is a nutritional marker, an antibiotic resistance marker, a fluorescent marker, a bioluminescent marker, or a counterselection marker. 66. The method of claim 65, further comprising performing positive selection for expression of a nutritional marker, wherein growth of the host yeast cells in a selective nutrient-deficient environment indicates that the target membrane protein of interest is activated. 67. The method of claim 66, wherein the nutritional marker is HIS3, HIS7, ARG6, LEU2, URA3 and TRP1. 68. The method of claim 65, further comprising performing positive selection for expression of an antibiotic resistance marker, wherein growth of the host yeast cells in the selective medium containing the antibiotic indicates that the target membrane protein of interest is activated. 69. The method of claim 68, wherein the antibiotic resistance marker indicates resistance to an antibiotic selected from the group consisting of geneticin, zeocin, hygromycin B, nourseothricin and bialaphos. 70. The method of claim 65, further comprising performing positive selection for expression of a fluorescent marker, wherein detecting fluorescence emitted by the host yeast cells indicates that the target membrane protein of interest is activated. 71. The method of claim 70, wherein the fluorescent marker is selected from the group consisting of green fluorescent protein, red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein and orange fluorescent protein. 72. The method of claim 65, further comprising performing positive selection for expression of a bioluminescent marker, wherein detecting bioluminescence emitted by the host yeast cells indicates that the target membrane protein of interest is activated. 73. The method according to claim 72, where the bioluminescent marker is luciferase or aequorin. 74. The method of claim 65, further comprising performing negative selection on expression of a counterselection marker, wherein the reduction in activity of the target membrane protein of interest upon binding of the exposed antibody to the target membrane protein of interest is detectable by growth of host yeast cells in the medium, containing an agent that selects against cells expressing a counterselection marker. 75. The method of claim 74, wherein the counter-selection marker is selected from the group consisting of CAN1, URA3, MET15, TRP1 and TK. 76. The method of screening the yeast periplasmic display library of claim 27 for an antibody that modulates the activity of a GPCR target of interest, the method comprising culturing at least a subset of yeast host cells of the yeast periplasmic display library of claim 27 in an environment where detection activation or inhibition of the pheromone response in at least one yeast host cell compared to a control yeast host cell that does not have antibody exposed in the periplasmic space, indicates that the exposed antibody in the at least one yeast host cell binds and modulates GPCR activity. 77. The method of claim 76, wherein the target GPCR of interest is a human GPCR. 78. The method of claim 77, further comprising contacting the human GPCR with a ligand. 79. The method of claim 78, wherein the GPCR has constitutive ligand-independent activity. 80. Method according to any one of paragraphs. 76-79, wherein the yeast host cell is a FAR1 strain containing both genomic copies of FAR1, wherein inhibition of the pheromone response by an antibody acting as an antagonist that binds and inhibits GPCRs in the yeast host cell results in cell cycle arrest and growth of the host yeast cell. 81. Method according to any one of paragraphs. 76-79, wherein the host yeast cell is a Δfar1 strain lacking both genomic copies of FAR1, containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene, wherein activation of the pheromone response by an antibody acting as an agonist that binds and activates GPCR in the host yeast cell, leading to increased reporter gene expression. 82. The method of claim 81, wherein the reporter gene is a nutritional marker, an antibiotic resistance marker, a fluorescent marker, a bioluminescent marker, or a counterselection marker. 83. The yeast periplasmic display library according to any one of claims. 1-22 or the method according to any paragraph. 48-53 or 64-75, wherein the host yeast cell genus is selected from the group consisting of Saccharomyces , Candida , Pichia , Kluyveromyces and Yarrowia . 84. The method of claim 83, wherein the host yeast cell genus is Saccharomyces. 85. The method of claim 84, wherein the Saccharomyces species is Saccharomyces cerevisiae . 86. A yeast host cell for use in a yeast display library, comprising: a) antibody for display in the periplasmic space of the host yeast cell, b) periplasmic anchor protein, wherein the periplasmic anchor protein is associated with the antibody such that the antibody is exposed in the periplasmic space; And c) a target membrane protein of interest, wherein the membrane protein of interest is localized to the plasma membrane of the host yeast cell and is accessible to the antibody exposed in the periplasmic space of the host yeast cell. 87. The yeast host cell of claim 86, wherein the antibody and the periplasmic anchor protein are non-covalently linked together through molecular binding interactions in a complex or are linked through a covalent non-peptide bond in a complex. 88. The yeast host cell of claim 86, wherein the antibody and the periplasmic anchor protein are covalently linked together in a fusion protein. 89. Yeast host cell according to any one of paragraphs. 86-88, wherein the periplasmic anchor protein further comprises a signal sequence that causes the periplasmic anchor protein to be transported into the periplasm of the host yeast cell, to the plasma membrane or to the cell wall, such that the antibody is exposed in the periplasm. 90. Yeast host cell according to any one of paragraphs. 86-88, wherein the periplasmic anchor protein comprises a membrane-spanning transmembrane domain or membrane-bound protein domain that exposes the antibody to the periplasm. 91. Yeast host cell according to any one of paragraphs. 86-88, where the periplasmic anchor protein is a protein that binds to the inner surface of the cell wall so that the antibody protrudes into the periplasm. 92. Yeast host cell according to any one of paragraphs. 86-88, wherein the periplasmic anchor protein is large enough such that the periplasmic anchor protein and associated antibody are retained in the periplasm. 93. Yeast host cell according to any one of paragraphs. 86-88, wherein the target membrane protein of interest is selected from the group consisting of a receptor, an ion channel and a transporter. 94. The yeast host cell of claim 93, wherein the receptor is a G protein coupled receptor (GPCR). 95. Yeast host cell according to any one of paragraphs. 86-88, further comprising a recombinant polynucleotide encoding an engineered Gα subunit capable of being activated by a GPCR, wherein the activated engineered Gα subunit is capable of activating a detectable pheromone response in a host yeast cell. 96. The yeast host cell of claim 95, wherein the engineered Gα subunit is a chimeric G protein alpha (Gα) subunit containing the N-terminal domain of the yeast Gα subunit and the C-terminal domain of the exogenous Gα subunit. 97. The yeast host cell of claim 96, wherein the yeast Gα subunit belongs to the Gα i, Gα q, Gα s, or Gα o family of G proteins. 98. The yeast host cell of claim 96, wherein the exogenous Gα subunit is a mammalian Gα subunit. 99. The yeast host cell of claim 98, wherein at least five C-terminal residues of the yeast Gα subunit are replaced with corresponding C-terminal residues of a mammalian Gα subunit such that the chimeric Gα subunit is capable of being activated by a mammalian GPCR. 100. The yeast host cell of claim 99, wherein at least 20 C-terminal residues of the yeast Gα subunit are replaced with corresponding C-terminal residues of the mammalian Gα subunit such that the chimeric Gα subunit is capable of being activated by a mammalian GPCR. 101. Yeast host cell according to any one of paragraphs. 86-88, wherein the yeast host cell is a FAR1 strain containing both genomic copies of FAR1, for selecting antagonist antibodies to a GPCR target of interest. 102. Yeast host cell according to any one of paragraphs. 86-88, wherein the host yeast cell is a Δfar1 strain lacking both genomic copies of FAR1 containing a pheromone-inducible PRM1 promoter operably linked to a reporter gene. 103. Yeast host cell according to any one of paragraphs. 86-88, wherein the host yeast cell genus is selected from the group consisting of Saccharomyces , Candida , Pichia , Kluyveromyces and Yarrowia . 104. The yeast host cell of claim 103, wherein the genus of the yeast host cell is Saccharomyces. 105. The yeast host cell of claim 104, wherein the Saccharomyces species is Saccharomyces cerevisiae . 106. A method of localizing an antibody to the periplasmic space of a host yeast cell, comprising binding the antibody to a periplasmic anchor protein such that the antibody is localized to the periplasmic space. 107. The method of claim 106, wherein the antibody and the periplasmic anchor protein are non-covalently linked together through molecular binding interactions in a complex or are linked through a covalent non-peptide bond in a complex. 108. The method of claim 106, wherein the antibody and the periplasmic anchor protein are covalently linked together in a fusion protein. 109. The method according to any one of paragraphs. 106-108, wherein the periplasmic anchor protein further comprises a signal sequence that causes the periplasmic anchor protein to be transported into the periplasm of the host yeast cell, to the plasma membrane or to the cell wall, such that the antibody is exposed in the periplasm. 110. Method according to any one of paragraphs. 106-108, wherein the periplasmic anchor protein contains a membrane-spanning transmembrane domain or membrane-associated protein domain that exposes the antibody to the periplasm. 111. The method of claim 110, wherein the membrane-associated protein domain is a plasma membrane-anchoring glycosylphosphatidylinositol (GPI) domain. 112. The method according to any one of paragraphs. 106-108, where the periplasmic anchor protein is a protein that binds to the inner surface of the cell wall so that the antibody protrudes into the periplasm. 113. The method according to any one of paragraphs. 106-108, wherein the periplasmic anchor protein is large enough such that the periplasmic anchor protein and associated antibody are retained in the periplasm. 114. The method according to any one of paragraphs. 106-108, where the antibody is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a humanized antibody, a nanoantibody, a recombinant antibody fragment, a Fab fragment, a Fab' fragment, an F(ab') ₂ fragment, _{an Fv} fragment, and scFv fragment. 115. Method according to any one of paragraphs. 106-108, wherein the host yeast cell genus is selected from the group consisting of Saccharomyces , Candida , Pichia , Kluyveromyces and Yarrowia . 116. The method of claim 115, wherein the host yeast cell genus is Saccharomyces. 117. The method of claim 116, wherein the Saccharomyces species is Saccharomyces cerevisiae .

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