A yeast phenotypic screening method for isolating functional antibodies against G protein-coupled receptors (GPCRs)

Abstract

The present invention provides a method for isolating and identifying a functional antibody or antigen binding protein or fragment thereof that can bind to a transmembrane target protein and activate the activity of the transmembrane target protein, comprising the following steps: providing a yeast cell transformed with a yeast expression vector encoding a library of antibodies or antigen binding proteins or fragments thereof, wherein the yeast cell expresses the transmembrane target protein; expressing the library of antibodies or antigen binding proteins or fragments thereof in the yeast cell, wherein the expressed antibodies or antigen binding proteins or fragments thereof are secreted into the periplasmic space of the yeast cell; culturing the yeast cell in a selective medium or at a restrictive temperature, or under a combination of a selective medium and a restrictive temperature; and detecting a predetermined phenotype in the yeast cell, wherein the detection or manifestation of a predetermined phenotype indicates that a functional antibody or antigen binding protein or fragment thereof binds to the transmembrane target protein and leads to the activation of the activity of the transmembrane target protein. The present invention also provides an antibody or antibody fragment identified as a G protein coupled receptor (GPCRs) activator by the method of the present invention.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention belongs to the field of antibody separation and identification through yeast phenotype screening. Background Art

[0002] G protein-coupled receptors (GPCRs), also known as seven-transmembrane proteins, are a unique family of receptors with similar structures and diverse functions. Members of this large family are present in all eukaryotic organisms from yeast to humans, and all have a core structure unique to the family. The core structure consists of seven transmembrane α-helices connected by six protruding interhelical loops, three of which protrude into the extracellular space with the N-terminus, and the other three interhelical loops and the C-terminus are exposed to the cytoplasm. The extracellular and intracellular domains of GPCRs contain the action site of the ligand and the association site of the molecules of the cell signal transduction system, respectively, making the receptor like a molecular device that can sense peripheral signals and transmit information to the cell to initiate various corresponding activities. Different GPCRs respond to different ligands. These ligands are of various types and sizes, including proteins, peptides, lipids, amino acids and nucleic acid derivatives, ions and even photons. The binding of ligands to GPCRs on the extracellular side triggers profound conformational changes in the latter, which in turn activates the signal transduction network coupled to the receptors on the intracellular side, leading to a series of important physiological processes, such as the perception of light, smell and taste, the regulation of psychological and nervous system states, metabolic homeostasis, immune system regulation, etc. There are at least 800 GPCR genes in the human genome, many of which are associated with diseases, including immune and nervous system diseases, cancer and inflammation, and metabolic diseases. Therefore, this receptor family has long been the most prominent and prolific target in the pharmaceutical industry.

[0003] In recent decades, monoclonal antibodies have evolved from common laboratory reagents and diagnostic tools to a new class of drugs with amazing performance and performance in the clinical and pharmaceutical markets. Monoclonal antibody technology (such as antibody production and antibody artificial modification) has greatly changed the scene of drug development with the help of research results on disease-related biological targets. Unlike many synthetic chemical drugs, antibodies are highly specific to their targets and are generally easy to generate. Therefore, potential biological targets identified in laboratory studies can be directly and often decisively evaluated for their clinical importance by using their monoclonal antibodies to activate or inhibit the function of the target in relevant in vitro and in vivo models (assuming that the antibody must be able to access the target in vivo). Generally, the evaluation studies of such targets using small molecule compounds are more complex, time-consuming and costly. Monoclonal antibodies also show other superior properties over small molecule drugs, such as low toxicity and high stability. Therefore, after completing the proof of concept by means of antibody interference, antibody products can achieve a higher success rate in clinical trials and approval processes. And compared with small molecule drugs, their time to market is much shorter. Today, the annual market value of antibody drugs exceeds $80 billion and is expected to grow to $140 billion by 2024. Antibody drugs have become the preferred treatment modality for a variety of health problems, including cancer and autoimmune diseases, because they are more effective or there are no other options.

[0004] To date, antibody drugs targeting GPCRs have been rare and progress has been slow. This may seem unusual, as GPCRs are widely considered to be one of the most studied drug targets in the pharmaceutical industry. In fact, it reflects the severity of the problems that researchers face when trying to generate clinically effective antibodies against GPCRs. As seven-pass transmembrane proteins, GPCRs are inherently difficult to develop functional antibodies for, as their function depends on their elaborate and complex spatial conformation, which in turn depends on their positioning in the cell membrane. Various strategies have been developed to address this challenge. These include increasing the stability of GPCRs by mutation or modification in vivo or in vitro to improve the purification of homogeneous conformation-retaining antigens; and immunization or screening using linear peptides of extracellular loops and termini, or using whole cell forms and membrane fractions of transfected cell lines that highly express the target receptor on the cell surface, or native antigens in DNA form. Each of these approaches has its own advantages and limitations, and no one method is universally applicable. For example, linear antigen selection, which is often the first choice, may work well for receptors whose N-termini are directly involved in ligand binding (e.g., some chemokine receptors), but it is not suitable for most GPCRs. Thermal stabilization of receptors by mutation can lock receptors in a conformation, but it may not be conducive to the generation of antibodies with receptor modulatory activity. The best form of antigen is of course the antigen embedded in the cell membrane, because it is the natural form most likely to produce functional antibodies. However, since the GPCR protein expressed in artificially modified cell lines is still a small proportion relative to the endogenous membrane protein, immunization with whole cells, cell membrane fractions, and DNA is usually a very inefficient method, and a large number of screening or counter-screening are required to accurately identify any true GPCR binders. For example, the monoclonal antibody PA14 against CCR5 was originally obtained by whole cell immunization, but it took about 10,000 hybridomas to screen before it was obtained.

[0005] In most cases, the search for functional antibodies for any target, whether GPCRs or otherwise, is a two-step process, as all current antibody discovery technologies, whether monoclonal or combinatorial, are limited to antigen-antibody binding. Whether the binders also have positive or negative receptor modulatory activity still requires further investigation. In the CCR5 example mentioned above, of the six specific CCR5-interacting antibodies identified from 10,000 hybridomas, only one, PA14, was found to be an antagonist, and none was an activator. It is no surprise that if a sufficient number of structurally appropriate antigens were available, it would be more likely that both activator and antagonist antibodies would be discovered. Unfortunately, this is not the case for a large number of GPCRs; for many clinically important GPCRs, the discovery of functional antibodies remains difficult, time-consuming, and resource-intensive.

[0006] Phenotypic screening can directly isolate functional antibodies without affinity selection steps. Although there are no examples of functional antibodies to GPCRs obtained by this method, there have been a number of reports that this method can be used to find functional antibodies to other receptors and cell surface antigens. For example, an efficient phenotypic screening method called "autocrine screening" has successfully generated a series of activating antibodies for growth factor receptors (e.g., erythropoietin (Epo) receptor, thrombopoietin (Tpo) receptor, granulocyte colony stimulating factor (G-CSF) receptor, fibroblast growth factor (FGF) receptor, etc.) using a combinatorial antibody library carried by a lentiviral vector. However, this mammalian phenotypic screening method is unlikely to be applicable to GPCRs, because efficient screening methods such as blocking cell death, although conveniently applied to growth factor receptors, are unlikely, or at least not so easy, to be directly applied to most GPCR receptors in mammalian cell systems.

[0007] Currently, there is no efficient method for isolating and identifying functional antibodies (especially activator antibodies) of GPCRs. Therefore, it is necessary to develop a rapid, reliable and low-cost method that can overcome the above shortcomings. Summary of the invention

[0008] In one aspect, a method for isolating and identifying a functional antibody or antigen-binding protein or a fragment thereof that can bind to a transmembrane target protein and activate the activity of the transmembrane target protein is provided, comprising the following steps:

[0009] a) providing a yeast cell transformed with a yeast expression vector encoding a library of antibodies or antigen-binding proteins or fragments thereof, wherein the yeast cell expresses the transmembrane target protein;

[0010] b) expressing the library of antibodies or antigen binding proteins or fragments thereof in yeast cells, wherein the expressed antibodies or antigen binding proteins or fragments thereof are secreted into the periplasmic space (periplasmic space) of the yeast cells;

[0011] c) culturing the yeast cells in a selective medium or at a restrictive temperature, or a combination of a selective medium and a restrictive temperature; and

[0012] d) detecting a predetermined phenotype of the yeast cell, wherein the appearance of the predetermined phenotype indicates that a functional antibody or antigen binding protein or a fragment thereof binds to the transmembrane target protein and leads to activation of the activity of the transmembrane target protein.

[0013] On the one hand, an antibody or antigen binding protein or a fragment thereof is provided, which is an activator of a transmembrane target protein identified according to the method disclosed herein, wherein the transmembrane target protein is human G protein coupled receptor (GPCR) GLP-1R, or yeast GPCR, i.e., α-factor receptor Ste2p.

[0014] In one aspect, an antibody or antigen-binding protein or fragment thereof identified according to the method disclosed herein is provided, comprising: (i) a heavy chain variable domain, including a VHCDR1 having an amino acid sequence of GYSFTSYW (SEQ ID NO: 1), a VHCDR2 having an amino acid sequence of IYPGDSDT (SEQ ID NO: 2), and a VHCDR3 having an amino acid sequence of CARLGYYDSSGYYSEDY (SEQ ID NO: 3); and (ii) a light chain variable domain, including a VLCDR1 having an amino acid sequence of SGRSGFA (SEQ ID NO: 4), a VLCDR2 having an amino acid sequence of VNSDGSH (SEQ ID NO: 5), and a VLCDR3 having an amino acid sequence of QTWGTGIWV (SEQ ID NO: 6).

[0015] definition

[0016] The following definitions are provided to help understand the present invention. They are general definitions for this purpose only and should not be construed as limiting the scope of the present invention.

[0017] The term "antibody or antigen-binding protein or fragment thereof" as used herein refers to a protein molecule that can selectively bind to an epitope of a target antigen in vivo and in vitro, and encompasses natural and artificially modified antibodies, antibody fragments, and proteins unrelated to antibodies and their fragments (e.g., protein domains and oligopeptides).

[0018] The term "antigen" as used herein refers to a target (or target) protein that can be recognized and reacted to by antibodies in a selective manner in vivo and in vitro. Preferred antigens include proteins that are present in the cell membrane and act as sensors, receptors or transporters for specific substances outside the cell. Further preferred antigens are a family of transmembrane proteins called G protein coupled receptors (GPCRs).

[0019] The term "antibody" as used herein has the broadest meaning, and refers to various forms of natural or artificially modified immunoglobulin molecules and immunologically active fragments thereof, including monoclonal antibodies, recombinant antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, conjugated antibodies, chimeric antigen receptors (CARs), single variable domains, domain antibodies, antigen binding fragments, immune effector fragments, double-chain antibodies, multispecific antibodies, human antibodies, camel antibodies, single-chain variable region fragments (scFv), disulfide bond-linked variable region fragments (sdFv), Fab fragments, F(ab') fragments and anti-idiotype (anti-Id) antibodies (including, for example, anti-idiotype antibodies of the antibodies of the present invention), and epitope binding fragments of any of the above. Immunologically active antibody fragments can also be fused with any polypeptide fusion partner including an Fc region or a fragment thereof. Examples of Fc variant fusions include scFv-Fc fusions, variable region (e.g., VL and VH)-Fc fusions, or scFv-scFv-Fc fusions. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0020] The term "single variable domain" refers to a variable domain of an antibody that independently binds an antigen or epitope, as opposed to different variable regions or domains (e.g., V H 、V HH 、V L ). A single variable domain may include a complete antibody variable domain or a modified variable domain. Examples of modified variable domains include: variable domains in which one or more residues in the antigen-interacting motif are converted to other sequences, and variable domains that are truncated or include N-terminal or C-terminal extensions.

[0021] The term "domain" as used herein refers to a protein folding structure with its own unique structural features. Typically, a domain is responsible for a certain independent function of a protein, and in many cases, can be added, removed or transferred to other proteins without causing loss of function of the rest of the protein and / or the domain.

[0022] Antigen binding fragments or antigen binding domains can be transferred to other suitable proteins or domains by arranging one or more complementary determining regions (CDRs) on a non-antibody protein backbone to produce a protein molecule that can obtain binding activity against the same target antigen. Examples of backbone proteins include cytotoxic T lymphocyte antigen 4 (CTLA-4), lipocalin, Staphylococcus A protein (SpA), Affibody, Avimer, GroE1 protein, transferrin, heat shock protein (GroES) and fibronectin / adnectin.

[0023] The antigen binding fragment or immune effector fragment may comprise a portion of a heavy chain and / or light chain variable region sequence. The fragment length is at least 5, 6, 8 or 10 amino acids. Alternatively, the length of these fragments is at least 15, at least 20, at least 50, at least 75 or at least 100 amino acids.

[0024] "CDR" refers to the complementary determining region of an antibody or its fragment. They are all hypervariable regions (HVRs) of the heavy and light chains of immunoglobulins. There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Therefore, "CDR" as used herein refers to any one or all of the three heavy chain CDRs and three light chain CDRs.

[0025] Antigen binding proteins can also be polypeptides or oligopeptides. An oligopeptide can contain 2 to 50 amino acids, preferably more than 10 amino acids. For example, an oligopeptide can be a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. A polypeptide can contain more than 50 amino acids.

[0026] When used in the context of an antibody or antigen binding protein or fragment thereof, the term "functional" refers to the ability of such molecules to bind to their target antigen and elicit a biological response mediated by the target antigen. "Functional" is also used to describe antibodies or antigen binding proteins or fragments thereof that have natural biological activity or any specific activity not previously identified. Examples of functional antibodies or antigen binding proteins or fragments thereof include such antibodies or antigen binding proteins or fragments thereof that can bind to receptors in the cell membrane and activate receptor-mediated signal transduction pathways. Functional antibodies or antigen binding proteins or fragments thereof may be activators or antagonists.

[0027] The term "library" refers to a combinatorial deoxyribonucleic acid (DNA) library consisting of a mixture of polynucleotides with different sequences. They encode polypeptides or oligopeptides with different sequences (e.g., antibody polypeptides or polypeptides or oligopeptides not derived from antibodies). The library can be used to screen antibodies or antibody fragments or polypeptides or oligopeptides with specific structures or functions. The sequence differences between library members constitute the diversity of the library. The library can be embodied as a mixture of polynucleotides, or in the form of a population of organisms or cells, such as a population of yeast cells, wherein each cell contains one or more copies of an expression vector, which contains different polynucleotide sequences encoding antibodies or antibody fragments or polypeptides or oligopeptides not derived from antibodies that can be expressed in yeast cells. Therefore, the host cell population represents a library encoding and expressing antibodies or polypeptide mixtures. Examples of combinatorial libraries include various combinatorial antibody libraries, covering single-chain and single-domain libraries (e.g., scFv and VHH), Fab libraries and full-length antibody libraries, as well as combinatorial peptide libraries. Combinatorial antibody libraries may further include immune libraries, natural libraries, semi-synthetic libraries, synthetic libraries, and combinations thereof. Combinatorial peptide libraries may further include random or partially random polypeptide or oligopeptide libraries.

[0028] The term "library" is used interchangeably with the term "pool."

[0029] The term "polynucleotide encoding an antibody or a fragment thereof" used herein may consist of one or more polynucleotide chains each encoding a polypeptide chain of an antibody or a fragment thereof.

[0030] "Vector" as used herein refers to any element of a vector that can act as a gene transfer, gene expression, or replication or integration of an exogenous polynucleotide in a host cell. The vector can be an artificial chromosome or a plasmid, and can be integrated into the host cell genome or exist in the form of an independent genetic element (e.g., an episome, a plasmid). The vector can direct the expression of a polypeptide library in a host cell. The vectors of the present invention include yeast expression vectors, 2μ vectors, and vectors containing centromeres.

[0031] A "shuttle vector" (or bifunctional vector) is any vector in the art that can replicate in more than one organism. For example, a shuttle vector that can replicate in both E. coli and S. cerevisiae can be constructed by ligating sequences from an E. coli plasmid with sequences from a yeast 2μ plasmid.

[0032] The expression vector is capable of expressing the polypeptide encoded by the loaded polynucleotide.

[0033] As used herein, the term "transmembrane domain" (TMD) refers to a domain of a peptide, polypeptide or protein that crosses the cell membrane.

[0034] As used herein, the term "promoter" refers to a region of DNA that initiates transcription of a specific gene.

[0035] As used herein, the term "detectable marker" or "reporter gene" refers to a detectable marker, such as a gene or phenotype, that is expressed only when a signal transduction pathway is activated.

[0036] The term "secretion signal" or "secretion signal peptide" as used herein refers to a short peptide sequence (usually 16-30 amino acids) located at the N-terminus of a protein, which directs the protein to translocate through the secretory pathway to a cellular destination, such as the endoplasmic reticulum, Golgi apparatus, endosomes, cell membrane or extracellular space.

[0037] As used herein, the term "periplasmic space" or "periplasm" refers to the space between the cell membrane and the cell wall of a yeast cell.

[0038] As used herein, the term "about" in the context of formulation ingredient concentrations generally refers to ±5% of the stated value, more typically ±4% of the stated value, more typically ±3% of the stated value, more typically ±2% of the stated value, even more typically ±1% of the stated value, and even more typically ±0.5% of the stated value.

[0039] Throughout the text of this application, certain embodiments are expressed in the form of ranges. This expression should be understood to be purely for the purpose of convenience and simplicity, rather than a rigid limitation of the range. Therefore, the expression of a range should be understood to have explicitly included all possible subranges and individual values ​​within the range. For example, the expression of a range of 1 to 6 should be considered to have explicitly included subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6 and 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 3, 4, 5 and 6. This rule applies regardless of the breadth of the range. Sequence Listing <110> Kangti Pte Ltd <120> A yeast phenotypic screening method for isolating functional antibodies against G protein-coupled receptors (GPCRs) <130> 31842SG2 <160> 86 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Gly Tyr Ser Phe Thr Ser Tyr Trp 1 5 <210> 2 <211> 8 <212> PRT <213> Saccharomyces cerevisiae <400> 2 Ile Tyr Pro Gly Asp Ser Asp Thr 1 5 <210> 3 <211> 17 <212> PRT <213> Saccharomyces cerevisiae <400> 3 Cys Ala Arg Leu Gly Tyr Tyr Asp Ser Ser Gly Tyr Tyr Ser Glu Asp 1 5 10 15 Tyr <210> 4 <211> 7 <212> PRT <213> Saccharomyces cerevisiae <400> 4 Ser Gly Arg Ser Gly Phe Ala 1 5 <210> 5 <211> 7 <212> PRT <213> Saccharomyces cerevisiae <400> 5 Val Asn Ser Asp Gly Ser His 1 5 <210> 6 <211> 9 <212> PRT <213> Saccharomyces cerevisiae <400> 6 Gln Thr Trp Gly Thr Gly Ile Trp Val 1 5 <210> 7 <211> 131 <212> PRT <213> Saccharomyces cerevisiae <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Pro Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Tyr Tyr Asp Ser Ser Gly Tyr Tyr Ser Glu Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala 115 120 125 Pro Thr Leu 130 <210> 8 <211> 127 <212> PRT <213> Saccharomyces cerevisiae <400> 8 Gln Pro Val Leu Thr Gln Ser Ser Ser Ala Ser Ala Ser Leu Gly Ala 1 5 10 15 Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gly Arg Ser Gly Phe Ala 20 25 30 Ile Ala Trp His Gln Gln His Pro Glu Lys Gly Pro Arg Tyr Leu Met 35 40 45 Asn Val Asn Ser Asp Gly Ser His Thr Lys Gly Asp Gly Ile Pro Asp 50 55 60 Arg Phe Ser Gly Ser Ser Ser Glu Ala Glu Arg Tyr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Phe Asp Asp Glu Val Asp Tyr Tyr Cys Gln Thr Trp Gly 85 90 95 Thr Gly Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg 100 105 110 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Ala Val Leu 115 120 125 <210> 9 <211> 15 <212> DNA <213> Saccharomyces cerevisiae <400> 9 aaaattggta ttata 15 <210> 10 <211> 15 <212> DNA <213> Saccharomyces cerevisiae <400> 10 caatacgaac tctta 15 <210> 11 <211> twenty one <212> DNA <213> Artificial Sequence <400> 11 attctgaggt tgctgctttg g 21 <210> 12 <211> twenty two <212> DNA <213> Artificial Sequence <400> 12 tgtcttggtc taccgacgat ag 22 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 tcgccgcaat gtctactacc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 gcccaattgt gcttgctgaa 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <400> 15 tgcctgcttg ccgaatatc 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 atatcacggg tagccaacgc 20 <210> 17 <211> twenty three <212> DNA <213> Artificial Sequence <400> 17 caggtbcagc tggtgcagtc tgg 23 <210> 18 <211> twenty three <212> DNA <213> Artificial Sequence <400> 18 carrtscagc tggtrcartc tgg 23 <210> 19 <211> twenty three <212> DNA <213> Artificial Sequence <400> 19 cagrtcacct tgaaggagtc tgg 23 <210> 20 <211> twenty three <212> DNA <213> Artificial Sequence <400> 20 sargtgcagc tggtggagtc tgg 23 <210> twenty one <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty one gaggtgcagc tgktggagwc ysg 23 <210> twenty two <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty two caggtgcarc tgcaggagtc ggg 23 <210> twenty three <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty three cagstgcagc trcagsagts sgg 23 <210> twenty four <211> twenty three <212> DNA <213> Artificial Sequence <400> twenty four gargtgcagc tggtgcagtc tgg 23 <210> 25 <211> twenty three <212> DNA <213> Artificial Sequence <400> 25 caggtacagc tgcagcagtc agg 23 <210> 26 <211> twenty one <212> DNA <213> Artificial Sequence <400> 26 aagggttggg gcggatgcac t 21 <210> 27 <211> twenty three <212> DNA <213> Artificial Sequence <400> 27 gacatccaga tgacccagtc tcc 23 <210> 28 <211> twenty three <212> DNA <213> Artificial Sequence <400> 28 gmcatccrgw tgacccagtc tcc 23 <210> 29 <211> twenty three <212> DNA <213> Artificial Sequence <400> 29 gatrttgtga tgacycagwc tcc 23 <210> 30 <211> twenty three <212> DNA <213> Artificial Sequence <400> 30 gaaatwgtgw tgacrcagtc tcc 23 <210> 31 <211> twenty three <212> DNA <213> Artificial Sequence <400> 31 gacatcgtga tgacccagtc tcc 23 <210> 32 <211> twenty three <212> DNA <213> Artificial Sequence <400> 32 gaaacgacac tcacgcagtc tcc 23 <210> 33 <211> twenty three <212> DNA <213> Artificial Sequence <400> 33 gawrttgtgm tgacwcagtc tcc 23 <210> 34 <211> twenty four <212> DNA <213> Artificial Sequence <400> 34 acactctccc ctgttgaagc tctt 24 <210> 35 <211> twenty three <212> DNA <213> Artificial Sequence <400> 35 cagtctgtgc tgactcagcc acc 23 <210> 36 <211> twenty three <212> DNA <213> Artificial Sequence <400> 36 cagtctgtgb tgacgcagcc gcc 23 <210> 37 <211> twenty one <212> DNA <213> Artificial Sequence <400> 37 cartctgccc tgactcagcc t 21 <210> 38 <211> twenty three <212> DNA <213> Artificial Sequence <400> 38 tcctatgwgc tgacwcagcc acc 23 <210> 39 <211> twenty three <212> DNA <213> Artificial Sequence <400> 39 tcttctgagc tgactcagga ccc 23 <210> 40 <211> twenty three <212> DNA <213> Artificial Sequence <400> 40 cacgttatac tgactcaacc gcc 23 <210> 41 <211> twenty three <212> DNA <213> Artificial Sequence <400> 41 cagcytgtgc tgactcaatc ryc 23 <210> 42 <211> twenty one <212> DNA <213> Artificial Sequence <400> 42 ctgcctgtgc tgactcagcc c 21 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <400> 43 cagsctgtgc tgactcagcc 20 <210> 44 <211> twenty three <212> DNA <213> Artificial Sequence <400> 44 aattttatgc tgactcagcc cca 23 <210> 45 <211> twenty three <212> DNA <213> Artificial Sequence <400> 45 cagrctgtgg tgacycagga gcc 23 <210> 46 <211> twenty three <212> DNA <213> Artificial Sequence <400> 46 cwgccwgkgc tgactcagcc mcc 23 <210> 47 <211> twenty three <212> DNA <213> Artificial Sequence <400> 47 tgaacattct gtaggggcca ctg 23 <210> 48 <211> twenty three <212> DNA <213> Artificial Sequence <400> 48 tgaacattcc gtaggggcaa ctg 23 <210> 49 <211> 47 <212> DNA <213> Artificial Sequence <400> 49 gcactaatta atgtggcctc aggacaggtb cagctggtgc agtctgg 47 <210> 50 <211> 47 <212> DNA <213> Artificial Sequence <400> 50 gcactaatta atgtggcctc aggacarrts cagctggtrc artctgg <210> 51 <211> 47 <212> DNA <213> Artificial Sequence <400> 51 gcactaatta atgtggcctc aggacagrtc accttgaagg agtctgg <210> 52 <211> 47 <212> DNA <213> Artificial Sequence <400> 52 gcactaatta atgtggcctc aggasargtg cagctggtgg agtctgg 47 <210> 53 <211> 47 <212> DNA <213> Artificial Sequence <400> 53 gcactaatta atgtggcctc aggagaggtg cagctgktgg agwcysg 47 <210> 54 <211> 47 <212> DNA <213> Artificial Sequence <400> 54 gcactaatta atgtggcctc aggacaggtg carctgcagg agtcggg 47 <210> 55 <211> 47 <212> DNA <213> Artificial Sequence <400> 55 gcactaatta atgtggcctc aggacagstg cagctrcags agtssgg <210> 56 <211> 47 <212> DNA <213> Artificial Sequence <400> 56 gcactaatta atgtggcctc aggagargtg cagctggtgc agtctgg 47 <210> 57 <211> 47 <212> DNA <213> Artificial Sequence <400> 57 gcactaatta atgtggcctc aggacaggta cagctgcagc aggacagg <210> 58 <211> 42 <212> DNA <213> Artificial Sequence <400> 58 ggaagatcta gaggaaccac caagggttgg ggcggatgca ct <210> 59 <211> 77 <212> DNA <213> Artificial Sequence <400> 59 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cggggcggtgg tggggacatc 60 biteccc agtctcc 77 <210> 60 <211> 77 <212> DNA <213> Artificial Sequence <400> 60 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tggggmcatc 60 crgwtgaccc agtctcc 77 <210> 61 <211> 77 <212> DNA <213> Artificial Sequence <400> 61 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tggggatrtt 60 gtgatgacyc agwctcc 77 <210> 62 <211> 77 <212> DNA <213> Artificial Sequence <400> 62 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tggggaaatw 60 gtgwtgacrc agtctcc 77 <210> 63 <211> 77 <212> DNA <213> Artificial Sequence <400> 63 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tggggacatc 60 gtgatgaccc agtctcc 77 <210> 64 <211> 77 <212> DNA <213> Artificial Sequence <400> 64 ggtggttcctctgatcttcctcctctggt ggcggtggct cgggcggtgg tggggaaacg 60 acactcacgc agtctcc 77 <210> 65 <211> 77 <212> DNA <213> Artificial Sequence <400> 65 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tggggawrtt 60 gtgmtgacwc agtctcc 77 <210> 66 <211> 48 <212> DNA <213> Artificial Sequence <400> 66 cttgtcgtca tcgtctttgt agtcgaagac agatggtgca gccacagt 48 <210> 67 <211> 77 <212> DNA <213> Artificial Sequence <400> 67 ggtggttcctctgatcttcctcctctggt ggcggtggct cgggcggtgg tgggcagtct 60 gtgctgactc agccacc 77 <210> 68 <211> 77 <212> DNA <213> Artificial Sequence <400> 68 60. ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tgggcagtct gtgbtgacgc agccgcc 77 <210> 69 <211> 75 <212> DNA <213> Artificial Sequence <400> 69 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cggggcggtgg tgggcartct 60 gccctgactc agcct 75 <210> 70 <211> 77 <212> DNA <213> Artificial Sequence <400> 70 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cggggcggtgg tgggtcctat 60. gwgctgacwc agccacc 77 <210> 71 <211> 77 <212> DNA <213> Artificial Sequence <400> 71 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cggggcggtgg tgggtcttct gagctgactc aggaccc 77 <210> 72 <211> 77 <212> DNA <213> Artificial Sequence <400> 72 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tgggcacgtt 60 atactgactc aaccgcc 77 <210> 73 <211> 77 <212> DNA <213> Artificial Sequence <400> 73 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tgggcagcyt 60 gtgctgactc aatcryc 77 <210> 74 <211> 75 <212> DNA <213> Artificial Sequence <400> 74 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tgggctgcct 60 gtgctgactc agccc 75 <210> 75 <211> 74 <212> DNA <213> Artificial Sequence <400> 75 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tgggcagsct 60 gtgctgactc agcc 74 <210> 76 <211> 77 <212> DNA <213> Artificial Sequence <400> 76 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cggggcggtgg tgggaatttt atgctgactc agcccca 77 <210> 77 <211> 77 <212> DNA <213> Artificial Sequence <400> 77 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cggggcggtgg tgggcagrct 60 gtggtgacyc aggagcc 77 <210> 78 <211> 77 <212> DNA <213> Artificial Sequence <400> 78 ggtggttcct ctagatcttc ctcctctggt ggcggtggct cgggcggtgg tgggcwgccw 60 gkgctgactc agccmcc 77 <210> 79 <211> 48 <212> DNA <213> Artificial Sequence <400> 79 cttgtcgtca tcgtctttgt sggygggaac 48 <210> 80 <211> 36 <212> DNA <213> Artificial Sequence <400> 80 atctcagcgg ccgcactaat taatgtggcc tcagga <210> 81 <211> 33 <212> DNA <213> Artificial Sequence <400> 81 cgcagtaagc ttgtcgtcat cgtctttgta gtc 33 <210> 82 <211> 51 <212> DNA <213> Artificial Sequence <400> 82 atgtttttgg cagtctttac acttctggca ctaattaatg tggcctcagg a 51 <210> 83 <211> 88 <212> DNA <213> Artificial Sequence <400> 83 gataagattt aaatataaaa gatatgcaac tagaaaagtc ttatcaatct ccggatcctc 60 attacttgtc gtcatcgtct ttgtagtc 88 <210> 84 <211> 308 <212> PRT <213> Saccharomyces cerevisiae <400> 84 Met Thr Met Pro His Arg Arg Met Phe Leu Ala Val Phe Thr Leu Leu 1 5 10 15 Ala Leu Ile Asn Val Ala Ser Gly Gln Val Gln Leu Val Gln Ser Gly 20 25 30 Ala Glu Val Lys Lys Pro Gly Glu Pro Leu Lys Ile Ser Cys Lys Gly 35 40 45 Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met 50 55 60 Pro Gly Lys Gly Leu Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser 65 70 75 80 Asp Thr Arg Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala 85 90 95 Asp Lys Ser Ile Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Ala 100 105 110 Ser Asp Thr Ala Met Tyr Tyr Cys Ala Arg Leu Gly Tyr Tyr Asp Ser 115 120 125 Ser Gly Tyr Tyr Ser Glu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 130 135 140 Val Ser Ser Gly Ser Ala Ser Ala Pro Thr Leu Gly Gly Ser Ser Arg 145 150 155 160 Ser Ser Ser Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Gln Pro Val 165 170 175 Leu Thr Gln Ser Ser Ser Ala Ser Ala Ser Leu Gly Ala Ser Val Lys 180 185 190 Leu Thr Cys Thr Leu Ser Ser Gly Arg Ser Gly Phe Ala Ile Ala Trp 195 200 205 His Gln Gln His Pro Glu Lys Gly Pro Arg Tyr Leu Met Asn Val Asn 210 215 220 Ser Asp Gly Ser His Thr Lys Gly Asp Gly Ile Pro Asp Arg Phe Ser 225 230 235 240 Gly Ser Ser Ser Glu Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu Gln 245 250 255 Phe Asp Asp Glu Val Asp Tyr Tyr Cys Gln Thr Trp Gly Thr Gly Ile 260 265 270 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Gln Pro Lys 275 280 285 Ala Ala Pro Ser Val Thr Leu Phe Pro Ala Val Leu Asp Tyr Lys Asp 290 295 300 Asp Asp Asp Lys 305 <210> 85 <211> 24 <212> PRT <213> Saccharomyces cerevisiae <400> 85 Met Thr Met Pro His Arg Arg Met Phe Leu Ala Val Phe Thr Leu Leu 1 5 10 15 Ala Leu Ile Asn Val Ala Ser Gly 20 <210> 86 <211> 8 <212> PRT <213> Saccharomyces cerevisiae <400> 86 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 BRIEF DESCRIPTION OF THE DRAWINGS

[0040] A better understanding of the present invention will be facilitated by reference to the following detailed description taken in conjunction with the accompanying drawings, while considering the present invention by way of non-limiting examples and drawings.

[0041] Figure 1 A yeast phenotypic screening system for isolating activating antibodies for G protein-coupled receptors (GPCRs) is shown. The screen shown uses a scFv library, but is applicable to other formats of libraries, including other antibody libraries, such as Fab libraries, and peptide libraries in general. The antibody library is introduced into yeast cells by gap repair recombination between a gapped vector and a PCR-generated antibody library, or as a pre-made library in a single molecule plasmid vector. Once inside the cell, the antibodies are expressed and secreted into the periplasmic space between the cell membrane and the cell wall. Due to their molecular size, most antibodies remain in the periplasmic space. The screen will identify antibodies that recognize GPCRs on the cell membrane and activate their signaling activity, resulting in expression of the CDC26, neoR, or zeoR genes, allowing the cells to grow at 37°C or in medium containing antibiotics, or both.

[0042] Figure 2 Plasmid maps are shown. The scFv library was inserted into the NotI-SacII sites of pKT047 or pKT103, or the NotI-AscI sites of pKT131, replacing the CH1 sequence and TEMP sequence, respectively. The scFv inserted into pKT047 has a GPI anchor and a V5 epitope tag located at the C-terminus of the scFv sequence, while the scFv inserted into pKT103 has no membrane anchor but contains a FLAG tag at the C-terminus provided by the PCR primers. Similarly, the scFv inserted into pKT131 has no anchor and contains a 6xHIS tag at the C-terminus. The maps are not drawn to scale.

[0043] Figure 3The high temperature survival of YKT099 cells dependent on GPCR is shown. YKT099 (pFUS2::CDC26pFIG1::neoR) and YKT112 (pFUS2::CDC26pFIG1::neoRste2△) were cultured in YPD medium at 28°C until the late logarithmic phase, diluted with medium, and 250 μl of about 10 3 The cells were plated on YPD plates with or without G418 or α-factor. The concentrations of G418 and α-factor were 20 μg / ml (G20) and 80 ng / ml, respectively. The plates were then placed at 37°C. A control plate of each strain was incubated at 28°C to determine the total number of cells inoculated on each plate. The plates were scanned after 3 days of incubation.

[0044] Figure 4 GPCR-dependent G418 resistance of YKT099 cells is shown. Cells from different cell lines grown in YPD at 28°C were streaked at late log phase on YPD plates containing the indicated concentrations of G418 (G50 = 50 μg / ml, etc.). In addition, α-factor was added at 80 ng / ml to one set of plates (bottom row). All plates were then incubated at 30°C. Drug-free control plates were incubated at 28°C. Plates were scanned after 3 days of incubation.

[0045] Figure 5 GPCR-dependent bleomycin resistance of YKT113 cells is shown.

[0046] Streak cells of different cell lines grown in YPD at 28°C in late log phase on YPD plates containing indicated concentrations of zeocin (Z10 = 10 μg / ml, etc.). At the same time, α-factor at a concentration of 80 ng / ml was added to one set of plates (last row). All plates were then placed at 28°C. The plates were scanned after 3 days of incubation.

[0047] Figure 6 Isolation of human antibody fragments that activate yeast GPCR signaling pathways is shown. YKT099 cells transformed with control vector pKT103 and five scFv-containing plasmids (Q5, W4, W5, W8, and W19) isolated as positive in screening were cultured to late logarithmic phase in SC-Leu medium at 28°C. Undiluted cultured cells were streaked onto SC-Leu plates containing the indicated concentrations of G418 (G20 = 20 μg / ml, etc.) and the plates were incubated at the indicated temperatures. Plates were scanned after 3 days of culture.

[0048] Figure 7The amino acid sequence of Q5 is shown. The amino acid sequence of Q5 is indicated in single-letter code. FLO1SP: signal peptide of Flo1p; HC: heavy chain; LC: light chain; CDR: complementarity determining region; FLAG: FLAG epitope tag. The CDR region is indicated in red. Germline alleles are identified according to the International Immunogenetic Information System (IMGT) (www.imgt.org).

[0049] Figure 8 It was shown that Q5 supported the growth of YKT099 under high temperature and in the presence of G418 by activating the yeast GPCR signaling pathway. YKT099 cells containing the Q5 expression plasmid pKT112 and the control vector pKT103 were cultured in SC-Leu medium at 28°C to late logarithmic phase. Undiluted cultured cells were streaked onto SC-Leu plates containing the indicated concentrations of G418 (G100 = 100 μg / ml, etc.) and grown at 30°C. Control plates without drug (A) were cultured at 28°C. At the same time, the cultured cells were diluted approximately 10 3 250 μl of each diluted culture was plated on SC-Leu plates with or without G418 and the plates were incubated at the indicated temperatures (B). YKT112 containing pKT112 was similarly cultured and plated for comparison (B). All plates were scanned after 3 days of incubation.

[0050] Fig. 9 Activation of pFIG1::GFP expression by Q5 in YKT099 cells is shown. YKT099 cells containing a Q5 expression plasmid (pKT112) and a control vector (pKT103) were cultured in SC-Leu medium at 28°C for 48 hours. The cells were then diluted 10-fold with medium and grown for another 4 hours at 28°C before being harvested for microscopic examination.

[0051] Fig.10 Activation of pFIG1::LacZ expression in YKT109 cells by Q5 is shown. YKT109 cells that have integrated the FIG1 promoter-driven β-galactosidase gene (pFIG1::lacZ) into the genome were transformed with the control vector pKT103, the Q5 expression plasmid pKT112, and a plasmid containing a randomly selected scFv sequence from the library transformant G3. The transformed cells were spotted onto SC-Leu plates and allowed to grow for 2 days. The plaques grown on the plates were then moved onto filter paper, which was used to perform the β-galactosidase filter lift assay. Color development was allowed to occur for 2 hours.

[0052] Fig.11Activation of α-factor-induced gene expression in YKT099 cells by Q5 is shown. RT-qPCR was used to measure the expression of native FUS1 and pFIG1::neoR genes, both of which are α-factor-inducible, in YKT099 cells containing the Q5 expression plasmid pKT112 or the control vector pKT103. Primers used to detect transcripts of these genes and transcripts of the ACT1 gene as an internal control are given in Materials and Methods.

[0053] Fig.12 The cell surface localization of Q5-GFP is shown. BJ5465 cells containing pKT112 (Q5) or pKT119 (Q5-GFP) were cultured to late logarithmic phase in SC-Leu medium at 28°C. The cells were diluted 5-fold and then grown for another 5-6 hours at 28°C before being harvested for microscopic examination.

[0054] Fig.13 The ability of Q5 with or without a GPI anchor to activate signaling pathways was compared. YKT099 cells containing Q5 expressed as free form (pKT112) or with a GPI anchor attached to the C-terminus (pKT113) were cultured to late logarithmic phase in SC-Leu medium at 28°C. The cells were diluted with medium and 250 μl of each dilution was plated on SC-Leu plates containing the indicated concentrations of G418 (G300 = 300 μg / ml, etc.) and the plates were incubated at different temperatures as indicated in the figure. The plates were scanned after 3 days of culture.

[0055] DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0056] In a first aspect, the present invention relates to a method for isolating and identifying a functional antibody or antigen-binding protein or a fragment thereof that can bind to a transmembrane target protein and activate its activity, comprising the following steps:

[0057] a) providing a yeast cell transformed with a yeast expression vector encoding a library of antibodies or antigen binding proteins or fragments thereof, wherein the yeast cell expresses the transmembrane target protein;

[0058] b) expressing the library of antibodies or antigen binding proteins or fragments thereof in yeast cells, wherein the expressed antibodies or antigen binding proteins or fragments thereof are secreted into the periplasmic space of the yeast cells;

[0059] c) culturing the yeast cells in a selective medium or at a restrictive temperature, or a combination of a selective medium and a restrictive temperature; and

[0060] d) detecting a predetermined phenotype in the yeast cell, wherein the appearance or detection of the predetermined phenotype indicates that a functional antibody or antigen binding protein or a fragment thereof binds to the transmembrane target protein and leads to activation of the activity of the transmembrane target protein.

[0061] In one embodiment, the transmembrane target protein is a G protein coupled receptor or GPCR.

[0062] In another embodiment, the functional antibody or antigen binding protein or fragment thereof is an activator of a GPCR.

[0063] In one embodiment, the yeast cells in step a) may comprise one or more reporter genes, wherein the expression of one or more reporter genes may be activated by a GPCR-mediated signal transduction pathway. In a preferred embodiment, the GPCR-mediated signal transduction pathway is based on a yeast mating factor, i.e., an α-factor signal transduction pathway. It is generally understood that one or more reporter genes may be induced by a yeast mating factor signal transduction pathway. In some embodiments, the reporter gene may be an antibiotic resistance gene, a temperature-sensitive complementary gene, a fluorescent protein gene, or a combination of these genes. Professionals in the art are familiar with suitable antibiotic resistance genes, temperature-sensitive complementary genes, and fluorescent genes that can be used in the present invention. In a preferred embodiment, the antibiotic resistance gene is a G418 resistance gene (neoR) or a zeocin resistance gene (zeoR). In another preferred embodiment, the temperature-sensitive complementary gene is CDC26. And in another preferred embodiment, the fluorescent protein gene is GFP, RFP, YFP, BFP, or CFP. In another preferred embodiment, the reporter gene is LacZ.

[0064] In one embodiment, the predetermined phenotype is: cell growth in the presence of one or more antibiotics due to expression of an antibiotic resistance reporter gene; cell growth at a restrictive temperature due to expression of a temperature-sensitive complementary reporter gene; fluorescence due to expression of a fluorescent protein reporter gene; or a combination of the above phenotypes. In a preferred embodiment, the antibiotic resistance reporter gene is the G418 resistance gene neoR and the bleomycin resistance gene zeoR. In another preferred embodiment, the temperature-sensitive complementary reporter gene is CDC26, and the restrictive temperature may be between about 30°C and about 40°C. In another preferred embodiment, the fluorescent protein gene is GFP, RFP, YFP, BFP or CFP.

[0065] In some embodiments, the transmembrane target protein in step a) is a G protein coupled receptor or GPCR. In one embodiment, the transmembrane target protein is the Ste2 protein of yeast Saccharomyces cerevisiae. In another embodiment, the transmembrane target protein is the human GPCR receptor GLP-1R.

[0066] In one embodiment, the antigen binding protein is an antibody-related molecule capable of interacting with a target antigen and having one or more complementary determining regions (CDRs). In another embodiment, the antigen binding protein is a polypeptide or oligopeptide capable of interacting with a target antigen and comprising an amino acid sequence that is not derived from an antibody. In a preferred embodiment, the antigen binding protein is a single-chain variable region fragment (scFv) or an antigen binding fragment (Fab).

[0067] In one embodiment, the functional antibody or antigen binding protein or fragment thereof is a G protein coupled receptor activator.

[0068] In one embodiment, the transmembrane target protein (e.g., GPCR) may be expressed on the cell membrane of yeast cells. It is generally understood that the binding of an activator to a transmembrane target protein may activate the cellular activity of the transmembrane target protein and cause an intracellular reaction downstream of the target protein, such as in the case of a GPCR, such a downstream reaction is the activation of a signal transduction system and the expression of one or more reporter genes. Therefore, in one embodiment, the expression of one or more reporter genes is induced by the binding of an activator to a transmembrane target protein.

[0069] The method of the present invention also relates to a library of antibodies or antigen binding proteins or fragments thereof expressed in yeast cells, particularly in the periplasmic space of yeast cells. The library of antibodies or antigen binding proteins or fragments thereof may comprise single chain variable region fragments (scFv), disulfide bond-linked variable fragments (scFv), Fab fragments and F(ab') fragments, V H dAb or cFvs. In one embodiment, the library of antibodies or antigen binding proteins or fragments thereof may comprise single chain variable fragments (scFv), single domains (VHH), Fab fragments, polypeptides or oligopeptides. In another embodiment, the peptide or oligopeptide library is a random or partially random peptide library not derived from an antibody sequence.

[0070] In another embodiment, the nucleic acid sequence encoding the antibody or antigen binding protein or fragment thereof further comprises a secretion signal sequence, which helps localize the antibody or antigen binding protein or fragment thereof to the periplasmic space of yeast cells.

[0071] The secretion signal sequence may be a secretion signal sequence of yeast mating factor (α-factor) or a secretion signal sequence of Flo1p, Suc2p or Aga2p. In a preferred embodiment, the secretion signal sequence may be encoded by the yeast mating factor gene MFα1 or FLO1 gene.

[0072] In some embodiments, the antibody or antigen binding protein or its fragment can be coupled to an anchoring element so as to remain in the periplasmic space of the yeast cell. In another embodiment, the antibody or antigen binding protein or its fragment can be fused to a transmembrane domain (TMD) so as to remain in the periplasmic space of the yeast cell. In a preferred embodiment, the anchoring protein can be a glycosylphosphatidylinositol (GPI) attachment protein or a transmembrane domain (TMD). In one embodiment, the antibody or antigen binding protein or its fragment can be further connected to a tag. Examples of such tags include, but are not limited to, polyhistidine, glutathione S-transferase (GSTs), calmodulin binding peptides, intein-chitin binding domains, streptavidin / biotin-based tags, tandem affinity purification tags. In a preferred embodiment, the tag is an epitope tag. In another preferred embodiment, the epitope tag is a FLAG tag or a 6xHIS tag.

[0073] The library can be assembled using methods known in the art. The library of antibodies or antigen binding proteins or fragments thereof can be inserted into plasmid vectors such as pKT047, pKT103 or pKT131. In one embodiment, the nucleic acid sequence encoding the antibody or antigen binding protein or fragment thereof is connected to a yeast promoter. In a preferred embodiment, the promoter can be a constitutive promoter (e.g., TEF1 promoter) or an inducible promoter (e.g., GAL1 promoter).

[0074] It is generally understood that libraries can be constructed from a variety of cell sources. For example, libraries can be constructed from animal bone marrow cells, spleen and lymph node cells, or circulating leukocytes. In a preferred embodiment, libraries can be constructed from human peripheral blood mononuclear cells.

[0075] The diversity or complexity of the library ranges from 10 5 Up to 10 12 , for example 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In a preferred embodiment, the library diversity of single chain variable region fragments (scFv), single domain (VHH) or Fab fragments is greater than 10 7 In another preferred embodiment, the diversity of the polypeptide or oligopeptide is greater than 10 5 These numbers refer to the diversity of the original library without any form of target-directed enrichment.

[0076] In some embodiments, the oligopeptide library comprises peptides greater than 5 amino acids in length.

[0077] Yeast cells of the present invention can be transformed using methods generally known in the art. In one embodiment, yeast cells can be transformed by gap repair transformation. In other embodiments, yeast are transformed using electroporation (EP) or heat shock methods using pre-made libraries in plasmid form.

[0078] The yeast cell of the present invention can be a budding yeast cell or a filamentous yeast cell. Examples of yeast cells include, but are not limited to, Saccharomyces cerevisiae, fission yeast, Candida albicans, Pichia pastoris, and Aspergillus species. In one embodiment, the yeast can be Saccharomyces cerevisiae, fission yeast, or Pichia pastoris. In a preferred embodiment, the yeast is Saccharomyces cerevisiae or fission yeast.

[0079] Yeast cells of the present invention may be cultured using culture media and conditions commonly known in the art. The culture media and culture conditions may be modified to select one or more predetermined phenotypes. The selective culture media may include one or more antibiotics. In one embodiment, the selective culture media may include bleomycin antibiotic and / or G418 antibiotic. The temperature of the selective medium can also range from about 25-40° C., for example, approximately at least about 25.5° C., about 26° C., about 26.5° C., about 27° C., about 27.5° C., about 28° C., about 28.5° C., about 29° C., about 29.5° C., about 30° C., about 30.5° C., about 31° C., about 31.5° C., about 32° C., about 32.5° C., about 33° C., about 33.5° C., about 34° C., about 34.5° C., about 35° C., about 35.5° C., about 36° C., about 36.5° C., about 37° C., about 37.5° C., about 38° C., about 38.5° C., about 39° C., about 39.5° C., and about 40° C. In a preferred embodiment, the temperature of the selective medium is between 30° C. and 40° C.

[0080] In one embodiment, the functional antibody or antigen-binding protein or fragment thereof is a monoclonal antibody, a recombinant antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody and a conjugated antibody, a single variable domain, a domain antibody, an antigen-binding fragment, an immune effector fragment, a single-chain variable fragment, a single-chain antibody, a monovalent antibody lacking a hinge region, a minibody, a diabody, a polypeptide or oligopeptide comprising an amino acid sequence not derived from an antibody.

[0081] The present invention also provides an antibody or antigen binding protein or a fragment thereof as an activator of the GPCR separated and identified according to the method of the present invention; wherein the GPCR is human GLP-1R or yeast α-factor receptor Ste2p.

[0082] The present invention also provides an antibody or antigen-binding protein identified by the method disclosed herein, comprising: (i) a heavy chain variable domain, including a VHCDR1 having an amino acid sequence of GYSFTSYW (SEQ ID NO: 1), a VHCDR2 having an amino acid sequence of IYPGDSDT (SEQ ID NO: 2), and a VHCDR3 having an amino acid sequence of CARLGYYDSSGYYSEDY (SEQ ID NO: 3); and (ii) a light chain variable domain, including a VLCDR1 having an amino acid sequence of SGRSGFA (SEQ ID NO: 4), a VLCDR2 having an amino acid sequence of VNSDGSH (SEQ ID NO: 5), and a VLCDR3 having an amino acid sequence of QTWGTGIWV (SEQ ID NO: 6).

[0083] In one embodiment, the antibody or antigen binding protein comprises a heavy chain variable region comprising the amino acid sequence QVQLVQSGAEVKKPGEPLKISCKGS GYSFTSYW IGWVRQMPGKGLEWMGI IYPGDS DTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYY CARLGYYDSSGYYSEDY WGQGTLVTVSSGSASAPTL (SEQ ID NO: 7); and a light chain variable region comprising the amino acid sequence QPVLTQSSSASASLGASVKLTCTLS SGRSGFA IAWHQQHPEKGPRYLMN VNSDGSH TKGDGIPDRFSGSSSEAERYLTISSLQFDDEVDYYC QTWGTGIWV FGGGTKLTVLRQPKAAPSVTLFPAVL(SEQ IDNO:8).

[0084] It is generally understood that there may be variations in CDR, variable heavy chain and variable light chain sequences. In some embodiments, the antibody or antigen binding protein or fragment thereof comprises heavy and light chain CDR regions that are about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the heavy and light chain CDR regions of (i) and (ii).

[0085] In other embodiments, the antibody or antigen binding protein or fragment thereof comprises a heavy chain variable region and a light chain variable region that are about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0086] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the two sequences (i.e., % homology = number of identical positions ÷ total number of positions x 100), wherein the number of gaps and the length of each gap need to be taken into account, and these two parameters must be introduced to achieve optimal alignment of the two sequences.

[0087] The present invention can be appropriately used in practice in the absence of any one or more factors or any one or more limitations not specifically specified herein. Therefore, the terms "including", "comprising", "containing" and the like should be understood to have a broad sense without any restrictive meaning. In addition, the terms and expressions used herein are only illustrative without any self-limitation, and the use of such terms and expressions does not exclude any equivalents of the features shown and described or parts thereof. It must be recognized that various modifications can be made within the scope of the present invention as described. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, professionals in the art can still modify and change the various inventions embodied in the present invention disclosed herein, and these modifications and changes should be regarded as still within the scope of the present invention.

[0088] The invention has been described broadly and generally herein. Each of the narrower species and subgeneric groupings falling within the generic scope are also included within the scope of the invention. This includes the general description of the invention, with accompanying limitations or negative limitations removing any subject matter from the generic category, whether or not the removed material is specifically enumerated herein.

[0089] Other embodiments are within the following claims and non-limiting examples. In addition, where various features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0090] Experimental Section

[0091] Non-limiting examples and comparative examples of the present invention will be further described by reference to specific examples, which should not be construed as limiting the scope of the present invention in any way.

[0092] Materials and methods

[0093] Yeast strains, vectors, and growth conditions

[0094] All yeast strains used in this study were derived from BJ5465, and their genotypes are detailed in Table 1. Gene knockout and gene integration used the URA3 circular knockout method. To construct YKT068, the SST2, FAR1, and CDC26 genes in BJ5465 were knocked out in sequence. A 650 bp fragment of the CDC26 gene starting from the start codon was amplified from wild-type genomic DNA by PCR and cloned into an integration vector, connected downstream of the FUS2 promoter and inserted into the genome. Then, the GFP coding sequence driven by the FIG1 promoter (-600 to -1) was inserted into the HIS3 sequence located in the pep4 gene in BJ5465. In addition, to prepare YKT069, the coding sequence of the STE2 gene in YKT068 was knocked out. The promoter region of STE2 was not affected by the gene knockout operation. The gene coding sequence of FIG1 in YKT068 was replaced with the LacZ coding sequence to prepare YKT109. YKT099 and YKT112 were made from YKT068 and YKT069, respectively, by inserting a FIG1 promoter-driven neomycin resistance gene (neoR) at the ADE2 gene. Similarly, YKT113 and YKT114 were made from YKT068 and YKT069, respectively, by inserting a FIG1 promoter-driven bleomycin resistance gene (Sh ble or zeoR) at the ADE2 gene.

[0095] Table 1 summarizes the yeast strains generated in this study.

[0096]

[0097] To prepare the GLP-1R expression strain, the last 15 nucleotides of the coding sequence before the stop codon of the GPA1 gene in the chromosome of YKT069 were directly replaced from AAAATTGGTATTATA (SEQ ID NO: 9) to CAATACGAACTCTTA (SEQ ID NO: 10) using a PCR product. The resulting strain was used as a host cell to receive the integration of the GLP-1R DNA clone. The natural signal peptide sequence at the N-terminus of the GLP-1R gene was replaced by the yeast alpha factor signal peptide (PPASP). It was followed by the TEF1 terminator sequence. The gene integration of GLP-1R was site-directed to the ste2 gene, whose start codon was immediately downstream of the STE2 promoter. The FIG1 promoter-driven neoR was then integrated into the ADE2 gene of the resulting strain to generate YKT106.

[0098] All vectors used in this study were derived from the universal shuttle vector pRS315, and these vectors with the pKT prefix are listed in Table 2. To prepare pKT047, an oligonucleotide sequence encoding the Flo1p signal peptide (Flo1SP) with a PmeI site added immediately upstream of the ATG codon was cloned into the SpeI-NotI site of pRS315 with a NotI site downstream of the SP sequence to generate pRS315-Flo1SP. A 490 bp DNA fragment of the TEF1 promoter (pTEF1) was obtained by PCR and cloned into the SmaI-SpeI site of pRS315-Flo1SP immediately upstream of Flo1SP to generate pTEF1-Flo1SP. pKT047 ( was prepared by cloning a chemically synthesized DNA fragment containing, in sequence, a 342 bp CH1 domain, a SacII restriction site, a V5 epitope tag, a 171 bp glycosylphosphatidylinositol (GPI) anchor from Flo1p containing a flexible linker, and a 146 bp HIS5 terminator sequence into the NotI-SacI sites of pTEF1-Flo1SP. Figure 2 To generate pKT103, the SacII-SacI fragment of pKT047 containing the V5 tag-GPI anchor-HIS5 terminator was replaced with the 310 bp TEF1 terminator ( Figure 2 pKT131 was prepared by replacing the Flo1 signal peptide coding sequence in pKT103 with a 267 bp fragment containing the precursor α-factor signal peptide.

[0099] Table 2 summarizes the plasmids generated in this study.

[0100]

[0101]

[0102] Prepare rich medium (YPD), synthetic complete (SC) medium and selection medium (dropout media) according to standard methods. Add antibiotics to the medium shortly after autoclaving. Spread the α-factor aqueous solution on the YPD plate to make the plate containing α-factor. The volume of each plate medium is calculated as 25 ml. The permissive temperature of the temperature-sensitive mutant strain is 28 to 30°C, and the restrictive temperature is 37°C. The former is used to propagate cells, and the latter is used for phenotypic analysis.

[0103] RT-qPCR

[0104] Total ribonucleic acid (RNA) was isolated from yeast cells using the QIAGEN RNeasy minikit kit according to the manufacturer's instructions. cDNA was synthesized using iScript reverse transcription master mix for RT-qPCR (Bio-Rad). SsoAdvanced TM Universal Green Supermix and CFX Connect TM RT-PCR reaction was performed using a real-time PCR detection system. Amplification conditions were 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Primers used to detect the expression of ACT1 (as an internal control), FUS1 and neoR were: ACT1-RT-5': 5'-ATTCTGAGGTTGCTGCTTTGG-3' (SEQ ID NO: 11), ACT1-RT-3': 5'-TGTCTTGGTCTACCGACGATAG-3' (SEQ ID NO: 12); FUS1-RT-5': 5'-TCGCCGCAATGTCTACTACC-3' (SEQ ID NO: 13), FUS1-RT-3': 5'-GCCCAATTGTGCTTGCTGAA-3' (SEQ ID NO: 14); Neo-RT-5': 5'-TGCCTGCTTGCCGAATATC-3' (SEQ ID NO: 15), Neo-RT-3': 5'-ATATCACGGGTAGCCAACGC-3' (SEQ ID NO: 16). The relative mRNA expression levels in the test cells and control cells were calculated using the double ΔCt method.

[0105] Construction of human antibody library

[0106] Human mononuclear cells (MNCs) were purchased from Stemcell Technologies (Singapore, Cat. No. 70025). Total RNA was extracted using TRIzol reagent (Invitrogen, Cat. No. 15596-026). 8Each cell was suspended in 1 ml TRIzol and incubated at room temperature for 5 minutes, occasionally inverting the tube. 0.2 ml chloroform was added, the tube was vortexed for 15 seconds, left at room temperature for 3 minutes, and then centrifuged at 12,000g for 15 minutes at 4°C. The aqueous phase was transferred to a new 1.5 ml tube and 0.5 ml isopropanol was added. After incubation at room temperature for 10 minutes, RNA was precipitated by centrifugation at 12,000g for 10 minutes at 4°C. The pellet was washed with 1 ml 75% ethanol and collected again by centrifugation at 7,500g for 5 minutes at 4°C. The pellet was air-dried at room temperature for 10 minutes and dissolved in 30 μl DEPC water. The tube was placed in a 58°C heater for 10 minutes to dissolve the RNA. mRNA was isolated from total RNA using the Oligotex mRNA Mini Kit (Qiagen, Cat. No. 70022) according to the manufacturer's instructions.

[0107] Other polyA-mRNA prepared from human peripheral leukocytes was purchased from Clontech (Cat. No. 636170). The mRNAs from both sources were evaluated for mRNA length of 9 housekeeping genes by PCR and confirmed to be of good quality. Therefore, the mRNAs from both sources were combined for library construction.

[0108] The PCR and construction of the scFv library are basically according to the published scheme. The primers used to prepare the scFv library are listed in Table 3. The first-chain cDNA was generated from the mRNA mixture of the above two sources using SuperScript III reverse transcriptase (Life Technologies, Cat. No. 18080) and random hexamer primers. The heavy chain, light chain κ and light chain λ (VH, Vκ and Vλ) variable region genes were amplified by PCR using GoTaq DNA polymerase (Promega, Cat. No. M3001). The product of the first round of PCR was then used as a template for the second round of PCR, in which the 5' primer of the light chain and the 3' primer of the heavy chain shared the overlapping sequence of the joint region. In addition, the FLAG epitope tag was attached to the 3' primer of the light chain, and a partial sequence of the signal peptide of FLO1 was included in the 5' primer of the heavy chain. Each variable region PCR reaction was performed in a volume of 50 μl, first, heating at 95°C for 2 minutes, then 30 cycles at 95°C for 30 seconds, 57°C for 1 minute, and 72°C for 1 minute, and finally, a final extension at 72°C for 5 minutes. In the third round of PCR, the heavy and light chains were combined and amplified using 5' and 3' outer primers. The combined PCR reaction contained an equimolar mixture of the mixed heavy chain (VH) DNA and the mixed light chain (Vκ or Vλ) gene set from the second round of PCR and cycled 8 times without primers (95°C for 45 seconds, 58°C for 30 seconds, and 72°C for 60 seconds). This was followed by a final round of PCR with 25 cycles (95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 60 seconds) using outer primers and a final extension at 72°C for 15 minutes. The final PCR reaction created a full-length scFv gene set. The scFv pool was subjected to agarose gel electrophoresis, and DNA bands of desired length were purified from the gel using an EZ-10 spin column DNA gel extraction kit (Bio Basic, Singapore, Cat. No. BS654).

[0109] Table 3 summarizes the primers used for scFv library construction3.

[0110]

[0111]

[0112]

[0113] Second round PCR primers (VLλ):

[0114]

[0115]

[0116] To clone the full-length scFv DNA into pRS315, the final scFv PCR product and pRS315 were digested with NotI and HindIII, respectively, at 37°C overnight. The digested scFv DNA and vector were then purified using EZ-10 spin columns as described above, mixed together in a ligation reaction at a vector / insert molar ratio of 1:3, and left overnight at room temperature in the presence of T4 DNA ligase (NEB, Cat. No. M0202). The ligated DNA was transduced into E. coli Top 10 cells by electroporation using a MicroPulser electroporator (Bio-Rad). The cells were then transferred to 2xYT medium and incubated at 37°C for 1 hour with shaking. 20 μl of the cultured cells were serially diluted and plated on 2xYT plates to obtain colony counts. The remaining cells were incubated for an additional 2 hours. The cells were then harvested and resuspended in 2xYT, aliquoted, and stored as glycerol stocks at -80°C. When necessary, aliquots of cells were taken and DNA was prepared using Gene JET Plasmid Maxi Kit (Thermo Fisher Scientific, Cat. No. K0491).

[0117] To generate a pre-made antibody library in the yeast vector pKT131, a final PCR reaction was performed with outer primers containing NotI and AscI sites, respectively, to generate the above-mentioned full-length scFv gene pool. The PCR product was digested with NotI and AscI overnight at 37°C and ligated with a pKT131 vector that was similarly digested with restriction enzymes at a vector / PCR product ratio of 1:3. The ligated DNA was then introduced into E. coli Top 10 cells by electroporation. 5×10 9 independent clones.

[0118] Antibody library screening

[0119] Transformation by gap repair:

[0120] The scFv library prepared in pRS315 was amplified by PCR using DreamTaq DNA polymerase (Thermo Fisher Scientific). The following mixture was prepared in a 50 μl reaction volume on ice: 5 μl of 10X DreamTaq buffer, 1 μl of dNTP mix (final 200 μM), 2.5 μl of gap repair forward primer (5'TGTTTTTGGCAGTCTTTACACTTCTGGCACTAATTAATGTGGCCTCAGGA3' (SEQ ID NO:82), final 0.5 μM), 2.5 μl of gap repair reverse primer (5'GATAAGATTTAAATATAAAAGATATGCAACTAGAAAAGTCTTATCAATCTCCGGATCCTCATTACTTGTCGTCATCGTCTTTGAGTC3' (SEQ ID NO:83), final 0.5 μM), 10 μl (100 ng) of pRS315-scFv library DNA, 0.25 μl (1.25 U) of DreamTaq DNA polymerase, and 28.75 μl of H2O. First, the mixture was heated at 95°C for 2 minutes; then, 5 cycles were performed at 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute; then, 25 cycles were performed at 95°C for 30 seconds, 65°C for 30 seconds, and 72°C for 65 seconds; and finally, a final extension was performed at 72°C for 6 minutes. As described above, the amplified scFv was purified by agarose gel electrophoresis.

[0121] To prepare host cells for transformation, overnight cells of yeast strain (e.g., YKT099) were grown in YPD at 28°C at an OD of 600 = 0.3 into 50 ml of fresh YPD and allowed to grow for another 5 to 6 hours at 28°C until OD 600 Reach 1.3-1.6. Collect cells by centrifugation at 3,000g for 4 minutes at 4°C and wash twice with 50ml of ice-cold water and once with ice-cold electroporation buffer (1M sorbitol, 1mM CaCl2). Then pellet cells by centrifugation and resuspend in 20ml of 0.1M LiAc, 10mM DTT and shake at 28°C for 30 minutes. Collect cells by centrifugation at 3,000g for 4 minutes and wash once with ice-cold electroporation buffer, resuspend in 0.5ml of ice-cold electroporation buffer and keep on ice until use.

[0122] 4 μg of NotI-SacII digested DNA pKT103 DNA and 12 μg of PCR amplified scFv DNA were added to 400 μl of yeast competent cells, and the mixture was transferred to a pre-cooled 0.2 cm electroporation dish. The electroporation dish was kept on ice for 5 minutes and then electroporated using a MicroPulser electroporator (Bio-Rad) at 2.5 kV and 25 μF. The cells were transferred to 8 ml of a 1:1 mixture of 1 M sorbitol and YPD medium and gently shaken at 28°C for 1 hour. The cells were centrifuged at 3,000 g for 5 minutes and then resuspended in 20 ml of 2xYPD medium. 10-fold serial dilutions were inoculated on leucine-deficient screening plates to determine the transformation efficiency. The remaining cells were pelleted by centrifugation and resuspended in 2 ml of leucine-deficient selection medium (SC-Leu), and 200 μl of the suspension was then plated on each SC-Leu plate, where the SC-Leu plates were supplemented with 200-400 μg / ml G418 (to be cultured at 30°C) or 20-40 μg / ml G418 (to be cultured at 37°C).

[0123] Transformation of pre-made libraries:

[0124] Aliquots of overnight cultured cells were collected at OD 600 =0.4 inoculated in 50 ml of YPD. Grow cells for 4.5 to 5 hours. Collect cells by centrifugation at 3000 g for 3 minutes and wash twice with ice-cold water and once with ice-cold 1 M sorbitol. Resuspend the cell pellet in 20 ml of 0.1 M LiAc / 10 mM dithiothreitol (DTT) and incubate at 30° C. for 30 minutes. Centrifuge the cells and wash once with ice-cold 1 M sorbitol and then resuspend in 1 M sorbitol in a final volume of 400 μl.

[0125] Add 10 μg of library DNA to every 400 μl cells. Mix gently and transfer the mixture to a precooled BioRad Gene Pulser electroporation dish (0.2 cm). Place the electroporation dish on ice for 5 minutes until electroporation is performed. Electroporation of cells is performed at 1.5 kV (usually for 3.5 to 4.5 milliseconds). Immediately transfer cells from the electroporation dish to 20 ml YPD containing 0.5 M sorbitol. Cultivate cells at 30 ° C for 1 hour and then plate on selective medium.

[0126] Example 1

[0127] A brief overview of the platform

[0128] Figure 1The platform shown is a typical phenotypic screening based on autocrine signaling. The antibody library is introduced into the cell through efficient transformation and secreted into the periplasmic space between the cell wall and the cell membrane. Most of the antibodies are retained in the periplasmic space due to their size. When the activator antibody encounters its epitope on the receptor (Ste2p or human GPCR receptor), the antibody binds to the receptor and activates the receptor, triggering a series of kinase reactions. The Stel2p transcription factor is activated at the end of the series of kinase reactions. The activated Stel2p binds to the promoter region of a group of α-factor induced genes and activates their transcription. The promoters of two such genes FUS2 and FIG1 were selected to drive the expression of the reporter gene CDC26 and the antibiotic resistance gene (neomycin resistance gene neoR and zeoR), so that the cells can grow in the presence of antibiotics or at 37°C or in the presence of both conditions.

[0129] Example 2

[0130] Construction of libraries, vectors and host strains

[0131] The scFv library was first 10 The diversity of the library is comparable to or better than most phage display antibody libraries, prepared in the universal shuttle vector pRS315. Sequence analysis of randomly selected library clones showed that more than 80% of the library members contained the antibody sequence correctly incorporated into the vector. For screening, the library was introduced into the screening cells by scFv library PCR and gap repair of the vector pKT103. This process resulted in the scFv fragments being linked to the FLO1 secretion signal and placed under the control of the TEF1 promoter.

[0132] To construct strains suitable for this platform, certain modifications to the Ste2p signaling pathway were necessary. First, as in other experiments using the yeast α-factor signaling pathway for heterologous GPCR studies, two genes, far1Δ and sst2Δ, needed to be knocked out in order to maintain cell division and enhance the sensitivity of signal detection when activated. One of the many consequences of α-factor signaling is cell division cycle arrest, for which the multifunctional and non-essential protein Far1p is responsible. Knockout of SST2 increases receptor-G protein coupling, thereby enhancing the signaling of this pathway. In order to facilitate sensitive and convenient signal detection, the antibiotic resistance genes neoR or zeoR and the temperature-sensitive cdc26 gene were used as reporter genes in this study, rather than the previously commonly used histidine-deficient method. The CDC26 gene encodes a subunit of the anaphase-promoting complex (APC). Loss of CDC26 function is lethal under high temperature (37°C) conditions. Cells with chromosomal cdc26 knockout and wild-type CDC26 under the control of the FUS2 promoter (YKT068 and YKT099) were sensitive to 37°C temperature in the absence of α-factor, but grew normally in the presence of α-factor ( Figure 3 Similarly, cells carrying the neoR or zeoR genes under the control of the FIG1 promoter (YKT099 or YKT113) could grow in medium containing the antibiotics G418 or bleomycin, respectively, only in the presence of α-factor ( Figure 4 and 5 ). In all these cases, α-factor-supported cell growth was also abolished if the receptor gene STE2 was knocked out. These data suggest that these strains are adequate for the envisioned screening protocol ( Figure 1 ). By employing multiple and convenient selection criteria, this study demonstrates the potential of this screening platform for efficient discovery of GPCR-activating antibodies.

[0133] It should be noted that any drug resistance gene and any mutation that can achieve temperature sensitivity can be used instead of the neoR, zeoR and cdc26 reporter genes mentioned above as long as the background level is low or controllable.

[0134] Example 3

[0135] Activating antibodies against Ste2p

[0136] This platform was first used to search for antibodies that can act on and activate the yeast GPCR receptor Ste2p. YKT099 was transformed with the scFv library, and the transformed cells were plated on SC-Leu plates (for selection of plasmids) that also contained 20-40μg / ml or 300-400μg / ml of G418 and cultured at 37°C or 30°C, respectively. The "high temperature / low drug" selection conditions favored the expression of CDC26, while the "low temperature / high drug" conditions favored the expression of neoR. Colonies grown on the plates under each selection scheme were collected and pooled together, with each pool containing about 10 individual colonies. Plasmids were extracted from the pooled cells and used to transform YKT099 again. This time, transformants were screened using a reverse selection scheme. That is, if the first round of screening used high temperature / low drug selection, the second round of screening would use low temperature / high drug selection, and vice versa. This enrichment process was repeated again. Typically, after three rounds of enrichment, almost all transformants are positive for growth on the selection plates. Colonies are collected for the final time, and their plasmid DNA is extracted and analyzed.

[0137] From a total of 5 × 10 8 Approximately a dozen plasmids were identified from each transformant, which reproducibly produced colonies on the selection plates. Figure 6 Five of the isolates are shown, and when tested with different selection conditions, these isolates showed different degrees of activity. For example, W5 and W8 supported cell growth at 37°C with about the same potency as the other three isolates, but showed a significant difference at high concentrations of G418 and 30°C ( Figure 6 One of the isolates that showed stronger activity, Q5, was selected for further analysis.

[0138] The Q5 gene encodes a 308-amino acid protein, including the Flo1 signal peptide at the N-terminus and the FLAG epitope tag at the C-terminus. The amino acid sequence of Q5 is shown in Figure 7 As shown. The Q5-expressing plasmid pKT112 showed strong activity in supporting YKT099 cell survival under selection conditions. YKT099 cells containing pKT112 were able to grow healthily in all G418 concentrations tested (up to 600 μg / ml). Figure 8 A). Similarly, pKT112 can also enable YKT099 cells to grow normally at 37°C ( Figure 8B). Since the CDC26 and neoR genes in YKT099 are expressed from two different α-factor inducible promoters (pFUS2 and pFIG1), the reason for cell survival is most likely due to the activation of transcription initiated by Ste12p, and it is unlikely that some other cell physiological changes caused by Q5 that are unrelated to the α-factor signaling pathway, such as increased cell heat resistance or reduced antibiotic membrane permeability, have enhanced resistance to the lethal effects of the selection conditions. Another piece of evidence supporting this idea is that the strain lacking the α-factor receptor Ste2p (YKT112) cannot survive under the same conditions even if it contains Q5 ( Figure 8 B). This suggests that the function of Q5 is mediated through the Ste2p signaling pathway.

[0139] To further demonstrate the function of Q5 in promoting α-factor-inducible gene expression, the expression of two other reporter genes, GFP and LacZ, were studied in YKT099 and YKT109, respectively. Both of them were driven by the FIG1 promoter, and it was found that both genes were significantly activated by the Q5-containing plasmid pKT112 ( Fig. 9 and 10 When transcripts of neoR and FUS1 genes were directly analyzed by real-time quantitative PCR, they were found to be increased 10-fold and 2-fold, respectively, in YKT099 by Q5 ( Fig.11 ).

[0140] To examine the cellular localization of Q5, the GFP coding sequence was spliced ​​into the C-terminus of Q5 in pKT112. The resulting plasmid pKT119 was introduced into BJ5465 cells, and it was found that approximately 30-50% of the cells clearly showed cell surface localization of GFP ( Fig.12 ), while the control cells (pKT112) containing Q5 but no GFP showed no GFP signal ( Fig.12 This cellular localization pattern of Q5 is consistent with our expectations and is consistent with its role as a Ste2p receptor activator.

[0141] In summary, the human antibody fragment Q5 isolated by the autocrine screening platform was demonstrated to be an activator of the yeast GPCR receptor Ste2p, because it was able to activate the expression of α-factor-induced genes in a Ste2p-dependent manner. These data indicate that the yeast autocrine screening method is effective and convenient for identifying functional antibodies for GPCR receptors.

[0142] Example 4

[0143] Attenuation of Q5 activity via GPI anchor

[0144] Previously, Ishii et al. demonstrated an autocrine signaling system in yeast using the human GPCR SSTR5 and its ligand somatostatin 14. They anchored the ligand to the cell surface using a 41-amino acid glycosylphosphatidylinositol (GPI) attachment domain from the yeast cell wall protein Flo1p. We constructed the vector pKT047 using the same GPI anchor element (Flo42 containing a flexible linker) and tested its effect in antibody library screening. Screening with pKT047 yielded fewer positive clones with weaker activity than those generated by screening with pKT103. In order to directly compare the activities of anchored and unanchored activator antibodies, Q5 was transferred from pKT112 to pKT047 and fused to the C-terminus of Q5 with the Flo42 anchor (pKT113). Fig.13 As shown in Figure 2, under selection conditions, the activity of Q5 in supporting the growth of YKT099 cells was significantly weakened due to the presence of GPI ( Fig.13 ). This result shows that the movement of Q5 in the periplasmic space may play an important role in its interaction with Ste2p and its activation. Since the apparent molecular weight of the Q5 protein is 30kD, it may not require the help of an anchor to retain a considerable amount of the protein product in the periplasmic space. However, when similar screening is performed using smaller protein constructs (e.g., single domain antibody fragment libraries or oligopeptide libraries), it may be necessary to use membrane or cell wall anchoring mechanisms (e.g., transmembrane segments or GPI anchor elements) to limit these molecules to the periplasmic space, because small molecular proteins may freely permeate the yeast cell wall.

[0145] In summary, we have constructed a yeast phenotypic screening system using the principle of autocrine signaling to isolate and identify human antibody molecules that can activate GPCR receptors. The effectiveness of this platform was demonstrated by isolating antibody fragments that can activate the Ste2p-mediated α-factor signaling pathway in yeast. Compared with other antibody discovery platforms, the platform described in this study is more convenient, efficient and cost-effective for the isolation of functional antibodies targeting GPCR proteins. It is conceivable that if the reverse operation is performed, this yeast autocrine screening system can also be used to isolate antagonistic antibodies.

[0146] Equivalence

[0147] The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. The description of the present invention and the examples given can obviously be modified and changed in various ways without departing from the basic principles of the present invention. All these modifications and changes are included in the scope of this application.

Claims

1. An antibody or antigen-binding protein or antigen-binding fragment capable of binding to a GPCR receptor Ste2p and activating the Ste2p activity, comprising: (i) a heavy chain variable domain, comprising a VHCDR1 having an amino acid sequence of GYSFTSYW (SEQ ID NO: 1), a VHCDR2 having an amino acid sequence of IYPGDSDT (SEQ ID NO: 2), and a VHCDR3 having an amino acid sequence of CARLGYYDSSGYYSEDY (SEQ ID NO: 3); and (ii) a light chain variable domain, comprising a VLCDR1 having an amino acid sequence of SGRSGFA (SEQ ID NO: 4), a VLCDR2 having an amino acid sequence of VNSDGSH (SEQ ID NO: 5), and a VLCDR3 having an amino acid sequence of QTWGTGIWV (SEQ ID NO: 6).

2. The antibody or antigen-binding protein or fragment thereof according to claim 1, wherein the heavy chain variable region amino acid sequence is QVQLVQSGAEVKKPGEPLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGII YPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLGYYDSSGYYSEDYWGQGTLVTVSSGSASAPTL (SEQ ID NO: 7); and wherein the light chain variable region amino acid sequence is QPVLTQSSSASASLGASVKLTCTLSSGRSGFAIAWHQQHPEKGPRYLMNVNSDGSHTKGDGIPDRFSGSSSEAERYLTISSLQFDDEVDYYCQTWGTGIWVFGGGTKLTVLRQPKAAPSVTLFPAVL (SEQ ID NO: 8).

3. The antibody or antigen-binding protein or fragment thereof according to claim 2, comprising a heavy chain variable region and a light chain variable region that are 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences described in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

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