WO2009009045A2 - Bibliothèques d'échappement de polypeptides cibles - Google Patents

Bibliothèques d'échappement de polypeptides cibles Download PDF

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WO2009009045A2
WO2009009045A2 PCT/US2008/008367 US2008008367W WO2009009045A2 WO 2009009045 A2 WO2009009045 A2 WO 2009009045A2 US 2008008367 W US2008008367 W US 2008008367W WO 2009009045 A2 WO2009009045 A2 WO 2009009045A2
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library
phage
binding
target polypeptide
protein
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PCT/US2008/008367
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WO2009009045A3 (fr
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Richard A. Lerner
Sydney Brenner
Tobin J. Dickerson
Kim D. Janda
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The Scripps Research Institute
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the invention provides methods for generating a library of binding pairs of escape variants of a target polypeptide and cognate antagonist agents.
  • the methods entail first mutagenizing the target polypeptide to generate a library of variant polypeptides, and then identifying one or more members of the library of variant polypeptides which retain the ability to bind to a binding partner of the wildtype target polypeptide. This is followed by contacting the identified variants with the binding partner in the presence of a library of known compounds which antagonize binding between the target polypeptide and the binding partner. This allows identification of one or more escape variants of the target polypeptide whose binding to the binding partner is not antagonized by the library of known compounds.
  • a library of candidate antagonist agents is screened to identify at least one cognate antagonist agent which antagonizes binding between an escape variant and the binding partner.
  • These steps can be repeated for additional rounds to identify more escape variants and cognate binding partners.
  • a different library of variants of the target polypeptide is employed.
  • an escape variant identified in the previous round can be used to generate a library of variant polypeptides for a subsequent round of selection.
  • the cognate antagonist agent identified in the previous round is combined with the library of known compounds used in the previous round.
  • the methods can further entail determining nucleotide sequence of a polynucleotide which encodes the identified escape variants.
  • the library of variant polypeptides is generated in a replicable genetic package.
  • Preferred replicable genetic package systems are phage display and yeast surface display.
  • the library of known antagonist agents and the library of candidate antagonist agents are polypeptides.
  • the library of known antagonist agents is provided in solution, and the library of candidate antagonist agents is displayed in a replicable genetic package (e.g., phage display).
  • the libraries of antagonist agents comprise antibodies.
  • the antibodies can be single chain variable region fragments (scFvs), single domain antibodies (dAbs), Fab fragments, F(ab') 2 fragments, Fv fragments or Fd fragments.
  • the antagonist agents are small molecule organic compounds.
  • the libraries of agents are provided in a combinatorial library.
  • the library of variant polypeptides is generated by error-prone PCR.
  • the binding partner is present on a cell surface or immobilized on a solid support.
  • the target polypeptide can be a viral protein (e.g., influenza hemagglutinin), and the binding partner is a corresponding receptor present on the surface of a host cell (e.g., sialic acid present on red blood cell).
  • the invention provides a library of binding pairs of escape variants of a target polypeptide and cognate antagonist agents.
  • the library is produced by combining the escape variant and the cognate antagonist agent binding pair(s) identified in each round of selection in the methods described herein.
  • the invention provides a library of escape variants of a target polypeptide which comprises or consists of the escape variant(s) identified in each round of selection, and a library of cognate antagonist agents which comprises or consists of the cognate antagonist agent(s) identified in each round of selection.
  • the invention provides methods for identifying an agent which antagonizes a specific interaction between a target polypeptide and a binding partner.
  • the methods involve (a) expressing the target polypeptide in a replicable genetic package; (b) contacting the replicable genetic package with the binding partner in the presence of a library of candidate antagonist; and (c) identifying an antagonist agent which antagonizes the binding between the target polypeptide and the binding partner.
  • the replicable genetic package employed in the methods is phage display.
  • the binding partner is usually present on a cell surface or immobilized on a solid support. Some of these methods are directed to a target polypeptide which is a viral protein and a binding partner which is a host receptor.
  • the candidate agents employed in the methods are antibodies. In some other methods, the candidate agents are small molecule organic compounds.
  • the invention provides phagemid vectors for displaying a multimeric viral protein on phage.
  • the vectors comprise a hydrophilic signal sequence and a suppressible stop codon for conditional expression of a fusion of a viral polypeptide with a phage coat protein.
  • hydrophilic signal sequence is one which encodes a signal peptide comprising or consisting of the sequence shown in SEQ ID NO: 1.
  • the suppressible stop codon used in the vectors can be, e.g., a suppressible amber codon.
  • the suppressible stop codon is located 5' to a coding sequence of the phage coat protein.
  • An example of viral protein that can be displayed with these vectors is influenza hemagglutinin.
  • kits for displaying a multimeric viral protein on phage can contain (a) a phagemid vector containing a hydrophilic signal sequence and a suppressible stop codon for conditional expression of a fusion of a viral polypeptide with a phage coat protein, and (b) a host cell for expressing the phagemid vector and producing phage.
  • the kits can further contain an instruction sheet and a helper phage for producing phage in the host cell.
  • the instruction can provide a protocol for cloning a sequence encoding the target polypeptide into the phagemid vector, and a protocol for producing in the host cell phage displaying a multimeric viral protein.
  • FIG. 1 is a schematic diagram of an immunological checkmate analysis. Phage displaying trimeric hemagglutinin (HA) are bound to red blood cells (black ball, upper left) to attain the phage "down" state. Addition of a collection of known neutralizing antibodies yields the phage "up" state (middle left), after which mutagenesis of the phage-HA clones and selection for binding to red blood cells yields a HA library comprised of the original clones as well as mutant HA molecules that retain the ability to bind red blood cells (red phage, bottom). Subsequent addition of neutralizing antibodies isolates the phage "down” escape mutant (middle right).
  • HA hemagglutinin
  • FIG. 2 is a diagram showing interconnected rounds of checkmate selection of phage displayed hemagglutinin and phage displayed antibodies.
  • FIG. 3A-3B show specificity of phage escape analysis.
  • A Results of plate- based phage escape assay highlighting the specific nature of the observed competition.
  • B Results of optical density-based phage escape high-throughput assay. The nomenclature in the legend is protein coated onto a plate followed by the competing antigen. Note that in the presence of specific antagonists such as ATF2 or the TI-JlPi 53 - 103 peptide, an increase in optical density is observed, signifying escape of phage from the plate surface and subsequent infection of bacteria.
  • Ligand binding epitopes of proteins can mutate rapidly as shown by viral mutations that lead to escape from neutralizing antibodies.
  • the present invention is predicated in part on the pioneering work of the present inventors to recreate in vitro the evolutionary competition between viral mutations that allow escape from antibody binding and host mutations that generate new neutralizing antibodies to the mutated viral antigen.
  • the inventors developed a replicable genetic package (e.g., phage display) based method that allows rapid analysis of molecules that perturb the binding of proteins to their ligands. Because the system can amplify by replication, single molecule sensitivity can be achieved. When combinatorial polypeptide or small molecule libraries are studied, very large numbers of binding events can be analyzed simultaneously.
  • such libraries may be used in a sequential phage escape format where cycles of phage binding and release of mutants are driven by antibodies or small molecules where the difficulty of escape increases at each cycle.
  • sequencing of the viral mutants allows annotation of the allowed trajectory of escape.
  • sequencing of the antibody perturb ants charts the chemistry of the immune system response to the viral challenge.
  • checkmate analysis Such analysis of competing mutations is termed herein checkmate analysis. When viral systems are studied, a checkmate analysis allows experimental evaluation of the evolutionary contest between viruses and the immune system. It can predict which antibodies and small molecule ligands should be generated in anticipation of viral mutations before these mutations create viral epidemics.
  • the present invention provides methods for generating a library of binding pairs of escape variants of a target polypeptide and cognate antagonist agents. Also provided are methods for identifying novel antagonists which interrupt or inhibit a binding or physical interaction between a target polypeptide and a cognate binding partner. Escape libraries of target polypeptides and cognate antagonist agents are also provided in the invention. Further, the invention provides phagemid vectors and related kits for displaying a functional multimeric target protein (e.g., a multimeric viral protein). [0018] The following sections provide more detailed guidance for practicing the present invention.
  • analog is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog can exhibit the same, similar, or improved utility.
  • Methods for synthesizing and screening candidate analog compounds of a reference molecule to identify analogs having altered or improved traits are well known in the art.
  • the term "antagonize” refers to the ability of a compound (e.g., an antibody or a small molecule compound) to disrupt or perturb a specific interaction or binding that is already formed between a target polypeptide (e.g., a viral protein) and a cognate binding partner (e.g., a cellular receptor). It also refers to an activity of the compound to inhibit or prevent the target polypeptide and its cognate binding partner to bind to each other.
  • the compound antagonizes the specific interaction by binding to an epitope on the target polypeptide that is the same as or spatially close to the epitope that is recognized by the binding partner.
  • antibody refers to polypeptide chain(s) which exhibit a strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes.
  • antibodies or antigen-binding fragments used in the invention can have sequences derived from any vertebrate, camelid, avian or pisces species. They can be generated using any suitable technology, e.g., hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof.
  • antibody as used in the present invention includes intact antibodies, antigen-binding polypeptide fragments and other designer antibodies that are described below or well known in the art (see, e.g., Serafini, J Nucl. Med. 34:533-6, 1993).
  • An intact "antibody” typically comprises at least two heavy (H) chains (about 50- 70 kD) and two light (L) chains (about 25 kD) inter-connected by disulfide bonds.
  • the recognized immunoglobulin genes encoding antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • Each heavy chain of an antibody is comprised of a heavy chain variable region (V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C HI , C m and C H3-
  • Each light chain is comprised of a light chain variable region (V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the first component (CIq) of the classical complement system.
  • V H and V L regions of an antibody can be further subdivided into regions of hypervariability, also termed complementarity determining regions (CDRs), which are interspersed with the more conserved framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • the locations of CDR and FR regions and a numbering system have been defined by, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, U.S. Government Printing Office (1987 and 1991).
  • Antibodies to be used in the invention also include antibody fragments or antigen- binding fragments which contain the antigen-binding portions of an intact antibody that retain capacity to bind the cognate antigen.
  • antibody fragments include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L and C HI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C HI domains; (iv) a Fv fragment consisting of the V L and VH domains of a single arm of an intact antibody; (v) disulfide stabilized Fvs (dsFvs) which have an interchain disulfide bond engineered between structurally conserved framework regions; (vi) a single domain antibody (dAb) which consists of a V H domain (see, e.g.,
  • Antibodies suitable for practicing the present invention also encompass single chain antibodies.
  • the term "single chain antibody” refers to a polypeptide comprising a V H domain and a V L domain in polypeptide linkage, generally linked via a spacer peptide, and which may comprise additional domains or amino acid sequences at the amino- and/or carboxyl-termini.
  • a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.
  • a single chain variable region fragment (scFv) is a single-chain antibody.
  • a scFv has the two domains joined (e.g., via recombinant methods) by a synthetic linker. This enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules.
  • Antibodies that can be used in the practice of the present invention also encompass single domain antigen-binding units which have a camelid scaffold. Animals in the camelid family include camels, llamas, and alpacas. Camelids produce functional antibodies devoid of light chains.
  • the heavy chain variable (V H ) domain folds autonomously and functions independently as an antigen-binding unit.
  • Fabs antigen-binding molecules
  • scFvs single chain variable fragments
  • the various antibodies or antigen-binding fragments described herein can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies, or identified using phage display libraries. Methods for generating these antibodies or antigen-binding molecules are all well known in the art. For example, single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990; and U.S. Pat. No. 4,946,778).
  • scFv antibodies can be obtained using methods described in, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988.
  • Fv antibody fragments can be generated as described in Skerra and Pl ⁇ ckthun, Science 240:1038-41, 1988.
  • Disulfide- stabilized Fv fragments (dsFvs) can be made using methods described in, e.g., Reiter et al., Int. J. Cancer 67:113-23, 1996.
  • single domain antibodies can be produced by a variety of methods described in, e.g., Ward et al., Nature 341 :544-546, 1989; and Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85, 1996.
  • Camelid single domain antibodies can be produced using methods well known in the art, e.g., Dumoulin et al., Nature Struct. Biol. 11 :500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J MoI Biol. 332:643-55, 2003.
  • antigen-binding fragments e.g., Fab, F(ab')2 or Fd fragments
  • Fab, F(ab')2 or Fd fragments can also be readily produced with routinely practiced immunology methods. See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998.
  • binding member or "binding partner” in its various forms refers to a molecule that participates in a specific binding interaction with a target polypeptide.
  • binding pairs refers to two cognate compounds or molecules which specifically interact with each other. Examples of binding pairs include antibodies/antigens, enzymes/substrates, receptor/ligands and the like.
  • a binding member as used herein can be a binding domain, i.e., a subsequence of a protein that binds specifically to a target polypeptide.
  • Binding affinity is generally expressed in terms of equilibrium association or dissociation constants (K a or Kj, respectively), which are in turn reciprocal ratios of dissociation and association rate constants (k d and k a , respectively).
  • K a or Kj equilibrium association or dissociation constants
  • k d and k a reciprocal ratios of dissociation and association rate constants
  • contacting has its normal meaning and refers to combining two or more agents (e.g., a compound and a phage-di splayed polypeptide) or combining agents and cells. Unless otherwise indicated, contacting as used herein typically occur in vitro, e.g., mixing a library of phages with a binding partner or mixing a phage displayed polypeptide with a library of candidate agents in a test tube or other container.
  • An escape library refers to the collection of at least one variant or mutant (e.g., escape polypeptide or escape variant) of a target polypeptide (e.g., a viral protein) which the wildtype polypeptide could evolve or mutate into and which maintains the activity for binding to a cognate binding partner (e.g., a cellular receptor or host receptor) of the target polypeptide.
  • a target polypeptide e.g., a viral protein
  • a cognate binding partner e.g., a cellular receptor or host receptor
  • Escape variants of a target polypeptide can occur due to natural evolutionary force or the pressure exerted by the presence of antagonizing agents (e.g., antibodies or small molecule inhibitors) which antagonize binding between the target polypeptide and the cognate binding partner.
  • an escape library of a target polypeptide can contain at least 2, 5, 10, 25, 50, 100, 10 4 , 10 5 , 10 6 , or more 10 7 escape variants of the target polypeptide.
  • a "fusion" protein or polypeptide refers to a polypeptide comprised of at least two polypeptides and a linking sequence or a linkage to operatively link the two polypeptides into one continuous polypeptide.
  • the two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature.
  • interaction or “interacts” when referring to the interaction between a target polypeptide and a binding partner refers to specific binding between the two molecules. Unless otherwise indicated, the terms “physical interaction” and “binding” are used interchangeably herein.
  • a "ligand” is a molecule that is recognized by a particular antigen, receptor or target polypeptide.
  • ligands that can be employed in the practice of the present invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, enzyme substrates, small molecule binding compounds, and monoclonal antibodies.
  • Linkage refers to means of operably or functionally connecting two biomolecules (e.g., polypeptides or polynucleotides encoding two polypeptides), including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding. "Fused” refers to linkage by covalent bonding.
  • a “linker” or “spacer” refers to a molecule or group of molecules that connects two biomolecules, and serves to place the two molecules in a preferred configuration with minimal steric hindrance.
  • mutagenesis refers to a process of introducing changes (mutations) to the base pair sequence of a coding polynucleotide sequence and consequential changes to its encoded polypeptide.
  • the term as used herein refers to mutations artificially introduced to the molecules as opposed to naturally occurring mutations caused by, e.g., copying errors during cell division or that occurring during processes such as meiosis or hypermutation.
  • Mutagenesis can be achieved by a number of means, e.g., by exposure to ultraviolet or ionizing radiation, chemical mutagens, or viruses.
  • mutagenesis can result in mutants or variants that contain various types of mutations, e.g., point mutations (e.g., silent mutations, missense mutations and nonsense mutations), insertions, or deletions.
  • point mutations e.g., silent mutations, missense mutations and nonsense mutations
  • insertions or deletions.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • polynucleotide or “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • Polynucleotides of the embodiments of the invention include sequences of deoxyribopolynucleotide (DNA), ribopolynucleotide (RNA), or DNA copies of ribopolynucleotide (cDNA) which may be isolated from natural sources, recombinantly produced, or artificially synthesized.
  • a further example of a polynucleotide is polyamide polynucleotide (PNA).
  • PNA polyamide polynucleotide
  • the polynucleotides and nucleic acids may exist as single-stranded or double-stranded.
  • the backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • the polymers made of nucleotides such as nucleic acids, polynucleotides and polynucleotides may also be referred to herein as nucleotide polymers.
  • Polypeptides are polymer chains comprised of amino acid residue monomers which are joined together through amide bonds (peptide bonds).
  • the amino acids may be the L-optical isomer or the D-optical isomer.
  • polypeptides refer to long polymers of amino acid residues, e.g., those consisting of at least more than 10, 20, 50, 100, 200, 500, or more amino acid residue monomers.
  • polypeptide as used herein also encompass short peptides which typically contain two or more amino acid monomers, but usually not more than 10, 15, or 20 amino acid monomers.
  • Proteins are long polymers of amino acids linked via peptide bonds and which may be composed of two or more polypeptide chains.
  • protein refers to a molecule composed of one or more chains of amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene coding for the protein. Proteins are essential for the structure, function, and regulation of the body's cells, tissues, and organs, and each protein has unique functions. Examples are hormones, enzymes, and antibodies. In some embodiments, the terms polypeptide and protein may be used interchangeably.
  • receptor broadly refers to a molecule that has an affinity for a given ligand. Receptors can be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Receptors may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of receptors which can be employed by this invention include, e.g., cell membrane receptors, antigens or antigenic determinants or epitopes (such as on viruses, cells or other materials), polynucleotides, and polypeptides.
  • replicaable genetic package refers to a cell, a spore, a phage or a eukaryotic virus (a display medium) on the surface of which an exogenous biomolecule (i.e., one that is not naturally present thereon) is displayed.
  • the replicable genetic package can be eukaryotic or prokaryotic.
  • the exogenous biomolecule e.g., a short peptide or a target polypeptide
  • the exogenous biomolecule is usually obtained from an organism or species that is different from the display medium (i.e., being heterologous) or artificially generated (e.g., a recombinant polypeptide such as a single chain antibody fragment).
  • a display library of replicable genetic package is formed by introducing polynucleotides encoding exogenous polypeptides or peptides to be displayed into the genome of the display medium to form a fusion protein with an endogenous package surface protein that is normally expressed from the outer surface of the display medium.
  • replicable genetic package or “replicable genetic package system” is used interchangeably with the term “replicable display platform.”
  • target refers to a molecule or compound of interest which specifically interacts with a cognate binding partner.
  • target molecule for practicing the present invention is a polypeptide.
  • a cell has been "transformed” by exogenous or heterologous polynucleotide when such polynucleotide has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming polynucleotide may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming polynucleotide has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a "variant" of a target polypeptide refers to a molecule which has a structure that is derived from or similar to that of the target polypeptide. Typically, the variant is obtained by mutagenesis of the target polypeptide in a controlled or random manner. As detailed herein, methods for performing mutagenesis of a polypeptide are well known in the art, e.g., site-specific mutagenesis, error-prone PCR, restriction digestion and relegation, and polynucleotide shuffling.
  • a "vector” is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment.
  • Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to as "expression vectors”.
  • the invention employs a checkmate analysis to generate a library of escape variants of a target polypeptide and/or a library of cognate antagonist agents.
  • a library of mutants or variants viral proteins and a library of candidate antibodies expressed in replicable genetic package systems are used to challenge each other.
  • Successful members of each collection can be easily replicated and deconvoluted as a consequence of the inherent phenotype-genotype link engendered by the replicable genetic package (e.g., phage display), thus allowing a detailed chemical map of the trajectories of viral escape and antibody response.
  • phage escape technology described herein to validate checkmate analysis as a useful tool to identify antagonists of specific protein target-binding partner interactions with clinical importance (e.g., JNK3-ATF2 and hemagglutinin-sialic acid).
  • the two requirements for a checkmate analysis are the expression in a replicable genetic system (e.g., phage) of a functional target polypeptide (e.g., a viral protein neutralization target), and a library of antagonist agents (e.g., an antibody or small molecule library) that binds to epitopes on this target.
  • a replicable genetic system e.g., phage
  • a functional target polypeptide e.g., a viral protein neutralization target
  • antagonist agents e.g., an antibody or small molecule library
  • phage display library can be prepared as described in Barbas et al., Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).
  • methods for expressing a target polypeptide or a library of variant polypeptides in a cell surface display platform are provided in, e.g., Boder and Wittrup, Nat Biotechnol. 15:553-7, 1997; and Feldhaus et al., Nat Biotechnol. 21 :163-70, 2003.
  • a cell surface display platform e.g., yeast display
  • Boder and Wittrup Nat Biotechnol. 15:553-7, 1997
  • Feldhaus et al. Nat Biotechnol. 21 :163-70, 2003.
  • display platforms that can be employed in the practice of the present invention are described in more detail below. In addition to teachings of the art, the specific methods described in the Examples below can also be employed in the practice of the present invention.
  • a specific multimeric viral protein i.e., influenza hemagglutinin
  • influenza hemagglutinin a specific multimeric viral protein
  • the methods and techniques provided in the Examples are broadly applicable to other target polypeptides, especially other multimeric viral proteins.
  • many other viral proteins are also assembled in a membrane and can be multimeric.
  • the diagram shown in Figure 2 illustrates the general concept underlying checkmate analysis.
  • a library of variant polypeptides expressed in a replicable display platform or replicable genetic package are first contacted with a cognate binding partner to identify variants which maintain the ability to bind to the binding partner.
  • the binding partner is usually present on a physical surface to enable easy separation and identification of variant polypeptides which maintain the binding activity.
  • the physical surface can be a cell surface or a physical solid support (beads, plates, and etc.).
  • the binding partner is a cellular receptor
  • the analysis can utilize a cell that expresses the cellular receptor on its surface, as demonstrated in the Examples below.
  • the cell can be a primary cell or a cultured cell line which naturally expresses the cellular receptor. Alternatively, it can be a cell which transiently expresses an exogenous receptor of a given target polypeptide (e.g., a viral protein). As described below, many target polypeptides which bind to a cellular receptor and cells expressing the receptor are known in the art.
  • a given target polypeptide e.g., a viral protein
  • the binding partner can be immobilized on a physical support medium to allow the cognate binding variant polypeptides to be separated from non- binding variant polypeptides.
  • the solid support can be made of any material suitable for immobilizing the binding partner that is also compatible with the checkmate analysis described herein. Compatibility can be determined using methods and materials known to those having skill in the surface or materials chemistry arts. Suitable materials include, e.g., plastics (such as polymers and copolymers) and glasses (such as formed from quartz, or silicon; and metals). Examples of suitable solid supports that can be used in the practice of the present invention include, e.g., beads, filter membranes, tubes, microtiter plates, reaction chambers, nanoparticles.
  • the variant polypeptides of a target polypeptide which retain the specific binding with the binding partner are probed with a library of agents (e.g., antibodies or small molecule compounds) which are known to be able to antagonize the binding between the target polypeptide and the binding partner.
  • agents e.g., antibodies or small molecule compounds
  • the library of known antagonist agents employed in the first round of selection can contain just one member. It can also harbor 2, 5, 10, 25, 50, 100 or more members.
  • these known antagonist agents are provided in a solution or liquid phase for interaction with the library of variant polypeptides expressed in a replicable display platform.
  • Variant polypeptides which are resistant to the activity of the known antagonists are identified as escape variants of the target polypeptide.
  • the identified escape variants are screened against a library of candidate antagonist agents (e.g., a combinatorial library of antibodies or small molecule compounds).
  • a library of candidate antagonist agents e.g., a combinatorial library of antibodies or small molecule compounds.
  • the library of candidate antagonist agents can contain at least about 10 2 , 10 3 or 10 4 diverse members. However, the library typically contains at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 members.
  • the library of candidate antagonist agents is provided in a replicable display format (e.g., a phage library of antibodies).
  • the escape variant is typically expressed as a free polypeptide (as opposed to phage expression) to perform this step of selection.
  • the coding sequence of the escape variant in the display vector needs to be subcloned for expression as a free polypeptide.
  • soluble polypeptide can be expressed from the same display vector that allows expression of both soluble target sequence and its fusion with a phage coat protein.
  • the phagemid vector described below can accomplish such a goal.
  • Cognate agents which antagonize binding between a soluble escape polypeptide and its binding partner can be readily identified.
  • the library of candidate antagonist agents can be screened with the escape variant bound by the binding partner (e.g. a host receptor) that is present on a cell surface or immobilized on a solid support.
  • a cognate antagonist agent of the escape variant is identified when a phage displayed candidate antagonist agent binds to the same epitope on the escape variant to which the binding partner binds.
  • the escape variant bound phage is released from the binding partner one cell surface or a solid support and present in solution.
  • phage can be readily separated from free phage also present in liquid phase by a number of means, e.g., immunoprecipitation or affinity chromatography using an antibody that recognizes the target polypeptide.
  • candidate antagonist agents can also be screened for ability to inhibit or prevent the formation of the binding between the escape variant and its binding partner.
  • the escape variant can be contacted with the binding partner prior to, simultaneously with or subsequent to addition of the library of candidate antagonist agents.
  • the library of candidate antagonists is a combinatorial library not provided in a replicable display platform.
  • the identified escape variant can also remain displayed in a replicable display platform (e.g., phage display) to screen the library of candidate agents for cognate antagonist.
  • a replicable display platform e.g., phage display
  • the phage displayed escape variant that is bound to the binding partner e.g., a host receptor
  • a solid support e.g., a bead
  • the spatially addressable compound library e.g., compounds in solution provided in microtiter plate wells.
  • the phage displayed escape variant is released into the liquid phase if a compound is able to bind to the escape variant and dissociate the escape variant from the cell or solid support. Presence of the phage in the liquid phase can be then determined by, e.g., infecting a host cell and conferring the cell a phenotype such as drug resistance that is carried by the phage. Presence of a phage in the liquid phase also allows identification of the cognate antagonist agent by the spatial location of the released phage.
  • the identified escape variants can be mutagenized to create a further library of variant polypeptides.
  • the further library of variant polypeptides to be used in a subsequent round of selection can be generated by mutagenizing the original target polypeptide with a different means.
  • the goal is to obtain variant polypeptides that are not identical to the library of variants used in the previous rounds of selection. For example, when a previous round of selection employed variant polypeptides produced by error-prone PCR, a subsequent round of selection can use variant polypeptides generated by a chemical mutagen or by DNA shuffling.
  • each subsequent round of screening typically utilizes a pool of known antagonist agents to which cognate antagonist agents identified from the previous rounds are added.
  • the library of candidate antagonist agents is a replicable display library (e.g., phage library)
  • the identified antagonist agents from each round of selection need to be expressed in soluble form before being added to the pool of known antagonist agents.
  • the library of candidate antagonist agents employed in each subsequent round of screening can be the same library as that used in the previous rounds. However, each subsequent round of screening typically employs a library of candidate antagonist agents which comprise more and/or additional members relative to the library of candidate antagonist agents employed in the previous rounds. This serves to raise the level of difficulty with which the target polypeptide can escape at each subsequent cycle.
  • the screening can have as many rounds of selection as needed to generate a desired library of binding pairs of escape variants and cognate antagonists. For example, at least 2, 5, 10, 25 or 50 rounds of selection and checkmate analysis can be performed for a target polypeptide. Alternatively, the selection and screening can conclude when no or very few (e.g., less than 2 or 5) new escape variants are identified in a given round of selection.
  • the identified escape variants and cognate antagonists are deconvoluted using methods described herein. For example, identify, (e.g., structure information) of an escape variant polypeptide and its cognate polypeptide antagonist can be determined by, e.g., sequencing analysis of the escape variants of the target polypeptide and cognate polypeptide antagonists. For other types of antagonist agents such as small molecule compounds, identity of the cognate antagonists can be easily ascertained, e.g., via the use of a combinatorial library of candidate agents that is spatially addressable or labeled with appropriate tags. By combining the escape variants identified from each round of selection, a library of escape variants of the target polypeptide is generated.
  • identify, (e.g., structure information) of an escape variant polypeptide and its cognate polypeptide antagonist can be determined by, e.g., sequencing analysis of the escape variants of the target polypeptide and cognate polypeptide antagonists.
  • identity of the cognate antagonists can be easily ascertained, e.g
  • a corresponding library of cognate antagonist agents of the escape variants is similarly obtained by combining the antagonists identified in each round of selection. Escape variants and cognate antagonists collected from each round of selection also form a library of cognate binding pairs. Typically, these libraries can each contain about 5, 10, 10 2 , 10 3 or more members. [0061] Other than identifying escape variants of a target polypeptide and cognate antagonists, methods and compositions of the invention are also suitable for screening for novel antagonists of a specific interaction between a target polypeptide and a binding partner.
  • interaction between a target polypeptide expressed in a replicable genetic system (e.g., phage) and a binding partner is subject to competition of a library of candidate agents or compounds (e.g., antibodies or small molecule compounds).
  • candidate agents or compounds e.g., antibodies or small molecule compounds.
  • Agents which are able to antagonize (e.g., disrupt or inhibit) the interaction are identified via the same techniques described above.
  • candidate agents can also be analyzed and/or quantified with a number of other assays well known in the art.
  • antagonist agents can be identified by their ability to perturb or inhibit the binding between the target polypeptide and the binding partner or by the detection of an activity or phenotype associated with a displayed target polypeptide that is not bound by the binding partner.
  • One exemplary method for analyzing binding between the target polypeptide and a binding partner is fluorescent microscopy as demonstrated in the Examples below for phage displayed hemagglutinin and host receptor on red blood cells.
  • Other suitable assays include, e.g., labeled in vitro protein-protein binding assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like. See, e.g., U.S. Pat. Nos.
  • sandwich competition assay see, e.g., Stahli et al., Methods in Enzymology 9:242-53, 1983
  • solid phase direct biotin-avidin EIA see, e.g., Kirkland et al., J. Immunol. 137:3614- 9, 1986
  • solid phase direct labeled sandwich assay see, e.g., Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Press (1988)
  • direct labeled RIA see, e.g., Moldenhauer et al., Scand. J. Immunol. 32:77-82, 1990).
  • such an assay can involve the use of the binding partner bound to a solid surface or cells bearing the binding partner (e.g., a host cellular receptor) and a labeled target polypeptide displayed on a replicable genetic package (e.g., a phage).
  • the candidate agents can be un-labeled or labeled with a different compound (e.g., a different fluorescent dye).
  • Competitive inhibition is measured by determining the amount of the target polypeptide remaining bound to the solid surface or cells in the presence of the candidate agents. Usually the candidate agents are present in excess.
  • Antagonist agents identified by competition assay include those binding to the same epitope on the target polypeptide as the binding partner and also those binding to an adjacent epitope sufficiently proximal to the epitope bound by the binding partner for steric hindrance to occur.
  • an antagonist agent when an antagonist agent is present in excess, it will perturb or inhibit specific binding of the binding partner to the target polypeptide by at least 50 or 75%.
  • an antagonist agent is identified if it can inhibit or disrupt the binding between the target polypeptide and the binding partner with an IC 5O or EC50 that is about the same as or lower than that of a known antagonist of the binding (e.g., an antibody), e.g., lower than 120%, 100%, 75% or 50% of the IC 50 Or EC 50 of the known antagonist.
  • An antagonist agent can also be defined by having an IC 50 Or EC 50 that is at least 10 fold, 10 fold, 50 fold or 100 fold lower than a control non-reactive agent (e.g., BSA or NaCl).
  • the antagonist can inhibit binding between the target polypeptide (or a variant) with IC 5O or EC 5O that is lower than l ⁇ M, preferably lower than 500 nM, 250 nM, 100 nM, and most preferably lower than 50 nM.
  • a control non-reactive agent may be able to antagonize or perturb the same binding with an IC 50 or EC 50 that is at least 5 ⁇ M, 25 ⁇ M, 100 ⁇ M, 0.5 mM, 1 mM or higher.
  • the invention is suitable to examine the binding interactions between various types of target polypeptides and their cognate binding partners. These include, e.g., viral protein/host receptor, enzyme/substrate, host protein/host receptor, host protein/polynucleotide, and protein/small molecule compound. For example, any protein which has a known cognate small molecule binding partner can be employed in the invention to identify additional compounds which compete with the known binding partner.
  • the binding interaction between the target polypeptide or its variants and the cognate binding partner is not particularly limited so long as binding can be achieved, e.g., electrostatic, ionic, hydrophobic, van der waals, covalent, adhesion, and the like.
  • the invention provides methods for identifying escape members of a viral surface or core protein (target polypeptide) and for identifying antagonists which antagonize (e.g., perturb, disrupt, inhibit or suppress) a binding between the viral protein (or its escape variants) and a host receptor of the viral protein (i.e., the cognate binding partner).
  • target polypeptide e.g., the viral protein
  • antagonists which antagonize (e.g., perturb, disrupt, inhibit or suppress) a binding between the viral protein (or its escape variants) and a host receptor of the viral protein (i.e., the cognate binding partner).
  • the interaction of a virus with its cellular receptor initiates a chain of dynamic events that will enable entry of the virus into the cell.
  • glycoproteins or surface antigens, e.g., GP 120 and GP41
  • capsid proteins or structural proteins, e.g., P24 protein
  • HIV surface antigens or core proteins of hepatitis A, B, C, D or E virus (e.g. small hepatitis B virus surface antigen (SHBsAg) and the core proteins of hepatitis C virus, NS3, NS4 and NS5 antigens); glycoprotein (G-protein) or the fusion protein (F-protein) of respiratory syncytial virus (RSV); surface and core proteins of herpes simplex virus HSV-I and HSV-2 (e.g., glycoprotein D from HSV-2).
  • Host receptors for many viral proteins are also known.
  • Specific examples of viral protein/host receptor interactions include, e.g., influenza hemagglutinin/sialic acid; rhinovirus surface/ICAM-1 ; and hepatitis C major envelope protein (E2)/CD81. Any of these target/binding partner interactions can be employed in the practice of the present invention.
  • a number of specific examples of viral protein/host receptor interactions that are amenable to the methods of the present invention are provided below.
  • HIV uses CD4 on host cell as the primary receptor for HIV (Klatzmann et al, Nature 312:767-768, 1984). Viral infection is initiated by the binding of viral gpl20 to CD4 which is followed by a conformational change in gpl20 which enables it to interact with a co- receptor.
  • .Target cells for HIV infection include T helper cells, dendritic cells, including Langerhans' cells in the skin and in mucous membranes, and cells of the monocyte- macrophage lineage, including microglial cells in the brain.
  • the non-enveloped capsids can interact with a variety of cellular proteins (Bergelson et al, Proc. Nat. Acad. Sci. USA 91:6245-6248 1994; Kuhn, Curr. Topics Microbiol. Immunol. 223, 209-226, 1997; Shafren, J. Virol. 72:9407-9412, 1998; Triantafilou et al, J. Gen. Virol. 80:2591-2600, 1999; and Ward e/ al, EMBO J. 13:5070-5074, 1994).
  • CAR coxsackie virus-adenovirus receptor
  • Poliovirus replicate initially in cells of the oropharyngeal and enteric tract.
  • the human poliovirus receptor belongs to the Ig superfamily. It contains three Ig-like extracellular domains, and domain 1 of hPVR is sufficient to bind the virus (Racaniello, Proc. Natl. Acad. Sci. USA 93: 11378-81, 1996).
  • the viral receptor-binding sites are amino acids located on the floor and at the rim of the canyon-like depressions in the capsid (Liao & Racaniello, J. Virol. 71 :9770-9777, 1997).
  • PVR proteins are expressed in many cells and tissues, including the small intestine, lung, liver, heart, neurons of the spinal cord and the motor end-plate of skeletal muscles.
  • ⁇ -herpesviruses Members of the poliovirus receptor family are also used by ⁇ -herpesviruses as cell entry mediators. In most cases, five viral surface proteins (gB, gC, gD, gH and gL) interact with cellular receptors and mediate virus entry (reviewed in, e.g., Spear, Semin. Virol. 4:167- 180, 1993).
  • Several different cellular surface molecules have been identified as receptors which may act together, consecutively or independently, to effect the uptake of herpesviruses. Such cellular surface molecules include, e.g., heparan sulfate.
  • Heparan sulfate proteoglycans are used by several viruses as initial attachment receptors, ⁇ -herpesviruses attachment to cells is normally mediated by gB and gC. However, gD is required for successful entry and is supposed to be involved in the activation of the fusogenic activity of the viral proteins gB and gH/gL. Wild-type strains of HSV-I and -2 use binding sites generated by sulfotransferases (3-OST-3) on heparan sulfate (Shukla et al., Cell 99:13-22, 1999). Similar to ⁇ - herpesviruses, interaction with heparan sulfate has been demonstrated for HIV-I (Mondor et al, J.
  • Epstein-Barr virus (EBV) glycoproteins gp350/220 mediate the attachment of virus to cellular receptors. These heavily glycosylated envelope proteins are the product of a single EBV gene, which is expressed as alternatively spliced RNAs (Beisele/ al., J. Virol. 54:665-74, 1985).
  • CD21 the human receptor for the complement protein C3dg, has been identified as a cellular receptor for EBV.
  • CD21 is a single transmembrane protein of approximately 145 kDa containing 15 to 16 complement control protein (CCP) domains. The first two amino-terminal CCP domains of CD21 bear the virus-binding sites (Lowell et al., J. Exp. Med. 170:1931-46, 1989).
  • CD21 is expressed by B cells, follicular dendritic cells and a subset of thymocytes.
  • Measles virus uses envelope glycoproteins hemagglutinin (H), as a tetramer, and the fusion protein (F), probably as a trimer, to interact with receptors on the target cell surface (Langedijk et al., J. Virol. 71 :6155-67, 1997).
  • a complement regulatory protein containing four CCP domains, CD46 was identified as a receptor for vaccine strains of MV (Gerlier et al., Trends Microbiol. 3:338-345, 1995).
  • CD46 is expressed on almost all human cells except erythrocytes and cells in the CNS such as oligodendrocytes and a proportion of neurons and astrocytes.
  • Binding sites for MV H protein have been mapped to the first two extracellular CD46 domains (Casasnovas et al., EMBO J. 18:291 1-22, 1999; and Hsu et al., J. Virol. 72:2905-16, 1999).
  • Lymphocytic choriomeningitis virus employs a glycoprotein G to interact with cellular receptor.
  • the envelope glycoprotein G is cleaved to an external Gl and a transmembrane G2 glycoprotein.
  • the cellular receptor for LCMV has been identified (Cao et al., Science 282:2079-81, 1998).
  • Coronaviruses of serogroup 1 interact specifically with aminopeptidase-N (APN) as the attachment receptor (KoIb et al, J. Gen. Virol. 77:2515-21, 1996).
  • APN is a type II transmembrane glycoprotein and belongs to the family of membrane- bound metal lopeptidases (Ashmun et al., Blood 79:3344-9, 1992). The protein is found in large amounts on the microvillar membrane of the small intestine and is also present on renal proximal tubule epithelium, synaptic membranes of the CNS and cells of the granulocytic and monocytic lineage.
  • a cellular receptor for hepatitis A virus has been identified by screening a cDNA expression library of African green monkey kidney cells with an infection-inhibiting antibody.
  • HAV cellular receptor 1 (HAVcr-1) is a class I integral mucin-like membrane glycoprotein of unknown function (Kaplan et al., EMBO J. 15:4282-96, 1996).
  • HBV hepatitis B virus
  • HBV BF 50 kDa binding factor
  • HBV BF is a neutral metalloproteinase which shares substrate specificity with a family of membrane-type matrix metalloproteinases.
  • HBV Treatment of HBV with the metalloproteinase results in cleavage of the N-terminal part of the pre-S2 envelope protein, and probably induces a conformational change in the pre-Sl domain that enables cell membrane attachment and virus entry into T lymphocytes.
  • the invention provides methods for identifying escape variants of an enzyme and methods for identifying antagonists which inhibit interactions between the enzyme (or escape variants) and a cognate substrate.
  • enzymes include, e.g., kinases (e.g., protein kinases such as JNK3) and proteases.
  • Protein kinases include serine/threonine protein kinases, tyrosine protein kinases (e.g., receptor tyrosine kinases), histidine protein kinases, and apartic acid/glutamic acid protein kinases.
  • proteases include, e.g., serine proteases, threonine proteases, cysteine proteases, aspartic acid proteases such as plasmepsin, metalloproteases, and glutamic acid proteases.
  • Other types of enzymes can be studied with the methods of the invention include, e.g., epoxide hydrolases, transaldolases, lipases and esterases, phosphatases, acylases, transketolases, Baeyer-Villigerases and ⁇ -lactamase.
  • a library of variants which contains mutations in the active or catalytic site can be examined with checkmate analysis to identify cognate inhibiting agents.
  • regions of allosteric control or higher order protein-protein interactions can be located. Identification of such escape variants could be useful in defining active site topology and/or profiling escape trajectories of enzymes important to cancer phenotypes or protein changes leading to antibiotic resistance in bacteria. Identification of escape variants of such enzymes or proteins and cognate antagonist agents can greatly benefit cancer therapy and treatment of bacterial infection. There are many known examples of mutations in enzymes which lead to resistance to cancer therapy.
  • BCR-ABL kinase mutations have been associated with resistance to a small molecule inhibitor (STI571) in chronic myeloid leukemia (see, e.g., Shah et al., Cancer Cell 2:117-125, 2002; and Gorre et al., Science 293:876-80, 2001).
  • Mutations in a GTPase, KRAS have been linked to resistance of lung adenocarcinomas to drugs gefitinib and erlotinib (Pao et al., PLoS Med. 2:el7, 2005).
  • Point mutations in EGFR, a tyrosine receptor kinase, also confer resistance to gefitinib in a non- small-cell lung cancer see, e.g., Giaccone, Nat. Clin. Pract. Oncol. 2:296-7, 2005.
  • mutations in certain enzymes have been found to be the cause of a number of incidences of antibiotic resistance. For example, it was shown that a Serl30Gly substitution in TEM beta-lactamase leads to resistance to inhibitors of the enzyme (Thomas et al., Biochemistry 44:9330-8, 2005).
  • escape variants of an enzyme are identified by monitoring their enzymatic activity on a substrate in the presence of a known inhibitor of the enzymatic activity.
  • the substrate can be immobilized on a solid support as described herein.
  • Variants maintaining the enzymatic activity can be then screened with a library of candidate inhibitors to identify agents which inhibit the enzymatic activity of the variants.
  • variant polypeptides of the enzyme are first identified which are able to bind to a substrate of the enzyme. Such variants are then examined with one or more known inhibitors of the binding activity to identify escape variants.
  • the escape variants are further screened with a library of candidate inhibitors to identify agents which inhibit the binding between the variants and the substrate.
  • the substrate employed is typically a substrate analog which maintains binding to the enzyme but does not support the catalytic reaction of the enzyme. The use of such an analog ensures that binding between the enzyme and the substrate can be maintained during the selection process and avoid likely dissociation between the substrate and the enzyme upon catalysis of the substrate.
  • substrate analogs for a given enzyme can be generated in accordance with techniques well known and routinely practiced in the art. See, e.g., Methods in Enzymology, Preparation and Assay of Enzymes and Substrates, Sidney et al.
  • inhibitors e.g., inhibitors of ⁇ -lactamases
  • suitable assays for analyzing the binding activity or enzymatic activity of the enzymes are also known.
  • many kinases and corresponding substrates are known, e.g., tyrosine kinases such as BCR-ABL and cognate substrates including c-Crk II, CRKL and pl30Cas.
  • enzyme/substrate pairs include ⁇ -lactamase and ⁇ -lactam antibiotics such as penicillins, cephalosporins, cephamycins and carbapenems; amino acid decarboxylase and amino acids; acyl transferase and an acyl moiety; sucrase/sucrose; maltase/maltose; and pyruvate decarboxylase and pyruvate. See, e.g., Badalassi et al., Angew. Chem. Int. Ed. 39:4067, 2000; Gonzalez-Garcia et al., Chem. Eur. J. 9:893, 2003; Leroy et al., Adv. Syn. Catal.
  • enzymes and their variants can be expressed in a replicable genetic package (e.g., a phage) using methods well known in the art.
  • a replicable genetic package e.g., a phage
  • Soumillion et al. J. MoI. Biol. 237:415, 1994
  • Siemers et al. reported construction of a ⁇ -lactamase phage display library to investigate the importance of a postulated cephalosporin-binding region for the design of enhanced ⁇ -lactamase enzymes.
  • suitable target polypeptides also include certain host proteins or polypeptides which interact with cellular receptors to initiate signaling transduction or otherwise regulate cellular activities.
  • host protein/host receptor binding interactions include, e.g., TNF ⁇ /TNF-receptor and Myc/Max. These interactions are all well known in the art. See, e.g., Tang et al., Biochemistry 35:8216- 25, 1996; Locksley et al., Cell. 104:487-501, 2001 ; Amati et al., Nature 359:423-6, 1992; and Marchetti et al., J. MoI. Biol. 248:541-50, 1995.
  • vascular endothelial growth factor VEGF
  • TGF transforming growth factor
  • FF fibroblast growth factor
  • PDGF platelet derived growth factor
  • insulin-like growth factor insulin receptor
  • MHC proteins e.g. class I MHC and class II MHC protein
  • CD3 receptor T cell receptors
  • cytokine receptors e.g., interleukin receptors
  • G-protein coupled receptors chemokine receptors.
  • the employed target polypeptide is a host polypeptide which specifically interacts with a polynucleotide, e.g., a transcription factor.
  • a polynucleotide e.g., a transcription factor.
  • transcription factors that are well known and characterized (including their binding target polynucleotide sequences) in the art. See, e.g., Meyyappan et al., Biol. Signals. 5:130-
  • target polypeptides or variants thereof can be used in the practice of the present invention.
  • the target polypeptides and variants are expressed in or associated with the surface of the replicable package via a non-natural linkage (e.g., by recombinant fusion expression).
  • a polynucleotide molecule encoding the target polypeptide can be altered at one or more selected codons.
  • An alteration is defined as a substitution, deletion, or insertion of one or more nucleotides in the polynucleotide encoding the target polypeptide that results in a change in the amino acid sequence of the polypeptide.
  • the alterations will be by substitution of at least one amino acid with any other amino acid in one or more regions of the molecule.
  • the alterations may be produced by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated mutagenesis (e.g., Zoller et al., Methods Enzymol. 154:329-50, 1987), cassette mutagenesis (e.g., Well et al. Gene 34:315 1985), error-prone PCR (see, e.g., Saiki et al., Proc. Natl. Acad. Sci. USA. 86:6230-4, 1989; and Keohavong and Thilly, Proc. Natl. Acad. Sci. USA., 86:9253-7, 1989), and DNA shuffling (Stemmer, Nature 370:389-91, 1994; and Stemmer, Proc. Natl. Acad. Sci. 91 :10747-51, 1994).
  • the target polypeptide and/or its variants are typically expressed in a replicable genetic package system.
  • some of the candidate antagonist agents detailed in the following section e.g., antibodies or polypeptides
  • the invention preferably employs a phage based or a yeast cell based replicable genetic package system to display the library of variant polypeptides or the library of candidate antagonist agents.
  • Preferred phage display systems are filamentous phage such as M 13, fd, fl, or engineered variants thereof.
  • Non-cell based display platforms include, e.g., eukaryotic virus display (see, e.g., Han et al., Proc. Natl. Acad. Sci. USA 92: 9747-9751, 1995), spores (see, e.g., Donovan et al., J. MoI. Biol. 196: 1-10, 1987), and ribosome based display platform (see, e.g., Hanes et al., Nat. Biotechnol. 18: 1287- 92, 2000).
  • cells other than yeast can also be used.
  • prokaryotic cells have been developed to express exogenous polypeptides on the outer surface of the cells.
  • these include, e.g., E. coli, S. typhimurium, P. aeruginosa, B. subtilis, P. aeruginosa, V. cholerae, K pneumonia, N. gonorrhoeae, N. meningitides, etc. See, e.g., U.S. Pat. No. 5,571,69S; Georgiou et al., Nat. Biotechnol. 15: 29-34, 1997; Wu et al., FEMS Microbiol. Lett. 256: 119-25, 2006; Lee et al., Appl.
  • the target polypeptide or its variants are expressed in a bacterial phage based replicable genetic package system.
  • phage display huge display libraries containing up to 10 10 individual members can be created from batch- cloned gene libraries. Most applications of phage display libraries aim at identifying polypeptides that bind to a given target molecule.
  • the enrichment of phages that present a binding protein (or peptide) is achieved by affinity selection of a phage library on the immobilized target. In this "panning" process, binding phages are captured whereas nonbinding ones are washed off. In the next steep, the bond phages are eluted and amplified by reinfection of E. coli cells. The amplified phage population can, in turn, be subjected to the next round of panning. See, e.g., WO 91/19818; WO 91/18989; WO 92/01047; WO 92/06204; WO 92/18619; Han et al., Proc. Natl. Acad. Sci. USA 92: 9747-51, 1995; Donovan et al., J. MoI. Biol. 196: 1-10, 1987.
  • phage populations expressing the target polypeptide and/or its variants can be first enriched with appropriate procedures and then subjected to further analysis as described herein.
  • variants polypeptides that have been expressed in a phage display library can be enriched for ability to bind to a binding partner of the target polypeptide. Enriched phage populations displaying such variant polypeptides are then examined with a library of known compounds which are able to inhibit or disrupt binding between the target polypeptide and the binding partner.
  • additional screening to identify cognate antagonist agents of the escape variants and further rounds of selections can be performed as described herein.
  • the enriched phage population displaying the target polypeptide can be subject to interaction with the binding partner (e.g., a ligand). Phages bound by the binding partner are then challenged with a library of candidate agents to identify agents which are able to disrupt the binding between the binding partner to the displayed target polypeptide. Alternatively, enriched phages displaying the target polypeptide can be examined for ability to bind to the binding partner in the presence of candidate agents. Agents which inhibit the interaction between the target polypeptide and the binding partner are identified as antagonists of the binding.
  • the binding partner e.g., a ligand
  • the target polypeptide is typically expressed as a soluble protein before put into contact with the binding partner and the library of candidate agents.
  • phage display in the present invention preferably employs E. coli filamentous phage such as Ml 3, fd, fl, and engineered variants thereof.
  • fd-tet An example of engineered variants of these phages is fd-tet, which has a 2775-bp BgHl fragment of transposon TnIO inserted into the BamWl site of wild-type phage fd. Because of its TnIO insert, fd-tet confers tetracycline resistance on the host and can be propagated like a plasmid independently of phage function as the displaying replicable genetic package.
  • the phage virion consists of a stretched-out loop of single- stranded DNA (ssDNA) sheathed in a tube composed of several thousand copies of the major coat protein pVIII (product of gene VHI or "gVIH").
  • pill product of gene III or "gill"
  • pi V product of gene IV or "gIV”
  • pVII product of gene VII or "gVII”
  • pIX product of gene IX or "glX"
  • polypeptides including random combinatorial amino acid libraries, randomly fragmented chromosomal DNA, cDNA pools, antibody binding domains, receptor ligands, etc., may be expressed as fusion proteins, e.g., with pill or pVIII, for selection in phage display methods.
  • Phage system has been employed successfully for the display of functional proteins such as antibody fragments (scFv or Fab'), hormones, enzymes, and enzyme inhibitors, as well as the selection of specific phage on the basis of functional interactions (antibody - antigen; hormone - hormone receptor; enzyme - enzyme inhibitor). See, e.g., Paschke, Appl. Micbiol. Biotechnol.
  • phage display platforms can be grouped into two classes on the basis of the vector system used for the production of phages.
  • True phage vectors are directly derived from the genome of filamentous phage (M 13, fl, or fd) and encode all the proteins needed for the replication and assembly of the filamentous phage (Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382, 1990; Scott and Smith, Science 249:386-390, 1990; Petrenko et al., Protein. Eng.
  • the library is ether cloned as a fusion with the coat protein originally present in the phage genome or inserted as fusion gene cassette with an additional copy of the coat protein.
  • the former vector system produces phages exclusively presenting the fusion coat protein, whereas the latter system yields phages that present the wild type and the fusion coat protein on the same phage particle.
  • the second group of phage display platforms utilizes phagemid vectors (see, e.g., Marks et al., J. MoI. Biol. 222:581-597, 1991; and Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991) which produce the fusion coat protein.
  • a phagemid is a plasmid that bears a phage-derived origin of replication in addition to its plasmid origin of replication.
  • the phage-derived origin of replication is also known as intergenic region.
  • the intergenic region contains a 78-nucleotide hairpin section (packaging signal), which promotes the packaging of the ssDNA in the phage coat.
  • packet signal packaging signal
  • the production of phages containing the phagemid genome can only be achieved when additional phage derived proteins are present.
  • these proteins are simply provided by superinfecting phagemid-carrying cells with a helper phage.
  • helper phage provides all the proteins and enzymes required for phagemid replication, ssDNA production and packaging, and also the structural proteins forming the phage coat.
  • helper phage acts on the phagemid DNA and on the helper phage genome itself. Therefore, two distinct types of phage particles with different genotypes are produced from cells bearing phagemid and helper phage DNA: (1) those carrying the phagemid genome and (2) those carrying the helper phage genome. Phage particles containing the helper phage genome are useless in phage display processes even if they present the desired phenotype because they do not contain the required genetic information. The fraction of phages containing helper phage genome can be reduced to -1/1,000 by using a helper phage with a defective origin of replication or packaging signal, which leads to preferential packaging of the phagemid DNA over the helper phage genome. Independent of the genotype, phagem id-based display platforms usually yield phages with a hybrid phenotype displaying wild type and fusion coat protein on the same particle.
  • Fusion of the target polynucleotide sequence and the phage polynucleotide can be accomplished by inserting the phage polynucleotide into a particular site on a plasmid that also contains the target polynucleotide gene, or by inserting the target polynucleotide into a particular site on a plasmid that also contains the phage polynucleotide.
  • the fusion polypeptides typically comprise a signal sequence, usually from a secreted protein other than the phage coat protein, a polypeptide to be displayed and either the gene IN or gene VIII protein or a fragment thereof effective to display the polypeptide.
  • the gene NI or gene VIN protein used for display is preferably from (i.e., homologous to) the phage type selected as the display vehicle. Exogenous coding sequences are often inserted at or near the N-terminus of gene NI or gene VIN although other insertion sites are possible.
  • Either a phage system or a phagemid system can be used to display the target polypeptides in the practice of the present invention.
  • vectors for expressing candidate library of proteins in phage display are Ml 3 phage vectors.
  • vectors include, but are not limited to, fUSE5, fAFFl, fd-CATl, m663, 33, 88, Phagemid, pHENl, pComb3, pComb ⁇ , plantar 5E, p8V5, and ASurfZap.
  • a particularly preferred vector is a phagemid vector which allows conditional expression of the fusion between the target polypeptide and the phage coat protein. Examples of such vectors are described in more detail below. Some other filamentous phage vectors have been engineered to produce a second copy of either gene III or gene VIII. In such vectors, exogenous sequences are inserted into only one of the two copies.
  • exogenous polypeptide sequences are cloned into phagemid vectors which encode a phage coat protein and phage packaging sequences but which are not capable of replication.
  • Phagemids are transfected into cells and packaged by infection with helper phage.
  • Use of phagemid system also has the effect of diluting fusion proteins formed from coat protein and displayed polypeptide with wildtype copies of coat protein expressed from the helper phage. See, e.g., Garrard, WO 92/09690.
  • the sequences to be displayed on the surface of phage particles can comprise amino acids encoding one or more tag sequences.
  • tag sequences can facilitate identification and/or purification of fusion proteins.
  • tag sequences include, but are not limited to, glutathione S transferase (GST), maltose binding protein (MBP), thioredoxin (Tax), calmodulin binding peptide (CBP) , poly-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and poly-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • Other suitable tag sequences will be apparent to those of skill in the art.
  • the vector with inserted exogenous gene can be transformed into a suitable host cell.
  • Prokaryotes are the preferred host cells for phage vectors.
  • Suitable prokaryotic host cells include, e.g., E coli strain JM109, E coli strain JMlOl, E. coli K12 strain 294 (ATCC number 31,466), E. coli strain W31 10 (ATCC number 27,325), E. coli strain X 1776 (ATCC number 31,537), E. coli strain TGl (Zymo Research), and E. coli XLl-Blue cells (Stratagene, La Jolla, CA).
  • E. coli such as HBlOl, NM522, NM538, NM539, and cells from many other species and genera of prokaryotes
  • bacilli such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella trphimurium or Serratia marcesans
  • various Pseudomonas species may all be used as hosts.
  • Phage particles displaying a library of variant polypeptides or candidate antagonist agents can be produced by culturing host cells that have been transformed with the recombinant phagemid or phage vectors, in accordance with the procedures described herein or that is well known in the art.
  • host cells e.g., XLl -Blue E. coli cells
  • harboring vectors encoding the fusion polypeptides can be grown under suitable conditions (e.g., at 37°C in superbroth-medium containing 1% glucose and appropriate antibiotics) to allow propagation of phage particles. If needed, a helper phage is also added.
  • the phage particles released into the growth medium can be then harvested in the form of phage medium at that time.
  • the harvested phage particles can be then used directly in subsequent screening.
  • the phage particles can also be precipitated (e.g., by centrifugation) and resuspended in a different solution (e.g., PBS, pH 7.4) for the subsequent screening.
  • the harvested phage particles can be first enriched before being used in subsequent screening.
  • phage displayed variants of a target polypeptide can be enriched by affinity selection or panning, using a cognate binding partner of the target polypeptide.
  • the enriched phage library can again be propagated and amplified in host cells prior to screening for escape variants with a library of known antagonists.
  • phage particles bound by the cognate binding partner can be directed analyzed with the known antagonists.
  • further enrichment and amplification of phage particles displaying escape variants may also be needed.
  • Detailed procedures for carrying out each of these steps are well known in the art. See, e.g., Barbas et al., Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).
  • the target polypeptide and its variants are expressed in a yeast display platform.
  • Yeast display (or yeast surface display) is a well established system for protein engineering (Boder and Wittrup, Yeast surface display or screening combinatorial polypeptide libraries, Nat Biotechnol. 15:553-7, 1997).
  • a target polypeptide is expressed as a fusion to the Aga2p mating agglutinin protein, which is in turn linked by two disulfide bonds to the Agalp protein covalently linked to the cell wall.
  • Expression of both the Aga2p-polypeptide fusion and Agalp are under the control of the galactose-inducible GALl promoter, which allows inducible overexpression.
  • the expressed fusion polypeptides can also contain one or more peptide tags or epitope tags (e.g., c-myc and HA), allowing quantification of the library surface expression by, e.g., flow cytometry.
  • yeast display has been employed in a number of successful applications, including engineering a high monovalent ligand-binding affinity for an engineered protein (Boder et al., Proc. Nat. Acad. Sci. 97:10701-10705, 2000). Many other successful applications of yeast display libraries have also been reported in the art. For example, Furukawa et al. (Biotechnol Prog. 22:994-7, 2006) described a yeast cell surface display platform for homo-oligomeric protein by coexpression of native and anchored subunits. Similarly, Shibasaki et al. reported development of combinatorial bioengineering using yeast cell surface display (Biosens. Bioelectron. 19: 123-30, 2003).
  • Nakamura et al. (Appl Microbiol Biotechnol. 57:500-5, 2001) described development of novel whole-cell immunoadsorbents by yeast surface display of the IgG-binding domain.
  • Kim et al. (Yeast. 19:1 153-63, 2002) reported cell surface display platform using novel GPI-anchored proteins in yeast Hansenula polymorpha.
  • yeast surface displayed libraries of target polypeptides are well known in the art.
  • yeast surface displayed libraries of variant polypeptides in the present invention can be generated in accordance with the teachings described in, e.g., U.S. Pat. Nos. 6,300,065; 6,423,538; 6,300,065; and U.S. Patent Application 20040146976. Additional teachings of yeast display platforms are provided in many other prior art references. These include, e.g., Feldhaus et al., Nat Biotechnol. 21 : 163- 70, 2003; Bhatia et al., Biotechnol Prog.
  • each plasmid contains a polynucleotide encoding a member of a library of variant target polypeptides or a library of candidate antagonist agents (e.g., a library of scFv fragments derived from a na ⁇ ve antibody library or a combinatorial antibody library) can be fused to Aga2p.
  • candidate antagonist agents e.g., a library of scFv fragments derived from a na ⁇ ve antibody library or a combinatorial antibody library
  • the yeast strain used must have the Agal gene stably integrated under the control of a galactose inducible promoter.
  • EBYlOO Invitrogen, Carlsbad, CA
  • Other vectors that can be employed for constructing a yeast surface display library of target polypeptides in the present invention include the pPNLS vector (Bowley et al., Protein Eng. Des. SeI. 20:81-90, 2007).
  • the displayed polypeptides are labeled with, e.g., an epitope tag, to facilitate subsequent selection.
  • the epitope tag e.g., c-myc or HA
  • the epitope tags enables subsequent labeling of the fusion polypeptide, e.g., via a fluorescently labeled antibody which specifically recognizes the epitope tag (e.g., an anti-HA monoclonal antibody).
  • a fluorescently labeled antibody which specifically recognizes the epitope tag (e.g., an anti-HA monoclonal antibody).
  • polypeptide epitope tags polypeptide sequences described herein or well known in the art can also be used in the invention. See, e.g., U.S. Patent Application 20040146976.
  • candidate antagonist agents can be employed in the present invention to identify novel antagonists of a target polypeptide or cognate antagonist agents of escape variants of a target polypeptide.
  • candidate antagonist agents which are antibodies or antigen-binding fragments.
  • candidate agents suitable for the invention can also be of other chemical classes noted below, as well as derivatives, structural analogs or combinations thereof.
  • candidate agents comprise functional groups necessary for structural interaction with polypeptides or proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • Candidate agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Combinatorial libraries of many types of candidate agents can be synthesized in a step-by-step fashion. Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642.
  • ESL encoded synthetic libraries
  • Antibody or polypeptide libraries can also be generated by a replicable genetic package system as described above (e.g., phage display or yeast surface display).
  • Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field.
  • Known pharmacological agents can be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
  • Combinatorial libraries of peptides, antibodies or other compounds can be fully randomized, with no sequence preferences or constants at any position.
  • the library can be biased, i.e., some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, or to purines.
  • the candidate antagonist agents employed are intact antibodies or antigen-binding fragments of antibodies.
  • Antigen binding fragments that can be used include, e.g., single chain variable region fragments (scFvs), single domain antibodies (dAbs), Fab fragments, F(ab')2 fragments, Fv fragments and Fd fragments.
  • An antibody library to be used in the invention can comprise unrelated antibodies from a na ⁇ ve antibody library (see, e.g., McCafferty et al., Nature 348:552-4, 1990). Libraries of na ⁇ ve antibodies (e.g., scFv libraries from human spleen cells) can be obtained as described in Feldhaus et al., Nat.
  • the antibody library can comprise antibodies which are derived from a specific antibody, e.g., by recombination, DNA shuffling or mutagenesis (Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-82, 1991). For example, Griffiths et al.
  • EMBO J 13:3245-60, 1994 described a library of human antibodies generated from large synthetic repertoires (lox library). Further, some embodiments of the invention employ libraries of antibodies that are derived from a specific scaffold antibody. Such antibody libraries can be produced by recombinant manipulation of the reference antibody using methods described herein or otherwise well known in the art. For example, Persson et al. (J. MoI. Biol. 357:607- 20, 2006) described the construction of a focused antibody library for improved hapten recognition based on a known hapten-specific scFv.
  • Antibody libraries can be single or double chain.
  • a single chain antibody display library is used.
  • Single chain antibody libraries can comprise the heavy or light chain of an antibody alone or the variable domain thereof.
  • the members of single-chain antibody libraries are formed from a fusion of heavy and light chain variable domains separated by a peptide spacer within a single contiguous protein. See e.g., Ladner et al., WO 88/06630; McCafferty et al., WO 92/01047. While expressed as a single protein, such single-chain antibody constructs can actually display on the surface of bacteriophage as double-chain or multi-chain proteins.
  • double-chain antibodies may be formed by noncovalent association of heavy and light chains or binding fragments thereof.
  • the diversity of antibody libraries can arise from obtaining antibody-encoding sequences from a natural source, such as a nonclonal population of immunized or unimmunized B cells. Alternatively, or additionally, diversity can be introduced by artificial mutagenesis as discussed herein for other proteins.
  • double-chain or multi-chain antibodies display libraries can be employed. Production of such libraries is described by, e.g., Dower, U.S. Pat. I No.
  • candidate agents used in the present invention are polypeptides or short peptides. These can be naturally occurring polypeptides or their fragments. Such candidate agents can be obtained from a natural source, e.g., a cell or tissue lysate. Libraries of polypeptide agents can also be prepared, e.g., from a cDNA library commercially available or generated with routine methods. The candidate agents can also be short peptides, e.g., peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides can be digests of naturally occurring proteins, random peptides, or "biased" random peptides.
  • the candidate agents are small molecules (e.g., molecules with a molecular weight of not more than about 1,000).
  • combinatorial libraries of small molecule candidate agents as described above can be readily employed to screen for small molecule antagonists of an interaction between a target polypeptide and a binding partner.
  • the agents are typically provided in a format that can be easily deconvoluted or spatially addressed.
  • a library of candidate antibodies can be provided in a replicable genetic package in the practice of the present invention, phage display or yeast surface display.
  • a library of polypeptide or short peptide agents can also be prepared in a replicable display platform described above.
  • the agents are provided in non-biological display platforms.
  • candidate agents can be attached to a non-nucleic acid tag that identifies the agent.
  • a tag can be a chemical tag attached to a bead that displays the agent or a radiofrequency tag (see, e.g., U.S. Pat.
  • a library of candidate agents e.g., small molecule organic compounds, polypeptides, or nucleotides
  • the library of agents can then be put into contact with the target polypeptide and the cognate binding partner. Identity of a candidate agent which competes with the binding partner for binding to the target polypeptide can then be immediately identified from their spatial location.
  • a phagemid vector for displaying a multimeric target protein on phage is provided.
  • a functional multimer on the surface of a bacteriophage e.g., M 13
  • the co-expression of soluble monomers and a fusion of a monomer attached to a phage coat protein e.g., pill
  • the vectors of the invention employ a suppressible stop codon (e.g., an amber codon) between the target polypeptide coding sequence and the phage coat protein sequence.
  • coding sequence for a viral target polypeptide can be cloned at a site that is 5' to the phage coat protein with the suppressor codon located between the two proteins.
  • the phagemid vectors of the invention also contain a hydrophilic signal sequence instead of hydrophobic signal sequence often present in phage display vectors. The presence of a hydrophilic signal sequence can prevent unintended membrane association of soluble monomers of the target polypeptide.
  • the hydrophilic signal sequence can be one that encodes the dgal signal peptide sequence
  • MNKKVLTLSAVMASMLFGAAAHA (SEQ ID NO: 1).
  • Other suitable hydrophilic signal sequences can be readily synthesized by modifying known signal sequences . See, e.g., Karlstrom et al., Vet. Microbiol. 104:179-88, 2004; Strobel et al., MoI. Biotechnol. 24:1-10, 2003; Bauer et al., Virol. 167:166-75, 1988; and Thammawong et al., Appl. Microbiol. Biotechnol. 69:697-703, 2006.
  • the phagemid vectors of the invention can also contain other elements usually present in an expression vector, e.g., transcriptional regulatory sequences such as a promoter and selection markers such as a drug resistance gene.
  • transcriptional regulatory sequences such as a promoter
  • selection markers such as a drug resistance gene.
  • Many known vectors can be used in the construction of the phagemid vectors of the present invention, including vectors which harbor a suppressible stop codon located 5' to the sequence encoding the phage coat protein. Examples of such vectors include pComb3 (Williamson et al., J Virol 72:9413-8, 1998) and pCGMT (Gao et al., Proc. Natl. Acad. Sci. USA 99:12612-6, 2002).
  • a Ml 3 based phagemid vector for displaying multimetic viral proteins can be constructed by replacing the relative hydrophobic pelB signal sequence from phagemid pCGMT with a hydrophilic signal sequence described herein.
  • Any target polypeptide which forms multimeric functional protein e.g., a viral protein described herein
  • the resulting expression/selection vector can be transfected into a suitable host cell which has appropriate suppressor activity. Suitable host cells include E. coli cells. Specific E.
  • coli cell lines which allow expression of both soluble monomers and fusion proteins from the phagemid vectors include TGl (Zymo Research) and XLl -blue (Stratagene). Phage displaying the target polypeptide are produced when the cells are cultured in the presence of a helper phage under appropriate conditions as described herein.
  • a hydrophilic signaling peptide enables the expressed soluble monomers to be efficiently routed to the periplasmic space. Given the extremely small volume of this space ( ⁇ 1.0 x 10 "15 L), very few molecules (e.g., a few hundred) will be present at micromolar concentrations and thus thermodynamically drive the assembly of a multimeric protein on the phage surface.
  • kits for displaying a multimeric target polypeptide on phage typically also contain a host cell with suitable suppressor activity for expressing both soluble monomers of a cloned target polypeptide and fusions of the target polypeptide with a phage coat protein. Examples of such host cells are described above. Further, the kits can optionally contain other materials that are necessary for cloning a target polypeptide-coding sequence into the phagemid vector and for producing phage displaying the desired multimeric target protein. For example, the kits can include a helper phage which is needed to produce phage particles in the host cell.
  • kits include an instruction which provides relevant information for using the phagemid vector to express and display a multimeric target protein.
  • the information can include, e.g., protocols for cloning a target sequence into the vector, transfecting the resulting expression vector into the host cell, and growing the host cell to produce phage particles that display the multimeric target protein.
  • protocols can be based on the specific disclosures provided herein and knowledge well known in the art.
  • This Example describes materials and methods employed in expressing functional hemagglutinin on phage and analyzing red blood cell agglutination mediated by the phage bound hemagglutinin.
  • OD 60 O 0.5-0.7, after which time 1.0-3.0 x 10 1 1 pfu/mL VCSM13 helper phage was added and cultures were rocked gently at 37°C for 30 min then shaken at 37°C for 1 h. Kanamycin was added to a final concentration of 70 ⁇ g/mL and cultures were grown overnight at 28°C.
  • aqueous solutions were prepared using distilled, deionized water (Mediatech Inc) and each step carried out by transferring each grid through individual droplets on Parafilm. Pioloform coated 400 mesh, nickel grids were first rinsed in 0.05% aqueous Bacitracin (Sigma) then while still wet, incubated for 10 min on droplets of either wild-type or HA-expressing phage both diluted 1 :20 in water. The samples were blocked in 2% BSA in TBS (25 mM Tris base, 137 mM NaCl, pH 8.2) for 20 min, followed by 2 min in murine anti-HA monoclonal antibody 12CA5 (9.3 mg/mL) diluted 1 :50 in 0.5% BSA in TBS.
  • TBS 25 mM Tris base, 137 mM NaCl, pH 8.2
  • the grids were washed in 0.2% BSA in TBS (6 x 1 min), incubated in goat- anti-mouse polyclonal antibody tagged with 6 nm gold (1 : 100 for 5 min) (Jackson ImmunoResearch), washed in TBS (6 x 1 min) then water (1 x 1 min). To stabilize the samples, the grids were floated on 0.2% aqueous glutaraldehyde (5 min), washed in water (4 x 1 min), then 0.1% aqueous ammonium acetate (2 x 1 min) and finally negatively stained in 2% aqueous uranyl acetate (2 min).
  • Red blood cell agglutination Chicken erythrocytes were separated from whole blood in heparin (Rockland Immunochemicals) with the Accuspin System-Histopaque-1077 (Sigma). Following isolation and washing, 25 ⁇ L of chicken or human red blood cells were added to 975 ⁇ L PBS and this cell suspension was used to make 1 : 1 serial dilutions in PBS in a V-bottom 96-well plate (50 ⁇ L total volume). The plate was centrifuged at 1,000 x g for 5 minutes and the supernatant discarded.
  • the plate was then washed 2 times by adding 50 ⁇ L PBS, resuspending the cells, centrif ⁇ ging at 1,000 x g for 5 min and discarding the supernatant. Finally, a 50 ⁇ L aliquot of a 1:1,000 dilution of goat anti-mouse polyvalent immunoglobulin (Sigma) was added to each well and the plate was incubated at 37°C with shaking overnight. Subsequently, the plate was centrifuged at 1,000 x g for 5 min and visually checked for red blood cell agglutination.
  • Poly-D-lysine coverslips were prepared by incubating coverslips in 50 ⁇ g/mL Poly-D-lysine (Sigma) for 1 h at 37°C and allowing them to air dry. Cells were rinsed twice and attached to poly-D-lysine coated coverslips via centrifugation. Coverslips were mounted onto slides with the Slowfade Anti-fade kit (Invitrogen) and imaged on Bio-Rad (Zeiss) MRC 1024 laser scanning confocal microscope.
  • This Example describes the general principle and exemplified procedures of checkmate analysis.
  • phage escape libraries are the experimental centerpiece of the present invention, while the binary protocol of a checkmate analysis is a powerful algorithm for generating the most useful information from such libraries.
  • phage escape libraries can involve a starting population of hemagglutinin containing phage that bind to a solid support (termed phage "down") and an antibody or small molecule collection that prevents attachment (termed phage "up").
  • the hemagglutinin is then mutated and the escape variants (phage "down") which still preserve binding capacity are selected. These can be used to screen for new variants of the antagonists that can capture the escape mutants.
  • the viral and antibody variants are deconvoluted and annotated.
  • the challenge to the virus escalates as the collection of new antibodies and small molecule antagonists grows and are added to each cycle.
  • the sequence analysis of successful viral mutants provides a map of escape routes the virus can use and similarly, the sequences of the antibodies provide information about the chemical basis of a successful immune response. For this to succeed, a functional viral protein must be robustly displayed as a fusion protein with a phage coat protein.
  • phagemid vectors have been constructed in such a fashion that the inserted gene is located 5' to the pill coat protein with an amber suppressor codon between the two proteins to allow for rapid conversion from a selection vector to an expression vector (Huse et al., Science 246: 1275-81, 1989; and Gao et al., Proc. Natl. Acad. Sci. USA 99: 12612-6, 2002).
  • This vector design perfectly fulfills our requirements for a checkmate analysis.
  • the relative hydrophobic pelB signal sequence from phagemid pCGMT was replaced with the more hydrophilic dgal signal sequence (SEQ ID NO: 1).
  • HA monomers would be expressed and efficiently routed to the periplasmic space. Given the extremely small volume of this space ( ⁇ 1.0 x IO 15 L), only a few hundred molecules will be present at micromolar concentrations and thus thermodynamically drive the assembly of trimeric HA.
  • each phage can only possess a limited number of HA trimers (usually one), and thus, in order to achieve efficient agglutination, adjacent phage molecules must be crosslinked on the red blood cell surface to mimic the native interaction.
  • HA trimers usually one
  • a multivalent system can be generated that crosslinks both sialic acid residues on the same cell as well as adjacent red blood cells.
  • Such antibody or small molecule collections could be stored and might find use because they would be quickly available to help stop the spread of newly emerging highly pathogenic virus such as avian influenza that has escaped surveillance.
  • An important practical outcome of immunological checkmate analyses is that one could build amalgamated antibodies that contain consensus sequences that were selected on the basis of response to several cycles of virus escape.
  • the success of an antibody collective in anticipating the routes of virus escape depends on the diversity that is allowed in the protein loops that are the targets of virus neutralization. If there is total degeneracy in the viral protein loops, the problem will be very difficult. However, one expects that there will be structural constraints on viral proteins and the allowed diversity may be somewhat limited, even for protein loops.
  • phage escape technology can operate at the level of single molecules. Also, the approach can be readily implemented in other display systems, such as yeast, that can allow expression of more complicated receptors such as GPCRs.
  • checkmate analyses are not limited to viral systems but can be applied to any perturbable protein-ligand interaction. When iterative cycles of binding and escape are studied, phage escape libraries and checkmate analysis provide information about critical regions in proteins. One also can envisage using this methodology in high-throughput screening procedures where organic compounds or small peptides rather than antibodies are used to disrupt the interaction between protein and ligand.
  • This Example describes mutagenesis of hemagglutinin (HA), generation of a mutant HA library for phage escape analysis, and their use in phage escape analysis.
  • HA hemagglutinin
  • Wild type HA fragment was mutagenized as follows. The wildtype HA is described in Emerging Infect. Dis. 1 1 : 1515-21 (2005 and accession number EF541403 in the NCBI Database.
  • the vector dgalxx used in the cloning and phage display of the mutant HA molecules was based on pcomb3 and pCGMT vectors (Barbas et al., Methods 8, 94-103, 1995; and Gao et al., Proc Natl Acad Sci USA, 94: 1 1777-82, 1997). Sequence of the vector is shown in SEQ ID NO:2.
  • PCR reactions were performed with the following conditions using reagents from the GeneMorph II Random Mutagenesis Kit (Stratagene): 1 ng DNA template, IX Mutazyme Buffer, 250 ng primers, 800 pmol DNTPs, 2% Mutazyme II.
  • QIAquick PCR Purification Columns (Qiagen) were used to purify the product and clean DNA was eluted in EB (Qiagen).
  • PCR DNA and Dgal_pcgmt-xho-xma were doubly digested separately with Xmal and Xhol (New England Biosciences) restriction enzymes under the following conditions: 4 ⁇ g DNA, IX NEBuffer 4, 4 ⁇ L Xmal, and 4 ⁇ L Xhol per 20 ⁇ L reaction volume digested at 37° C overnight.
  • Appropriate fragments were gel extracted by running on a 1 % agarose gel, excising bands and purifying with QIAquick gel purification kit (Qiagen). No more than 400 mg of gel was loaded through each column and each column was eluted with 50 ⁇ L EB. Fragments were combined and PCR purified as above to concentrate.
  • Phage Preparation/Library Rescue A 250 ⁇ L aliquot of each of an original library glycerol stock was inoculated into 1 L SB/glu/tet/carb media. Cells were grown to an
  • coli cells (ER2738 or TG-I). The culture was rocked gently to allow for phage infection at 37° C for 45 minutes and then centrifuged at 3000 rpm in a tabletop centrifuge to pellet. Cells were taken up in a final volume of 1.5 mL, 350 ⁇ L of which was plated on 150 mm LB/glu/tet/carb plates. Plates were grown overnight (-12-14 hours) at 30° C. Plates were scraped the next day as described previously for library preparation. Glycerol stocks were aliquoted and stored at -80° C until used to inoculate cultures for the next round of panning. This cycle was repeated for a total of four to five panning rounds prior to sequencing of phage clones.
  • bacteriophage was prepared as described above. Petri dishes (50 mm) of confluent MDCK cells were blocked and phage treated as above for erythrocyte panning, with the exception that incubations were performed while cells were adhered to the dish and not in solution. Consequently, all incubations were performed on an orbital rocker for each step rather than a tube inverter.
  • cells were washed five times with PBS, and washed ten times for all subsequent panning rounds. Phage was eluted as described and neutralized with the appropriate amount of Tris-HCl (pH 8.0). This process was continued for a total of five rounds of panning.
  • Example 4 Application of phage escape to study JNK3/ATF2 interaction
  • This Example describes the use of phage escape technology disclosed herein to examine the interactions between c-jun NH 2 -terminal kinase 3 (JNKs) and its substrate ATF2 (activating transcription factor 2).
  • JNK c-jun NH 2 -terminal kinases
  • the c-jun NH 2 -terminal kinases (JNK) are members of the MAP kinase family, a group of serine/threonine kinases which play important roles in the integration of external signals from cytokines and cellular stress and the ultimate activation of downstream effectors (e.g., transcription factors).
  • JNK3 The JNK subfamily composed of 3 distinct genes, JNKl, JNK2, and JNK3, as well as at least 10 splice isoforms, all of which share high sequence homology. While JNKl and JNK2 have high tissue distribution, JNK3 is primarily expressed in CNS neurons, with reduced levels found in the heart and testes. In the adult brain, JNK3 is primarily localized to pyramidal neurons in the CAl, CA4 and subiculum regions of the hippocampus, and to layers three and four of the neocortex, areas that are affected by neurodegenerative disorders. Furthermore, neuronal cells from JNK3-negative mice have been shown to possess resistance to A ⁇ -induced apoptosis, the characteristic lesion of Alzheimer's disease.
  • ATF2-GST Fusion Protein Expression The ATF2-GST fusion protein was cloned into the pDEST15 vector as an N-terminal fusion of GST with the first 1 15 amino acids of human ATF2[1-115] (kind gift of P. LoGrasso). The cloned expression vector was transformed into the BL21 strain of E. coli. Fusion protein expression was induced with IPTG and bacterial lysates were purified using glutathione sepharose beads. The ATF2-GST fusion protein product was identified by SDS-PAGE as a band of -54 kDa size while native GST appeared as a doublet of -40 kDa.
  • JNK3 Phagemid Preparation and Phage Expression Plasmid vectors containing JNK3 residues 39-422 were obtained (kind gift of P. LoGrasso) and cloned into the pCGMT phagemid to yield a fusion protein containing JNK3 [39-422] fused to a fragment of Ml 3 bacteriophage coat protein 3 (pill). The TG-I strain of bacteria was transformed and expressed this JNK3-pIII phagemid. Bacterial suspensions of these cells were osmotically lysed to produce a periplasmic fraction. This cellular compartment would contain an enriched proportion of the expressed fusion protein.
  • JNK3 Catalysis and Enzymatic Activity Bacterial expression of chimeric proteins can result in changes to key intramolecular interactions. These disruptions can, in turn, result in loss of physiologic functions including catalytic activity or protein-protein interactions.
  • JNK3 protein expressed as a fusion with pill maintained the physiologic function of interacting with and phosphorylating its major substrate, c-jun.
  • the JNK family of kinases represents a critical step in the signal transduction cascade of the MAP kinses, integrating external cues from cytokines and cellular stress, and activating downstream effectors including transcription factors, like c-jun and ATF2.
  • JNK3-pIII represents such a small fraction of expressed protein with at most 1-2 copies per phage particle (by comparison, there are -2,700 copies of pVIII), in order to concentrate and purify the chimeric protein, it was first immunoprecipitated with anti-JNK antibody beads to remove the other phage proteins that are present in excess. This enriched JNK3-pHI population was then activated by incubation with both MKK4 and MKK7b in the presence of ATP.
  • JNK3-pIII protein could be identified by Western blot.
  • Treatment of the enriched JNK3-pIII proteins with activated MKK4 and MKK7b resulted in specific phosphorylation of JNK3-pIII as detected with phospho-JNK specific antibodies.
  • incubation of the activated JNK3-pIII proteins resulted in specific binding and catalytic phosphorylation of c-jun.
  • the phage displayed protein was recognized by appropriate upstream kinase partners and could also subsequently phosphorylate its cognate substrate in an identical manner to recombinantly expressed protein.
  • Phage escape analysis ofJNK3/ATF2 interaction The power of phage escape technology is predicated on the phenotypic expression of a chimeric phage protein on the coat of a viral particle that contains the exact genotypic information for that same chimeric protein.
  • JNK3 can be expressed and displayed as a fusion with the pill coat protein on the Ml 3 bacteriophage.
  • the phage particle can then interact with its physiologic binding partner ATF2 that is bound to a solid substrate. This interaction can be specifically disrupted by competition with test compounds, releasing phage particles and enabling their "escape.” Only those molecules that specifically antagonize the desired protein-protein interaction (conditions 2 and 3) can cause the release of phage from the surface.
  • Sensitivity of phage escape The incorporation of phenotype and genotype within a single phage particle coupled with the ability of the bacteriophage to replicate allows an unparalleled degree of sensitivity. Theoretically, a single phage "escape" event can be detected as multiple rounds of replication will exponentially amplify the signal. This signal amplification is unparalleled amongst analytical technologies, save the polymerase chain reaction.
  • serial titers of known phage plaque forming units (pfu) were incubated with host TG-I bacteria. Bacteria infected with the phage acquired specific antibiotic resistance and could replicate exponentially.
  • FIG. 3A The plates in Figure 3A display results from the study, showing specific competition of JNK3 phage off the solid substrate by ATF2-GST versus GST alone.
  • a solution of JNK3 phage from a dilution of 10 9 pfu/mL was used.
  • the bottom row demonstrates that as little as a ten-fold dilution results in a situation where no phage is detectable in the wild-type GST condition, in effect delineating a background escape of zero.
  • the ATF2-GST results in high levels of phage release, resulting in a high signal to noise ratio.
  • TI-JIP 153.163 a characterized JNK inhibitory peptide
  • TNF- ⁇ Tumor necrosis factor- ⁇
  • IL-12 Tumor necrosis factor- ⁇
  • IL-12 a well-studied cytokine with ubiquitous roles in systemic inflammation processes and thus, a highly recognized target for drug discovery. Indeed, inhibition of the TNF- ⁇ inflammatory signaling pathway has been heavily studied for application to a wide range of autoimmune diseases including rheumatoid arthritis, asthma, and Crohn's disease. However, no small molecule inhibitor of TNF- ⁇ has yet been approved for clinical use. As detailed, we have undertaken studies to examine this protein-protein interaction for its clinical relevance, using the phage escape format disclosed herein.
  • P hagemid Preparation and Phage Expression The human gene encoding the mature soluble form of TNF- ⁇ (157 amino acids, 17 kDa) was cloned into two different phagemid vectors, enabling TNF- ⁇ to be displayed on the pill coat protein of the filamentous M 13 bacteriophage.
  • One vector contains a relatively hydrophobic signal sequence (pelB) while the second contains a more hydrophilic signal sequence (dgal) previously utilized in our phage escape library approach for influenza hemagglutinin.
  • TNF- ⁇ phage was produced in both systems and each provided preparations with good phage titers.
  • TNF- ⁇ displaying phage Characterization of the TNF- ⁇ displaying phage was performed by SDS-PAGE followed by western blot analysis with commercially available anti-phage and anti-TNF- ⁇ antibodies. We found that many of the purchased antibodies provided no detectable signal, even in the case where an anti-TNF- ⁇ antibody was used to probe against a standard sample of soluble TNF- ⁇ which was purchased from a commercial supplier. Therefore, screening a number of different commercially available antibodies was necessary to determine those antibodies capable of effectively identifying the target protein by Western blot analysis. After optimizing conditions with the standard soluble TNF- ⁇ , visualization of this protein at the appropriate molecular weight ( ⁇ 17 kDa) was ultimately confirmed.
  • TNF- ⁇ monomeric (inactive) versus trimeric (active) TNF- ⁇ .
  • One representative assay capable of distinguishing between active and inactive forms of TNF- ⁇ is a cytotoxicity activity assay conducted with fibroblasts cells. L-929 fibroblasts cells were acquired and cultured successfully. Currently, evaluation of potential biologically active TNF- ⁇ phage can be conducted with this particular assay. Thereafter, small molecules capable of disrupting the interactions between active phage-displayed TNF- ⁇ and its cognate cellular receptors can be screened.
  • Example 6 Additional materials and methods employed in validating checkmate analysis [00159] This Example describes some additional materials and methods that were employed in Examples 4 and 5 for validating checkmate analysis.
  • the pCGMT phagemid was modified to incorporate the dgal signal sequence as described in Dickerson et al., Proc. Natl. Acad. Sci. USA ,104:12703-8, 2007.
  • the 3' Aval site was mutated into a Xhol site using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA).
  • JNK N-terminal domains [residues 1-365 (Jnkl) and 39-422 (Jnk3)] were PCR amplified from sd-dtopo plasmids (kind gift of P. LoGrasso) and cloned into the 5' Xmal and 3' Xhol sites of the modified pCGMT phagemid. All sequences were verified.
  • Jnkl (1-365) andJnk3 (39-422) Phage Production The pCGMT plasmids containing the Jnk-P3 fusion proteins and amp R gene were transformed into a chemically competent TG-I strain of E. coli, using standard procedures. Phage protocols follow the standard procedures as outlined in Barbas et al. Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). SB media containing 100 ⁇ g/mL carbenicillin (Sigma, St. Louis, MO) and 2% fructose was inoculated with overnight cultures at 1:100 dilution.
  • the bacterial cultures were grown at 37 0 C with shaking at 280 rpm until they reached an OD ⁇ oo of 0.6-0.9, when they were infected with VCSM 13 helper phage (final concentration 10 10 -10 ⁇ pfu/mL).
  • the phage was allowed to adsorb to the bacteria at 37 0 C with gentle rocking for 30 minutes, followed by shaking at 280 rpm for 1 hour.
  • Kanamycin was added to a final concentration of 70 ⁇ g/mL, and the cultures incubated overnight at 30 0 C with shaking.
  • Phage Plate Titering Phage handling protocols are as described in Barbas et al. (2001). In short, infection competent TG-I strain of E. coli was maintained on M9 minimal media agar plates (Teknova, Hollister, CA). A single colony was picked into 3 mL SB media and grown overnight at 37 0 C with shaking. Fresh SB media was inoculated with 1 : 100 overnight culture and grown at 37 0 C to an OD 60O of 0.5 to 0.7. For adsorption, 10 ⁇ L of phage-containing solution (in PBS) was mixed with 100-200 ⁇ L of TG-I cells for 30 minutes at 37 0 C.
  • sterile 96-well V-bottom polystyrene plates were utilized, product #3896 (Costar/Corning, Lowell, MA).
  • the bacteria infected with phage were plated onto LB plates containing 100 ⁇ g/mL carbenacillin. Plates were incubated overnight at 37 0 C to allow colony formation and growth.
  • SDS gel electrophoresis and Western blotting Denatured SDS protein gel electrophoresis was accomplished using standard procedures. The Novex mini cell electrophoresis system and pre-cast NuPAGE Novex Bis-Tris SDS gels with MOPS buffer system were utilized throughout (Invitrogen, Carlsbad, CA). Protein samples were combined with 4X SDS loading buffer and 1/10 volume 1 M dithiothriotol (DTT) and denatured by heating to 95 0 C for 3-5 minutes. Gels ran at a constant voltage of 150 V for 1.5 to 2 hours.
  • DTT dithiothriotol
  • TG-I bacteria containing Jnk-P3 expressing phagemid were grown from overnight cultures by inoculation 1 : 100 into fresh SB media containing 100 ⁇ g/mL carbenicillin. When the culture reached an OD 6O o of 0.5 to 0.7, protein expression was induced using 0.1 mM IPTG for 2 hours at 30 °C with shaking. The bacteria were pelleted by centrifugation at 16,000 x g at 4 0 C for 20 minutes and washed in ice-cold PBS. The pellets were frozen at -70 0 C overnight and stored.
  • Frozen pellets were thawed on ice with addition of 1 mL B-Per reagent per 100 mL culture (Pierce, Rockford, IL) and Complete protease inhibitor cocktail without EDTA (Roche, Basal, Switzerland). Cells were sonicated at maximum wattage using the microtip (Branson 450 sonifier, Danbury, CT) for 3 bursts of 20 seconds separated by 1 minute rest periods on ice. After centrifugation at 16,000 x g at 4 0 C for 20 minutes, this intracellular supernatent was reserved. The periplasmic fraction was obtained from bacterial cells that were osmotically shocked and fractionated.
  • ⁇ 1 mL of 20 OD ⁇ oo bacterial cells were pelleted and resuspended in 350 ⁇ L ice-cold 0.75 M sucrose/100 mM Tris-HCl (pH 8.0) with 100 ⁇ g/mL lysozyme.
  • the drop-wise addition of 700 ⁇ L of ice-cold 1 mM EDTA was followed by a 10 minute incubation on ice.
  • 50 ⁇ L of 0.5 M MgCl 2 was added and mixed with an additional 10 minute incubation on ice.
  • the mixture was centrifuged at 16,000 x g at 4 0 C for 20 minutes to pellet the spheroplast fraction.
  • the resulting supernatent consisted of the periplasmic fraction and was stored at -20 0 C.
  • ATF2 (1-115) GST Fusion Protein A plasmid containing the first 1 15 amino acids of human ATF2 fused to the C-terminal of GST, pDEST15 Biotin ATF2 (kind gift of P. LoGrasso), was obtained. This plasmid was maintained in E. coli strain BL21 (Invitrogen, Carlsbad, CA). A single colony was inoculated into LB media supplemented with 100 ⁇ g/mL carbenicillin and incubated overnight at 37 °C with shaking. Fresh LB media with carbenicillin was inoculated 1 : 100 with the overnight culture and grown at 37 0 C with shaking to an OD ⁇ oo of 0.5 to 0.7.
  • Protein expression was induced using 0.1 mM IPTG (Sigma, St. Louis, MO) for 3 hours at 28 0 C with shaking.
  • the bacteria were pelleted by centrifugation at 5,000 x g at 4 0 C for 20 minutes and washed in ice-cold PBS. The pellets were frozen at -70 0 C overnight and stored. Frozen pellets were thawed on ice with addition of 1 mL B-Per reagent per 100 mL culture (Pierce, Rockford, IL) and Complete protease inhibitor cocktail without EDTA (Roche, Basal, Switzerland).
  • the protein-bound glutathione-sepharose beads were washed with ice-cold PBS containing protease inhibitors. The washed beads were then eluted with 10-50 mM reduced glutathione in PBS or 50 mM Tris (pH 8.0). The eluted protein solution was dialyzed against PBS using a 1OK MWCO Slide-A-Lyzer (Pierce, Rockford, IL) at 4 0 C. The protein solution was concentrated using Centricon YM- 10 concentrator (Amersham Biosciences, Pittsburgh, PA) and stored in aliquots at -20 °C to -70 ⁇ C.
  • Catalytic Assay The bacterially expressed Jnk3-P3 was first immunoprecipitated from the bacterial periplasmic fraction. Previously fractionated periplasm from Jnk3-P3 expressing cells was added to 50 ⁇ L anti-Jnk antibody coupled-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) in the amount of -700 ⁇ g protein in a total volume of 1 mL PBS with protease inhibitors (and all subsequent solutions contained protease inhibitors). This was incubated overnight at 4 0 C on a rotator. The anti-Jnk agarose beads were centrifuged at 1000 x g at 4 0 C for 1 minute.
  • the beads were washed with 1 mL PBS, then 1 mL Lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerolphosphate, 1 mM Na 3 VO 4 , and 1 ⁇ g/mL leupeptin), and twice with 1 mL Kinase buffer (25 mM Tris pH 7.5, 5 mM ⁇ - glycerolphosphate, 2 mM DTT, 0.1 mM Na 3 VO 4 , 10 mM MgCl 2 ).
  • 1 mL Lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -
  • the washed beads were split equally into two groups: 1) immunoprecipitated Jnk3-P3 eluted from anti-Jnk beads, and 2) immunoprecipitated Jnk3-P3 associated with anti-Jnk beads.
  • 100 ⁇ L 0.2 M glycine pH 2.2 (Sigma, St. Louis, MO) was added to the 120 ⁇ L bead volume and incubated for 2 minutes at room temperature.
  • the solution was neutralized with addition of 20 ⁇ L 1 M Tris pH 9.0 and neutralization checked with pH paper.
  • the eluted protein supernatent was removed after pelleting the beads with centrifugation at 1000 x g at 4 0 C for 1 minute.
  • a buffer solution exchange was performed by concentrating the 220 ⁇ L of eluted protein in a Microcon YM-IO concentrator centrifuged at 14,000 x g at 4 0 C for 20 minutes to a final volume of 10 ⁇ L. To finish the buffer exchange, the concentrated protein elution was added to 100 ⁇ L of kinase buffer.
  • the immunoprecipitated Jnk3 constructs (both eluted and bead-associated) were activated in vitro with addition of activated MKK4 and MKK7 ⁇ (Upstate Biotechnology, Waltham, MA) to final concentration of 150 nM for both kinases in the presence of 200 ⁇ M ATP (Cell Signaling Technology, Danvers, MA).
  • the in vitro activation reaction was incubated at 30 °C for 4 hours.
  • the Jnk3 catalysis was assayed using the SAPK/JNK (Nonradioactive) Assay Kit (Cell Signaling Technology, Danvers, MA).
  • the activated Jnk3 proteins were incubated with 20 ⁇ L immobilized c-jun fusion protein bead slurry in 500 ⁇ L Lysis buffer overnight at 4 0 C on a rotator.
  • the activated Jnk3 protein/c-jun bead slurry was washed twice with 500 ⁇ L Lysis buffer and twice with 500 ⁇ L Kinase buffer.
  • the pellets containing the activated Jnk3 protein/c-jun bead slurry were then resuspended in 50 ⁇ L Kinase buffer supplemented with 200 ⁇ M ATP and incubated at 30 0 C for 4 hours. Products were analyzed by SDS gel electrophoresis and Western blotting.
  • Wells were washed five times with 160 ⁇ L of PBS at room temperature. The wells were then blocked with either 50 ⁇ L of 5% BSA in PBS or 5% skim milk (Becton Dickinson, Sparks, MD) in PBS at 37 °C for 1 hour covered with Seal Plate membrane. The blocking step was followed by five additional PBS washes at room temperature. The wells were then incubated with 25 ⁇ L of various titers of Jnk3 phage (ranging from 10 12 to 10 2 pfu/mL) in blocking buffer at 37 0 C for 1 hour covered with Breath-Easy permeable membrane (Diversified Biotech, Boston, MA).
  • the wells were then repeatedly washed with PBS at room temperature (six times). Finally, the competition compounds were diluted in blocking buffer at various concentrations: 5% BSA, 1.2 ⁇ M GST, 0.6-1.2 ⁇ M ATF2-GST, 9-900 nM SP600125 (AG Scientific, San Diego, CA), and Jnk Inhibitor Peptide, 40 nM-4 ⁇ M TI-JIPi 5 3 -163 (Calbiochem, La JoI Ia, CA). 50 ⁇ L of the competing compound solution was incubated in the wells at 37 0 C for 1 hour covered with Breath-Easy permeable membrane. The competing solution and any eluted phage were recovered and stored at 4 0 C. Phage titering was done by standard plating techniques or modified spectrophotometric/fluorescent techniques.
  • Phage Optical/Fluorescent Titering Bacterial preparation is identical to traditional plate titering methods as described above. For simple optical light scattering assays, 10 ⁇ L of phage-containing solution (in PBS) was mixed with 100 ⁇ L of TG-I cells in sterile, clear untreated 96-well plates, product #3370 (Costar/Corning, Lowell, MA). Positive controls consisted of known titers of Jnk3 phage in PBS, 5% skim milk in PBS, or 5% BSA in PBS. Negative controls consisted of PBS, 5% skim milk in PBS, 5% BSA in PBS, as well as various concentrations of competition compounds in blocking buffer.
  • the OD 6 Oo was measured using the SoftMaxPro software version 1.2.0 running the SpectraMax 250 UV/Vis plate spectrophotometer. Initial time measurements were made, and the bacteria and phage were incubated at 37 0 C for 30 minutes covered with Breath-Easy permeable membrane (as with all subsequent steps). After 30 minutes, a solution of 1000 ⁇ g/mL carbenacillin in PBS was added to the wells (final concentration of 100 ⁇ g/mL), and spectrophotometric measurements made. The plates were then covered and incubated at 37 0 C with shaking. Additional measurements were made at 1 - 24 hours.

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Abstract

La présente invention a trait à des procédés permettant d'identifier des variants d'échappement d'un polypeptide cible. Les procédés impliquent typiquement les étapes consistant à générer une bibliothèque de variants de polypeptides du polypeptide cible sur une plate-forme d'affichage pouvant être répliquée (par exemple, un affichage de phage) ; identifier les variants de polypeptides qui maintiennent la capacité de se lier à un partenaire de liaison connu du polypeptide cible, et examiner les variants de polypeptides identifiés en vue d'identifier les variants d'échappement dont la liaison au partenaire de liaison n'est pas antagonisée par une bibliothèque de composés connus qui perturbent la liaison entre le polypeptide cible et le partenaire de liaison. Les procédés peuvent en outre comprendre l'analyse d'une bibliothèque d'agents antagonistes candidats en vue d'identifier un agent antagoniste lié qui antagonise la liaison entre la variant d'échappement et le partenaire de liaison. Des variants d'échappement supplémentaires peuvent être identifiées en créant une bibliothèque de variants de polypeptides supplémentaires du variant d'échappement identifié, et en soumettant les variants de polypeptides supplémentaires au processus d'analyse. Lors de chaque cycle d'analyse suivant, l'agent antagoniste identifié est combiné à la bibliothèque de composés connus employés lors du précédent cycle d'analyse. L'invention a également trait à des procédés liés permettant d'identifier de nouveaux agents antagonistes d'un polypeptide cible. La présente invention a également trait à des vecteurs et à des kits permettant d'afficher une protéine multimérique fonctionnelle cible sur la surface d'un phage.
PCT/US2008/008367 2007-07-10 2008-07-08 Bibliothèques d'échappement de polypeptides cibles WO2009009045A2 (fr)

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WO2012158739A1 (fr) * 2011-05-17 2012-11-22 Bristol-Myers Squibb Company Procédés améliorés pour la sélection de protéines de liaison
WO2014047500A1 (fr) * 2012-09-21 2014-03-27 Alexion Pharmaceuticals, Inc. Analyses permettant de rechercher par criblage des antagonistes du composant c5 du complément
WO2016064673A1 (fr) * 2014-10-20 2016-04-28 The Scripps Research Institute Procédés basés sur la proximité pour la sélection de partenaires de liaison

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US6943000B2 (en) * 1997-10-03 2005-09-13 University Of Massachusetts JNK3 modulators and methods of use
US20060084608A1 (en) * 2000-08-18 2006-04-20 Human Genome Sciences, Inc. Binding polypeptides and methods based thereon
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US6943000B2 (en) * 1997-10-03 2005-09-13 University Of Massachusetts JNK3 modulators and methods of use
US6277489B1 (en) * 1998-12-04 2001-08-21 The Regents Of The University Of California Support for high performance affinity chromatography and other uses
US7135287B1 (en) * 1999-10-02 2006-11-14 Biosite, Inc. Human antibodies
US20060084608A1 (en) * 2000-08-18 2006-04-20 Human Genome Sciences, Inc. Binding polypeptides and methods based thereon
US20050136428A1 (en) * 2003-06-27 2005-06-23 Roberto Crea Look-through mutagenesis

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012158739A1 (fr) * 2011-05-17 2012-11-22 Bristol-Myers Squibb Company Procédés améliorés pour la sélection de protéines de liaison
US9347058B2 (en) 2011-05-17 2016-05-24 Bristol-Myers Squibb Company Methods for the selection of binding proteins
EP3318880A1 (fr) * 2011-05-17 2018-05-09 Bristol-Myers Squibb Company Procédés améliorés pour la sélection de protéines de liaison
US10150962B2 (en) 2011-05-17 2018-12-11 Bristol-Myers Squibb Company Methods for the selection of binding proteins
US11060085B2 (en) 2011-05-17 2021-07-13 Bristol-Myers Squibb Company Methods for the selection of binding proteins
US11913137B2 (en) 2011-05-17 2024-02-27 Bristol-Myers Squibb Company Methods for the selection of binding proteins
WO2014047500A1 (fr) * 2012-09-21 2014-03-27 Alexion Pharmaceuticals, Inc. Analyses permettant de rechercher par criblage des antagonistes du composant c5 du complément
WO2016064673A1 (fr) * 2014-10-20 2016-04-28 The Scripps Research Institute Procédés basés sur la proximité pour la sélection de partenaires de liaison
EA034306B1 (ru) * 2014-10-20 2020-01-27 Зе Скриппс Рисёрч Инститьют Основанные на близости расположения способы выбора партнеров для связывания

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