WO1999030151A1 - Technique de cellules pour criblage de bibliotheques combinatoires - Google Patents

Technique de cellules pour criblage de bibliotheques combinatoires Download PDF

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WO1999030151A1
WO1999030151A1 PCT/EP1998/007922 EP9807922W WO9930151A1 WO 1999030151 A1 WO1999030151 A1 WO 1999030151A1 EP 9807922 W EP9807922 W EP 9807922W WO 9930151 A1 WO9930151 A1 WO 9930151A1
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polypeptide
target compound
cells
interest
selection
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PCT/EP1998/007922
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English (en)
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Alexander Von Gabain
Aaron Hirsh
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Intercell Biomedizinische Forschungs- Und Entwicklungs Gmbh
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Priority claimed from EP97121451A external-priority patent/EP0922957B1/fr
Application filed by Intercell Biomedizinische Forschungs- Und Entwicklungs Gmbh filed Critical Intercell Biomedizinische Forschungs- Und Entwicklungs Gmbh
Priority to AU18769/99A priority Critical patent/AU749042B2/en
Priority to EP98963539A priority patent/EP1036321A1/fr
Priority to JP2000524659A priority patent/JP2001526054A/ja
Priority to CA002310381A priority patent/CA2310381A1/fr
Publication of WO1999030151A1 publication Critical patent/WO1999030151A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • 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
    • 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
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • 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

Definitions

  • Combinatorial chemistry (reviewed in Janda, 1994), in which a diverse library of laboratory-constructed molecules is screened for structures that exhibit properties of interest, is a relatively novel and potentially powerful technology, promising useful application in three general areas.
  • combinatorial chemistry may be used to identify molecules with highly specific adhesive or enzymatic activities useful in biomolecular research, clinical therapy, or diagnostics.
  • Second, by screening a library for molecules that interact specifically with a known compound of significant interest, combinatorial chemistry may assist in identification of the known compound's native interactants (e.g. Phizicky.et al . , 1995) .
  • combinatorial chemistry may be used for epitope mapping, serving to elucidate the surface of structural interaction between two molecules of interest (e.g. Phizicky et al., 1995) .
  • polypeptide combinatorial chemistry and polypeptide interaction identification that resolves or circumnavigates many of the difficulties encountered by other technologies.
  • polypeptide is used herein to comprise all types of peptides or proteins, including glycoproteins, proteoglycans, lipoproteins, acetylated proteins or (other) processed peptide or protein forms.
  • a method for identifying and isolating at least one of the following: a polypeptide of interest, a target compound or a gene of a polypeptide of interest or a gene of a target compound, wherein at least one of said polypeptide of interest or said target compound is unknown comprising the steps a) constructing a cell population by engineering it to express a membrane polypeptide which has said polypeptide of interest in its extra- or intracellular domain, said extra- or intracellular domain being exhibited in one or more copies on each cell's surface directly or indirectly interacting with a selection site specific for a selection agent, such that upon binding of said target compound to said polypeptide of interest said selection site of cells which exhibit such a binding of said target compound to said polypeptide of interest is modified in a manner that said selection agent may be used for selecting those cells which exhibit said binding of said target compound to said polypeptide of interest from cells which do not exhibit said binding, b) exposing said cell population to said target compound or a population of said target compounds for a
  • a library of polypeptides is exhibited in one or more extra- or intracellular domains of a membrane polypeptide expressed by a cell population.
  • Selective pressures exerted by selection agents eliminate those cells whose polypeptide sequences fail to bind to a preselected target compound.
  • cells whose sequences bind the target compound are selected and e.g. amplified by growth.
  • polypeptide imprinting is used to refer to the use of the method according to the present invention to select cells exhibiting polypeptides that bind the target compound or exhibit specific properties.
  • prokaryotic and eukaryotic polypeptides may be imprinted, serving e.g. creation or discovery of polypeptide sequences with high affinity for a predetermined target, interaction studies, or epitope mapping.
  • polypeptides may be imprinted to exhibit the following specific properties: multivalent binding to a predetermined target; high affinity for a target and low affinity for one or more molecules closely related to the target; inhibitory activity in a biological pathway (antagonism) ; excitatory activity m a biological pathway (agonism) ; or enzymatic activity.
  • the present invention may be performed by displaying the polypeptide to be imprinted (the polypeptide of interest) in the extra- or intracellular domain of a membrane protein.
  • This invention is the product of the confluence of three distinct areas of research: i. the study of prokaryotic and eukaryotic cellular membranes and their complex constituents; ii. the study of the evolution of microbial populations under the selective pressures of toxins, bacteriocins, or viral parasites; and iii. the design and screening of polypeptide combinatorial libraries.
  • a polypeptide with promising properties can immediately be identified, amplified and isolated, making the present method extremely sensitive. It is possible to select one polypeptide that binds to a ligand out of a background of 10 6 non specific polypeptides as a minimum, but in there is no evidence for an upper limit on sensitivity regarding the present method per se.
  • the present method all cells of the combinatorial library of polypeptides and/or target compounds are screened at once, rather than serially, providing results in far less time than many alternative technologies.
  • the present invention is easy to use, because the important parameters are easily adjustable for the man skilled in the art. Once a set of cells is selected, the genes for the polypeptides or the target compounds (if these are also expressed by the cells) are easily isolated.
  • the polypeptide of interest may be displayed by any of the wide ranges of cell types known and used for displaying foreign proteins in extra- or intracellular domains (e.e. on the cell surface) . This allows the present method to avoid the biases of any one expression system.
  • the polypeptide is preferably expressed in a membrane protein which is biochemically appropriate for the target compound or for the system wherein the isolated polypeptide/target pair is intended to be applied (e.g. as a vaccine or as a therapeutic) .
  • Combinatorial chemistry which is one of the major components of the present invention, consists of two or three steps, often performed in iterative cycles.
  • the first step is the generation of a diverse population of molecules, the so- called combinatorial library.
  • the molecules in the combinatorial library are partitioned according to their exhibition of properties sought by researchers, such as affinity for a particular compound or enzymatic activity.
  • the molecules found to exhibit the desired properties are replicated or resynthesized.
  • the cycle may then be repeated, beginning either at the first step, with the introduction of new variation to the molecular population, or at the second step, with the partitioning of the population according to the exhibition of desired properties.
  • the immune system is essentially a genetically coded combinatorial library, complete with steps of variation, selection, and replication: A vast diversity of antibodies is displayed on the surface of B-cells (variation) , and specific recognition of antigen by any given cell triggers that cell's proliferation (partitioning and replication) . It is not surprising, then, that the first in vitro genetically-coded combinatorial libraries were directly appropriated from the immune system: B-cells were immortalized through hybridization to mouse myeloma lines (Marks et al . , 1991) . Later, in order to circumnavigate difficulties of hybridization, and to utilize a greater part of the antibody repertoire, E. coli were engineered to express and secrete the variable chains of antibodies.
  • Combinatorial libraries of fusion-phage are composed of filamentous bacteriophage or phagemid that have been engineered to bear semi-random exogenous proteins as sandwich fusions near the end of the adsorption protein pill (Janda, 1994, Marks et al., 1991) .
  • the method of screening these libraries for phage that exhibit affinity for a target compound is known as "affinity purification": the target is immobilized on a solid surface or column, over which the phage library is washed; phage bound to the target are eluted, amplified by growth in bacterial culture, and then subjected to yet another round of partitioning. This cycle is usually iterated until a tractable number of clones are isolated.
  • Phage libraries have been used for epitope mapping (Phizicky et al., 1995) -viz. pinpointing the region of a larger protein that is involved in binding a target compound of interest-as well as for deriving novel ligands to targets. Entire single- chain variable-fragment antibodies (scFv's) have been inserted into phagemid pill (Marks et al . , 1991), and libraries of scFv's or semi-random oligopeptides have been screened for binding to a variety of medically significant polypeptides, nucleic acids, and molecules not previously known to bind proteins .
  • scFv's single-chain variable-fragment antibodies
  • fusion phage technology has been responsible for significant strides forward in combinatorial chemistry - such as the recognition that "tagged" libraries would permit screening of a vastly increased number of sequences - phage libraries themselves have not escaped several besetting difficulties.
  • affinity purification of phage libraries factors besides affinity for the target compound contribute significantly to each clone's selection.
  • the collection of clones selected from a single library by affinity purification can vary dramatically with the technique used for eluting phage from the solid substrate of target compound (Phizicky et al . , 1995) .
  • phage adsorption protein, pill may fail to provide exhibited proteins with a sufficiently stable platform. Many polypeptides acquire structural rigidity only in certain constraining conformations; otherwise they remain extremely plastic. Therefore, to be functionally active structures, many polypeptides will require a foundation other than pill .
  • LamB intercalates the outer membrane even when exogenous polypeptide sequences have been inserted into one or more of the protein's extracellular domains.
  • the E. coli population was placed in a suspension of iron oxide particles, binding was allowed to occur, and a magnet was used to recover bacteria that had adhered to metal (Brown, 1992) .
  • the selected portion of the population was amplified by bacterial growth, and the partitioning procedure was repeated. After four cycles of selection and amplification, fresh variation was introduced according to a strategy that would not erase sequence- information accrued thus far. Another four cycles of selection and amplification then yielded four clones, three of which bore a consensus sequence for binding iron oxide.
  • the combinatorial library m LamB introduced the promising possibility of using bacterial outer membrane proteins as platforms for presenting libraries of diverse polypeptides
  • the selection procedure applied to the library was labor-intensive and very limited in its applicability.
  • the target compound, iron oxide was conveniently chosen to allow screening for magnetism - a procedure that could not be applied to screening for the ma-jonty of interesting interactions that polypeptides exhibit.
  • the present invention offers a user-friendly, efficient, and precise method of selection, capable of screening libraries of all sorts of polypeptides presented on the platform of a membrane polypeptide .
  • membrane polypeptides bounding any prokaryotic or eukaryotic cell present an extremely complex surface to the extra- or intracellular environment.
  • These molecules - referred to here as membrane polypeptides - exhibit a vast range of distinctive conformational, chemical, and electrical properties.
  • the membrane polypeptides are here categorized into several structural families. The conformational and functional differences between these families are relevant to this invention because membrane polypeptides provide the underlying structures that are used to display exogenous sequences, and are selected for interaction with the target compound.
  • this invention often capitalizes on the intrinsic properties of membrane polypeptides by imprinting them to exhibit properties or perform functions that are similar to their natural ones.
  • the deliniation of structural families presented here should also provide a profile of the vast range of properties exhibited by polypeptides on the cell membranes, for it is in part this range that makes the capabilities of this invention so broad.
  • Single-pass transmembrane polypeptides (reviewed in Singer, 1990) , also called bitopic polypeptides, have one intra- and one extra-cellular domain, and a midsection that passes through the membrane. The terminal domains fold into tertiary structures, while the transmembrane section often forms a hydropathic ⁇ -helix, in which no more than 2 of the 20 transmembrane residues may be ionic. Singer (Singer, 1990) divides bitopic polypeptides into types la, lb, and II (figure 1) . Types la and lb exhibit the amino terminus on the extracellular side of the outer membrane.
  • Type la polypeptides are ubiquitous, and in eukaryotes they are the predominant type. Because the transmembrane sequence of type la polypeptides is typically closer to the carboxyl terminus, the extracellular domain is often large. Consequently, type la polypeptides are competent for functions involving high-affinity adhesion or highly specific binding to especially extracellular targets.
  • type la polypeptides are receptors for growth factors and for peptide hormones; immune system antigen recognition molecules; and molecules responsible for cell adhesion (Singer, 1990, Staunton et al . , 1990) . Proclivity for adhesion or highly specific binding to extracellular targets make type la polypeptides suitable for use in many embodiments of this invention, especially those in which a polypeptide is imprinted to bind to an extracellular target.
  • Type lb polypeptides do not have a cleavable signal sequence.
  • the transmembrane stretch is typically close to the amino terminus, and therefore there are few extracellular residues (Singer, 1990) . This makes type lb polypeptides suitable for sophisticated cytoplasmic functions, and interactions, but poorly suited for interactions with targets in the extracellular medium.
  • Type II bitopic polypeptides are oriented with their carboxyl terminus on the exterior side of the membrane (figure 1) . As in type la bitopic polypeptides, the bulk of type II molecules is usually on the exterior of the cell. Type II polypeptides are typically enzymes with extracellular active sites, such as glycosyl transferases, or highly specific extracellular receptors, such as the asialoglycoprotein receptor (Singer, 1990) . Competence for extracellular enzymatic or receptor activity makes type II bitopic polypeptides suitable for use in certain embodiments of this invention, in which extracellular domains are imprinted to bind or enzymatically modify targets in the extracellular medium.
  • Multipass transmembrane polypeptides (reviewed in Singer, 1990), also called polytopic polypeptides, traverse the lipid bilayer in two to fifteen or more passes. Like thread stitched into cloth, the polypeptide begins on one side of the membrane, passes through it, loops into the space on the other side of the membrane, then once again traverses the membrane to expose either its end or yet another loop leading to more transmembrane passes (figure 1) . Although referred to and often illustrated as "a loop", each exposed region of a multipass polypeptide exhibits not only a unique sequence of amino acids, but also a distinctive tertiary structure.
  • multipass ones are suspended in the lipid bilayer by the hydrophobicity of transmembrane strands.
  • these strands do not invariably twist into ⁇ -helices, because when multiple strands neighbor one another, they can adopt an alternative strategy for hydrogen bonding between peptide bonds: if each strand adopts the secondary structure of a ⁇ - strand, these strands can curl together into a closed cylinder, allowing hydrogen bonds to form between the peptide bonds on separate strands.
  • the resultant tertiary structure is called a ⁇ -barrel (figure 1) . It is extremely stable, and can be released from the outer membrane without denaturing tertiary structure.
  • the extremely stable, polytopic ⁇ -barrel OmpA is imprinted to bind to various targets.
  • Polytopic polypeptides that traverse the membrane in ⁇ - helical conformation often function as specific receptors for messengers or hormones, such as yeast mating pheromone and many others. These and other ⁇ -helical polytopic receptors are categorized by Singer (Singer, 1990) as type III polypeptides. Their capacity to bind target molecules specifically, and sometimes multivalently, makes ⁇ -helical polytopic receptors ideal for use in many embodiments of this invention. Moreover, selection systems according to the present invention are easy to construct when the membrane polypeptides involved in the selection are polytopic.
  • An anchor consists of a lipid molecule, such as dimyristyl glycerol or alkylacyl glycerol, adorned by a multi-residue sugar complex, which is, in turn, covalently linked to the membrane polypeptide.
  • glycolipid anchor a well-defined ammo terminal signal sequence, which enables transport into the lumen of the endoplasmic reticulum, and a carboxy terminal glycolipid- addition sequence, which is not precisely defined but generally consists of a short hydrophobic domain located 10-12 residues carboxy terminal to a pair of small residues (Cross, 1990) .
  • carboxy terminal glycolipid- addition sequence which is not precisely defined but generally consists of a short hydrophobic domain located 10-12 residues carboxy terminal to a pair of small residues (Cross, 1990) .
  • glycolipid-anchored outer membrane proteins cannot perform cytosolic or periplasmic functions. Instead, they provide protection from the extracellular environment, or function as enzymes or adhesms recognizing exclusively extracellular substrates.
  • At least six leukocyte surface proteins, many other eukaryotic proteins belonging to the immunoglobulin superfamily (Cross, 1990) , and the trypanosome variable surface glycoprote (the protective protein that the trypanosome sequentially alters to avoid detection by the host immune system) are anchored by glycolipids.
  • the diverse array of surface- exposed proteins is adorned by yet another tier of complex molecules with distinctive properties, namely, the saccha ⁇ des (reviewed Lis et al . , 1993).
  • the oligosaccharides adorning glycoprote s generally consist of less than 15 residues. However, owing to the diversity of residues and covalent linkages that permit assembly into branched as well as linear structures, oligosaccharides exhibit thousands of distinct structures.
  • the polysaccharides of integral membrane proteoglycans are larger than oligosaccharides, and can contain dozens of residues.
  • the oligo- or polysaccharide molecule attached to a particular site in a polypeptide is not a simple function of the local ammo acid sequence.
  • the same polypeptide sequence receives distinct saccharide chains when expressed in different cells, and separate copies of a polypeptide expressed in a single cell type can have different sugars attached at a given site. Because sugar residues are sequentially appended, each by a particular enzyme to which the protein is exposed in the course of the processes of translation, folding, and export to the outer membrane, the glycosylation at a site is largely a product of the extent and duration of the site's exposure to each enzyme.
  • Exposure of a site is, in turn, affected by the way a protein folds and the rate at which it moves through the steps from translation to insertion. Consequently, the glycosylation' at a site can be altered in unpredictable ways by changes to the local sequence or to the sequence elsewhere in the polypeptide (Lis et al . , 1993) .
  • glycoproteins and proteoglycans are involved in the intercellular recognition and adhesion underpinning sperm-egg interactions, blood clotting, and inflammation (Lis et al., 1993, Singer, 1990). The roles they play, via adhesion, in these important processes make the glycoproteins and proteoglycans ideal for use in certain embodiments of this invention.
  • the membrane protein is an ⁇ -helical polytopic receptor, a glycolipid-anchored polypeptide, a polytopic integral membrane polypeptide, a membrane-bound isoform of a secreted polypeptide, a bitopic integral membrane polypeptide, a glycoprotein or a proteoglycan .
  • insertion of polypeptides into the membrane and translocation of secreted polypeptides across the membrane are performed by the same cellular machinery (Singer, 1990) .
  • integral membrane polypeptides inserted into the endoplasmic reticulum (ER) have the same cleavable or noncleavable amino terminal signal sequences as completely translocated polyeptides. This is consistent with the fact that insertion and secretion depend on the same cytoplasmic signal recognition protein (SRP) , which transfers both inserted and secreted polypeptides to the SRP receptor on the ER membrane.
  • SRP cytoplasmic signal recognition protein
  • type III polypeptide intercalation into the membrane also has implications that are significant to this invention.
  • the cellular machinery that inserts bitopic polypeptides and translocates secreted ones is also responsible for intercalating ⁇ -helical multipass polypeptides into the outer membrane. This is clear from studies that show that l) type III polypeptides sometimes have the same signal sequences as bitopic and secreted polypeptides, and n) membrane intercalation of type III polyeptides requires the same gene products as insertion of bitopic polypeptides.
  • inducting gene fusion have shown that the cellular machinery for inserting polypeptides in the outer membrane intercalates most type III polypeptides by translocating one hydrophilic extracellular loop at a time. The polypeptide is thus intercalated as a series of independently inserted segments (Smger, 1990) .
  • Most embodiments of this invention require a topological map of the imprinted polypeptide, and a technique for introducing new sequences to specified extracellular domains of the imprinted polypeptide. Both of these needs are met by gene fusion techniques.
  • each embodiment of this invention also requires a technique for inserting exogenous peptide sequences into one or more extra- or intracellular sites in the imprinted polypeptide.
  • exogenous peptide sequences may replace existingsections of the extra- or intracellular domains of membrane.
  • oligonucleotides rich m restriction endonuclease sites were inserted into the ompR gene at sites corresponding to protein loops II and IV, and the altered protein was expressed and exhibited normally (Freudl, 1989) .
  • influenza haemaglutmm, beta-galactosidase, foot and mouth disease viral protein 1 (FMDV-VP1), malaria antigens, and several other antigenic polypeptides up to 56 ammo acids in length were successfully displayed m OmpA loops III and IV (Georgiou et al., 1997).
  • two lines of evidence demonstrated that these exogenous polypeptides adopted stable folded structures.
  • extracellular domains of a number of membrane proteins accept exogenous peptide sequences without exhibiting disturbance to normal sorting and insertion (Boyd, 1994, Boder and Wittrup, 1997).
  • Higher eukaryotic membrane polypeptides have also exhibited the capacity to accept foreign inserts at extracellular domains.
  • gene fusion studies may insert extra glycosylation sites to function as reporters: only polypeptide domains on the luminal side of the ER are exposed to glycosylating enzymes, and luminal domains become extracellular domains.
  • Extra glycosylation sites were inserted into extramembranous regions of mouse muscle nicotinic acetylcholine receptor: the inserted sites did not disrupt normal sorting or insertion of the outer membrane polypeptide; and, sites inserted in extracellular receptor domains were in fact glycosylated (Chavez et al . , 1991).
  • gene fusion mapping techniques contribute to this invention in four ways.
  • these techniques have been widely used to determine the topology of countless outer membrane polypeptides, many of which are described above and may be imprinted by this invention.
  • these techniques may be used to provide topological maps for polypeptides that have not yet been mapped, but may be candidates for imprinting by this invention.
  • gene fusion techniques provide this invention with commonly practiced methods for inserting an exogenous peptide sequence into a specified region of a membrane polypeptide; the use of these techniques in the invention, as well as the straightforward extension that enables the insertion of variable sequences, is described below.
  • secreted and membrane-bound isomorphs can use the same amino terminal signal sequence, so in many cases, the only alteration required for conversion of a polypeptide to its isomorph is a change at the polypeptide ' s carboxy terminal. Attachment of a 20 residue hydrophobic sequence to the carboxy terminal of a secreted polypeptide is often sufficient to construct an isomorphic polypeptide that is anchored by a transmembrane span; this technique for converting a- secreted polypeptide into a membrane-bound one is described in the literature (Singer, 1990) .
  • transmembrane or secreted polypeptides can be converted to glycolipid anchored variants by addition of carboxy terminal signal sequences from the precursors of glycolipid anchored polypeptides; this technique is also described in the literature (Cross, 1990).
  • a glycolipid anchor not only attaches an exogenous polypeptide to a cell membrane, but also directs sorting of the anchored alien polypeptide.
  • a single glycolipid anchor sequence is all that is required to direct an exogenous polypeptide to the membrane, translocate it, and anchor it (Cross, 1990) .
  • two or more outer or inner membrane polypeptides are fused to construct a chimeric multipass polypeptide comprising extra- and/or intracellular domains of both constituent polypeptides.
  • This manipulation has been successfully performed with a variety of prokaryotic and eukaryotic polypeptides.
  • E. coli ompF-ompC chimeric protein 3601 the region of ompF comprising extracellular loops 1 through 5 is fused to the region of o pC comprising extracellular loops 6 and 7.
  • the extra- or intracellular domain of the membrane polypeptide contains a highly variable region, which is, upon binding to a target compound, identified and isolated as the polypeptide of interest or a section thereof.
  • a target compound identified and isolated as the polypeptide of interest or a section thereof.
  • the membrane polypeptide can be a homologous protein for the cell (adapted for the present method) or - which is preferred - a heterologous polypeptide for the host cells.
  • the membrane polypeptide is genetically engineered to contain the variable domain and the selection site residing adjacent to each other on the cellular surface.
  • the target compound can be any material, e.g. chemically produced substances, proteins, nucleic acids, complex products derived from biological sources or mixtures or (combinatorial) libraries of these materials.
  • Preferred substance classes for the target compound are known drugs (or derivatives thereof) (e.g. to investigate further binding patterns of such compounds to polypeptides) , polypeptides (to identify and isolate protein/protein interactions) , antibodies or antigens, vaccines or pathogens, etc..
  • the present invention is performed using an intracellular domain of the membrane protein, i.e. the polypeptide of interest is displayed at the inner surface (including the periplasma) , it is essential for the target compound to be present inside the cell. This can be achieved by transportation or diffusion of the target compound from outside into the cell (which is, however, only possible with a few or small target compounds.
  • An alternative way of bringing also the target compound into the cell is by coexpression of the polypeptide of interest and the target compound the same cell. Systems to enable such a coexpression of two (or more) exogenous proteins one and the same cell are known m the art. It is clear that this way of performing the invention is restricted to target compounds that can be produced by the host cell, e.g. proteins or nucleic acids.
  • any suitable selection system known m the art may be adapted to the method according to the present invention.
  • These selection systems are known to the skilled man in the art in principle, the adaption of any suitable selection system to the system according to the present invention will be explained below.
  • a critical point for the present invention is how the binding of the target compound with the polypeptide of interest influences the selection site:
  • the binding between the polypeptide of interest-portion of the membrane polypeptide and the target compound results m a signal which influences the status of the selection site.
  • This influence may be a direct or an indirect interaction of the membrane polypeptide or the bound target compound with the selection site.
  • a direct interaction may e.g. be performed by a ste ⁇ cal hindrance of the selection site upon binding of the target molecule, i.e. the site, where the selection agent should bind, is not accessible anymore, because of the physical presence of the target molecule.
  • such a sterical hindrance may be induced by a conformational change of the membrane polypeptide upon binding of the target compound to the polypeptide of interest-portion of the membrane polypeptide.
  • the selection site is present m the vicinity of the membrane polypeptide, e.g. by covalent bond to the membrane polypeptide.
  • the selection site can also be a part of the membrane polypeptide itself. In such a case, the sterical properties of the system are clearly defined and easily adjustable by common protein engineering.
  • a preferred embodiment of the present invention is a method, wherein the interaction between the extra- or intracellular domain with the polypeptide of interest and the selection site is a sterical interaction, especially a sterical interaction which does not allow the selection agent to interact with the selection site after binding of the target compound to the polypeptide of interest.
  • the selection site is immediately adjacent (especially via covalent bonds) to the extra- or intracellular domain of the polypeptide of interest.
  • the selection site is an adhesion domain for the selection agent, e.g. an adhesion domain which is also part of the membrane polypeptide displaying the polypeptide of interest.
  • the membrane protein exhibits an additional domain on the surface of the cell, which additional domain is adjacent to both, the variable domain and the selection site and this additional domain also interacts, preferably by binding, with the target compound.
  • Indirect interaction can be performed e.g. by messenger molecules, which are released upon binding and induces a change of the selection site by binding or by contact. It is essential that the change in selection specificity of the cell is in direct correlation to the binding of the target compound to the polypeptide of interest, i.e. the binding immediately induces the (direct or indirect) interaction with the selection site.
  • the mode of selection in the method according to the present invention is not critical, positive and/or negative selection protocols are applicable.
  • the selection agent is a toxic agent.
  • the polypeptides of the (outer) membrane are ports of adhesion for an array of toxic agents that kill or retard multiplication of the cells they enter.
  • the term "toxic agent” denotes any agent that impairs or inhibits growth of the cells.
  • Most of the classical selection systems applied molecular biology fall under this type of selection system. Due to its nature, this selection system is preferably applied the pupilextracellular" embodiment of the present invention, wherein the polypeptide of interest is located m an extracellular domain of the membrane polypeptide.
  • Binding by the agent to the adhesion domain is the first step m a process that culminates in cell lysis or inhibition of cell growth and multiplication. If this first step is impeded by mutations m the adhesion domain, by repression of the receptor, or by the interference of an inhibitor occluding the adhesion domain, a selection agent's invasion of the cell is blocked.
  • the cell is rendered immune.
  • the cell may become resistant but not immune: reduction of the number of receptors may slow the entry of agents, and thus reduce the rapidity or intensity of the agent's harmful effects.
  • a selection agent may be employed that has been alterered such that it is different from the corresponding naturally occurmg agent with regard to specificity and mode of action on the cell.
  • pathogenic agents like bacteriocins and bacteriophages - for which the above mentioned pathway via an adhesion domain is quite common - can be altered such that they change their preference for a specific adhesion domain.
  • design strategies to make pathogens adhere to a great variety of adhesion domains.
  • the selection agent is a pathogenic agent, such as a bacteriocm, a bacteriophage or a toxin or virus of an eukaryotic cell.
  • Bacteriocins are bacterially produced proteins that are harmless to the strains that express and secrete them, but fatal to other, often closely related, bacteria (bacteriocins are reviewed m Pattus et al., 1990) .
  • the most extensively studied of the bacteriocins are the colicins, which are expressed by E . coli , and are encoded by plasmids that also code for the proteins that confer colicm immunity upon colicm-producmg cells.
  • the colicins are composed of three structural domains, each of which is responsible for one of three consecutive steps of colicm activity: l. recognition of an adhesion domain; n. translocation across the cellular membrane; and in. cell destruction (Pattus et al . , 1990).
  • the colicins kill by two principal mechanisms: l. the formation of a pore m the cytoplasmic membrane, resulting m membrane permeabilization and de-energization (colicins El, A, B, la, lb, and K) ; n. enzymatic cleavage of DNA or 16S ribosomal RNA (colicins E2 and E3) (Pattus et al . , 1990).
  • the kinetics of cell death when bacteria are exposed to colicins suggest that the toxic molecules enter the cell in discrete, fatal doses, which may comprise only a single molecule, but may also consist of a number of active molecules imported one concerted step (Pattus et al . , 1990) .
  • the important point here is that a single dose kills a single cell.
  • the adhesion domain of colicm A is somewhat different. Loops 1 through 5 are required for colicinA to bind its receptor, while L6 and L7 are superfluous (Fourel et al., 1990).
  • the chimeric ompF-ompC protein 3601 which was described above, comprises loops 1-5 from ompF and loops 6 and 7 from the distinct ompC .
  • E. coli SM1005 expressing 3601 are totally susceptible to colicm A, but totally immune to colicm N.
  • the number of receptors per cell is important for an easy handling of this invention's selection of specific cells from a highly variable population.
  • the functional receptors of colicins are generally present on the surface of the cell thousands of copies.
  • Bacteriophages are a ubiquitous and extraordinarily diverse group of viral parasites of bacteria. There are twelve families of phage, comprising 3500 distinct isolates of known morphology, and infecting over 100 bacterial genera. Their vast variation is evident even at the level of nucleic acid: depending on species, phage contain single stranded DNA, double stranded DNA, single stranded RNA, or double stranded RNA. A wide range of morphologies is usually divided into four categories: tailed; cubic; filamentous; and pleomorphic. Extensive reviews of morphology, genetics, ecology, and life- cycle are available in the literature (Calendar, 1988).
  • phages bind to specific adhesion domains of receptors on the host surface.
  • a single outer membrane protein may be the receptor for multiple phage species, whose adhesion domains overlap to varying extents.
  • Figure 3 shows the adhesion domains of various phages, as well as one of the colicins, that utilize E. coli outer membrane protein A as their receptor. The figure also shows the receptor domains that are manifestly not utilized by the phages: alterations to these regions of the receptor do not affect phage adsorption, and thus do not confer phage- resistance to the bacterial cell.
  • Phage receptors like those of bacteriocins, are often present m 100s to 1000s of copies per cell, but this number can be manipulated through use of a plasmid-encoded receptor regulated by an mducible heterologous promoter. As was stressed above, regulation of the level of expression of the imprinted polypeptide is important to this invention. Protocols for the construction and manipulation of plasmids with inducible promoters are described m the literature.
  • phage multiplication commences.
  • the strategies for multiplication, and the effects of multiplication upon the host vary widely across phage species. Immediately following infection, some but not all species of phage confer immunity to subsequent infections by other individuals of the same phage species. If the infecting phage is a virulent one, it quickly modifies normal bacterial syntheses in order to appropriate them to its own multiplication and release into the extracellular space. Distinct strategies for release have very different effects upon the host.
  • the Inoviridae (a family of filamentous phages) are liberated by extrusion: new phage are secreted through the bacterial membrane (Calendar, 1988).
  • the bacteriophages exert such a wide range of effects upon their hosts that even among phage species using the same receptor there may be several different consequences of infection. Moreover, the effects of a phage upon its host can be manipulated. Mutagenesis followed by artificial selection for phage virulence or benevolence can intensify or mitigate a phage ' s deleterious effect on its host. Insertion or deletion of phage genes, including insertion of genes that confer metabolic capabilities or antibiotic resistance upon infected bacteria, are commonly practiced means of manipulating the impact that phage have upon their bacterial hosts. Another way to modify a phage ' s effects upon its host is to alter the host. For example, certain groEL mutations m E .
  • coli produce bacterial strains that are susceptible to T4, T5, lambda, and 186 phage infection, but do not permit the intracellular assembly of mature phage particles (Zeilestra-Ryalls et al . , 1993) .
  • infected bacteria are retarded, or even killed, but they do not yield infectious phage particles; m short, these bacteria are a dead-end for the phage that infect them.
  • the effects of phage upon their hosts can be modified by the preparation of phage ghosts (phage depleted of DNA) , by methods described the literature (Boulanger et al . , 1988) . Because they do not carry a genome, phage ghosts do not infect cells.
  • Toxins and viruses of eukaryotic cells recognize specific adhesion domains of their receptors on eukaryotic cell- surfaces.
  • the receptors of at least thirty different species of virus of eukaryotic cell lines, representing eleven different viral families, are described in the literature, and for most of these pathogens, the viral adhesion domains, which may comprise oligosaccharides, glycosylated peptide sequences, or sequences unadorned by sugars, have been characterized (Tyler et al., 1996).
  • the receptors of eukaryotic pathogenic agents like those of bacteriocins and bacteriophage, are commonly cloned onto vectors and expressed from inducible promoters, allowing manipulation of the number of receptors expressed per cell (Staunton et al . , 1990).
  • HRVs Major group human rhinoviruses
  • HRVs provide an example of the pathogenic agents that affect eukaryotic cells and are suitable for use in this invention.
  • HRVs are members of Pi cornaviridae, small protein-encapsidated viruses with RNA genomes. Of 100 serotypes of rhinovirus, 89 use an extremely well-characterized glycoprotein, ICAM-1 (CD-54) as their receptor. HRV serotype 14 is well studied , easily propagated in the lab, and therefore suitable for use in this invention (Staunton et al . , 1990, Couch, 1996).
  • Major group HRVs grow efficiently in many different lines of human cell culture. The most commonly used tissues are WI-38 diploid fibroblast, fetal tonsil, MRC, HeLa and COS cell lines.
  • HRVs form plaques on monolayers of a variety of cell lines exposed by any one of the described techniques for exposure to viral agents. Infected cells yield 10 to 200 plaque forming units per cell after 10 to 12 hours of incubation (Couch, 1996) .
  • the structure of HRV14 has been solved by X-ray crystallography (Staunton et al., 1990) .
  • the capsid is 30 n in diameter, exhibiting icosahedral symmetry and a canyon, 12- 30 A wide and 25 A deep, that runs along the 5-fold axis and contains the pathogen's receptor binding site.
  • the receptor is the well-characterized member of the immunoglobulin superfamily, ICAM-1 (CD-54), a 532 residue, 19nm x 2-3 nm rod comprising 5 immunoglobulin-like domains arranged in a line with a hinge between domains 2 and 3 (figure 4) .
  • Viral binding involves insertion of the distal immunoglobulin domain (Dl) into the viral canyon.
  • ICAM-1 bears multiple N-linked oligosaccharides, and variation in glycosylation between cell-types results in ICAM- l's molecular mass heterogeneity of 76 to 114 kD. It functions as the cell-surface ligand for lymphocyte function-associated antigen 1 (LFA-1), and binding between ICAM-1 and LFA-1 contributes to leukocyte adhesion in several immunological and inflammatory processes (Staunton et al . , 1990). Cytokines secreted by T-lymphocytes and monocytes, including IFN-g, TNF- a and b, and IL-a and b, induce expression of 3.5 x 10 6 copies per endothelial cell. However, uninduced cells exhibit much lower levels of ICAM-1, and in the CDM8 expression system,
  • ICAM-1 can be exogenously regulated through control of an inducible promoter (Staunton et al . , 1990) which makes this system excellentely suitable for the function of this invention.
  • the method is carried out in the form of a high-throughput screening assay wherein said cell population is exposed to a multiplicity of target compounds from which an unknown binding partner for a polypeptide of interest is to be identified and isolated.
  • the target compound in this embodiment may be a single definite compound, a mixture of compounds or, to have a large variety of binding possibilities tested, a (combinatorial) library of compounds .
  • Such a high-throughput assay is especially suited for screening of organisms for which comprehensive or complete genomic information is available, such as Mycobacterium tuberculosis or Helicobacter .
  • the method according to the present invention is usually carried out by performing steps a) to f) as described above. It may be necessary to repeat the selection process, e.g., with an increase in the selection pressure. This is suitable if, e.g., the number of isolated, positive clones is too large to handle.
  • steps b) through e) at least once, preferably comprising an increase in the selection pressure in the repeated steps.
  • the method can be carried out by steps b) through e) until the cell population comprises a desired number of distinct clones .
  • background is used to refer to cells that fail to bind the target compound but nonetheless remain in the selected population. In general, it is desireable to minimize the number of background cells, so that CISTEM reliably eliminates cells whose imprinted domains fail to bind the target compound, while selecting those cells whose imprinted domains do bind the target.
  • the CISTEM cell population is supplemented with "helper cells", whose function is propagation and amplification of the selective agent. This amplification of the selective agent increases the intensity of selection, and thereby reduces residual background cells.
  • helper cells lack a selectable marker that is present in the rest of the CISTEM cell population; this permits efficient elimination of the helper cells after they have served their purpose of amplifying the selective agent.
  • Supplemental cells may also be used in other ways to regulate the action of the selective agent.
  • heat-killed cells bearing the agent's receptor may be added to the CISTEM cell population. These cells bind the agent, removing them from selective activity in the remaining CISTEM cell population.
  • step d) When repeating the steps, new sequence variations can be introduced in the variable regions of clones selected in step d) before steps b) to e) are repeated.
  • the screening test may also be (additionally) performed by functional parameters.
  • the polypeptide of interest binds to the target compound and affects its function. Then, the polypeptides exhibited by such clones selected in step d) and amplified in step e) are screened by their ability to affect the function of the target compound.
  • the method according to the present invention comprises a step wherein the cell population is placed in a nutrient medium in which each cell's supply of a vital compound depends upon that cell's variable region acting enzymatically upon a compound structurally similar, but not identical to the target compound .
  • This invention provides a method for identifying and isolating a polypeptide of interest or a target compound or a gene of said polypeptide or target using the detection of the interaction between a polypeptide of interest and a target compound.
  • at least one of the interactants is unknown.
  • the method allows for identification of the unknown interaction partner, i.e. a polypeptide of interest and/or the target compound.
  • both interactants may be unknown, e.g. when an unknown ligand, upon binding at a receptor site, exerts a certain effect on the cell which needs to be antagonized or interfered with and both the ligand and the receptor domain to which it binds need to be identified.
  • CISTEMs an acronym for Cellular Imprinting by Selection: Teleological Evolution of Molecules.
  • a CISTEM applies selective pressure to a population of genetically engineered cells in order to amplify the frequency of cells in which a membrane polypeptide shows prescribed properties. This process is - as stated above - termed "imprinting" the polypeptide.
  • the binding of the target compound prevents the action of the selection agent via intramolecular interaction (in the membrane polypeptide), e.g. by occluding the selection agent from its adhesion domain.
  • This may comprise binding, adhesion, structural changes or chemical modifications of the variable domain which has an impact on the adhesion domain.
  • Such impact on the adhesion domain may, inter alia, be a conformational change of the adhesion domain that prevents the harmful action of the selection agent.
  • the invention is useful in at least three general areas.
  • the invention may be employed to identify polypeptides with properties that are useful in medical diagnostics, therapeutics, research, laboratory procedures, or other applications.
  • polypeptides that may be identified by the method disclosed here are polypeptides that interact with, in particular bind with high affinity and specificity to, virtually any given target compound, polypeptides that adhere to their respective target compounds at multiple epitopes, polypeptides that bind their respective target compounds but show no affinity for compounds closely related to the targets, enzymatic polypeptides, and polypeptides that show biological activities, such as antagonism or agonism of a receptor.
  • this invention may assist in the identification of the known compound's native interactants.
  • this invention may be used to analyse the interaction between a known polypeptide of interest and a known target molecule. By identifying which sequences in the polypeptide of interest interact with the target molecule, the invention assists in mapping the polypeptide epitopes that play a role in the interaction.
  • the method of the invention may be used to screen a population of target compounds and to identify those compounds that bind to a known polypeptide.
  • cells displaying a given section in the extra- or intracellular domain of the membrane polypeptide are used in a screening assay as test cells to screen for e.g. pharmaceutically active compounds that interact with the polypeptide of interest.
  • This invention may also be used to identify polypeptides with more sophisticated properties of interaction with the target compound than prescribed binding affinity.
  • the molecules that may be identified by this method are the following: polypeptides that exhibit multivalent binding; polypeptides that bind tightly to one molecule but show no affinity for a second, closely related molecule; polypeptides that affect the functional activity of the target compound to which they bind; and enzymatic polypeptides.
  • the invention is based on the interaction of three CISTEM components: (1) a genetically engineered population of prokaryotic or eukaryotic cells; (2) a target compound; and (3) a selection agent (figure 5) .
  • a genetically engineered population of prokaryotic or eukaryotic cells On the surface of each cell in the population, there are - at least - two polypeptide domains involved in the operation of the CISTEM: the cellular domain carrying either a given polypeptide whose interaction with an unknown target compound is to be determined or, in the case of an unknown polypeptide, the imprinted domain, and the selection site, e.g. the adhesion domain.
  • the imprinted domain is highly variable, because the CISTEM cell population is engineered to display up to at least 10 12 distinct oligopeptides at the imprinted domain.
  • the oligopeptides may be adorned by diverse oligosaccharides or other structures resulting from post- translational processing of the polypeptide
  • the imprinted domain on each cell's surface is neighboring the selection site, e.g. on an adhesion domain, the structure to which the CISTEM' s selection agent must bind in order to exert its effects upon the cell.
  • this may be an organism or molecule, and it may be fatal to cells or merely retardative of their growth, but the first step in the agent's destruction or retardation of a cell is necessarily binding between the agent and its adhesion domain.
  • the two important domains - namely, the imprinted domain and the adhesion domain - are expressed in multiple copies on the surface of each cell, but they are - in the case of direct interaction - normally adjacent to each other: more specifically, they must be sufficiently close to each other that the target compound, when interacting with the imprinted domain, either extends far enough over the adhesion domain that adhesion of the selection agent is at least partially impeded, or exerts its effect on the selection site in a different way (e.g. by intramolecular steric alteration of the selection site) .
  • the target compound is the molecule or set of molecules with which the polypeptide sequences identified by the CISTEM must interact in the prescribed manner in order to exhibit the properties or perform the functions desired of them.
  • the CISTEM identifies polypeptides that interact with, e.g. bind to, and optionally also structurally or functionally alter, the target compound.
  • a target compound can be any molecule, especially a protein, carbohydrate, saccharide, glycoprotein, proteoglycan, hormone, receptor, antigen, antibody, pathogen, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, small molecular compound, etc., without limitation.
  • Each CISTEM places only one requirement on the target compound: it must be effective in affecting the change in the selection site upon binding to the polypeptide of interest, e.g. that it is sufficiently large that its attachment to the imprinted domain results in occlusion of the adjacent adhesion domain or that its binding affects the structure of the membrane protein such that the selection site is sufficiently changed to prevent or to allow action of the selective agent.
  • each selected clone increases in frequency and in number as it continues to divide, while unprotected clones are afflicted by the selection agent.
  • the CISTEM selects and amplifies clones expressing structures that bind the target compound (figure 8) .
  • CISTEMs permit selection for a prescribed affinity or nature of interaction of the imprinted domain with the target. Multivalent binding may be selected, as may highly specific binding, binding at predetermined sites, or binding by specified structural motifs. Moreover, CISTEMs may be extended to select not only for binding, but also for functional activity; these extended CISTEMs may be used to create enzymes and other functionally active molecules.
  • CISTEMs for Cellular Imprinting by Selection: Teleological Evolution of Molecules.
  • a CISTEM consists of three components. They are: (1) an engineered population of cells; (2) a target compound; and (3) a selection agent (figure 5).
  • the CISTEM cell population may be prokaryotic or eukaryotic.
  • the unknown interactant is the polypeptide of interest
  • in the CISTEM cell population one extra- or intracellular domain (the "imprinted domain") of a membrane polypeptide (the "imprinted polypeptide") is engineered to exhibit highly variable peptide sequences.
  • the CISTEM cell population is eukaryotic, glycosylation sites within the imprinted domain may be adorned by variable complex saccharides. High sequence variability at the imprinted domain is achieved through the insertion of variable oligonucleotides at the sites in the polypeptide gene that correspond to translated regions in which variation is desired.
  • variable oligonucleotides may be cDNAs, random sequences, or any other form of oligonucleotides.
  • Techniques for inserting a gene for an exogenous oligopeptide at a targeted site in an outer membrane protein gene were described above, and an extension of these techniques, which allows the insertion of variable oligonucleotides (at least 10 5 to 1012 different sequences, instead of a single, stereotyped nucleotide sequence) , will be described in detail below.
  • a CISTEM' s selection agent may bind to an adhesion domain that is located immediately adjacent to the imprinted domain on the surface of the cells in the CISTEM population (figure 5) .
  • the adjacent extra- or intracellular domains in most of the membrane proteins used in this invention are separated by 3 - 30 A.
  • this arrangement of domains is the result of carefully choosing an imprinted polypeptide and a selection agent such that the imprinted domain is immediately adjacent to the adhesion domain.
  • membrane proteins are genetically altered in order to construct a hybrid polypeptide in which the imprinted domain is placed beside the adhesion domain.
  • the techniques for engineering membrane proteins, including the fusion of two separate polypeptides to form a chimeric polytopic polypeptide, were described above; their specific applications to embodiments of this invention are described below.
  • the target compound is any molecule or set of multiple, distinct molecules, which are simply added to the cell population according to the present invention or, in some embodiments of this invention, are secreted by the cells themselves.
  • the present invention identifies and selects cells in which the displayed polypeptide sequence interacts with the target compound, or some subset of molecules included in the target compound, and this interaction event is necessary to the functions to which the polypeptides are ultimately applied, such as enzymatic activity or alteration of the target compound's biological activity.
  • Each cell in the CISTEM population expresses multiple copies of its imprinted polypeptide, and binding is a dynamic process balancing on-rate and off-rate. Therefore even when a cell's imprinted polypeptide exhibits high affinity for the target compound, some copies of the imprinted polypeptide are left unprotected some of the time. Consequently, every cell in a CISTEM population, no matter how high its affinity for the target compound, has some probability of being hit by a selection agent. Nonetheless, this probability is lower when a cell's affinity for the target compound is higher.
  • each clone in a CISTEM is present in multiple copies (the CISTEM cell population is redundant) the probability that a single cell will be hit is also the fraction of that cell's clonal population that will be hit, and the differential elimination of clonal populations is the very basis of selection.
  • each selected clone increases in frequency and in number as it continues to divide, while unprotected clones are destroyed.
  • the CISTEM selects and amplifies clones expressing structures that bind the target compound (figure 8) .
  • the effect of the target compound relevant for the preferred embodiment of the invention, wherein the selection site is located on an adhesion domain is that, due to its size, it extends, upon interaction with the imprinted domain, from the imprinted domain to the adhesion domain.
  • This is 3 - 30 A for many imprinted polypeptides imprinted by this invention.
  • the interaction between the target compound and the imprinted domain may lead to a conformational and/or chemical change of the variable domain and/or the adhesion domain which prevents the selection agent from attachment.
  • the target compounds, their source, preparation, and use of CISTEMs with particular applications in this specific embodiment of the invention are described hereinafter. While the concentration of target compound in a CISTEM is also important to its function, this concentration depends on the nature of other CISTEM components, and is therefore discussed below.
  • the target compound is simply the molecule that is to be tagged and/or partitioned by the polypeptide.
  • the target compound is HIV gpl20, the extracellular subunit of a major HIV glycoprotein (Fantini et al., 1997).
  • the production of recombinant gpl20 in a soluble form that is suitable for use in CISTEMs is described in the literature (Fantini et al . , 1997) .
  • Another embodiment of the invention is used to design a diagnostic polypeptide for the direct detection of a different virus, Human T-cell Leukemia Virus type I (HTLV-1); here the target compound is the HTLV-1 surface glycoprotein gp46.
  • HTLV-1 Human T-cell Leukemia Virus type I
  • the target compound is the HTLV-1 surface glycoprotein gp46.
  • the production of authentic, glycosylated HTLV-1 gp46 in a recombinant vaccinia virus/T7 polymerase system is described in the literature (Arp et al . , 1996). Diagnostic assays for detecting human viral infection often bind not to the virus itself, but rather to the antibodies raised against the infectious agent.
  • One preferred embodiment of this invention imprints a polypeptide to exhibit high affinity for a monoclonal antibody, MAbT7.
  • the target compound in this CISTEM, MAbT7 itself, is commercially available (Novagene) .
  • the invention is also used to imprint polypeptides to serve as antagonists, agonists, or modulators of the functional action of biologically active molecules.
  • the target compound may be either of the molecules involved in the interaction to be antagonized.
  • HIV gpl20 is the viral glycoprotein that binds to the CD4 receptor, and thus mediates viral invasion of the human cell (Fantini et al . , 1997) .
  • the embodiment of this invention that imprints a polypeptide (ICAM-1, which is described above) to bind specifically to HIVgpl20 provides a polypeptide that could serve as an antagonist of the interaction between the viral glycoprotein and its host cell receptor.
  • polypeptides selected by a CISTEM are more likely to functionally antagonize a given molecular interaction when the CISTEM' s target compound is pared down, so far as is possible, to one interactant ' s active site. Structures on either interactant that are distinct and topologically distant from the active site are less promising as target compounds because a polypeptide that binds such structures may not interfere with the normal interaction of the active molecules.
  • one embodiment of this invention imprints a polypeptide (E. coli OmpA, which is depicted in figure 13/, and described further below) to function as an antagonist of the interaction between the eukaryotic cell-signalling proteins Ras and Raf (Block et al . , 1996).
  • Raf Activation of Raf, a protein kmase, by the immediately upstream protein, Ras, initiates pathways leading to the phosphorylation of transcription factors, the stimulation of numerous genes, and, ultimately, to cell proliferation and differentiation.
  • Constitutively active oncogenic mutants of Ras or Raf occur in a large number of human tumors (Block et al . , 1996) .
  • the target compound is the domain of Raf (ammo acids 51-131) that is known to be directly involved m binding to Ras (Raf's so-called Ras-bmdmg domain, or Raf-RBD) (Block et al., 1996).
  • Raf-RBD Ras-bmdmg domain
  • Raf-RBD rather than Raf m its entirety, as the target compound ensures that the polypeptides selected by the CISTEM bind to precisely that domain of Raf which interacts with Ras, rather than to Raf epitopes that are extraneous to the important cell-signalling interaction.
  • the target compound is the receptor or biomolecule that the imprinted polypeptide is intended to activate.
  • the target compound is a solubilized form of IL-6R: the expression of soluble human IL-6R in CHO cells, and the protein's purification, are described in the literature (Yasukawa et al . , 1990).
  • CISTEM selects polypeptides with affinity for IL-6R
  • a secondary screen is used to isolate polypeptides whose binding to the receptor results in transduction of the signal that is normally initiated by IL-6. This secondary screen is described in the passages relating to extensions of the basic CISTEM.
  • the target compound When this invention is used to create agonists, the target compound is pared, so far as is possible, to the active site of the receptor or biomolecule that is to be agonized. This is done because removal of extraneous epitopes from the target compound increases the probability that an imprinted polypeptide that binds the target will also be active as an agonist .
  • CISTEM is used to identify, or to create mimics of, specific antigens that are present in infectious pathogens or cancer. These antigens or antigen mimics are then often used to provoke a specific immune response against the pathogen or tumor.
  • the target compound is composed of more than one molecule: multiple monoclonal antibodies, specific immune receptors, such as cytotoxic T-lymphocyte receptors or MHC-I molecules, or polyclonal antisera derived from individuals exposed to a specific antigen, such as meningicoccal Opa proteins, to a specific pathogen, such as meningicocci, or to a tumor, such as melanoma.
  • Polyclonal antisera are divided from animals, human volunteers or actual patients. This composite target is used as an impression - a "negative", or “footprint", of the antigen - and from this footprint, the CISTEM is used as described in example 2 to identify the antigen or to create a mimic of the antigen.
  • An antigen-mimic is of interest, because a polypeptide that is imprinted to bind the antibodies composing sera against an antigen is likely to elicit an immune response very similar to the one provoked by the antigen.
  • Antibodies suitable for use as the target compound in a CISTEM for imprinting E are examples of the target compound in a CISTEM for imprinting E .
  • coli OmpA to mimic Neisserial protein Opa 5c include mAb B306, mAb A222/5b, and others described in the literature (Olyhoek et al . , 1991).
  • Antibodies for use as the target compound in a CISTEM for identifying tumor antigens are derived from the polyclonal serum of a cancer patient, the isolation of which is widely described in the literature.
  • polypeptides are imprinted to bind to transition state analogs.
  • the preparation and structure of these transition state analogs are described in the literature (e.g. Charbonnier et al . , 1995).
  • a phosphonate transition-state analog (structure and preparation described in Charbonnier et al . , 1995) is used as the target compound for imprinting E. coli OmpA to function as a catalyst of the hydrolysis of p-nitrophenyl esters.
  • the target compound is not a transition state analog, but rather a modified substrate.
  • the target is a molecule that is structurally very similar, but not identical, to the substrate upon which the imprinted polypeptide will ultimately act.
  • the reason for using a slightly modified structure, rather than the substrate itself, is the following: if the substrate itself were used as the target compound, imprinted polypeptides that not only bound the substrate, but also performed the desired enzymatic activity, would alter the substrate and then release it, rendering the underlying cell sensible to the selection agent. Thus, if the substrate itself were used as the target compound, the CISTEM would select against clones expressing polypeptides with the desired enzymatic activity.
  • the target compound must be an effective structural mimic of the substrate (i.e. polypeptides that bind the target must also be able to bind the substrate) , but it must be invulnerable to the prescribed enzymatic activity (i.e. polypeptides that act enzymatically on the substrate must be unable to act on the target compound) .
  • Imprinted polypeptides that show high affinity for a different peptide motif bind at most only a tmy fraction of the target polypeptides, and are therefore left vulnerable to the selection agent.
  • Several methods for the synthesis of this oligopeptide target compound are described m the literature
  • a secondary screen may be applied to isolate imprinted polypeptides that not only bind the target, but also perform the desired enzymatic reaction. Secondary screens, including the one used in the CISTEM that imprints polypeptides to hydrolyze the bond between two methionines, are described below.
  • this invention In addition to the creation of functionally active polypeptides, such as antagonists and enzymes, this invention's applications include interaction analysis (current techniques reviewed m Phizicky et al . , 1995). By screening a diverse library of polypeptides for sequences that interact specifically with a target compound of significant interest, this invention assists m the identification of the compound's native interactants. For example, although the structural interaction between Ras and Raf-RBD has been well- characterized through crystallography and mutational analysis
  • This CISTEM involves the following components: Loop IV of E.coli OmpA is used to display the polypeptides translated from fragments of cDNAs derived from a cell expressing Ras and Raf (the insertion of foreign oligonucleotides into the gene for OmpA is described below) ; Raf-RBD (see above for a more detailed description of this target compound and its preparation) serves as the target compound; and phage K3 serves as the (pathogenic) selection agent.
  • the appropriate concentrations of the target compound and the selection agent can easily be determined by the skilled man in the art after reading the present specification and are described below in the examples of OmpA imprinting CISTEMs.
  • This invention is also used for epitope mapping (Phizicky et al., 1995), serving to elucidate the surface of structural interaction between two molecules.
  • the target compound is one of the two molecules whose interaction is to be mapped.
  • the interactant that is most easily prepared in soluble form that serves as the target compound.
  • Raf-RBD serves as the target compound.
  • it is readily prepared in soluble form (Block et al., 1996).
  • one of the interacting proteins is dissected into multiple smaller domains which are displayed in the imprinted domain and selected by the CISTEM for their capability to bind to the other interactant.
  • multiple separate molecules compose the target compound.
  • Each molecule must meet the CISTEM' s size requirement and must be present in the CISTEM at a concentration sufficient to confer protection upon clones that bind to it with high affinity. (Selection by CISTEMs with multiple distinct target molecules strongly favors binding by imprinted domains to multiple distinct molecules, because the effective concentration of target compound rises significantly with each additional compound that the imprinted domain is capable of binding.)
  • the CISTEM assists m the identification of interacting pairs of molecules: polypeptides expressed m the imprinted domain are selected for their affinity for particular members of the library of molecules that composes the target compound.
  • the target compound consists of a library of polypeptides or phage-displayed polypeptides that are secreted by the CISTEM cell population itself.
  • the target compound is the interaction partner that is to be identified due to its interaction with a given polypeptide of interest, e.g. a section of a naturally occuring cell surface receptor which is displayed the extracellular domain.
  • the target compound is the unknown interactant.
  • the cells presenting the receptor domain of interest may be incubated with chemical compounds from a pool of substances. Surviving cells are indicators that the compound that has interacted with these cells is a candidate substance for interaction with the receptor domain.
  • cells are protected from the (pathogenic) selection agent m an autocrme fashion, due to binding between the imprinted domain and a polypeptide target that is secreted by the cell itself.
  • two separate libraries of polypeptides expressed from two separate libraries of genes, are screened for interaction with each other.
  • the cell population displays one library on the cell surface, exactly like the standard CISTEM cell population.
  • the cell population carries also a second library of genes. These genes reside in a vector that encodes a secreted polypeptide. Such vectors are described m the literature (Mollenkopf et al . , 1996) .
  • Each cell m the autocrme population displays one foreign polypeptide on its surface and secretes a second foreign polypeptide into the extracellular medium. In the event that these two polypeptides bind to each other, a cell protects itself from the pathogenic agent and is therefore positively selected.
  • polypeptide relates to all types of peptides or proteins synthesizeable by a host cell.
  • a polypeptide must comprise at least one extra- or intracellular domain that accepts exogenous oligopeptide insertions without disturbing translation or export of the polypeptide to the cell surface.
  • This capacity to accept insertions has been observed in numerous secreted polypeptides, which can be attached to the outer membrane by a genetically engineered anchor that is described m the literature (Smger, 1990; Cross, 1990) , as well as in each one of the dozens of bacterial, yeast, and mammalian membrane polypeptides that have been topologically mapped by gene fusion techniques.
  • a number of these polypeptides such as E. coli OmpA, E. coli LamB, and mammalian ICAM-1, their properties, and their capacities to exhibit exogenous sequences m extracellular domains, are described m the literature.
  • the membrane polypeptide may be autologous or heterologous for the cell.
  • the membrane polypeptide is heterologous.
  • all copies of the membrane protein gene a given clone must be altered m some wayFor example, if a plasmid- borne copy of the gene for the imprinted polypeptide bears a foreign insert at the imprinted domain, whereas the chromosomal copy of the gene remains wildtype, then proper selection by the CISTEM is hindered, because the selection agent and/or the target compound can bind to the wildtype copy of the polypeptide, as well as to the engineered copy.
  • the gene encoding the membrane protein is inactivated, e.g. by deletion. Standard molecular methods are available to the person skilled in the art.
  • Exogenous insertions at an extra- or intracellular domain are involved in two stages of each CISTEM.
  • an oligopeptide tag is inserted into the imprinted domain, and cell populations exhibiting this tag are used m preliminary rt ⁇ T ⁇ tr t*J 3 TJ o 3 ⁇ ⁇ Hi 3 P- ⁇ - ⁇ ⁇ TJ ⁇ cn C ⁇ PJ TJ ⁇ - O rt tr o ⁇ - TJ TJ TJ cn TJ ⁇ - rt
  • an amber-suppressing host such as E. coli UH203
  • CDM8 cDNA expression vector pCDl.8 may be used in conjunction with the COS cell expression system (Staunton et al . , 1990).
  • the vectors bearing the gene for the imprinted polypeptide are replicated and isolated. They may be replicated by a cell population, by PCR techniques, or by any other commonly employed methods described in the literature.
  • 10 to 10 copies of the vector are isolated. The exact number depends on the number of distinct clones that will compose the CISTEM cell population, and on the technique used in the step described below, the insertion of the variable oligonucleotides into the vectors .
  • the gene for a well-characterized oligopeptide tag is inserted into the vector at a genetic site corresponding to the imprinted domain of the polypeptide.
  • the oligonucleotide tag may replace a nucleotide sequence of the naturally occuring sequence. Replacement is accomplished by excising a natural coding region and by inserting the oligonucleotide tag in place thereof.
  • the tag may be any peptide sequence that is known to bind with high affinity and specificity to a ligand that is commercially available or easily produced in the laboratory by techniques described in the literature.
  • One example of a commercially available tag/ligand pair is the T7 sequence (Met -Ala -Ser-Met -Thr-Gly-
  • any common technique for inserting an oligonucleotide at a specific site m a vector can be used (e.g. Boyd, 1994). Several such techniques are referenced above, and one preferred procedure is described here m more detail.
  • the vector is cleaved at the two designated sites by digestion with restriction endonucleases Rl and R2, following the protocols and under the conditions specified by the suppliers of the enzymes. This yields a linearized vector with non- complementary sticky ends (RSI and RS2) and an excized fragment.
  • the linearized vector is isolated by standard electrophoretic size fractionation .
  • oligonucleotide template comprising the gene for the tag is synthesized using conventional technology, such as phosphoramitide chemistry, or can be obtained from commercial laboratories offering custom oligonucleotides.
  • the oligonucleotide is synthesized to read 5 ' -P--Rl--tag gene--R2- -3', in which P is the primer sequence for a proof-reading polymerase, such as Vent polymerase (New England Biolabs) , Rl and R2 are the restriction endonuclease sites mentioned above, and --tag gene-- is the genetic sequence encoding the oligopeptide tag.
  • oligonucleotide template is used to generate a double stranded oligonucleotide.
  • This oligonucleotide is digested with restriction endonucleases Rl and R2 under conditions described by the enzyme suppliers, yielding an oligonucleotide with sticky ends RSI' and RS2 ' .
  • This oligonucleotide which has sticky-ends complementary to the linearized vector, is ligated into the vector by a common ligation procedure familiar to those practiced in the art.
  • the result is that the vector's gene for the imprinted polypeptide bears the gene for the oligopeptide tag at a genetic site corresponding to the imprinted domain.
  • Oligonucleotide 1 5' - CG[ATGGCTAGCATGACTGGTGGACAGCAAATGGGT] - 3'
  • Oligonucleotide 2 5' - CTAG[ACCCATTTGCTGTCCACCAGTCATGCTAGCCAT]CGAGCT - 3'
  • the sequence in brackets comprises the gene segment encoding the oligopeptide that is recognized by the T7 antibody.
  • These two single-stranded oligonucleotides were annealed in MBI Fermentas Klenow buffer, forming the double stranded T7-tag gene segment with sticky ends complementary to the sticky ends on pEV218, linearized by Sad and Xbal, as described in the previous paragraph.
  • the double-stranded oligonucleotide was ligated into the linearized plasmid, the T7 tag gene segment was in frame with the ompA gene on pEV218.
  • the new construct was designated pEV218T7.
  • the vector is used to transform a population of cells lacking a chromosomal copy of the gene for the imprinted polypeptide.
  • Strains lacking chromosomal genes for outer membrane polypeptides are described in the literature (e.g., Brown, 1992, Freudl, 1989, and Staunton et al . , 1990), as well as below, in the passages relating to cell lines and media.
  • Conventional methods of transformation are used, and transformants are selected by their expression of the vector- borne selectable marker, such as a gene for ampicillin resistance.
  • E in which E .
  • coli OmpA was engineered to display the T7 polypeptide tag at loopIV, pEV218T7 was used to transform E . coli strain UH203 ⁇ l a c, s upF, ompA, recA, proAB, rpsL) (Freudl, 1989), according to a common method of electrotransformation, as follows.
  • variable oligonucleotides that are inserted into the vector, and, ultimately, translated into the variable regions exhibited by the CISTEM cell population.
  • the variable oligonucleotides must have conserved sequences at their termini, just as the tag gene described in the previous section was given defined termini RSI and RS2.
  • variable oligonucleotides with defined sequences at their termini are easily synthesized using solid-phase phosphoramidite chemistry (Brown, 1992): at defined terminal positions, only one of the four nucleotides is made available for addition to the strands being synthesized, whereas at variable positions, a mixture of multiple nucleotides is presented to the strands.
  • end addition of bases catalyzed by terminal transferase m the presence of nonlimitmg concentrations of multiple nucleotides, can append a highly variable sequence to a defined terminal.
  • Highly variable sequences may also be obtained by selecting fragments of defined length from digestions of natural cellular DNA or RNA preparations.
  • variable oligonucleotides are the cDNA library genes.
  • the size of the cDNA library that is screened by such a CISTEM is in some embodiments of this invention reduced by limiting the screen only to genes of interest, as determined by comparison of the cDNA library genes to sequences catalogued in genomic libraries.
  • cDNAs are prepared from tumor cells according to protocols described in the literature.
  • variable oligonucleotides are sections of the gene encoding the known polypeptide.
  • the oligonucleotides inserted into pEV218 are sections of the sequence encoding Ras.
  • stop codons are excluded from variable oligonucleotide inserts in order to prevent truncation of the imprinted polypeptide. This is achieved by synthesizing oligonucleotides to read Dl(NNP) n D2, where Dl and D2 represent the defined sequences that facilitate insertion of oligonucleotides into vectors; n represents the number of triplet codons that compose the variable section of each oligonucleotide; N represents all four nucleotides, each with equal probability; and P represents a subset of nucleotides, each with equal probability. NNP encodes all 20 amino acids, but precludes the occurrence of stop codons recognized by the CISTEM cell population.
  • the stop codons TAA and TGA are excluded from the oligonucleotide inserts. It is not necessary to preclude the occurrence of all three stop codons in inserted oligonucleotides, because the CISTEM cell population may be composed of stop codon- suppressing mutants, which are capable of translating one or more of the stop codons. Numerous suppressor strains are described in the literature (e.g. Brown, 1992, Freudl, 1989).
  • the inserted oligonucleotides may include consensus sequences for N-linked glycosylation (Chavez et al . , 1991) . These are synthesized as defined sequences interrupting the variable sequence of each oligonucleotide.
  • variable oligonucleotide inserts may include a mixture of defined and variable positions, sequences coding for fixed motifs, or statistical biases toward certain sequences or structures.
  • variable oligonucleotides are inserted at a prescribed site m the vectors constructed and amplified by the methods described above. Two preferred procedures for precisely targeted insertion are described here-one m which the number of oligonucleotides inserted into each vector is defined, and another in which the number of inserts is variable.
  • the available procedures for inserting oligonucleotides at specific vector sites are certainly not limited to the two described here. In fact, any of the gene fusion techniques used to insert a gene coding for a reporter moiety into the gene for a membrane protein may be used to the same effect as the procedures that will be described.
  • the site of insertion m the vector is located such that oligonucleotides inserted there are translated as part of the imprinted domain.
  • the site of insertion may be further specified (i.e. withm the sequence encoding the imprinted domain) according to structural analysis or prescriptions for a particular nature of interaction between the imprinted domain and the target compound.
  • both of the procedures outlined here make use of restriction endonuclease sites that are unique m the vector, and are located at or near the prescribed site of insertion. If a unique restriction site does not already exist at the site of insertion, one may be engineered using any one of a number of procedures described in the literature (e.g. Boyd, 1994, Brown, 1992 and Freudl, 1989) .
  • variable oligonucleotide To insert one and only one variable oligonucleotide per vector, two distinct restriction sites, each unique m the vector, must be located such that they flank a short restriction fragment comprising the prescribed site of insertion. If the sticky ends left by cleaving the vector with one endonuclease (Rl) at its unique site are RSI and RSI', while cleaving with the other endonuclease (R2) at its unique site leaves sticky ends RS2 and RS2 ' , then the variable oligonucleotides generated at step 3 are given defined and conserved sticky ends RSI and RS2 ' .
  • the vectors are digested by both restriction nucleases (Rl and R2 ) , and the digestion products are size-fractionated to isolate the vectors, each of which has non-complementary sticky ends RSI' and RS2.
  • the population of restriction-digested vectors is combined with a slightly smaller population of variable oligonucleotide inserts (vectors/insert » 1.5 - 2.0), and ligation is allowed to occur. Because the sticky ends of the vectors are non- complementary to each other, each vector can neither ligate shut, nor ligate to other vectors.
  • oligonucleotide inserts have non-complementary sticky ends RSI and RS2', they can neither self-ligate into loops, nor ligate to each other to form concatamers of multiple oligonucleotides.
  • the only ligation that can occur is the insertion of one variable oligonucleotide into each vector. After ligation has taken place, the vector population is size- fractionated to discard vectors that did not receive an insert. In the resultant vector population, each vector has one variable oligonucleotide at the prescribed site in the region encoding the imprinted domain.
  • This plasmid is digested with Sad (enzyme and reaction buffer from New England Biolabs) and Xbal (enzyme and reaction buffer from MBI Fermentas), under conditions described by the suppliers, yielding a fragment of the restriction site polylmker and the linearized plasmid.
  • the linearized plasmid is isolated by standard gel electrophoresis .
  • Single stranded oligonucleotides are synthesized by conventional technology to read 5'GGTCTAGA(VNN) n CGAGCTC3 ' , in which N represents all four nucleotides, each with equal probability; and V represents C, G, or A, each with equal probability.
  • a 5' primer and a proof reading polymerase such as the Vent polymerase, are used to generate double-stranded oligonucleotides from the single-stranded templates.
  • a proof reading polymerase such as the Vent polymerase.
  • These double-stranded, highly variable oligonucleotides are then digested with Sad and Xbal, under conditions described by the suppliers, yielding highly variable oligonucleotides with non-complementary sticky ends 5'-CTAG and AGCT-3'.
  • the highly-variable oligonucleotides are then ligated into the linearized plasmid (MBI ligation buffer, conditions described by supplier) ; they ligate only one oligo per plasmid, and in only one orientation.
  • Conventional electrophoretic size fractionation is used to isolate ligated plasmids containing an insert.
  • the product of this procedure is a population of plasmids (pEV218I) bearing, at the genetic site corresponding to loop IV of OmpA, a highly variable exogenous sequence free of TAA and TGA stop codons.
  • a restriction site that is unique in the vector is located at the prescribed site of insertion, i.e., at a genetic site corresponding to the imprinted domain of the imprinted polypeptide.
  • a unique restriction site does not already exist at the desired site, one may be engineered by conventional methods described m the literature, such as oligonucleotide-directed mutagenesis (e.g. Brown, 1992 and Freudl, 1989) .
  • variable oligonucleotides generated by the procedures described above are given defined and conserved complementary sticky ends RS and RS ' .
  • the vectors bearing the gene for the imprinted polypeptide are cleaved by R.
  • the vectors are then combined with an excess of variable oligonucleotide inserts. Several distinct ligation events can occur. First, the vectors, which have complementary sticky ends RS and RS ' , can ligate to other vectors or self-ligate, closing without an oligonucleotide insert.
  • variable oligonucleotides which also have complementary sticky ends RS and RS ' can l) self-ligate as a loop, n) ligate into a vector with sticky ends RS and RS ' , m) ligate to each other, forming a multi- oligonucleotide concatenation, which itself can perform ligations 1, 11, or m.
  • size fractionation can be used to discard all inserts that did not ligate into a vector and all vectors that did not receive inserts. This yields a population of vectors, each of which harbors between 1 and n variable oligonucleotides at the prescribed site m the gene encoding the imprinted polypeptide.
  • the vectors bearing variable oligonucleotide inserts are used to transform a population of cells that lack a gene for the imprinted polypeptide. Conventional methods of transformation may be used. Transformants are selected by their expression of the vector-borne selectable marker, such as a gene for antibiotic resistance. (Efficient transformation
  • the product of transformation is a population of cells, each of which harbors a distinctive oligonucleotide insertion at a genetic site corresponding to a point m the imprinted domain. After transformation, the cell population is amplified by growth a suitable medium.
  • m which the CISTEM cell population is engineered to display variable oligopeptides at loop IV of E. coli OmpA
  • transformation is performed m the following manner.
  • the variable plasmid population, pEV218I whose construction was described above, is used to transform E . col i strain UH203 ⁇ lac, supF, ompA, recA, proAB, rpsL) , according to a common method of transformation described in the literature (Freudl, 1989) .
  • the UH203 population is grown Luna broth supplemented with 100 mg Ap/ml, selecting for Ap resistance of transformants.
  • the imprinted polypeptide is induced with the compound appropriate to the vector's promoter.
  • the population of UH203 transformed with pEV218I is induced with lm IPTG) .
  • Each clone displays a distinctive oligopeptide at the imprinted domain. If the imprinted polypeptide is a glycoprotein or a proteoglycan, glycosylation sites in the imprinted domain bear diverse and unpredictable oligosaccharides.
  • a CISTEM is constructed wherein the imprinted polypeptide is a polytopic membrane protein in which the domain that accepts insertions is directly adjacent to the adhesion domain of a selection agent suitable for use in the CISTEM.
  • This preferred configuration is depicted in figure 5, and several of the polypeptide/selection agent combinations that exhibit this configuration are listed in table 1, and in the literature (Tyler et al . , 1996).
  • the CISTEM is assembled with a minimum of genetic manipulation of the imprinted polypeptide; the only necessary manipulation is the introduction of an oligopeptide tag, as well as variable peptide sequences, to the imprinted domain, as described above.
  • the list of polypeptides that may be imprinted is by no means limited to polytopic proteins exhibiting adjacent adhesion and imprinted domains.
  • two genetic manipulations are employed.
  • secreted proteins When secreted proteins are imprinted, they are first converted to their glycolipid- or transmembrane-anchored isoforms. Because translocation of secreted proteins and membrane insertion of integral proteins are handled by the same cellular machinery (reviewed in Singer, 1990, and above) , many secreted proteins may be converted to membrane-bound isoforms simply by carboxyl-terminal attachment of a hydrophobic transmembrane span or a signal sequence for glycolipid addition.
  • the gene-fragment encoding the 37 carboxyl terminal amino acids of decay accelerating factor can be fused to the gene for a secreted protein, such as human growth hormone, in order to target and anchor the protein to the apical surface of human intestinal cell lines.
  • a secreted protein such as human growth hormone
  • heparin-binding epidermal growth factor-like growth factor can be expressed as a longer, membrane-spanning isoform (HB-EGFTMII) comprising an extracellular domain that is structurally identical to the secreted polypeptide (Onu et al., 1994).
  • HB-EGFTMII membrane-spanning isoform
  • Other examples of and techniques for the conversion of secreted polypeptides into isoforms anchored by transmembrane spans are described in the literature (Singer, 1990) .
  • a second genetic manipulation of the imprinted polypeptide is performed when the domain that is to be imprinted lacks an adjacent adhesion domain. This occurs e.g. when the polypeptide chosen for imprinting is not polytopic, or when the selection agents that adhere to domains adjacent to the imprinted domain are unknown or poorly suited for use in the CISTEM.
  • the sequence for a functional adhesion domain may be fused to the gene for the extra- or intracellular domain, creating a gene for a chimeric polypeptide comprising both the functional adhesion domain and the variable domain.
  • the colicin A adhesion domain from E.
  • coli OmpF when fused to separate domains from another outer membrane protein, OmpC, remains functional as a colicin A receptor (Fourel et al . , 1990) .
  • a group of eukaryotic viral systems which are described in the literature (Chu et al . , 1995), provide further examples of the insertion of functional adhesion domains. In their application to embodiments of this invention, these systems operate as follows.
  • the antigen-binding site of an antibody (scA) is displayed on the surface of a virus, such as spleen necrosis virus, as described in the literature (Chu et al., 1995).
  • the antigen that is recognized by the scA is then expressed as an exogenous insert in the extracellular domain that will serve rt ⁇ TJ tr ⁇ ⁇
  • type la polypeptides are generally competent for functions involving high-affinity adhesion or specific binding to extracellular targets. They are therefore well-suited to be imprinted for use as highly specific markers or antagonists of small polypeptides, such as small peptide hormones.
  • Type II polypeptides are often enzymes with extracellular active sites, such as glycosyl transferases . Some type II polypeptides, then, are well suited to be imprinted to exhibit enzymatic activity. When this invention is used to imprint a polypeptide to serve as a functionally active effector, the preferable imprinted polypeptide is usually a membrane-bound isoform of a secreted protein, such as human growth hormone.
  • the imprinted polypeptide is a molecule that naturally exhibits functional or structural homology with molecules that perform functions similar or identical to the one desired of the imprinted polypeptide.
  • structural homologues to E . coli OmpA have been found in Haemophil us f Shi gella , Nei sseri a , Bordetell a , and Streptococcus . In these virulent bacteria, the OmpA homologues play a role in adhesion to host tissues, and are often recognized by the host's specific immune response.
  • OmpA The structural homology between OmpA and these antigenic adhesins makes OmpA well-suited for imprinting for use in applications requiring mimicry of homologous adhesins. Two such applications are competitive inhibition of microbial adhesion, and vaccination, described in greater detail below.
  • this invention can screen libraries of polypeptides expressed by eukaryotic cells.
  • the oligo- and polysaccharides adorning the imprinted domain are not varied in the same systematic manner as the underlying polypeptide sequence, because, as was described above, the saccharide molecule attached to a particular site on a polypeptide is not a simple function of the local amino acid sequence.
  • glycosylation at a site is altered in complex and unpredictable ways by changes to the local sequence or to the sequence elsewhere in the polypeptide (Lis et al., 1993). Therefore, when the glycosylation sites within the imprinted domain are preserved while immediately flanking regions are varied by the method described above, a semi- random diversity of sugar structures is appended to a systematic diversity of polypeptide sequences, creating a complex glycoprotein or proteoglycan library.
  • CISTEM' s target compound When a CISTEM' s target compound naturally interacts with processed, e.g. glycosylated polypeptides (i.e., in its natural setting, the target compound binds to the processed forms, e.g. to the glycoproteins or proteoglycans), it is preferred that the imprinted polypeptide be processed, especially glycosylated.
  • the CISTEM cell population be eukaryotic.
  • the adhesions that underpin inflammation, blood clotting, sperm-egg interactions, and intercellular recognition are all mediated by glycoproteins or proteoglycans. Therefore, for the identification of molecules for use as antagonists or agonists in these processes, CISTEMs with eukaryotic cell populations and glycosylated imprinted domains are preferred.
  • the CISTEM' s selection (pathogenic) agent must not require a specific glycosylation on its adhesion domain. Otherwise, unexpected changes in the sugars adorning the adhesion domain renders some cells immune to the pathogenic agent even if they do not bind the target compound at their imprinted domains.
  • the selection site especially an adhesion domain of a pathogenic agent, used in a CISTEM is well-characterized. In most cases, this characterization reveals whether or not the agent requires glycosylations for adhesion. This may be verified, moreover, by removing glycosylations from the receptor and testing for susceptibility to the agent; methods for removing glycosylations are described in the literature (Lis et al., 1993) .
  • polypeptides that exhibit structural integrity are preferred. It is particularly preferred that they not only maintain their properties when liberated from the cell surface, but also exhibit stability in the environmental- perhaps in vivo-conditions in which the imprinted molecules are ultimately applied.
  • prokaryotic polypeptides ⁇ -barrels are exceptionally stable; they can be denatured only by boiling in strong detergents.
  • glycosylation often adds structural stability to otherwise malleable protein structures (Lis et al . , 1993).
  • the structural integrity of imprinted domains may be heightened by genetic manipulations. Such manipulations may be performed before or after imprinting, and they may be used to increase the stability of structures that will be attached to, or liberated from, the membrane.
  • genetic manipulations of the imprinted polypeptide may also be used to introduce an element of rational structural design to a CISTEM.
  • Structural analysis of the target compound, its native ligands, or molecules that naturally perform functions similar to that desired of the imprinted polypeptide may guide the insertion of predetermined motifs into the imprinted polypeptide.
  • a tendency toward the formation of certain structural motifs, such as ⁇ -helices may be incorporated into imprinted domains by inserting variable oligopeptides that are not entirely random, but instead are statistically biased toward sequences that form the desired motifs.
  • Predetermined motifs may also be inserted into the conserved regions of imprinted domains.
  • polyproline or a-helices may be used to extend a variable region away from the cell surface, making it more accessible to a sunken epitope on the target compound.
  • genetic manipulation informed by structural analysis is by no means necessary to CISTEMs, it may be utilized in combination with CISTEMs to design polypeptides that are well-suited to the performance of desired functions.
  • the CISTEM cell population consists of cells that lack a chromosomal copy of the gene for the imprinted polypeptide, but are capable, upon transfection with an episomal or vector-borne copy of this gene, of translating, exporting, and inserting the imprinted polypeptide in the membrane.
  • CISTEMs which imprint E.
  • E. coli OmpA to bind to any one of several targets E. coli strain UH203 (Freudl, 1989), which is ompA ⁇ , composes the CISTEM cell population.
  • E. coli strain S1755 (Brown, 1992), which lacks the lamB gene, composes the cell population.
  • a eukaryotic example is provided by several preferred eukaryotic CISTEMs, which imprint human ICAM-1 to bind to any one of several targets.
  • COS-7 cells are used; this strain lacks ICAM-1, but is easily transfected with an ICAM-1 gene borne on the CDM8 expression vector (the COS-7/CDM8 expression system is described in Staunton et al., 1990).
  • the strains that compose CISTEM cell populations are preferably stop-codon suppressors; that is, they recognize one of the stop-codons, such as UAG, as a translatable codon.
  • stop-codon suppressors that is, they recognize one of the stop-codons, such as UAG, as a translatable codon.
  • any stop-codons recognized by the strain composing the CISTEM cell population must be excluded from the variable oligonucleotides that are inserted into the vector-borne gene for the imprinted polypeptide. (The synthesis of variable oligonucleotides free of certain stop- codons, and their insertion into the gene for the imprinted polypeptide, are described above) . Therefore, the synthesis of variable oligonucleotides is simplified when the CISTEM cell population is able to recognize one of the stop-codons. For example, E.
  • coli UH203 which is used in several preferred CISTEMs for imprinting OmpA, is supF, i.e., capable of translating the UAG stop-codon. Consequently, variable oligonucleotides inserted into the OmpA vector (pEV218), must be free of the stop codons UAA and UGA, but not of UAG. This is easily achieved by the method of synthesis described above.
  • S1755 the strains used in CISTEMs imprinting LamB, is supE44, i.e., capable of translating UAG.
  • the medium in which the CISTEM cell population is propagated depends simply on the strain composing the population. For example, E.
  • coli UH203 is grown in Luria broth or agar. When a strain contains a plasmid with an antibiotic resistance gene, the appropriate antibiotics are added to the medium or agar. Standard media for all commonly used strains of prokaryotic and eukaryotic cells are described in the literature and familiar to those practiced in the art. The conditions for transformation of CISTEM cells with the vector bearing the imprinted polypeptide also depend upon the cell type, and are described m more detail above, as well as in the literature. Induction of expression of the imprinted polypeptide requires incubation of the cell population with the compound that activates the imprinted polypeptide' s mducible promoter. As is described above, however, certain inducible promoters, such as the lac promoter/operator elements, are "leaky", allowing for the appropriate level of expression of the imprinted polypeptide without any induction.
  • the medium When the CISTEM cell population is incubated with a suspension or solution of the target compound, the medium generally contains only the minimal requisites for cellular sustenance m addition to the target compound.
  • the presence of abundant macromolecules other than the target compound can result m selection for clones whose imprinted domain bind not to the target, but rather to one of the constituents of the medium.
  • imprinting OmpA E . coli UH203 are incubated m the presence of any one of several target compounds in ATCC Medium 52.
  • m some CISTEMs, Luna broth was used.
  • CISTEMs for imprinting ICAM-1
  • COS cells are incubated with target compound m TC Minimal Medium Eagle or Minimal TC Medium 99.
  • target compound m TC Minimal Medium Eagle or Minimal TC Medium 99.
  • Minimal media for other commonly used prokaryotic and eukaryotic cell lines are described m the literature. The concentration of target compound and duration of incubation are important to CISTEM function, and are discussed m detail below.
  • the procedure demonstrates that the agent is competent to perform CISTEM selection.
  • this procedure is used to determine the experimental conditions that will allow reliable CISTEM selection.
  • multiple parameters may be varied, including the following: concentration of the target compound ( [T] ) with which the CISTEM cell population is incubated; duration of incubation with the target compound; initial multiplicity of infection (Mol)or concentration of the selection agent; duration of incubation with the selection agent; temperature; level of expression of the imprinted polypeptide.
  • concentration of the target compound ( [T] ) with which the CISTEM cell population is incubated duration of incubation with the target compound
  • initial multiplicity of infection (Mol)or concentration of the selection agent concentration of the selection agent
  • duration of incubation with the selection agent duration of incubation with the selection agent
  • temperature level of expression of the imprinted polypeptide.
  • concentration of target compound, [T] and initial multiplicity of infection, Mol, are the two parameters that are varied over a number of trials.
  • two different parameters - namely, level of expression of the imprinted polypeptide and duration of incubation with the selection agent - are varied.
  • the level of expression of the imprinted polypeptide is one of the varied parameters.
  • the method by which regulation of the imprinted polypeptide is achieved depends upon which heterologous promoter controls the gene for the imprinted polypeptide.
  • heterologous promoter controls the gene for the imprinted polypeptide.
  • ompA is under the control of the lac regulatory elements, which are induced by isopropyl-b-D-thiogalactopyranoside (IPTG) .
  • the ompA gene on pEV218T7 comprises a TAG stop codon corresponding to amino acid 7 (Freudl, 1989) of the polypeptide; therefore, when expressed in the amber-suppressing (supF) host E. coli UH203, ompA expression is regulated not only by the level of induction with IPTG, but also by the concentration of the suppressor tRNA, which is expressed from a plasmid. Low level expression of OmpA is achieved with 1.0 mM IPTG induction. On pSB1649, lamB is under the control of the lac regulatory elements, and expression is regulated as described in the literature (Brown, 1992) . Regulation of the expression of ICAM-1 from pCDl.8, the CDM8 vector construct used in a preferred eukaryotic embodiment of this invention, is also described in the literature (Staunton et al., 1990).
  • Two populations of cells (populations A and B) exhibiting the oligopeptide tag in the imprinted domain are grown in a medium appropriate to the cell type and the expression vector.
  • E. coli UH203 transformed by pEV218T7 are grown at 37°C in Luria broth supplemented with lOO ⁇ g/ml ampicillin, and 1.0 mM IPTG for induction of pEV218T7 expression; conditions for growth of other cell types are described above and in the literature.
  • a third population of cells (C) of the same strain as populations A and B, transformed by the same vector as populations A and B, but without the oligopeptide tag, is grown in the same medium.
  • E. coli UH203 transformed by pEV218, rather than pEV218T7 are grown in the conditions described above.
  • Populations B and C are each grown to generate three clonal subpopulations of 10 cells, Bl, B2, B3, and Cl, C2, C3.
  • Population A is grown to generate nine clonal subpopulations, Al , A2...A9, each comprising 10 cells.
  • Populations Al ...9 and C1...3 are incubated with the target compound, while populations B1...3 are incubated under identical conditions, but without the target.
  • Optimal conditions and time required for binding between the oligopeptide tag and the target are prescribed by the supplier of the tag/target system or in the literature (Phizicky et al., 1995).
  • the cell population expressing pEV218T7 is incubated with mAbT7 for approximately 60 minutes.
  • the concentration of target compound with which cells are incubated is systematically varied across populations Al ...9 and Cl ...3 in order to identify the concentration that is optimal for CISTEM selection (table 3) .
  • Populations A7...9 and C1...3 are incubated with the highest concentration of target, such as lOOx the concentration used with populations Al ...3.
  • C1...3 are exposed to the selection agent.
  • the technique of exposure depends upon the nature of the agent, and, for all well-known agents, is described in the literature. Methods of exposing prokaryotic cell populations to bacteriophages are described by Calendar (Calendar, 1988); methods of exposing prokaryotic cell populations to colicins are described by
  • Pattus et al . (Pattus et al., 1990); and methods of exposing eukaryotic cell populations to viruses are described in the literature (e.g. Staunton et al . , 1990, Tyler et al . , 1996).
  • concentration of agent is varied across the cell populations in order to identify optimal conditions for the CISTEM' s selection (table 3).
  • the concentration of target is not one of the parameters that is varied. Instead, this concentration is fixed, while two other parameters are varied.
  • Cell populations A2,5,8, B2, and C2 are exposed to a doubled concentration of the agent, and populations A3, 6, 9, B3, and C3 are exposed to yet another doubling of agent concentration.
  • Table 3 summarizes the variation of target and agent concentrations over the cell populations in this procedure for optimizing CISTEM selection.
  • the concentrations in this procedure may vary depending on the nature of the selection agent and the method of exposure, but several fundamental aspects of the procedure do not change from one CISTEM to another.
  • the selection agent has unimpeded access to its receptor, and therefore affects every cell.
  • Population B is susceptible to the agent because, although cells bear the tag at the imprinted domain, the population is not exposed to the tag's ligand. Therefore, the selection agent's site, e.g. the adhesion domain, remains exposed and functional.
  • Population C is susceptible because, although the cell population is exposed to the ligand, the ligand is not bound by the imprinted polypeptide, which lacks the ligand' s tag.
  • populations Al ...9 exhibit a range of resistance, extending even to immunity.
  • the imprinted domain harbors the oligopeptide tag, which binds the target ligand during incubation.
  • the adjacent adhesion domain is occluded from the selection agent, and the cells are protected.
  • Populations Al ...9 exhibit a range of resistance because they are exposed to a range of concentrations of target ligand. When the range of concentrations is broad enough, the range of resistance extends from partial susceptibility to complete immunity.
  • the concentration of selection agent that allows for functional CISTEM selection is found as follows. Ubiquitous pathogenic effect--!. e. every cell in a population is affected by the agent--is easily observed when cell populations are exposed to agents according to protocols described in the literature. For example, in exposing prokaryotic populations to virulent lytic phage, ubiquitous pathogenic effect results in confluent lysis of the population, with zero surviving colonies (Calendar, 1988) . The lowest concentration of selection agent at which every cell in the control populations (B and C) is affected by the agent is an appropriate level for CISTEM selection.
  • the minimum concentration required for ubiquitous effect could be more exactly determined by further trials with Mol's ranging from 5 to 10.
  • the threshold for a CISTEM can be set by adjusting the concentration of the target compound.
  • the target concentration used in the CISTEM is the lowest level that provides complete protection to population Ai in the presence of selection agent at the CISTEM' s Mol. (Complete immunity to the agent is easily observed when cell populations are exposed to agents according to common protocols described in the literature.)
  • population A5 exhibits complete immunity to the selection agent while population A2
  • CISTEM to select for cells whose imprinted polypeptides bind the target with an affinity equal to or greater than the affinity exhibited by the oligopeptide tag for its ligand.
  • 3 10 molecules per imprinted polypeptide is used to select for cells with imprinted polypeptides that exhibit an affinity for the target that is significantly higher than the affinity exhibited by the oligopeptide tag for its ligand.
  • target compound such as 10 molecules per imprinted polypeptide
  • CISTEM The application of CISTEM to cell populations yields cell clones that survive the selection procedure by virtue of the interaction between the polypeptide of interest and the target (s).
  • the sequence of the polypeptide of interest is encoded by the DNA sequence which has been inserted into the display domain.
  • the surviving cell population can be monoclonal or polyclonal. In the latter instance, surviving cells need to be plated out on solid medium plates to recover colonies derived from single cells (state of the art bacterial genetics) .
  • Amplification of monoclonal cell populations and the subsequent preparation of their relevant episomal or vector DNA is performed according to standard protocols of recombinant DNA methodology.
  • DNA sequence analysis of the inserted DNA tag provides the DNA sequence of the insert and thereby allows the prediction of the ammo acid sequence of the polypeptide of interest which has been expressed and displayed at the surface as integral part of the membrane protein.
  • the inserted DNA is derived from a pool of randomly synthesized oligonucleotides or from DNA fragments derived from natural occurmg mRNA (cDNA) or genomic DNA.
  • cDNA mRNA
  • genomic DNA The selected DNA and cognate polypeptide sequences can be scrutinized to match sequences of known proteins and their cognate genes.
  • sequence analysis can be employed to identify the polypeptide of interest and to attribute and/or relate its composition to known genes or DNA sequences and their predicted proteins.
  • Modifications and elaborations of the basic CISTEM achieve several extensions of its capabilities, including the following: 1. augmentation of the maximum affinity of selected polypeptides for the target compound; 11. specification of the number of binding sites joining a multivalent imprinted polypeptide to the target compound; 111. selection for imprinted polypeptides that exhibit both high affinity for the target compound and low affinity for a second compound; IV. selection not only for affinity, but also for functional activity; v. screening the polypeptides expressed from a cDNA library for binding to a target compound; vi . selection for imprinted polypeptides that exhibit affinity for one or more of a set of multiple, distinct target compounds.
  • variation is introduced not as an extension of the original oligonucleotide inserts, but rather as an oligomeric rearrangement of the original inserts (the procedure of oligomeric rearrangement of oligonucleotide inserts is described in Brown, 1992) or a limited mutagenesis of the original inserts (techniques for the limited mutation of oligonucleotide inserts are described m Balmt et al . , 1993; insertion of mutated oligonucleotides is performed according to the procedure described above for insertion of variable oligonucleotides into a targeted site in a vector) .
  • oligopeptide "sequence space" may be searched by a number of strategies, some of which have been applied to screening other combinatorial libraries (Balmt et al., 1993, Brown, 1992, Janda, 1994).
  • the vectors bearing fresh variation at a site corresponding to the imprinted domain are then used to transform a population of cells, according to common protocols cited above, creating a new CISTEM cell population. This population is then subjected to yet another round of selection by the CISTEM (figure 10).
  • TJ TJ ⁇ 3 3 ⁇ - ⁇ a ⁇ ⁇ ⁇ - ⁇ PJ ⁇ - ⁇ ⁇ tr ⁇ 3 ⁇ - O a ⁇ 3 O ⁇ 3 ⁇ 3 O ⁇ - 1 ⁇ 3 - O 3 J ⁇ cn cn ⁇ - a ⁇ 3 ⁇ cn Hi ⁇ a ⁇ TJ C ⁇ 3 X rt ttl ⁇ X C ⁇ cn ⁇ - 3 3 3 3 3 3
  • Certain embodiments of this invention select for imprinted polypeptides that exhibit high on-rate in their binding with the target compound.
  • the CISTEM cell population is not pre-mcubated with the target compound prior to introduction of the selection agent. Rather, the selection agent is introduced simultaneously with the target compound.
  • concentrations of agent and target compound are varied over multiple trials, as is described in the optimization procedures detailed elsewhere in this document. Concentrations are adjusted such that, upon simultaneous introduction of agent and target to the CISTEM cell population, clones whose imprinted polypeptides bind to the target relatively rapidly are protected, whereas clones whose imprinted polypeptide binds to the target more slowly are eliminated by the selection agent.
  • a CISTEM that imprints a polypeptide to show high affinity for a target compound but extremely low affinity for a second compound, which is here called the "repelled compound” is constructed as follows.
  • the size of the target compound, the size of the repelled compound, and the distance between the imprinted domain and the adhesion domain are altered, if necessary, to meet the following condition: when the target compound is bound to the imprinted domain, it occludes the adhesion domain, whereas when the repelled compound is bound to the imprinted domain, it is too small to occlude the adhesion domain. If this criterion is not met fortuitously (i.e.
  • the diameter of the target compound and the distance between the domains may be altered, as follows.
  • the target compound may be enlarged by creating a multimer. (The procedure for creating a multimer depends upon the nature of the target compound, but many techniques for joining multiple copies of a compound are commonly practiced and described m the literature.)
  • the distance between the imprinted domain and the adhesion domain may be increased by moving either domain to a site on the imprinted polypeptide that is topologically farther from the other domain. Transposing the imprinted domain simply involves inserting variable oligonucleotides, according to the procedure described above, at a genetic site corresponding to a different extracellular domain of the imprinted polypeptide.
  • the adhesion domain may be transposed to a different extracellular domain of the imprinted polypeptide without affecting its function as a receptor for the selection agent.
  • the procedure for transposing an adhesion domain is described above as well as m the literature (Chu et al . , 1995) .
  • the superfluous domains may be replaced by polyprolme linkers m order to ensure that extra- or intracellular structures do not impede proper occlusion of the adhesion domain by a target compound bound to the imprinted domain.
  • the CISTEM cell population is incubated with both the target compound and the repelled compound. Then the CISTEM' s selection agent is introduced, and this creates two selective pressures.
  • One pressure favors cells expressing imprinted polypeptides with higher affinity for the target compound (figure 12) : a target compound bound to an imprinted domain occludes the adjacent adhesion domain, and thus serves to protect the underlying cell from the selection agent.
  • a second selective pressure favors cells with lower affinity for the repelled compound; a repelled compound bound to an imprinted domain not only fails to occlude the adjacent adhesion domain, but also prevents binding by a target compound (figure 12) .
  • an imprinted polypeptide ' s affinity for the repelled compound effectively reduces its affinity for the target compound.
  • selection favors cells that exhibit imprinted domains with high affinity for the target compound and low affinity for the repelled compound (figure 12) .
  • a basic CISTEM may be coupled to a secondary screen for activity.
  • a CISTEM selects clones whose imprinted polypeptides bind the target compound m the prescribed fashion. From this population of clones, or from the imprinted domains extracted from such a population, a secondary screen selects the structures whose binding to the target compound exhibits the desired functional activity.
  • Imprinted domains liberated from the cell surface by detergent or by expression as a secreted isomorph (lsomorphic forms and their mterconversion are described above) may be subjected to any commonly practiced screens or assays for activity or modification of the target compound's activity.
  • one CISTEM imprints E. coli OmpA to bind with high affinity to soluble IL-6-Receptor (This target compound is described in more detail above) .
  • the imprinted polypeptides freed from the cell membrane by a conventional gentle detergent, which is known not to denature OmpA, are then subjected to a second screen for their capacity to agonize the cell-bound, functional IL-6-Receptor .
  • This screen for agonistic activity is described m the literature (Hibi et al . , 1990).
  • extremely strong selection for functional activity may be exerted upon the CISTEM' s selected set of cellular clones, and does not require liberation of the imprinted domains from the cellular membranes.
  • CISTEMs can be coupled to selection regimes very similar to genetic complementation.
  • each cell's supply of a vital compound such as a nutrient
  • the imprinted domain's functional activity either converts the target compound to a compound vital to the underlying cell, or secures the vital compound for the cell.
  • the target compound consists of a library of separate and distinct molecules, which is here called cryosomal target library".
  • a CISTEM m which a target library (rather than a single target compound) is used serves to select interacting pairs m which one member of each selected pair is the oligopeptide displayed at the imprinted domain of a protected clone, and the other member is a target library molecule that binds to this oligopeptide and thus serves to protect the clone.
  • the target library is large enough to meet the size requirement for CISTEM function, and is present in the medium at a concentration adequate to confer protection upon cells whose imprinted polypeptides bind to one or more molecules m the target library, the target library may comprise as many distinct molecules as is desired, and may be constructed according to any known procedure for making or isolating libraries of molecules.
  • CISTEMs involving target libraries two strategies are used, if necessary, to increase the concentration of each molecule m the target library to the minimum level required for effective CISTEM selection, i . e . , to achieve the concentration required to confer protection from the selection agent upon those cells whose imprinted polypeptides bind to one or more of the molecules m the target library.
  • the CISTEM cell population and the target library are each amplified, divided into fractions, and exposed to one another m microtiter wells. This allows the concentration of target molecules to be sufficiently high to confer protection upon cells whose imprinted polypeptides bind to one or more molecules in the target library.
  • the target library is secreted by the CISTEM cell population itself.
  • libraries of variable oligopeptides can be displayed as fusions to gene protein 3 of E. coli filamentous phage (M13, fl, or fd) (filamentous phage-display technology is described in Janda, 1994 and Phizicky et al . , 1995).
  • Filamentous phage create relatively avirulent infections m which phage particles are liberated from the host cell by extrusion, leaving the cell in tact. Therefore, a CISTEM cell population that secretes its own target library can be engineered as follows.
  • a cell population that lacks a gene for the imprinted polypeptide is transformed with a library of exogenous copies of this gene, m which variable oligonucleotides have been inserted at the genetic site corresponding to the imprinted domain.
  • E. coli UH203 is transformed with pEV218I, a library of ompA plasmids with variable oligonucleotides at the genetic site corresponding to extracellular loop IV of the membrane protein.
  • the resultant cell population displays a library of exogenous oligopeptides at the imprinted domain.
  • this cell population is then transformed with a second extra-chromosomal genetic element that encodes a secreted library of oligopeptides.
  • the OmpA- imprinting cell population for example, is transfected with a library of filamentous bacteriophage engineered to display exogenous oligopeptides at gene protein 3. (Many such libraries are described in the literature, reviewed m Janda, 1994 and Phizicky et al .
  • the CISTEM cell population expressing two extra- chromosomal genetic elements is then exposed to the CISTEM' s selection agent, following the same protocols for exposure as were described for the basic CISTEM.
  • a clone m the CISTEM' s population is protected only if the target library molecule that is secreted by the clone binds specifically to the clone' s imprinted domain. That is, a clone is protected by an autocrme loop.
  • K3 serves as the selection agent (figure 13) .
  • coli clone is protected from K3 only if the exogenous polypeptide displayed at loop IV of OmpA binds specifically to the M13 that the clone itself secretes.
  • the genes for both members of this interacting pair of oligopeptides are easily recovered from the protected cell.
  • a CISTEM with a target library selects for specifically interacting pairs of oligopeptides, as well as for the genes that encode them.
  • a CISTEM is constructed according to the procedure ust described, but a hemolysin library is used m place of the phage M13 library. Hemolysms are secreted bacterial polypeptides that tolerate exogenous insertions. Thus, the hemolysin gene can be used to carry exogenous genes, which encode polypeptides that are then secreted from the cell.
  • CISTEM Two possibilities for an intracellular CISTEM are depicted m figure 20.
  • the protective partner :partner interaction occurs within the cell.
  • Version A shows a typical CISTEM, wherein the two partners are transported to the cell surface.
  • version B the positive selection occurs, because the two partners cannot be transported to the cell surface and e.g. accumulate withm the cell and/or become substrates for protein degradation pathways .
  • a CISTEM for the identification and isolation of cancer antigens may be constructed as follows: First, a cDNA library is derived from a patient's tumor cells. Protocols for isolation of cells and derivation of the cDNA library are described m the literature. The tumor-derived cDNAs serve as the variable inserts m the construction of the CISTEM cell population: they are inserted into the imprinted polypeptide vector at a genetic site corresponding to the imprinted domain of the polypeptide. The cDNAs are inserted m all three reading frames m order to guarantee that polypeptides associated with the tumor are expressed by the CISTEM cell population.
  • Methods for inserting cDNAs into vectors are available m the art. These vectors are used to transform a population of cells lacking an endogenous copy of the gene for the imprinted polypeptide. When expression of the vector-borne gene is induced, each clone the CISTEM cell population displays one tumor-associated polypeptide, fused to the imprinted domain on the cell surface.
  • the target compound in a CISTEM for the identification of tumor antigens preferably consists of polyclonal antibodies, usually isolated from the same patient whose cells are used as the source for the cDNA library.
  • the tumor cells that supply cDNA and the antibodies that comprise the target compound are preferably autologous.
  • any clone m which the imprinted domain displays a tumor-associated polypeptide that binds autologous antibodies has a different status concerning the selection agent than a cell with no such binding. E.g. in case of binding, these cells may be protected from the selection agent.
  • any clones m which the imprinted domain displays a tumor polypeptide that is not recognized by autologous antibodies is eliminated.
  • this CISTEM identifies tumor-associated polypeptides that interact specifically with autologous antibodies. It is shown m the literature that such polypeptides are likely to be lmmunogenic, eliciting a tumor-specifIC humoral and cellular immune response.
  • Certain embodiments of this invention are used to determine whether certain molecules interact specifically with an imprinted polypeptide that has not been altered by the insertion of variable oligopeptides.
  • Any outer membrane polypeptide that is used as a receptor by a pathogenic agent including polypeptides engineered by the manipulations described above, may be used m these embodiments of the invention.
  • the imprinted polypeptide is not engineered to display highly variable oligopeptides, there is no Uber ⁇ mprmted domain", and the selection agent's adhesion domain may be located anywhere on the imprinted polypeptide.
  • the imprinted polypeptide is expressed from an mducible promoter.
  • the cell population, induced to express the imprinted polypeptide, is exposed to a target library, according to one of the methods described above for constructing target libraries and introducing them to CISTEMs. Either microtiter wells or the autocrme loop methods may be used to increase the concentration of target library molecules.
  • the CISTEM' s selection agent is then introduced according to procedures described for the basic CISTEM. Cells are protected only if molecules m the target library bind to the imprinted polypeptide, and thus prevent adhesion by the selection agent.
  • these embodiments of this invention provide an easy and straightforward method for assaying a set of molecules' affinity for the imprinted polypeptide.
  • preferred areas, for which the present method is especially suitable is the discovery and isolation of novel antigens for use m vaccines, tumors, novel targets for therapeutic intervention, novel diagnostic markers, or molecules useful for monitoring treatment efficacy.
  • novel antigens for use m vaccines, tumors, novel targets for therapeutic intervention, novel diagnostic markers, or molecules useful for monitoring treatment efficacy is also subject matter of the present invention.
  • the present invention relates to the cells/the cell population to be used m the present invention with a) a membrane polypeptide which has the polypeptide of interest m its extra- or intracellular domain and b) a selection site being specific for a selection agent, such that upon binding of a target compound to the polypeptide of interest the selection site of cells which exhibit such a binding of said target compound to said polypeptide of interest is modified m a manner that said selection agent may be used for selecting those cells which exhibit said binding of said target compound to said polypeptide of interest from cells which do not exhibit said binding.
  • the present invention also relates to a kit comprising the cells/cell population according to the present invention, the target molecule (and/or a library of target compounds) and one or more selection agents.
  • kits may be provided &J H- M o IVi O 3 T) ⁇ - ⁇ 3 3 cn 3 ⁇ ⁇ ⁇ - ⁇ ⁇ 3 3 PJ rt TJ ⁇ a ⁇ 3 Hi n
  • E. coli Outer membrane protein A is a 325-res ⁇ due polypeptide comprising an ammo-termmal, eight-stranded ⁇ - barrel with four extra-cellular domains, three periplasmic loops, and a roughly 150-residue periplasmic carboxyl-termmal moiety (figure 3) (Freudl, 1989, Morona et al . , 1985).
  • the fa- barrel conformation is extremely stable, and can be denatured only by boiling in strong detergents.
  • OmpA is among the most abundant proteins m the E. coli outer membrane. It is also widely conserved m bacterial evolution.
  • exogenous oligonucleotides inserted into the ompA gene at sites corresponding to extracellular domains of the polypeptide, are normally translated as part of the polypeptide, and do not disturb the polypeptide ' s insertion in the outer membrane.
  • oligopeptides encoded by these exogenous genetic insertions are displayed on the surface of the cell, adopt stable folded structures, and can exhibit enzymatic, agonistic, or antagonistic activities.
  • an E. coli population lacking wild-type OmpA was engineered to express a plasmid-encoded copy of OmpA, m which extracellular loop IV of the polypeptide displayed the T7 tag oligopeptide (Met-Ala-Ser- Met-Thr-Gly-Gly-Gln-Gln-Met-Gly) , which binds to the commercially available T7 antibody (Novagene) .
  • pEV218 In pEV218 (Freudl, 1989) , the ompA gene, which is under the IPTG- mducible control of the lac promoter, has been engineered to bear a polylmker rich in restriction endonuclease sites at a genetic region corresponding to extracellular loopIV of the polypeptide (figure 9) .
  • This plasmid was digested with Sad (enzyme and reaction buffer from New England Biolabs) and Xbal (enzyme and reaction buffer from MBI Fermentas), yielding a fragment of the restriction site polylmker and the linearized plasmid with sticky ends left by the restriction enzymes.
  • the linearized plasmid with sticky ends was isolated by standard gel electrophoresis.
  • Two single stranded oligonucleotides were synthesized by conventional technology (custom oligonucleotides are available from commercial laboratories, and methods for synthesizing single-stranded oligonucleotides are commonly use and described m the literature) to read as follows :
  • Oligonucleotide 1 5' - CG[ATGGCTAGCATGACTGGTGGACAGCAAATGGGT] - 3'
  • Oligonucleotide 2 5' - CTAG[ACCCATTTGCTGTCCACCAGTCATGCTAGCCAT]CGAGCT - 3'
  • the sequence m brackets composes the T7 tag gene sequence.
  • These two single-stranded oligonucleotides were annealed m Klenow reaction buffer (MBI catalogue) , forming the double stranded T7-tag gene with sticky ends complementary to the sticky ends on pEV218, linearized by Sad and Xbal as described m the previous paragraph.
  • MBI catalogue m Klenow reaction buffer
  • the double-stranded oligonucleotide was ligated (MBI Fermentas ligation buffer) into the linearized plasmid
  • the T7 tag gene was inserted, in frame, into the ompA gene on pEV218.
  • the new construct was designated pEV218T7.
  • the plasmid pEV218T7 was used to transform a population of the strain UH203 (lac, supF, ompA, recA, proAB, rpsL) (Freudl, 1989) , according to common methods of transformation described m detail above.
  • Coliphage K3 invades E. col i by binding to loops I, II, and III of OmpA. Loop IV is not used by the phage, and, indeed, exogenous oligopeptides at loopIV do not affect K3 susceptibility of the cell (the K3 adhesion domains are shown m figure 3; bacteriophages are reviewed briefly m the specification, and bacteriophage K3, as well as its receptor requirements, are described m Freudl, 1989 and Morona et al . , 1985) .
  • the following procedure was performed to fulfill two objectives: a) to conclusively demonstrate that cells whose imprinted polypeptides bind the target compound are protected from K3 attack, whereas cells whose imprinted polypeptides fail to bind the target are eliminated by the phage, and b) to achieve preliminary optimization of the duration of incubation of the CISTEM cell population with phage K3.
  • the principle of this experiment is illustrated m figure 14.
  • E . coli MC4100 (pEGFP-Nl), a kanamycm-resistant, ompA wild-type strain, which will here be referred to as U (for thoughUntagged"), was cultivated overnight m Luna broth (LB) supplemented with 25 ⁇ g kanamycm/ml .
  • E. coli UH203 (pEV218T7), which will here be referred to as T (for perhapsTagged”), is an ampicill -resistant, ompA strain that has been transformed, as described m the specification, by a plasmid encoding OmpA bearing the T7 tag sequence at the fourth extracellular loop of the polypeptide.
  • Mol Multiplicity of Infection
  • This experiment also serves m the preliminary optimization of CISTEM selection by phage K3.
  • m table 4 and figure 15 m the mixed population of U and T cells that were incubated with mAbT7, U cells were completely eliminated from the population after 30 mm. incubation with phage K3.
  • T cells by contrast, maintained a roughly stable population well beyond 30 mm. of incubation with the phage. This suggested that when starting with Mol - 10, the minimum time period for phage K3 to eliminate all cells that failed to bind the target would be 30 - 60 mm.
  • the preliminary, single-parameter optimizations of incubation time with mAbT7 and the duration of K3 selection allowed an initial test of the CISTEM' s capacity to select rare cells whose imprinted polypeptides bind the target from a background of cells whose imprinted polypeptides fail to bind the target.
  • the following procedure was performed.
  • E . coli strains U (Untagged) and T (Tagged) are described above.
  • U was cultivated overnight m LB supplemented with 25 ⁇ g kanamycm/ml.
  • T was cultivated overnight m LB supplemented with lOO ⁇ g ampicillm/ml and ImM IPTG to induce expression of pEV218T7, the plasmid encoding OmpA with the T7 tag at loopIV of the polypeptide.
  • Each overnight culture was diluted with LB to OD600 - 0.005.
  • a 50 ⁇ l aliquot of the U culture was combined with a 50 ⁇ l aliquot of the T culture to create a mixed population.
  • the mixed population was created with equal sized aliquots of U and T cultures, it was composed predominantly of U cells. The reason for this was probably that the very low level of OmpA expression induced in the T strain (only ImM IPTG was used) made this strain significantly less viable than the U strain under standard conditions of cultivation.
  • the mixed population was incubated at 37C with 68nM mAbT7 (described above) diluted in PBS for 60 min.. Following incubation, 100 ⁇ l aliquots of the cell population were plated on LB plates supplemented with 25 ⁇ g kanamycin/ml or lOO ⁇ g ampicillin/ml . Colonies were counted after overnight growth, showing that the population consisted of approximately 1 T cell for every 120 U cells
  • T cells 10 8 U to 10 4 T; 10 8 U to 10 3 T; 10 8 U to 10 2 T; and 10 9 U to 102 T. Each population is incubated for 60 min. at
  • T cells are created by combining aliquots, as described above. 10 nM mAbT7 is added to each population, and the populations are incubated for 60 mm. at 37°C. During the 60 mm. incubation, the population composition is monitored, as described above, by taking sample aliquots of 5 ⁇ l every 20 mm., plating, growing, and counting the numbers of U and T cells. After the removal of an aliquot at 60 mm., coliphage K3 is introduced at an Mol of 2. Over the next 180 mm., the populations are monitored by 5 ⁇ l aliquot sampling every 20 mm.. The [IPTG] and duration of incubation with K3 that result m the elimination of all U cells, with concomitant elimination of fewest T cells, are optimal parameter values for use in the CISTEM.
  • OmpA-imprinting CISTEM cell population OmpA plasmid pEV218 (described above, shown m figure 9, construction described m Freudl, 1989) , bears a polylinker rich in restriction endonuclease sites at a genetic region corresponding to extracellular loopIV of the polypeptide.
  • This plasmid is digested with Sad (enzyme and reaction buffer from New England Biolabs) and Xbal (enzyme and reaction buffer from MBI Fermentas) , under conditions described by the suppliers, yielding a fragment of the restriction site polylmker and the linearized plasmid.
  • the linearized plasmid is isolated by standard gel electrophoresis. 10 distinct, single-stranded oligonucleotides are synthesized by conventional technology (synthesis of variable oligonucleotides is described above) to read as follows:
  • N represents all four nucleotides, each with equal probability; and V represents C, G, or A, each with equal probability.
  • V represents C, G, or A, each with equal probability.
  • variable plasmid population pEV218I
  • UH203 ⁇ lac, supF, ompA, recA, proAB, rpsL This strain, its propagation and its transformation, are described more completely above, and in Freudl, 1989.
  • the UH203 population is grown in Luria broth supplemented with
  • the UH203 (pEV218I) population of 10 8 cells plus approximately 100 UH203 (pEV218T7) cells is incubated at 37C for 15 min. in 100ml LB medium supplemented with 100 ⁇ g Ap/ml and IPTG between 0.25 and 2.0 mM, as determined in the optimization trial described above. This yields a population of 10 8 clones, comprising 106 distinct clones, each of which displays a distinct, exogenous, oligopeptide sequence at loop
  • lOnM mAbT7 is added to the 100ml of cells. The cells are incubated with the mAb for 60 min. at 37°C.
  • the cell population is centrifuged to separate cells from phage and the products of lysis, and cells are resuspended in 100ml NB, without lOOmM
  • This cell population represents the set of clones selected by the CISTEM for binding to the target compound mAbT7. To amplify the numbers of selected clones, the selected cell population is grown to 10 cells at 37°C in 100ml NB .
  • OmpA The structural homology between OmpA and a wide range of antigenic adhesins makes OmpA well-suited for imprinting for use m applications requiring mimicry of homologous adhesins.
  • One such application is vaccination.
  • a polypeptide in this case, OmpA
  • OmpA polypeptide
  • the imprinted polypeptide may elicit an immune response very similar to the one provoked by the antigen (Folgori et al . , 1994).
  • coli OmpA to mimic Nei sseria meningi tidi s Opa proteins (Olyhoek et al., 1991) include mAb B33, which reacts with all Opa proteins, mAbs P322 and P514, which react with a broad subset of Opa proteins, and mAb B306, mAb A222/5b (Olyhoek et al . , 1991), and others described in the literature.
  • this CISTEM like the one described in Example 1, uses phage K3 to select from a cell population displaying variable inserts 20 amino acids in length at loop IV of OmpA.
  • this CISTEM imprints OmpA to bind to mAbT7
  • this CISTEM imprints OmpA to bind to antibodies against Neisseria meningi tidi s Opa proteins.
  • E. coli strains U (Untagged) and T (Tagged) are described above (in Example 1).
  • U was cultivated overnight in LB supplemented with 25 ⁇ g kanamycin/ml.
  • T was cultivated overnight in LB supplemented with lOO ⁇ g amplicillin/ml and 2mM IPTG to induce expression of pEV218T7, the plasmid encoding OmpA with the T7 tag at extracellular loopIV of the mature polypeptide.
  • Figure 17 shows the dynamics of phage titer and Mol over the course of CISTEM selection. Mol climbs exponentially and reaches a maximum near 100,000.
  • Figure 18 shows the dynamics of cell growth and death during CISTEM selection. The rapid exponential growth of phage results in a monotonic decline of U cells, irrespective of the presence of absence of mAbT7. By contrast, in the presence of mAbT7, T cells are protected from the extremely high Mol, and consequently they expand in number. Thus, specific interaction between the antibody and the epitope displayed at OmpA loopIV resulted in selection of cells exhibiting the epitope, while cells that did not exhibit the epitope were eliminated. This result is summarized in figure 19. iv: EXAMPLE 4: REDUCTION OF BACKGROUND THROUGH THE USE OF HELPER CELLS
  • background is used to refer to cells that fail to bind the target compound but nonetheless remain in the selected population. In general, it is desireable to minimize the number of background cells, such that CISTEM reliably eliminates cells whose imprinted domains fail to bind the target compound, while selecting those cells whose imprinted domains do bind the target compound.
  • the following experiment illustrates one strategy for reducing background.
  • the CISTEM cell population is supplemented with "helper cells", the function of which is propagation and amplification of the selective agent. This amplification of the selective agent increases the intensity of selection, and thereby reduces residual background cells.
  • helper cells lack a selectable marker that is present in the rest of the CISTEM cell population; this permits efficient elimination of the helper cells after they have served their purpose of amplifying the selective agent.
  • UH203 (pEV218T7) , the "T" strain, was cultivated overnight in LB supplemented with lOO ⁇ g ampicillin/ml and lmM IPTG to induce expression of the ompA plasmid.
  • UHFl(pEV218) was used as a background strain that does not exhibit the T7 epitope at loopIV of OmpA.
  • UHF1 is virtually isogenic with UH203, except that it carries a gene for Chloramphenicol resistance.
  • the plasmid pEV218 is identical to pEV218T7, except that it carries a restriction-enzyme site polylmker in place of the gene for the T7 tag.
  • UHF1 pEV218
  • UHF1 the "U” strain
  • pEV218 was cultivated overnight in LB supplemented with lOO ⁇ g ampicillin/ml and lmM IPTG to induce expression of pEV218.
  • To create mixed populations aliquots of the "T" and "U” strains were combined to compose populations with a ratio of T to U cells of approximately 1 to 100 (table 9) .
  • helper cells MC4100 (pEGFPNl) were used as helper cells.
  • lOO ⁇ l of MC4100 (pEGFPNl) at OD 600 0.5 were added to two mixed populations. Two other mixed populations received no helper cells.
  • the background proportion defined as U cells/ (U cells + T cells) is reduced from 34% in the absence of helper cells to 16% with the addition of lOO ⁇ l of helper cells at OD 6 oo.
  • This experiment thus illustrates the principle of helper cells: they are added to CISTEM cell population to modify the activity of the selective agent.
  • the E. coli strains UH203 (pEV218T7) , here called “T” cells, and UHF1 (pEV218) , here called “U” cells, were cultured overnight with lmM IPTG to induce expression of the ompA plasmids.
  • T cells E. coli strains UH203 (pEV218T7)
  • UHF1 pEV2128
  • U UHF1
  • Helper cells were added as described in the previous example.
  • One set of five mixed populations representing each of the serial dilutions of T cells (see table 10), was incubated for 1 hour at 37C with 340 nM mAbT7.
  • Two replicate sets of five mixed populations (a total of 10 populations, comprising two of each of the serial dilutions of T cells) were incubated under the same conditions without the antibodies.
  • phage K3 was added to the set of five mixed populations that had been incubated with the antibodies, as well as to one of the two sets of five mixed populations that were not incubated with the antibodies.
  • the presence of antibodies or phage is indicated by a +, whereas the absence of antibodies or phage is indicated by a -.
  • aliquots from each population were plated on agar containing the appropriate antibiotics, as described above. After overnight culture, colonies were counted to determine the composition of the CISTEM cell populations.
  • Table 10 shows not only the final numbers of cells, but also several indices of CISTEM' s performance in selecting T cells from a background of U cells. Enrichment, an index of the selection system's capacity for amplifying clones that bind the target compound while eliminating those that do not, is defined as ( T Cel l s /U Cel ls ) after selerr mn/ ( T Cel ls /U Cells ) befDre selection -
  • Yield an index of the proportion of all cells that bind the target compound that are successfully selected by the CISTEM, is estimated by comparing the number of T cells m the K3- populations with the number of T cells m the K3+, mAb+ populations. Yield is defined as (T cells) ⁇ +, Ab+/ (T cells) 3 -.
  • CISTEM One application of CISTEM is in mapping the epitopes involved in protem-protem interactions. To illustrate this application, CISTEM was used to select cells displaying the phage T7 polymerase epitope that is recognized by the T7-polymerase specific mAbT7 (described above) .
  • the plasmid pET17xb (Novagene) contains the entire gene for phage T7 polymerase. This plasmid was digested with micrococcal nuclease, according to protocols provided by the supplier, to yield fragments of a range of sizes from approximately 50 basepairs to 300bp.
  • the band corresponding to fragments between 60 and 75 bp in length was excized with a razorblade and the fragments were resuspended.
  • the fragments were treated with SI nuclease (conditions according to supplier) to remove the 3' phosphates left by micrococcal nuclease.
  • the fragments were then phosphorylated by T4 polynucleotidkmase .
  • the ompA plasmid pEV218 (described above) was cleaved by the restriction enzymes Ecll36II and Sail (conditions of digestion according to supplier) , at unique sites in the genetic region corresponding to the fourth extracellular loop of the mature polypeptide.
  • the linearized vector was dephosphorylated by calf intestinal phosphatase. Combining lOOng linearized vector with 200ng fragments, the fragments were ligated into the linearized vector by standard blunt-end ligation using T4 ligase. PEG was added to the ligation buffer to favor circularization. Circularization of the vector without insertion of a fragments creates a Sail site. Therefore, the products of ligation were treated with Sail to linearize vectors that did not contain a fragment. The circularized vectors containing fragments were isolated by electrophoresis, and used to transform the highly competent, commercially available strain XL-1 blue. Cells were plated on LB supplemented with lOO ⁇ g/ml ampicillin to select for transformants.
  • Colonies were grown in 250ml LB supplemented with ampicillin, as well as glucose for catabolic repression of low- level expression of the ompA plasmid. Plasmid DNA was extracted using the Qiagene kit. 7 ⁇ g of plasmid DNA was cleaved by Sail and used for transformation of UH203 (an ompA deficient strain described above) by electroporation and CaMg transformation. Cells were grown on LB supplemented with lOO ⁇ g ampicillin to select for transformants. This population of transformants will be refered to as UH203 (pEV218T7L) .
  • the UH203 (pEV218T7L) population was grown in overnight culture with lmM IPTG to induce expression of the ompA plasmid.
  • the cells were incubated for one hour with 680nM mAbT7, allowing for binding between the monocolonal antibody and the epitope presented at OmpA loopIV by certain members of the cell population.
  • Each polypeptide (column A) is listed with the selection agent (column B) that could be used in a CISTEM to imprint the polypeptide; the respective domains that compose the agent's adhesion domains (column C; numbering begins at the amino terminal) ; a domain that is interacting (e.g. for sterical reasons) with the adhesion domain and could be imprinted to bind a target compound (column D)
  • Table 3 Serial dilutions of selection agent and target concentrations in preliminary optimization of CISTEM selection. The cell populations listed in the table are described in the text.
  • Rhinoviruses propagation in cell culture.

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Abstract

Cette invention se rapporte à un procédé destiné à identifier et à isoler au moins un des composants suivants: un polypeptide d'intérêt et/ou un gène de ce polypeptide: un composé cible et/ou un gène de ce composé, le polypeptide d'intérêt, le composé cible, ou bien les deux à la fois étant inconnus. Le procédé consiste à: (1) afficher une bibliothèque de polypeptides d'intérêt sur la surface d'une population de cellules procaryotes ou eucaryotes; (2) exposer cette population à un composé cible; et (3) exposer ladite population à un agent sélecteur. L'interaction des trois composants 'grave' le polypeptide: en d'autres termes, cette interaction a pour effet de sélectionner les cellules dans lesquelles le polypeptide interagit avec le composé cible de la manière prescrite.
PCT/EP1998/007922 1997-12-05 1998-12-07 Technique de cellules pour criblage de bibliotheques combinatoires WO1999030151A1 (fr)

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AU18769/99A AU749042B2 (en) 1997-12-05 1998-12-07 Method and cells to screen combinatorial libraries
EP98963539A EP1036321A1 (fr) 1997-12-05 1998-12-07 Technique de cellules pour criblage de bibliotheques combinatoires
JP2000524659A JP2001526054A (ja) 1997-12-05 1998-12-07 組合せライブラリーをスクリーニングするための方法および細胞
CA002310381A CA2310381A1 (fr) 1997-12-05 1998-12-07 Technique de cellules pour criblage de bibliotheques combinatoires

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000045839A2 (fr) * 1999-02-01 2000-08-10 Cistem Biotechnologies Gmbh Procede de selection et de fabrication de preparations de vaccination et diagnostiques
WO2002102855A2 (fr) * 2000-11-17 2002-12-27 University Of Rochester Methodes in vitro de production et d'identification de molecules d'immunoglobulines dans des cellules eucaryotiques
AT410798B (de) * 2001-01-26 2003-07-25 Cistem Biotechnologies Gmbh Verfahren zur identifizierung, isolierung und herstellung von antigenen gegen ein spezifisches pathogen
US6706477B2 (en) 1997-09-22 2004-03-16 University Of Rochester Methods for producing polynucleotide libraries in vaccinia virus
WO2005001130A1 (fr) * 2003-06-09 2005-01-06 Applera Corporation Methodes pour selectionner des fractions de liaison proteinique
US7067251B2 (en) 2000-03-28 2006-06-27 University Of Rochester Methods of directly selecting cells expressing inserts of interest
EP1830190A2 (fr) * 2000-11-17 2007-09-05 University Of Rochester Procédés in vitro de production et d'identification de molécules d'immunoglobulines dans des cellules eucaryotes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
E.T. BODER ET AL.: "Yeast surface display for screening combinatorial polypeptide libraries", NATURE BIOTECHNOLOGY, vol. 15, no. 6, 1 June 1997 (1997-06-01), WASHINGTON DC USA, pages 553 - 557, XP002064402 *
M.C. KIEKE ET AL.: "Isolation of anti-T cell receptor scFv mutants by yeast surface display.", PROTEIN ENINEERING, vol. 10, no. 11, 1 November 1997 (1997-11-01), OXFORD UK, pages 1303 - 1310, XP002064403 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6706477B2 (en) 1997-09-22 2004-03-16 University Of Rochester Methods for producing polynucleotide libraries in vaccinia virus
US6872518B2 (en) 1997-09-22 2005-03-29 University Of Rochester Methods for selecting polynucleotides encoding T cell epitopes
WO2000045839A3 (fr) * 1999-02-01 2001-10-04 Cistem Biotechnologies Gmbh Procede de selection et de fabrication de preparations de vaccination et diagnostiques
WO2000045839A2 (fr) * 1999-02-01 2000-08-10 Cistem Biotechnologies Gmbh Procede de selection et de fabrication de preparations de vaccination et diagnostiques
US7067251B2 (en) 2000-03-28 2006-06-27 University Of Rochester Methods of directly selecting cells expressing inserts of interest
WO2002102855A3 (fr) * 2000-11-17 2003-06-26 Univ Rochester Methodes in vitro de production et d'identification de molecules d'immunoglobulines dans des cellules eucaryotiques
US7858559B2 (en) 2000-11-17 2010-12-28 University Of Rochester In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells
WO2002102855A2 (fr) * 2000-11-17 2002-12-27 University Of Rochester Methodes in vitro de production et d'identification de molecules d'immunoglobulines dans des cellules eucaryotiques
US8741810B2 (en) 2000-11-17 2014-06-03 University Of Rochester In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells
CN1306272C (zh) * 2000-11-17 2007-03-21 罗切斯特大学 筛选编码抗原特异性免疫球蛋白分子或其抗原特异性片段的方法
EP1830190A2 (fr) * 2000-11-17 2007-09-05 University Of Rochester Procédés in vitro de production et d'identification de molécules d'immunoglobulines dans des cellules eucaryotes
EP1830190A3 (fr) * 2000-11-17 2008-07-09 University Of Rochester Procédés in vitro de production et d'identification de molécules d'immunoglobulines dans des cellules eucaryotes
AT410798B (de) * 2001-01-26 2003-07-25 Cistem Biotechnologies Gmbh Verfahren zur identifizierung, isolierung und herstellung von antigenen gegen ein spezifisches pathogen
WO2005001130A1 (fr) * 2003-06-09 2005-01-06 Applera Corporation Methodes pour selectionner des fractions de liaison proteinique
JP2007501636A (ja) * 2003-06-09 2007-02-01 アプレラ コーポレイション タンパク質結合部分を選択するための方法

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