EP1570073A2 - Nachweis einer molekularen wechselwirkung durch reaktivierung eines autoinhibierten respondermoleküls (rair) - Google Patents
Nachweis einer molekularen wechselwirkung durch reaktivierung eines autoinhibierten respondermoleküls (rair)Info
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- EP1570073A2 EP1570073A2 EP03713528A EP03713528A EP1570073A2 EP 1570073 A2 EP1570073 A2 EP 1570073A2 EP 03713528 A EP03713528 A EP 03713528A EP 03713528 A EP03713528 A EP 03713528A EP 1570073 A2 EP1570073 A2 EP 1570073A2
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- European Patent Office
- Prior art keywords
- binding
- responder
- binding ensemble
- linked
- ensemble
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the yeast two-hybrid system has been used to identify cDNA translation products that interact with a protein of interest.
- This system uses fragments of a gene transcription factor fused to the protein of interest and candidate cDNA products, such that when an interaction occurs inside yeast cells, the fragments complement to activate expression of a responder gene.
- Another system based on complementation of fragments of the enzyme
- DHFR dihydrofolate reductase
- the current invention circumvents many of these limitations by using intact, natural proteins as responders, inhibitors, and reactivators.
- the full activity of the responder is available for more sensitive detection of the molecule or interaction of interest, and the stable components make these systems suitable for many applications for which fragment or subunit complementation systems are not practical.
- analyte-activated systems in which responder activation is directly coupled to interaction with a target analyte can form the basis of sensitive and convenient analyte assays.
- Such assays are homogeneous, requiring no manipulations other than mixing a clinical specimen with the components of a system of the invention, which include responder, inhibitor, and reactivator fused to molecules that bind the analyte, and in so doing shift the equilibrium of inhibitor binding from the responder to the reactivator, thereby activating the responder, such that the responder output is directly proportional to the absolute amount of analyte in the specimen.
- the current invention provides methods and systems for detecting a binding interaction.
- the systems comprise the following components: a responder protein, an inhibitor of the responder, a reactivator and binding ensemble members.
- the responder activity is inhibited by the inhibitor in the absence of a binding interaction between binding ensemble members, i.e., the responder is "automatically", or "auto"- inhibited.
- the invention provides a responder complex comprising a responder molecule, an inhibitor, and a binding ensemble member, which in some embodiments is a member of a plurality of candidate binding ensemble members.
- the components of the responder complex may be arranged in various configurations.
- the binding ensemble is linked to the responder molecule and the inhibitor is linked to the responder molecule.
- the responder molecule is linked to the member of the binding ensemble, and the inhibitor is linked to the member of the binding ensemble.
- the responder molecule is linked to the inhibitor and the binding ensemble member is linked to the inhibitor.
- Any of the linkages may be covalent or non-covalent.
- the covalent linkages further comprise a linker.
- the components of the responder complex are all linked by covalent linkages. In other embodiments, they are all linked by non-covalent linkages. In alternative embodiments, one linkage is non-covalent and the other is covalent.
- the invention provides a reactivator complex comprising a reactivator molecule and a binding ensemble member, which may be a member of a plurality of candidate binding ensemble members.
- the reactivator and binding ensemble member may be linked either covalently or non covalently.
- the covalent linkage may further comprise a linker.
- the invention also provides host cells, nucleic acids and expression vectors that encode responder and reactivator complexes in which the components are covalently linked.
- the invention provides systems comprising a responder complex and a reactivator complex, wherein the responder complex comprises a first member of a binding ensemble and the reactivator complex comprises a second member of the binding ensemble. Any of the responder complex configurations may be used in the systems. Further, the components of the reactivator and responder complexes may be covalently or non-covalently linked.
- the first member of the binding ensemble is a member of a plurality of candidate binding ensemble members.
- the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.
- the method further comprises a third member of the binding ensemble, wherein the first member of the binding ensemble and the second member of the binding ensemble interact with the third member of the binding ensemble.
- the third member may also be a member of a plurality of candidate binding ensemble members.
- all three binding ensemble members are members of a plurality of candidate binding ensemble members.
- the invention provides a method for detecting an interaction of binding ensemble members, comprising the steps of: providing a responder complex comprising a responder molecule, an inhibitor, and a first member of a binding ensemble; providing a reactivator complex comprising a reactivator molecule and a second member of the binding ensemble; combining the responder complex and the reactivator complex; and detecting an activity of the responder molecule, thereby detecting the interaction of the first and the second binding ensemble members.
- This process is termed "reactivation of an auto- inhibited responder," or "RAIR.” Any of the responder complex configurations may be used. Further, the components in the responder and reactivator complexes may be covalently or non-covalently linked.
- the method comprises a step of providing a
- the first member of the binding ensemble may be a member of a plurality of candidate binding ensemble members.
- the second member of the binding ensemble is a member of a plurality of candidate binding ensemble members.
- the third member may also be a member of a plurality of candidate binding ensemble members. In some embodiments, all three binding pair members or
- the invention provides a method of interaction mapping based on the RAIR process, comprising the steps of: providing a plurality of responder complexes comprising a responder molecule, an inhibitor and a binding ensemble member; providing a plurality of reactivator complexes comprising a plurality of candidate binding ensemble
- each candidate binding ensemble member is individually linked to a reactivator molecule; individually combining at least one member of the plurality of reactivator complexes with a responder complex; and detecting an activity of the responder molecule, thereby detecting the interaction of the binding ensemble member with at least one of the plurality of candidate binding ensemble members.
- the method may employ a
- the components of the responder and reactivator complex are linked covalently.
- the plurality of responder complexes is a plurality of one responder complex that comprises a particular binding ensemble member.
- the plurality of responder complexes may comprise complexes that are different, i.e., that
- 55 comprise at least two different candidate binding ensemble members.
- the invention provides a method of interaction mapping based on the RAIR process, comprising the steps of: providing a plurality of responder complexes comprising a plurality of candidate binding ensemble members; providing a plurality of reactivator complexes comprising a binding ensemble member linked to a reactivator
- the method may employ a responder complex that is in any of the configurations set forth above.
- the components of the responder and reactivator complex are linked covalently.
- the plurality of reactivator complexes is a plurality of a single reactivator complex that comprises a particular binding ensemble member.
- the plurality of responder complexes may comprise complexes that are different, i.e., that comprise at least two different candidate binding ensemble members.
- the invention provides a method for improving the affinity of a first binding pair member based on the RAIR process, the method comprising the steps of: providing a plurality of a first binding pair member; providing a plurality of a reactivator complex comprising a reactivator molecule and a second binding pair member; providing a plurality of responder complexes comprising a plurality of a responder molecule, a plurality of an inhibitor, and a plurality of candidate binding pair members, wherein the plurality of candidate binding pair members comprises variants of the first binding pair member; combining the reactivator complexes, the responder complexes, and the plurality of the first binding pair member, wherein a responder molecule is activated when a candidate binding pair member binds to a second binding pair member; and wherein the activity of the responder molecule is proportional to the affinity of the candidate binding pair members for the second binding pair member; and detecting an activity of the responder molecule corresponding to an affinity of the candidate
- the responder complex may be in any of the configurations described above. Often, the members of the responder and reactivator complexes are linked covalently. [15]
- the invention also provides a method for improving the affinity of a first binding pair member based on the RAIR process, the method comprising the steps of: providing a plurality of a first binding pair member; providing a plurality of a responder complex comprising a responder molecule, an inhibitor, and a second binding pair member; providing a plurality of reactivator complexes comprising a plurality of a reactivator molecule and a plurality of candidate binding pair members, wherein the plurality of candidate binding pair members comprise variants of the first binding pair member; combining the reactivator complexes, the responder complexes, and the plurality of the first binding pair member, wherein a responder molecule is activated when a candidate binding pair member binds to a second binding pair member; and wherein the activity of the responder molecule is proportional to the
- the responder complex may be in any of the configurations described above. Often, the members of the responder and reactivator complexes are linked covalently.
- the invention provides a method for isotropic selection of a plurality of binding molecules, also based on the RAIR process, the method comprising the steps of: providing a plurality of responder complexes comprising a responder molecule, an inhibitor, and a first member of a binding ensemble; providing a first plurality of reactivator complexes comprising a plurality of candidate binding ensemble members, wherein each candidate binding ensemble member is individually linked to a reactivator molecule; combining the plurality of responder complexes with the plurality of reactivator complexes; detecting a first set of binding molecules, each of which binds to the first member of the binding ensemble, by detecting a responder activity when one of the candidate binding molecules bind to the first member of the binding ensemble; providing a plurality of responder complexes, each comprising a responder
- the first plurality of reactivator complexes and the second plurality of reactivator complexes are the same.
- An additional method based on RAIR for isotropic selection of a plurality of binding molecules comprises the steps of: providing a first plurality of responder complexes comprising a plurality of candidate binding ensemble members wherein each candidate binding ensemble member is individually mixed with a responder molecule, and an inhibitor; providing a plurality of reactivator complexes comprising a reactivator linked to a first member of a binding ensemble; combining the first plurality of responder complexes with the plurality of reactivator complexes; detecting a first set of binding molecules, each of which binds to the first member of the binding ensemble, by detecting a responder activity when the candidate binding molecules bind to the first member of the binding ensemble; providing a second plurality of responder complexes comprising a plurality of candidate binding ensemble members wherein each candidate binding ensemble member is individually mixed with
- the steps comprise: providing a plurality of responder complexes comprising a plurality of candidate binding ensemble members wherein each candidate binding ensemble member is individually linked to the responder molecule and the inhibitor; providing a plurality of reactivator complexes comprising a plurality of candidate binding ensemble members, wherein each candidate binding ensemble member is individually linked to a reactivator molecule; providing a plurality of a first member of a binding ensemble; combining the plurality of responder complexes, the plurality of reactivator complexes and the plurality of the first member of the binding ensemble, wherein each combination is comprised of one responder complex, one reactivator complex, and the first binding ensemble member; and detecting a responder activity when a binding molecule from the first set of candidate binding molecules and a binding molecule from the
- Figure 1 illustrates interaction-mediated reactivation of an auto-inhibited responder (RAIR).
- RAIR auto-inhibited responder
- the responder is in a tri-partite fusion with a binding ensemble member and an inhibitor of the responder, such that the enzyme is constitutively inactive. No particular order of elements is preferred.
- a second binding ensemble member is fused to a reactivator.
- Fig. 1 depicts two types of reactivator, inhibitor-binding and responder-binding.
- the reactivator binds to the inhibitor with a higher affinity than that of the inhibitor for the responder, so that when the reactivator is docked to the responder - inhibitor complex by the subject molecule interaction, the binding equilibrium of the inhibitor is shifted from the responder to the reactivator, and the responder is thereby activated.
- the reactivator binds to the responder with higher affinity than that of the inhibitor for the responder, and without inhibiting it, so that when docked by the subject molecule interaction the reactivator displaces the inhibitor from the responder, and the latter is activated.
- Figure 2 illustrates interaction-mediated reactivation of BLIP - inhibited ⁇ -lactamase by an inhibitor binding reactivator, BIP (A.), and by a responder binding reactivator, BLIP- F142A (B.).
- BIP inhibitor binding reactivator
- B. responder binding reactivator
- BLIP- F142A competes with BLIP for binding to ⁇ -lactamase, but does not inhibit the enzyme.
- the affinity between inhibitor BLIP and ⁇ -lactamase is reduced by linker-induced strain and/or by using reduced-affinity mutations such as E104K, Q, D, or A in ⁇ -lactamase, or D49A in BLIP.
- FIG. 3 illustrates target-mediated reactivation of BLIP-inhibited ⁇ -lactamase.
- a third binding ensemble member, the target, to which the first two members are bound simultaneously is required to facilitate the docking of the reactivator, in this case BIP, to the BLIP - ⁇ -lactamase complex.
- Figure 4 illustrates an embodiment in which free wild-type ⁇ -lactamase is constitutively inhibited by wild-type BLIP fused to a first binding ensemble member, except when the first binding ensemble member interacts with other members of the ensemble, at least one of which is fused to a reactivator, in this case BIP.
- Figure 5 shows exemplary vectors for the expression in the E. coli periplasmic space of components for isotropic binding molecule selection of antibodies against all available epitopes on an antigen of interest using a ⁇ -lactamase RAIR system of the present invention.
- the antigen linked to the amino terminus of the BLIP - ⁇ -lactamase fusion, is co-expressed in E. coli cells with a library of antibody scFv fragments linked to the amino terminus of the reactivator, in this case BEP.
- the cells are then selected for growth in the presence of antibiotic.
- scFv are sub-cloned for expression as fusions to the amino terminus of the BLIP - ⁇ -lactamase fusion, and co-expressed with the free antigen and the scFv library - reactivator fusion.
- the cells are again selected for growth in the presence of antibiotic, such that surviving colonies will be expressing pairs of complementing scFv, which can activate ⁇ -lactamase only when bound simultaneously to the free antigen, lacp, inducible / ⁇ cUV5 promoter; clactp, constitutive mutant of the lacUV5 promoter; SP, signal peptide for translocation to the periplasmic space; tt, transcription terminator; kan, kanamycin resistance gene; cat, chloramphenicol resistance gene; pl5A ori and pBR322 ori, compatible plasmid origins of replication; fl ori, filamentous phage origin of replication
- Figure 6 shows a structural model of the reactivation of BLIP-inhibited ⁇ -lactamase by simultaneous binding of two antibody scFv fragments to non-overlapping epitopes on a free model antigen, in this case, the extra-cellular domain of the human B-cell activation antigen, CD40.
- the BLIP and ⁇ -lactamase structures were rendered from the x-ray crystal coordinates of Lim et al, 2001 (Nat.Struct.Biol. 8: 848ff).
- the scFv structures were rendered from the x-ray crystal coordinates of a model scFv (Eigenbrot et al, (1993) J.Mol.Biol 229: 969ff).
- FIG. 7 is a schematic showing affinity maturation by competition for limiting antigen using a ⁇ -lactamase RAIR system of the present invention.
- the molecule of interest i.e., the antigen, linked to the amino terminus of the BLIP - ⁇ -lactamase fusion, is co- expressed in E.
- coli cells with (a) a molecule which binds to the antigen ("competitor"), such as an antibody, and (b) a library of mutants of the competitor, linked to the amino terminus of the reactivator, in this case, BIP.
- the cells are then selected for growth in the presence of antibiotic at a concentration which is lethal for cells expressing the same binder as both the competitor and the reactivator fusion.
- antibiotic a concentration which is lethal for cells expressing the same binder as both the competitor and the reactivator fusion.
- Figure 8 shows exemplary vectors for affinity maturation of an antibody scFv fragment by competition for limiting antigen using a ⁇ -lactamase RAIR system of the present invention.
- the antigen is linked to the amino terminus of the BLIP - k ⁇ - lactamase fusion and library of "test variants" of the antigen-binding scFv is fused to the reactivator, while the scFv "competitor" is expressed from a separate vector.
- Figure 9 depicts selection of natural interactors from an expressed sequence library with a bait protein of interest using a ⁇ -lactamase RAIR system of the present invention.
- the bait protein is depicted as linked to the amino terminus of the BLIP - ⁇ -lactamase fusion, and the expressed sequence library is linked to the amino terminus of the reactivator, in this case BIP. Only cells expressing natural ligands of the bait protein will be able to reactivate ⁇ - lactamase and grow in the presence of the antibiotic.
- Figure 10 shows exemplary vectors for reactivation of auto-inhibited ⁇ -lactamase by the interaction of two leucine zipper helixes from the c-fos and c-jun subunits of the AP-1 transcription factor, and for inhibition of reactivation by competitive inhibition of the helix interaction by an additional copy of the c-jun helix.
- the c-fos helix is fused to the amino terminus of the BLIP - ⁇ -lactamase fusion and the c-jun helix is fused to the amino terminus of the reactivator, either BIP or BLIP-F142A.
- a second copy of the c-jun helix is expressed from the reactivator fusion vector as a fusion to the amino terminus of thioredoxin (for stability).
- the jun-trx fusion competes with the jun-reactivator fusion for binding to fos, thereby competitively inhibiting the reactivation of ⁇ -lactamase.
- Abbreviations are defined in the description of Figure 5.
- FIG. 11 A. Vectors for testing anti-CD40 antibody Fab fragments for their ability to mediate the reactivation of auto-inhibited ⁇ -lactamase when the latter is fused to CD40.
- the CD40 extra-cellular domain (CD40-ED) is expressed as an amino-terminal fusion to the BLIP - ⁇ -lactamase fusion, and co-expressed with three different Fabs fused to the BL1P- F142A reactivator, an anti-CD40 Fab (HB15), a higher-affinity variant of HB15 (HB15Y), and a negative control (anti-GST Fab).
- Each Fab was expressed from a dicistronic transcript with the light chain (LC) encoded by the upstream cistron and the Fd fragment encoded by the downstream cistron.
- the BLIP-F142A reactivator was fused to the amino terminus of the Fd fragment.
- Cells expressing the antigen fusion and each of the Fab fusions were scored for plating efficiency on increasing concentrations of antibiotic.
- the HB15 Fab gene was also inserted into the antigen-responder fusion vector to provide a non-activating competitor for binding to the antigen, and each Fab was tested for its ability to compete with HB15 to activate ⁇ -lactamase.
- IRES internal ribosome entry site for translation of the downstream cistron.
- CD40 - BLIP - ⁇ -lactamase fusion was split into two separate fusion proteins, each fused to an interacting leucine zipper helix and expressed from a separate gene.
- CD40 was fused to the amino terminus of the c-jun helix, and the c-fos helix was fused to the amino terminus of the BLIP - ⁇ -lactamase fusion. These fusions were then co-expressed with each of the anti- CD40 Fabs fused to the reactivator to test for the strength of ⁇ -lactamase reactivation when two docking interactions involving three binding ensemble members was required.
- Figure 12 provides an illustration of tri -molecular activation of auto-inhibited ⁇ - lactamase by the combined interactions of antigen with antibody and the c-fos/c-jun leucine zipper.
- the interaction of the c-fos and c-jun helixes docks the antigen to the BLIP - ⁇ - lactamase fusion, and the interaction of antigen and antibody docks the reactivator, in this case BLIP-F142A, to the auto-inhibited ⁇ -lactamase.
- Figure 13 shows enzymatic activities of ⁇ -lactamase reactivated in vitro by antigen- antibody interactions.
- B Reactions containing approximately 0.1 ⁇ M purified CD40 - BLIP - ⁇ -lactamase fusion and 0.1 ⁇ M of the indicated purified Fab/reactivator fusions in PBS were initiated with the addition of an excess ( ⁇ lmM) of the chromogenic ⁇ -lac
- FIG. 14 depicts structures of some anti-cancer drugs and their cephalosporin prodrugs.
- YW-200 is a DNA-binding tri-indole (Wang et al. (1998) US Patent 5,843,937), and YW-285 is its cephalosporin prodrug.
- Figure 15 illustrates the process of affinity maturation of an antigen-specific antibody using a cell-based selection system based on reactivation of an auto-inhibited reporter.
- Figure 16 shows vectors for expression of human CD40 and the anti-CD40 antibody KB2F10 as fusions to BLIP-inhibited ⁇ -lactamase and the reactivator BIP for affinity maturation of KB2F10 in Fab format.
- SP signal peptide for secretion into the bacterial periplasm
- lacp lactose operon promoter
- clacp constitutive mutant of the lactose operon promoter
- IRES internal ribosome entry site, for translation of the downstream cistron in a dicistronic cassette
- (G 4 S) 3 flexible hydrophilic 15-mer linker comprised of (Gly Ser) 3 ; tt, transcription terminator; kan, kanamycin resistance gene; ori, origin of replication; fl ori, filamentous phage origin of replication for transformation by phage transfection; cat, chloramphenicol resistance gene.
- Figure 17 shows graphs of plating efficiencies of E. coli cells expressing CD40 - BLIP - ⁇ -lactamase fusion with various positive and negative antibody control constructs to
- affinity matured in the context of binding ensemble members refers to a member that is derived from a reference binding ensemble member by, e.g., mutation,
- Antibody refers to a polypeptide comprising at least a heavy chain variable region
- immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
- Light chains are classified as either kappa or lambda.
- Heavy chains are classified as gamma, mu, alpha, delta, or epsilon,
- An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
- Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
- the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
- the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain variable regions respectively.
- antibody may also refer to any functional, i.e., capable of binding specifically to an epitope, VH and VL pair that are each linked in various configurations to other polypeptide(s) that may perform various functions, e.g., as responder, inhibitor, or stabilizer of the VH-VL complex.
- Antibodies exist, e.g., as intact immunoglobulins, as a number of well-characterized fragments produced by digestion with various peptidases, or as well-characterized fragments produced by recombinant gene expression.
- pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 (Fd fragment) by a disulfide bond.
- the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer.
- the Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature 348:552-554 (1990)).
- phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al, Nature 348:552-554 (1990); Marks et al, Biotechnology 10:779-783 (1992)).
- Binding refers to the non-covalent adherence of molecules to one another, for example, enzymes to substrates, antibodies to antigens, DNA strands to their complementary strands. Binding occurs because the shape and chemical natures of parts of the molecules surfaces are complementary.
- Binding affinity is generally expressed in terms of equilibrium association or dissociation constants (K a or K d , respectively), which are in turn reciprocal ratios of dissociation and association rate constants (k ⁇ and k a , respectively).
- K a or K d equilibrium association or dissociation constants
- k ⁇ and k a reciprocal ratios of dissociation and association rate constants
- a "binding ensemble member” refers to a molecule that participates in a specific binding interaction with another member of the binding ensemble.
- a binding ensemble often comprises two members, i.e., a binding pair, but can comprise three or more members.
- an antigen and two antibodies that recognize two different epitopes on the antigen and can be bound to the antigen at the same time comprise a binding ensemble.
- the "third” member e.g., an antigen, brings the first and second members of the binding ensemble, e.g., two antibodies, into proximity.
- a third member of a binding ensemble need not be a single molecule, e.g., a single protein or polypeptide, but may comprise multiple subunits.
- binding ensemble members may therefore bind to either the same subunit or different subunits.
- a cell and two antibodies that bind to two different epitopes on one cell surface protein or to two different cell surface proteins at the same time comprise a binding ensemble in which a "third" member, i.e., the cell, comprises multiple subunits.
- Binding ensemble members can include antibodies/antigens, receptors/ligands, biotin/avidin, and interacting protein domains such as leucine zippers and the like, as well as components of supra-molecular structures such as ribosomes, transcription complexes, cytoskeletal structures, signal transduction complexes, and metabolic complexes.
- binding ensemble member as used herein can be a binding domain, i.e., a subsequence of a protein that binds specifically to another member of the binding ensemble.
- the binding pair members can also be referred to as a binding pair member and a binding partner (or cognate binding partner).
- Binding ensembles can also include docking agents, i.e., members that are added to dock binding ensemble members to the responder and/or the inhibitor, or to the reactivator, such as, for example, biotin/avidin, antibody/antigen, or leucine zipper.
- a “complex” as used herein refers to an assemblage of components that are linked, either covalently or non-covalently, e.g., via a binding interaction. As appreciated by one of skill in the art, components that are linked by a binding interaction will typically be in an equilibrium, depending on the affinity and concentration of the components. [46] “Docking” and “dock” refer to a binding interaction between two molecules which brings other molecules into proximity, which other molecules are linked to the docking molecules.
- Domain refers to a unit of a protein or protein complex, comprising a polypeptide
- the function is understood to be broadly defined and can be binding to a binding partner, catalytic activity, structural activity, or can have a stabilizing effect on the structure of the protein.
- Domain also refers to a structural unit of a protein or protein complex, comprising one or more polypeptide sequences where that unit has a
- expression vector includes vectors which are capable of expressing nucleic acid sequences contained therein, i.e., any nucleic acid sequence which is capable of
- [5 effecting expression of a specified nucleic acid code disposed therein the coding sequences are operably linked to other sequences capable of effecting their expression.
- Some expression vectors are replicable in the host organism either as episomes or as an integral part of the chromosomal DNA.
- a useful, but not a necessary, element of an effective expression vector is a marker encoding sequence— i.e. a sequence encoding a protein which results in a
- Expression vectors are frequently in the form of plasmids or viruses. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which may, from time to time become known in the art.
- Heterologous when used with reference to portions of a protein, indicates that the protein comprises two or more domains that are not found in the same relationship to each other in nature.
- a protein e.g., a fusion protein or a conjugate protein, contains two or more domains from unrelated proteins arranged to make a new functional protein.
- Heterologous may also refer to a natural protein when it is found or expressed in an unnatural
- SO location such as when a mammalian protein is expressed in a bacterial cell.
- immunoglobulin variable region domain refers to any VH or VL domain used as a binding moiety without a companion VH or VL domain. As with antibodies, such domains may be linked in various configurations to other polypeptide(s) that may perform various functions, e.g., as responder, inhibitor, or reactivator.
- An "inhibitor” refers to a molecule that can inhibit the responder when both are in the responder complex. In one embodiment, the inhibitor, a binding ensemble member, and the responder are linked together. In an alternative embodiment, the inhibitor is linked to a binding ensemble member and the responder is free in solution.
- a "low-affinity inhibitor” is a relative term referring to an embodiment of the inhibitor where the inhibitor has a K d (equilibrium dissociation constant) for the responder which is at least ten-fold higher than the working concentration of the inhibitor, such that the inhibitor cannot bind to the responder to an appreciable extent without a heterologous mechanism for bringing the two together.
- interaction refers generally to attractive physical interactions, i.e., the binding of two or more molecules into a supra-molecular complex which is stable in the sense that each component has an affinity for at least one other member of the complex corresponding to a K d of ⁇ lmM.
- interaction when referring to the interaction of binding ensemble members generally refers to binding to one another. However, it may also refer to indirect interaction mediated by other molecules, usually additional binding ensemble members. Accordingly, a molecule that interferes with the binding interaction of binding ensemble members with one another decreases or prevents binding of a binding ensemble member to another member of the binding ensemble.
- Typical binding pairs include antibodies/antigens, receptor/ligands, subunits of multimeric proteins or supra-molecular structures.
- Binding or "interacting” as used herein refers to noncovalent associations, e.g., hydrogen bonding, ionic bonding, electrostatic bonding, hydrophobic interaction, Van der Waals associations, and the like.
- ligand refers to a molecule that is recognized by, i.e., binds to, a particular receptor.
- a molecule or macromolecular complex
- a ligand can be both a receptor and a ligand, typically when both are soluble or both are membrane-bound. However, when one is membrane-bound and the other is soluble, the former is commonly referred to as the receptor and the latter is the ligand.
- the binding partner having a smaller molecular weight is typically referred to as the ligand and the binding partner having a greater molecular weight is referred to as a receptor. More generally, the binding partners of non-receptor proteins may also be referred to as ligands.
- a "linker” or “spacer” refers to a molecule or group of molecules that covalently connects two molecules, such as a binding pair member and a responder or an inhibitor, and serves to place the two molecules in a preferred configuration, e.g., so that a responder can interact with an activator or inhibitor with minimal steric hindrance from a binding pair member, and a binding pair member can bind to a binding partner with minimal steric hindrance from the responder or inhibitor.
- a “flexible linker” refers to a peptide linker of any length in which the amino acid composition minimizes the formation of rigid structure by interaction of amino acid side chains with each other or with the polypeptide backbone.
- a “flexible linker” is rich in glycine.
- An example of such a linker has the composition (Gly 4 Ser) x , where "x" may typically vary from 1 to 10.
- "Link” or “join” or “fuse” refers to any method of functionally connecting peptides, typically covalently, including, without limitation, recombinant fusion of the coding sequences, and covalent bonding (e.g., disulfide bonding).
- a binding pair member is typically linked or joined or fused, often using recombinant techniques, at the amino-terminus or carboxyl-terminus by a peptide bond to a responder or to an activator or inhibitor of the responder.
- binding pair member may also be inserted into the responder or inhibitor at an internal location that can accept such insertions.
- the binding pair member can either directly adjoin the fragment to which it is linked or fused or it can be indirectly linked or fused, e.g., via a linker sequence.
- "Linked" may also refer to a non-covalent physical association, particularly one which is constitutive, i.e., does not require docking, under operating conditions.
- each component is typically linked to at least one other component, either covalently, e.g., via peptide linkage, or non-covalently, via high-affinity binding interaction.
- a "member” or “component” in the context of a responder system refers to a responder, a fragment or subsequence of a responder, a subunit of a responder, or an activator or inhibitor of the responder.
- the responder can be a complete polypeptide, or a fragment or subsequence thereof that retains responder activity.
- operably linked refers to a linkage of polynucleotide elements in a functional relationship.
- a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
- a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
- Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
- a "reactivator” as used herein refers to a molecule, typically a protein, that can displace an inhibitor from a responder and thereby activate the responder. In one
- the reactivator binds to the inhibitor. In an alternative embodiment, the reactivator binds to the responder. In another embodiment, the reactivator binds anywhere on the responder complex, e.g., at a junction between two components of the responder complex or to two components of the complex. In preferred embodiments, the binding of reactivator occurs only when it is brought into proximity with the inhibited responder by interaction of
- reactivator complex refers to a complex comprised of the reactivator and a binding ensemble member.
- the reactivator and the binding ensemble member may be complexed together by a binding interaction.
- the reactivator and binding ensemble member are part of the same polypeptide chain.
- the reactivator complex include a linker.
- Recombinant nucleic acid refers to a nucleic acid in a form not normally found in nature. That is, a recombinant nucleic acid is flanked by a nucleotide sequence not naturally flanking the nucleic acid or has a sequence not normally found in nature. Recombinant nucleic acids can be originally formed in vitro by the manipulation of nucleic acid by
- Recombinant polypeptide refers to a polypeptide expressed from a recombinant nucleic acid, or a polypeptide that is chemically synthesized in vitro.
- a “reference binding ensemble member” is a known binding ensemble member for which the practitioner wants to obtain a variant with higher binding affinity, i.e., an
- the term "responder” refers to any protein that produces a detectable signal, including, but not limited to detectable signals such as fluorescence, enzymatic activity, a selectable phenotype (e.g., antibiotic resistance), a screenable phenotype, or that produces an activity that results in a phenotypic change or provides a functional product that is detectable.
- detectable signals such as fluorescence, enzymatic activity, a selectable phenotype (e.g., antibiotic resistance), a screenable phenotype, or that produces an activity that results in a phenotypic change or provides a functional product that is detectable.
- reporter refers to a responder that produces a detectable signal, or that confers a selectable phenotype.
- responder complex refers to a complex comprised of the responder, the inhibitor, and a binding ensemble member.
- One or more members of the responder complex may be complexed with the others by a binding interaction.
- At least one member of the responder complex may also be in the same polypeptide chain as at least one other member. In a preferred embodiment, all three members of the responder complex are in the same polypeptide chain.
- single-chain antibody refers to a polypeptide comprising a V H domain and a V L domain in polypeptide linkage, generally linked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser] x ), and which may comprise additional amino acid sequences at the amino- and/or carboxyl-termini.
- a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.
- a scFv is a single-chain antibody.
- Single-chain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 contiguous amino acids substantially encoded by genes of the immunoglobulin superfamily (e.g., see The Immunoglobulin Gene Superfamily, A. F. Williams and A. N. Barclay, in Immunoglobulin Genes, T. Honjo, F. W. Alt, and T. H. Rabbitts, eds., (1989) Academic Press: San Diego, Calif, pp.361-387, which is inco ⁇ orated herein by reference), most frequently encoded by a rodent, non-human primate, avian, porcine, bovine, ovine, goat, or human heavy chain or light chain gene sequence.
- a functional single-chain antibody generally contains a sufficient portion of an immunoglobulin superfamily gene product so as to retain the property of binding to a specific target molecule, typically a receptor or antigen (epitope).
- target molecule is used to refer to a binding ensemble member which is being tested for its presence in a sample or specimen, e.g., a clinical, biological, or environmental sample, or which is being used as a "homing" device to co-locate auto- inhibited responder and reactivator at a specific location, e.g., at the site of a tumor in the body, or which is being used as a switch (wherein it may also be called a "switch molecule" to activate a responder, e.g., at a desired time.
- a "target molecule” binds to two members of a binding ensemble, one of which is linked to the auto-inhibited responder and the other of which is linked to the reactivator, such that the responder and reactivator are brought into mutual proximity, and the former is reactivated thereby.
- the target molecule can be any of a number of molecules including peptides, chemicals, carbohydrates, lipids, etc.
- a "scaffolded peptide” refers to a peptide, typically of up to about 20 amino acids in length, that is inserted into a natural protein at a location known to accept such insertions without interfering with the folding or native configuration of the protein (A Skerra, J Mol Recognit 13:167-87, 2000). Usually the location is on the surface of the protein. Often, the peptide is not a known natural sequence, and therefore is not expected to fold into a stable structure on its own, but generally assumes a random coil structure in solution. However, when inserted into the scaffold protein the peptide is expected to acquire some degree of stable structure by packing against the surface of the protein.
- Such structure generally improves the ability of the peptide to bind with high affinity to other molecules, such as other proteins.
- Many proteins may serve as scaffolds for random peptide libraries.
- surface loops between elements of secondary structure such as ⁇ -helixes or strands of a ⁇ - sheet may accept such insertions without significant perturbation of folding or structure.
- proteins that have been used as scaffolds include, but are not limited to, thioredoxin (or other thioredoxin-like proteins), nucleases (e.g., RNase A), proteases (e.g., trypsin), protease inhibitors (e.g., bovine pancreatic trypsin inhibitor), antibodies or structurally-rigid fragments thereof, and other domains of the immunoglobulin superfamily.
- nucleases e.g., RNase A
- proteases e.g., trypsin
- protease inhibitors e.g., bovine pancreatic trypsin inhibitor
- antibodies or structurally-rigid fragments thereof e.g., bovine pancreatic trypsin inhibitor
- the detection systems disclosed herein overcome many limitations of molecular detection systems in the art, such as component instability and weak activity, and allow applications that are not possible or practical with prior art systems, e.g., therapeutic applications or in vitro applications.
- This invention provides novel methods and systems for linking the activity of a responder protein to the interaction of two or more binding ensemble members of interest, either in vitro or in vivo, and thereby producing a signal, phenotype, or other functional output in response to the interaction of the binding ensemble members.
- the systems of the invention comprise the following components: a responder protein, an inhibitor of the responder, an inhibitor of the inhibitor, i.e., a "reactivator" of the responder protein, and interacting components that comprise the binding ensemble members.
- the system basically operates such that, in the absence of a binding interaction between binding ensemble members, the inhibitor inhibits the activity of the responder molecule.
- the reactivator displaces the inhibitor from the responder molecule. Detection of responder molecule activity, or of a desired level of responder molecule activity, thus indicates the presence of a binding interaction.
- the system components can be arranged in a number of different configurations. In one embodiment, a first member of a binding ensemble is linked via a flexible linker to a fusion of the responder molecule and an inhibitor of the responder; and a second binding ensemble member is linked to the reactivator molecule (see, e.g., Figure 1).
- the first member of the binding ensemble may be linked to a high- affinity inhibitor of the responder molecule with the responder being unlinked (see, e.g., Figure 4); or the binding ensemble member may be linked to the responder with the inhibitor being unlinked.
- the reactivator and the second binding ensemble member are complexed by a binding interaction rather than a covalent linkage.
- the responder protein is constitutively bound by the inhibitor, and is therefore inactive, when the binding ensemble members do not interact. The responder is thus an auto- inhibited responder.
- the inhibitor is displaced from the responder by the reactivator, so that the responder becomes functionally active.
- This process is referred to as "reactivation of auto-inhibited responder," or "RAIR.”
- Displacement of the inhibitor by the reactivator may be direct (e.g., competitively) or indirect (e.g., allosterically).
- the responder and inhibitor of the responder are fused to one another via a flexible linker, such that the responder is constitutively auto-inhibited (i.e., on the same conjugate molecule), or "masked".
- This masked responder and the reactivator are each fused, via flexible linkers, to different binding ensemble members, such that interaction of the binding ensemble members, either directly or via one or more additional binding ensemble members, facilitates displacement of the inhibitor from the responder by the reactivator, thereby causing functional reactivation of the responder (see, e.g., Figure 1).
- the components of the RAIR system may be deployed in vitro, as in an assay for an analyte in a biological specimen, or they may be expressed in cells, as in a selection system for protein-protein interactions or antibodies for a particular antigen.
- an enzyme responder which is activated by direct allosteric interaction with analyte, can be used in excess, so that equilibration is rapid and independent of the analyte concentration, and the analyte can be saturated to produce signal from every molecule.
- an enzyme responder which is activated by direct allosteric interaction with analyte
- microbial or viral pathogens where unique surface markers may be present in hundreds to thousands of copies per cell or particle, such enzymes, which would be activated by binding to the marker, can allow rapid detection of as little as a single cell or particle, whereas the sensitivity of equilibrium assays for such analytes would typically be much lower.
- Biosensors are devices for automated electronic or optical detection and quantification of analytes (Lowe (1989) Philos Trans R Soc LondB Biol Sci., 324:487-96). They are extremely useful in many different types of automated process control, from pharmaceutical and industrial purification processes to the monitoring of toxic substances in natural resources and the environment. Most current biosensor platforms are quite limited in the types of molecules they can detect. For example, most require enzymatic oxidation or other chemical transformation of the analyte. A few biosensors work by coupling specific analyte binding to the enzymatic generation of an electrical or optical signal, but these are generally limited to a small number of applications.
- the systems of this invention can be set up to couple the binding of any analyte, including small molecules, macromolecules, viruses, and cells, to the generation of electrical or optical signals by using an appropriate enzyme or fluorescent responder protein with an inhibitor and a reactivator linked to binding ensemble members that can be coupled by binding to the analyte (which is also a binding ensemble member), whereupon the responder is activated and an electrical or optical signal is generated which is directly proportional to the absolute amount of analyte in the sample.
- Systems of the present invention can also be used to activate effector molecules upon binding to specific cell surface molecules. This would allow the effector to become localized and activated at specific sites in the body for target-restricted activation of reagents for therapy or imaging.
- Antibody-Directed Enzyme Prodrug Therapy is a promising chemotherapeutic strategy for the treatment of cancer, in which a prodrug-activating enzyme, such as a ⁇ -lactamase, is targeted to the tumor by a tumor-specific antibody to which it is chemically or genetically conjugated. After unbound conjugate has cleared the circulation, an inactive prodrug, such as an anthracycline cephalosporin, is administered, which is converted to a potent tumor-killing cytotoxin at the site of the tumor by the remaining tumor-bound enzyme.
- a prodrug-activating enzyme such as a ⁇ -lactamase
- an inactive prodrug such as an anthracycline cephalosporin
- ADEPT The main problem with ADEPT is that the unbound conjugate must clear the circulation before the prodrug can be administered in order to minimize systemic toxicity. However, by the time the conjugate has cleared the circulation >90% of the tumor bound enzyme has been lost (Bagshawe (1995) Drug Devel Res 34: 220-30; Springer and Niculescu-Duvaz (1995) Anti-Cancer Drug Design 10: 361-72). In spite of this, ADEPT has been able to achieve higher active drug concentrations in the tumor than any other procedure (Sedlacek et al. in Contributions to
- systems of the invention can be targeted for activation by surface markers on other types of cells and tissues, such as pathogen-infected cells, transplants, and sites of inflammation or atherogenesis.
- the target-localized and activated enzymes can then be used to activate not just cytotoxins, but other types of therapeutic agents such as small molecule agonists or antagonists of biological response modifiers, as well as imaging reagents for precise localization of tissue with disease or other phenotype of interest.
- target-activated enzymes maybe used to deliver: (1) immune stimulants to tumors, (2) immuno-suppressants to sites of chronic inflammation or to organ transplants to inhibit rejection, (3) antibiotics to specific pathogens, (4) cytotoxins and anti-virals to virus-infected cells, (5) hormones and other pleiotropic agents to specific cells and/or tissues, or (6) neuro- transmitters and other neuro-modulators to specific nerves or tissues.
- target- activated enzymes may be used to deliver to any tissue any small molecule cytotoxin, hormone, steroid, prostaglandin, neuro transmitter, or agonist/antagonist of peptide hormone, cytokine, or chemokine, etc., which can be inactivated by conjugation to the appropriate substrate.
- Target-activated enzymes of the present invention can be used in vivo as sensors for rapid detection of the activation or inhibition of key steps in metabolic, signal transduction, cell cycle regulation, or gene expression pathways, enabling high-throughput cellular screens for inhibitors or activators of those pathways.
- screening for agonists or antagonists of receptor tyrosine kinases or G-protein-coupled receptors usually requires coupling receptor ligation to a selectable phenotype which results from de novo gene expression.
- Such multi-step signal generating mechanisms are prone to high rates of false positive and false negative selection, severely compromising their efficiency for high- throughput screening.
- target- activated enzymes of the present invention may be set up for activation by phospho-tyrosine-containing or GTP-bound signal transducers, so that a selectable phenotype is generated just downstream from receptor ligation.
- Interaction between the signal transducer and the enzyme may be designed to be either dependent on, or inhibited by phosphorylation or bound GTP/GDP, so that either receptor agonists or receptor antagonsists can be selected.
- Responders include any protein that produces a detectable signal, a selectable phenotype, or which performs a useful function in response to the interaction of binding ensemble members.
- enzymes such as ⁇ -lactamase may be used to generate a color signal from chromogenic or fluorogenic substrates, or to confer an antibiotic resistance phenotype on host bacterial cells, or to activate a cephalosporin prodrug to produce a cancer- killing drug upon interaction with, i.e., binding to a cancer marker.
- Other enzymes that can be used as responder proteins include those that hydrolyze chromogenic or fluorogenic substrates to yield a colored or fluorescent product.
- Non-enzymatic molecules can also be employed as responders using the methods of the invention.
- biological response modifiers such as insulin
- useful cellular functions such as glucose uptake in insulin-dependent diabetics, in response to the presence of subject molecules, such as glucose.
- Fluorescent proteins such as the green fluorescent protein (GFP) of Aequorea victoria (Chalfie et al, (1994) Science 263: 802-805) can also be employed as responders.
- GFP absorbs blue light and fluoresces green.
- GFP fluorescence can be quenched or shifted in cis by fusing it to a protein or other molecule that perturbs the chromophore.
- Reactivators can be fashioned from proteins or other molecules that bind to the quencher/shifters, thereby preventing them from binding to GFP. With such components GFP can be used in systems of the present invention.
- responder molecules can be found in U.S. Patent Nos. 6,294,330; 6,220,964; 6,342,345; and/or U.S. Patent Application Serial No. 09/526,106, filed on March 15, 2000, which are hereby inco ⁇ orated by reference.
- Inhibitors may include natural inhibitors or artificial inhibitors.
- inhibitors include polypeptides, nucleic acids, carbohydrates, other macromolecules, or small molecules (i.e., ⁇ 1 kDa MW).
- Many responders have known natural inhibitors which may be used in the invention.
- the ⁇ -lactamase Inhibitor Protein (BLIP; Strynadka et al, (1994) Nature 368: 657-660) is a 165 amino acid natural inhibitor of ⁇ -lactamase which may be used in the present invention when ⁇ - lactamase is the responder (see, e.g., Figure 2).
- proteases also have natural protein inhibitors, e.g., thrombin and anti-thrombin, trypsin has many inhibitors, etc.
- Other inhibitors known in the art include: pyridindolol for beta-galactosidase; urethane for firefly luciferase; the bacterial luciferase inhibitor reported in Kratasiuk et al, (1978) Biokhimiia 43(8): 1369-76; Src kinase specific inhibitor PP2; and Rho-kinase specific inhibitor Y-27632.
- Inhibitors may be selected from any of the small molecule/combinatorial libraries that are well-known in the art (see e.g., U.S. Patent Nos. 6,294,330, 6,343,257, 6,281,245, 6,274,716, 6,255,120, 6,239,152, 6,207,820, 6,191,256, 6,127,191, 6,093,798, 5,977,328, 5,965,718, 5,962,337, 5,958,792, 5,877,278, 5,792,821, 5,766,963, 5,663,046, 5,549,974, hereby inco ⁇ orated by reference).
- Inhibitors may also be selected from random peptide libraries or from natural populations of binding proteins with diverse binding specificities such as antibodies or immunoglobulin variable region domains. Random peptide libraries may be inco ⁇ orated into the sequence of a suitable scaffold protein such that upon folding of the scaffold protein the peptides are displayed or "scaffolded" on the protein surface (see, e.g., Skerra (2000) J Mol Recognit. 13:167-87). Many different proteins may be used as scaffolds. These proteins are typically small in size (e.g., less about 200 amino acids), rigid in structure, of known three dimensional configuration, and are able to accommodate insertions of peptides of interest into exposed loops without undue disruption of their structures.
- scaffold proteins are capable of stable expression in both prokaryotic and eukaryotic hosts.
- Such scaffold proteins can be stably expressed at high levels in various prokaryotic and eukaryotic hosts, or in suitable cell- free systems.
- the scaffold proteins are generally soluble and resistant to protease degradation.
- thioredoxin has been widely used.
- Peptide libraries typically of 6-20 random amino acids, can be inserted into the active site of thioredoxin without disturbing its stability.
- Thioredoxin has the further advantage that it is much smaller than most natural inhibitors, and is therefore less sterically constrained when access to the responder is restricted by linker lengths and the orientations of interacting subject molecules.
- Another good scaffold is the immunoglobulin domain, of which the antibody variable region domain is a prime example (Skerra (2000) JMol Recognit 13: 167-87).
- the immunoglobulin superfamily is one of the largest families of structurally homologous protein folds found in nature (Hawke et al. (1999) Immunogenetics 50:124-33). Immunoglobulin domains are comparable in size to thioredoxin and tolerate random peptide libraries in a number of exposed loops in the structure.
- Other scaffold proteins useful in the invention include RNase A, proteases (e.g., trypsin), and protease inhibitors (e.g., bovine pancreatic trypsin inhibitor).
- any of a variety of systems that detect protein-protein interactions such as bacteriophage display (Phase Display of Peptides and Proteins Kay, Winter, and McCafferty, Eds. (1997) Academic Press, San Diego) or ⁇ -lactamase fragment complementation (US Patent Application Serial No. 09/526,106) or the system of the invention (using a responder with an already defined inhibitor and reactivator) can be used to select inhibitors that can be used in the present invention.
- a thioredoxin- scaffolded peptide (trxpep) library can be displayed on the surface of filamentous bacteriophage, and panned against the immobilized responder molecule.
- Phage which bind to the responder can then be recovered, and the encoded trxpeps can be individually screened for their ability to inhibit the responder only when both are fused to interacting subject molecules. It is reasonable to expect that a substantial proportion of responder - binding trxpeps will also inhibit the function of the responder.
- the responder itself may be used to screen trxpep libraries for inhibitors. The only requirement is that a null responder phenotype be selectable.
- the responder and trxpep library can be fused to each member of a model binding pair, such as the leucine zipper helices from the c- fos and c-jun subunits of the AP-1 transcription factor, and expressed in cells.
- an inhibitor trxpep will, upon docking to the responder by the binding pair interaction, render the host cells colorless or non-fluorescent, and this can be detected by eye or by flow cytometry.
- an inhibitor mask that quenches or shifts GFP fluorescence in cis can be selected from a random peptide library fused to either end of GFP via a flexible linker.
- a selectable phenotype-conferring responder such as chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPT II), can be fused via linker to the carboxyl terminus of the GFP/peptide library to ensure that selected masks do not quench by destabilizing GFP, in which case the selectable phenotype would also be quenched (see, e.g., co-pending US Patent Application Serial No. 09/510,097).
- the GFP/peptide-responder library is expressed in bacterial cells and plated on solid medium containing the selecting antibiotic (e.g., chloramphenicol or kanamycin).
- GFP quenchers for use in the present invention can also be isolated from antibody, immunoglobulin variable region, or scaffolded peptide libraries by phage display methods (Phase Display of Peptides and Proteins Kay, Winter, and McCafferty, Eds. (1997)
- Reactivators are molecules that can displace an inhibitor from a responder directly or indirectly.
- the affinity of the reactivator for either the inhibitor or the responder is greater than the affinity of the inhibitor for the responder, so that the inhibitor is sterically excluded from binding with the responder (see, e.g., Figure 1).
- reactivators may be selected from libraries of random peptides, scaffolded random peptides, antibodies, or other natural binding proteins. Reactivators may also be engineered from responders by introducing mutations which disable responder activity without imparing inhibitor binding or the stability of the reactivator.
- a reactivator for ⁇ -lactamase may be engineered by mutating the active site serine (Ser) of the enzyme to alanine (Ala). This mutation renders ⁇ -lactamase catalytically inactive, but still fully capable of binding its natural inhibitor, BLIP. Because of the concentration effect, this BLIP Inhibitor Protein (BIP) is not able to activate ⁇ -lactamase at working concentrations when the latter is fused to BLIP.
- Ser active site serine
- Al alanine
- BIP BLIP Inhibitor Protein
- BIP acts as a reactivator, i.e., it displaces BLIP from ⁇ -lactamase, thereby activating the latter (see, e.g., Figure 2A).
- BIP affinity is reduced by, e.g., a D49A mutation in BLIP, and/or by an E104K, D, Q, or A mutation in ⁇ -lactamase, and/or by linker-induced strain, reactivation by BIP is favored, and may go to near completion.
- Reactivators may also be engineered from inhibitors of responders by introducing mutations which impair inhibition of the responder active site without interfering with reactivator binding to the responder.
- a reactivator for ⁇ -lactamase may be engineered by mutating phenylalanine (F) 142 of BLIP to alanine (A). This mutation impairs BLIP'S ability to block the ⁇ -lactamase active site without impairing its ability to bind to ⁇ - lactamase (Petrosino et al , (1999) J Biol Chem 274:2394-2400).
- BLIP-F142A is not able to activate ⁇ -lactamase at working concentrations when fused to BLIP.
- BLIP-F142A is displaces BLIP from ⁇ -lactamase, thereby activating the latter (see, e.g., Figure 2B).
- the affinity of the ⁇ -lactamase - BLIP complex is reduced by strain from, e.g., a short linker, replacement of BLIP by BLIP-F142A will be favored, and may even go to near completion.
- Reactivators can be selected from libraries of random peptides, scaffolded peptides, antibodies, or other binding molecules by fusing the library to a binding ensemble members, and co-expressing this library in appropriate cells under selective conditions with the appropriate responder complex.
- Cells exhibiting the screenable/selectable phenotype conferred by the activated responder protein, i.e., the activated responder protein, should be expressing reactivator molecules.
- the activated responder protein i.e., the activated responder protein
- a random hexameric peptide library biased for hydrophilic residues was fused via flexible linker to the amino terminus of an antibody Fab fragment which bound human CD40 antigen.
- Fab fragment which bound human CD40 antigen.
- Reactivators for masked or cis-quenched GFPs which can be used in systems of the present invention may be obtained in several ways.
- a non- fluorescent GFP mutant e.g., Ser65Ala, Heim and Tsien (1996) Curr Biol 6: 178-82
- the quencher re-equilibrates with the reactivator, and GFP fluorescence is restored.
- GFP reactivators can also be isolated from antibody, immunoglobulin variable region, or scaffolded peptide libraries using the methods of the present invention.
- the cis-quenched GFP and a library of binding molecules such as trxpeps can each be fused to binding ensemble members.
- useful reactivators can be identified and isolated by screening the resultant colonies for fluorescence at the wild-type GFP maxima.
- Binding ensemble members for most applications will be two or more protein molecules or stable fragments thereof which interact with one another.
- the binding ensemble members may comprise binding pairs from recognizable classes, such as receptor/ligand pairs or antigen/antibody pairs.
- the binding ensemble members may comprise subunits of higher order structures such as multi- subunit enzymes or other functional protein assemblies of the cell, such as those responsible for cytoskeletal or nuclear architecture.
- the binding ensemble members may also include proteins or stable fragments thereof that interact transiently or temporally in such intra- cellular processes as signal transduction, gene expression, and metabolism.
- the binding ensemble members may comprise combinations of proteins of interest with one or two additional binding proteins or domains, which provide additional docking functions.
- an ensemble might comprise three molecules where the first and second molecules are complexed with the responder and reactivator, respectively, and the third molecule binds to the first and second molecules (and the first and second molecules do not bind to each other).
- an ensemble might comprise a bait protein (fused to the responder), and a pair of docking domains, such as leucine zipper helixes, fused to an expressed sequence
- Binding proteins or domains may include antibody fragments, peptides, scaffolded peptides, or immunoglobulin variable region domains, which have been selected from diverse natural or artificial populations for their ability to bind to the proteins of interest. [95] Many applications will involve the use of unmodified target molecules to dock the
- Such applications include detection of analytes in clinical, biological, manufactured, or environmental specimens; detection of cell surface molecules for e.g., prodrug activation; and mapping the epitopes of target molecules.
- Such applications typically use two binding molecules which bind non-overlapping epitopes on the target molecule, or which bind to each other only in the presence of the target molecule.
- Non-protein subject molecules can also be used.
- small molecules such as hormones (e.g., steroids), vitamins, nutrients (e.g., sugars), or toxins (e.g., anthracyclines),
- small molecules selected from small molecule/combinatorial libraries can be used as binding ensemble members in vitro.
- small subject molecules When the system is deployed in cells, small subject molecules may be used by conjugating them to a chemical tag such as biotin.
- conjugates typically can diffuse freely into the bacterial periplasm, allowing them to serve, for example, as antigens for antibody selection using the
- An antibody library is linked to either the ⁇ -lactamase - BLIP fusion or to BIP, and a binder to the chemical tag, such as streptavidin, is linked to the other component.
- Antibodies that bind the small subject molecule are selected by their ability to activate ⁇ -lactamase, thereby conferring antibiotic resistance on the cells. [97] Further examples of subject molecules are found in U.S. Patent Nos. 6,294,330;
- the binding of inhibitor to responder must be of relatively low affinity, i.e., stable enough to maintain the responder in the inactive state, but sufficiently unstable that it can readily be displaced by the reactivator when the latter is docked to the responder complex by the interaction of binding ensemble members.
- the affinity of the responder - inhibitor interaction must be high enough to prohibit reactivation in the absence of binding ensemble interaction at working concentrations.
- tethered proteins typically have the equivalent of millimolar concentrations relative to one another, but are present in cells or are used in vitro at concentrations of micromolar or less.
- a K in the sub-millimolar range should satisfy the concentration effect condition for an optimally reactivatable responder - inhibitor fusion.
- mutations may be introduced into ⁇ -lactamase at Glul04 (to Lys, Gin, Asp, or Ala), or into BLIP at Asp 149 (to Ala) or at Phe 142 (to Ala) that reduce the affinity without appreciable effect on stability or on the enzymatic activity of ⁇ -lactamase.
- a low-affinity ⁇ -lactamase Glul04 (E104) mutant when fused to BLIP, can be fully reactivated by the high-affinity wild-type BIP, or by BLIP-F142A, which binds ⁇ -lactamase but does not inhibit (Petrosino et al, (1999) JBiol Chem 274:2394-2400), when they are docked together by a subject protein interaction.
- wild- type ⁇ -lactamase can be fully reactivated by interaction-mediated docking to BIP or BLIP- F142A.
- a further reduction in affinity between linked ⁇ -lactamase and BLIP, or between any linked responder and its inhibitor, may result from the strain effect.
- the strain effect reflects the fact that, depending on the length and stiffness of the linker, tethered proteins typically have restricted freedom of movement relative to one another, so that the association rate component of affinity is lower than it would be if the proteins were not fused, i.e., had complete freedom of movement, at the same concentration. Further, the linked proteins may also be under tortional strain when bound to one another, and this would increase the dissociation rate component of affinity. [100] This strain effect allows the affinity of the responder - inhibitor interaction to be modulated to some extent by adjusting the length of the linker.
- the responder - inhibitor interaction it is useful for the responder - inhibitor interaction to have a higher dissociation rate constant (k d ) than that of the bound complex of binding ensemble members, so that when the responder - inhibitor fusion is docked to the reactivator by interaction between the subject binding ensemble members, the reactivator has time to bind to the inhibitor before the binding ensemble proteins disengage. Since subject protein interactions will typically have k d S in the range of 10 "3 sec " ' or lower, it is useful for the responder - inhibitor k to be higher than this value.
- One way in which the k of a fused responder - inhibitor complex may be increased is to shorten the linker between them, thereby increasing the torsional strain.
- the linker between wild-type ⁇ -lactamase and BLIP is 15 amino acid residues in length (e.g., (Gly 4 Ser) ) ⁇ -lactamase is fully inhibited, but the tortional strain on the complex is such that the enzyme can be fully activated when the complex is docked to BIP by a subject protein interaction, even though both BIP and ⁇ -lactamase have the same affinity for BLIP.
- the BIP-BLIP interaction in the docked complex is under much less strain than the ⁇ -lactamase - BLIP interaction, so that the equilibrium is shifted far toward the latter interaction.
- the responder molecule is unlinked and the inhibitor and reactivator are each linked to a first and second subject molecule, such that when the subject molecules interact, the reactivator is docked to the inhibitor, preventing the inhibitor from binding to the responder, and thereby activating the latter (see, e.g, Figure 4).
- a high-affinity interaction between responder and inhibitor is necessary to ensure that the responder is maximally inhibited at working concentrations in the absence of a subject molecule interaction.
- the affinity of the reactivator for the inhibitor must be lower, so that it does not bind to the inhibitor at working concentrations in the absence of a subject molecule interaction.
- Such a result can be obtained with the following conditions: (1) working concentrations of the components in the micromolar range, as when stably expressed in cells, (2) a modest K d of ⁇ 0.1 ⁇ M for both the responder - inhibitor interaction and for the subject molecule interaction, (3) a K of ⁇ 10 ⁇ M for the inhibitor-reactivator interaction, and (4) an effective mutual concentration in the millimolar range for the inhibitor and reactivator when docked together.
- responder - inhibitor and reactivator conjugates can be joined by methods well known to those of skill in the art. These methods include both chemical and recombinant means.
- the means of linking responder- inhibitor fusion, and the reactivator molecules to the subject molecules comprises a heterobifuncitonal coupling reagent that ultimately contributes to formation of an intermolecular disulfide bond between the two moieties.
- Other types of coupling reagents that are useful in this capacity for the present invention are described, for example, in U.S.
- an intermolecular disulfide bond can be formed between cysteine residues present in each of the protein molecules to be linked.
- the cysteines can occur naturally or are inserted by genetic engineering.
- the means of linking moieties may also use thioether linkages between heterobifunctional crosslinking reagents or specific low pH cleavable crosslinkers or specific protease cleavable linkers or other cleavable or noncleavable chemical linkages.
- the means of linking the heterologous domains of the protein can also comprise a peptidyl bond formed between domains that are separately synthesized by standard peptide synthesis chemistry or recombinant means.
- the protein itself can also be produced using chemical methods to synthesize an amino acid sequence in whole or in part.
- peptides can be synthesized by solid phase techniques, such as, e.g. , the Merrifield solid phase synthesis method, in which amino acids are sequentially added to a growing chain of amino acids (see, Merrifield (1963) J. Am. Chem. Soc, 85:2149-2146).
- Equipment for automated synthesis of polypeptides is commercially available from suppliers such as PE Co ⁇ . (Foster City, CA), and may generally be operated according to the manufacturer's instructions.
- the synthesized peptides can then be cleaved from the resin, and purified, e.g., by preparative high performance liquid chromatography (see Creighton, Proteins Structures and Molecular Principles, 50-60 (1983)).
- the composition of the synthetic polypeptides or of subfragments of the polypeptide may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, Proteins, Structures and Molecular Principles, pp. 34-49 (1983)).
- nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the sequence.
- Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, ⁇ -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxy-proline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -mefhyl amino acids, N ⁇
- the amino acid can be D (dextrorotary) or L (levorotary).
- the responder-inhibitor and reactivator conjugates are joined via a linking group.
- the linking group can be a chemical crosslinking agent, including, for example, succinimidyl-(N-maleimidomethyl)-cyclohexane-l-carboxylate (SMCC).
- SMCC succinimidyl-(N-maleimidomethyl)-cyclohexane-l-carboxylate
- the linking group can also be an additional amino acid sequence(s), including, for example, a polyalanine, polyglycine or similar linking group.
- the coding sequences of each polypeptide in the fusion protein are directly joined at their amino- or carboxy-terminus via a peptide bond in any order.
- an amino acid linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
- Such an amino acid linker sequence is inco ⁇ orated into the fusion protein using standard techniques well known in the art.
- Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that can interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
- Typical peptide linker sequences contain Gly, Val and Thr residues.
- linker sequences which may be usefully employed as linkers include those disclosed in Maratea et al (1985) Gene 40:39-46; Mu ⁇ hy et al (1986) Proc. Natl. Acad. Sci. USA 83:8258-8262; U.S. Patent Nos. 4,935,233 and 4,751,180.
- the linker sequence may generally be from 1 to about 50 amino acids in length, e.g., 3, 4, 6, or 10 amino acids in length, but can be 100 or 200 amino acids in length. Linker sequences may not be required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
- Other chemical linkers include carbohydrate linkers, lipid linkers, fatty acid linkers, polyether linkers, e.g., PEG, etc.
- poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or hetero functional linkages.
- Other methods of joining the components of the responder, inhibitor and reactivator conjugates include ionic binding by expressing negative and positive tails, and indirect binding through antibodies and streptavidin-biotin interactions. (See, e.g., Bioconjugate Techniques, supra). The components can also be joined together through an intermediate interacting sequence.
- the moieties included in the conjugate molecules can be joined in any order, and the most favorable order is generally determined empirically to be that which produces the least steric impediment to responder reactivation upon the interaction of subject molecules which are linked or conjugated to the responder and the reactivator.
- a plurality of putative binding ensemble members are bound to their respective complex (either responder or reactivator) through the interaction of a pair of other binding ensemble, such as for example, antibody/antigen, receptor/ligands, biotin avidin, c-fos/c-jun, and interacting protein domains such as leucine zippers and the like.
- One member of the interaction pair is linked to the appropriate complex (either responder or reactivator) and a plurality of the other is linked individually to a plurality of putative binding ensemble members.
- the plurality of putative binding ensemble members can then be docked to the appropriate complex by the binding of the interaction pair.
- the interaction pairs of the above embodiment can be used to generate a plurality of putative binding ensemble members in vivo. This is done by using a vector (e.g., a transposon or viral based vector) that will insert into the host cell DNA and tag the proteins encoded in that DNA with one of the interaction pairs (e.g., c-fos). The appropriate complex is then placed into these tagged cells to make the plurality of complex (responder or reactivator) bound individually to the plurality of putative binding ensemble members.
- the tags will also be useful for identifying and obtaining the genes from the plurality of putative binding member clones that are positive in the interaction assay.
- the plurality of putative binding member clones that are positive in the interaction assay.
- putative binding ensemble members can be constructed by cloning random DNA fragments from suitable DNA and inserting these fragments into an expression vector so that the inserted DNA fragments will encode a protein that is fused to one member of the interaction pair. Examples of methods for generating tagged libraries for these embodiment are disclosed in Telmer et al. (2002) Biotechniques 32(2):422-24; Uzzau et al. (2001) Proc.
- the responder, inhibitor, and reactivator conjugates included in the systems of the invention are protein molecules that are produced by recombinant expression of nucleic acids encoding the proteins as a fusion protein. Expression methodology is well known to those of skill in the art. Such a fusion product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods
- Nucleic acids encoding the domains to be inco ⁇ orated into the fusion proteins of the invention can be obtained using routine techniques in the field of recombinant genetics (see, e.g., Sambrook and Russell, eds, Molecular Cloning: A Laboratory Manual, 3rd Ed, vols. 1- 3, Cold Spring Harbor Laboratory Press, 2001; and Current Protocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc. New York, 1997).
- nucleic acid sequences encoding the component domains to be inco ⁇ orated into the fusion protein are cloned from cDNA and genomic DNA libraries by hybridization with probes, or isolated using amplification techniques with oligonucleotide primers. Amplification techniques can be used to amplify and isolate sequences from DNA or RNA (see, e.g., Dieffenbach & Dveksler, PCR Primers: A Laboratory Manual (1995)).
- overlapping oligonucleotides can be produced synthetically and joined to produce one or more of the domains.
- Nucleic acids encoding the component domains can also be isolated from expression libraries using antibodies as probes. [116] In an example of obtaining a nucleic acid encoding a domain to be included in the conjugate molecule using PCR, the nucleic acid sequence or subsequence is PCR amplified, using a sense primer containing one restriction site and an antisense primer containing another restriction site. This will produce a nucleic acid encoding the desired domain sequence or subsequence and having terminal restriction sites.
- This nucleic acid can then be easily ligated into a vector containing a nucleic acid encoding the second domain and having the appropriate corresponding restriction sites.
- the domains can be directly joined or may be separated by a linker, or other, protein sequence.
- Suitable PCR primers can be determined by one of skill in the art using the sequence information provided in GenBank or other sources.
- Appropriate restriction sites can also be added to the nucleic acid encoding the protein or protein subsequence by site-directed mutagenesis.
- the plasmid containing the domain- encoding nucleotide sequence or subsequence is cleaved with the appropriate restriction endonuclease and then ligated into an appropriate vector for amplification and/or expression according to standard methods.
- polypeptides encoding the components of the conjugate molecules may be desirable to modify the polypeptides encoding the components of the conjugate molecules.
- One of skill will recognize many ways of generating alterations in a given nucleic acid construct. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques. See, e.g., Giliman and Smith (1979) Gene 8:81-97, Roberts et al. (1987) Nature 328: 731-734.
- the domains can be modified to facilitate the linkage of the two domains to obtain the polynucleotides that encode the fusion polypeptides of the invention.
- Catalytic domains and binding domains that are modified by such methods are also part of the invention.
- a codon for a cysteine residue can be placed at either end of a domain so that the domain can be linked by, for example, a disulfide linkage.
- the modification can be performed using either recombinant or chemical methods (see, e.g., Pierce Chemical Co. catalog, Rockford IL).
- linkers usually polypeptide sequences of neutral amino acids such as serine or glycine, that can be of varying lengths, for example, about 200 amino acids or more in length, with 1 to 100 amino acids being typical.
- the linkers are 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues or less in length.
- proline residues are inco ⁇ orated into the linker to prevent the formation of significant secondary structural elements by the linker.
- Linkers can often be flexible amino acid subsequences that are synthesized as part of a recombinant fusion protein. Such flexible linkers are known to persons of skill in the art.
- a flexible linker is a peptide linker of any length whose amino acid composition is rich in glycine to minimize the formation of rigid structure by interaction of amino acid side chains with each other or with the polypeptide backbone.
- a typical flexible linker has the composition (Gly 4 Ser) x .
- the recombinant nucleic acids encoding the fusion proteins of the invention are modified to provide preferred codons which enhance translation of the nucleic acid in a selected organism (e.g., yeast preferred codons are substituted into a coding nucleic acid for expression in yeast).
- the polynucleotide that encodes the fusion polypeptide is placed under the control of a promoter that is functional in the desired host cell.
- a promoter that is functional in the desired host cell.
- An extremely wide variety of promoters are available, and can be used in the expression vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends upon the cell in which the promoter is to be active.
- Other expression control sequences such as ribosome binding sites, transcription termination sites, enhancers, operators, and the like are also optionally included.
- Expression cassettes Constructs that include one or more of these control sequences are termed "expression cassettes.” Accordingly, the nucleic acids that encode the joined polypeptides are inco ⁇ orated for the desired level of expression in a desired host cell.
- Expression control sequences that are suitable for use in a particular host cell are often obtained by cloning a gene that is expressed in that cell.
- prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. (1980) 8: 4057), the tac promoter (DeBoer, et al, Proc. Natl Acad. Sci. U.S.A.
- promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. (1980) 8: 4057), the
- Standard bacterial expression vectors include plasmids such as pBR322-based plasmids, e.g., pBLUESCRIPTTM, pSKF, pET23D, ⁇ -phage derived vectors, pl5A-based vectors (Rose, Nucleic Acids Res. (1988) 16:355 and 356) and fusion expression systems such as GST and LacZ.
- Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc, HA-tag, 6-His tag, maltose binding protein, VSV-G tag, anti-DYKDDDDK tag, or any such tag, a large number of which are well known to those of skill in the art.
- fusion polypeptides in prokaryotic cells other than E. coli regulatory sequences for transcription and translation that function in the particular prokaryotic species is required.
- promoters can be obtained from genes that have been cloned from the species, or heterologous promoters can be used.
- the hybrid trp- lac promoter functions in Bacillus in addition to E. coli.
- suitable bacterial promoters are well known in the art and are described, e.g., in Sambrook et al. and Ausubel et al.
- Bacterial expression systems for expressing the proteins of the invention are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al, Gene 22:229-235 (1983); Mosbach et al, Nature 302:543-545 (1983). Kits for such expression systems are commercially available.
- telomeres for expression of fusion polypeptides in eukaryotic cells, transcription and translation sequences that function in the particular eukaryotic species are required.
- eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
- vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp series plasmids) and pGPD-2.
- Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus.
- eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
- Either constitutive or regulated promoters can be used in the present invention. Regulated promoters can be advantageous because the concentration of heterologous protein in the host cell can be controlled.
- An inducible promoter is a promoter that directs expression of a gene where the level of expression is alterable by environmental or developmental factors such as, for example, temperature, pH, anaerobic or aerobic conditions, light, transcription factors and chemicals.
- inducible promoters are known to those of skill in the art. These include, for example, the lac promoter, the bacteriophage lambda P L promoter, the hybrid trp-lac promoter (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc. Nat 7. Acad. Sci USA 80: 21), and the bacteriophage T7 promoter (Studier et al. (1986) J. Mol. Biol; Tabor et al. (1985) Proc. Natl. Acad. Sci. USA 82: 1074-8). These promoters and their use are discussed in Sambrook et al, supra.
- Inducible promoters for other organisms are also well known to those of skill in the art. These include, for example, the metallothionein promoter, the heat shock promoter, as well as many others.
- Translational coupling may be used to enhance expression.
- the strategy uses a short upstream open reading frame derived from a highly expressed gene native to the translational system, which is placed downstream of the promoter, and a ribosome binding site followed after a few amino acid codons by a termination codon. Just prior to the termination codon is a second ribosome binding site, and following the termination codon is a start codon for the initiation of translation.
- the system dissolves secondary structure in the RNA, allowing for the efficient initiation of translation. See Squires, et. al. (1988), J. Biol. Chem. 263: 16297- 16302.
- polynucleotide constructs generally requires the use of vectors able to replicate in host bacterial cells, or able to integrate into the genome of host bacterial cells. Such vectors are commonly used in the art.
- kits are commercially available for the purification of plasmids from bacteria (for example, EasyPrepJ, FlexiPrepJ, from Pharmacia Biotech; StrataCleanJ, from Stratagene; and, QIAexpress Expression System, Qiagen).
- the isolated and purified plasmids can then be further manipulated to produce other plasmids, and used to transform cells.
- the fusion polypeptides can be expressed intracellularly, or can be secreted from the cell.
- Intracellular expression often results in high yields. If necessary, the amount of soluble, active fusion polypeptide may be increased by performing refolding procedures (see, e.g., Sambrook et al, supra.; Marston et al, Bio/Technology (1984) 2: 800; Schoner et al, Bio/Technology (1985) 3: 151). Fusion polypeptides of the invention can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines.
- the host cells can be mammalian cells, insect cells, or microorganisms, such as, for example, yeast cells, bacterial cells, or fungal cells.
- the recombinant fusion polypeptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer- Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)).
- Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred.
- the nucleic acids that encode the fusion polypeptides can also include a coding sequence for an epitope or "tag" for which an affinity binding reagent is available.
- suitable epitopes include the myc and V-5 responder genes; expression vectors useful for recombinant production of fusion polypeptides having these epitopes are commercially available (e.g., Invitrogen (Carlsbad CA) vectors pcDNA3.1/Myc-His and pcDNA3.1/V5-His are suitable for expression in mammalian cells).
- Additional expression vectors suitable for attaching a tag to the fusion proteins of the invention, and corresponding detection systems are known to those of skill in the art, and several are commercially available (e.g., FLAG" (Kodak, Rochester NY).
- FLAG Kodak, Rochester NY
- Another example of a suitable tag is a polyhistidine sequence, which is capable of binding to metal chelate affinity ligands. Typically, six adjacent histidines are used, although one can use more or less than six.
- Suitable metal chelate affinity ligands that can serve as the binding moiety for a polyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli, E.
- modifications can be made to the protein domains without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or inco ⁇ oration of a domain into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, the addition of codons at either terminus of the polynucleotide that encodes the binding domain to provide, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences
- variants refers to a version of the polypeptide of interest that has been mutated.
- a population of variants is produced in order to select a version of the polypeptide of interest that has an enhanced property relative to the parent, e.g., increased binding affinity.
- a variant specifically binds the same binding partner as the parent binding pair member.
- Methods of introducing mutations are well known in the art (see, e.g., Sambrook and Ausubel, supra). For example, error-prone PCR or chemical mutagenesis can be performed to introduce mutations. In some embodiments, it may be desirable to introduce mutations at particular sites and then select for the enhanced property. In such instances, techniques such as site-specific mutagenesis may be performed.
- the methods and systems of the invention have many applications for which they offer distinct advantages over existing molecular interaction-sensing technologies such as two-hybrid systems and fragment or subunit complementation systems. These applications include but are not limited to the following: (1) selection of antibodies and other binding molecules for antigens of interest, (2) affinity maturation of antibodies and other binding molecules, (3) protein-protein interaction mapping for functional proteomics, (4) analyte detection assays for clinical diagnostics, expression profiling, food testing, environmental testing, and process monitoring, (5) high-throughput cell-based screening systems for agonists and antagonists of signal transduction pathways involved in disease, and (6) target- mediated activation of prodrugs and other therapeutic or diagnostic reagents at specific locations in the body.
- All the methods for selecting/screening for binding ensemble members employ at least one round of selection/screening, and may include additional rounds of selection screening.
- U.S. Patents 6,342,345, 6,294,330, and 6,270,964, and/or U.S. Patent application Serial Numbers 09/562,106, filed March 15, 2000 disclose applications for fragment based complementation systems that can be adapted to the whole enzyme reactivation systems of the invention. These patents and this patent application are hereby inco ⁇ orated by reference.
- the methods and systems of the invention may be used for the selection of antibodies and other binding molecules which bind with high affinity and specificity to molecules of interest, or "antigens".
- the antigen is linked to a responder-inhibitor fusion and the binding molecule library is linked to a reactivator molecule.
- the antigen may be linked to the reactivator, and the binding molecule library is linked to the responder-inhibitor fusion.
- Gene expression constructs for both fusion molecules are then introduced into a suitable cell line such that all cells express the antigen fusion and each cell expresses at least one binding molecule library member. The doubly-transformed cells are then screened for the phenotype conferred by the responder.
- binding molecules for multiple epitopes on an antigen In many applications it is desirable to obtain binding molecules for multiple epitopes on an antigen. For example, binding to a specific epitope on a cell surface receptor may be required for a desired biological activity in a therapeutic application, but the required epitope often is not known. Thus, binding molecules for multiple epitopes may need to be tested to find the desired bioactivity. This is the principal reason for failure of many attempts to develop therapeutic antibodies, because most antibody selection methods have strong epitope biases, which may exclude the required epitopes.
- the antibody responses of most animals to foreign antigen exhibit strong epitope biases, which are indifferent to required target epitopes for desired bioactivities.
- in vitro methods such panning of phage displayed antibody libraries against immobilized antigens have strong epitope biases because protein antigens typically denature when attached to non-biological surfaces, thus native epitopes are often lost or obscured.
- Antigen-specific binding molecules selected with some embodiments of the invention are also likely to be restricted with respect to the epitopes they bind on the antigen because binding to some epitopes may orient the reactivator in such a way that the responder-inhibitor complex is sterically inaccessible to it, thus binders to such epitopes may not be selectable.
- the antigen is subjected to a second round of selection in which the binding molecules selected in the first round replace the antigen as the fusion partner of the responder-inhibitor or the reactivator, whichever was originally used.
- the antigen is expressed free.
- These molecules are then co-expressed in the same host cells with the original binding molecule fusion library, such that each cell now expresses the free antigen with one or more first antigen-binding molecules fused to one responder activation component, and one or more binding molecule library members fused to the other responder activation component.
- An example of this application is the isolation of human antibody scFv fragments that bind specifically to a model antigen using E. coli cells expressing ⁇ -lactamase as the responder fused to its natural inhibitor BLIP, and a disabled ⁇ -lactamase mutant, BIP, as the reactivator.
- the antigen is fused via flexible linker (e.g. , (Gly Ser) 3 ) to the amino terminus of BLIP, which is fused in turn via similar linker to the amino terminus of ⁇ -lactamase.
- the antigen has a signal peptide sequence at its amino terminus to facilitate translocation of the tripartite fusion protein across the bacterial plasma membrane into the periplasmic space, where ⁇ -lactamase activity is required for antibiotic resistance.
- the ⁇ - lactamase - BLIP affinity is reduced by virtue of a D49A mutation in BLIP, and/or by an E104K, D, Q, or A mutation in ⁇ -lactamase, and/or by linker-induced strain, so that the enzyme is fully inhibited in cis, but can be readily reactivated when docked to BIP by an antigen-antibody interaction.
- a vector for expression of the gene for this tripartite fusion protein is illustrated in Figure 5, and may be constructed by using techniques standard in the art (see, e.g., Sambrook and Russell, eds, Molecular Cloning: A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001; and Current Protocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc. New York, 1997).
- a human non-immune antibody repertoire library may be assembled in single chain Fv format as described in the art (see, e.g., Marks et al, (1991) J. Mol Biol 222:581-97). Routinely, such libraries can have IO 9 or more distinct binding specificities.
- the coding sequences for this antibody scFv library are recovered by PCR and subcloned for expression as fusions via flexible linker to the amino terminus of BIP.
- the scFvs have signal peptides at their amino termini for translocation to the periplasmic space.
- a vector for expression of these fusion proteins which is compatible with the tripartite fusion protein expression vector, is illustrated in Figure 5, and may be constructed using techniques common in the art.
- This vector may be a phagemid vector which comprises both plasmid and bacteriophage origins of replication, and by virtue of the latter, can be encapsulated in phage particles and recovered as such from cells infected with helper phage (see, e.g., Sambrook and Russell, supra).
- the resulting large phagemid library can then be introduced efficiently into cells expressing the tripartite antigen fusion by high-mulitplicity transfection. Each transfected cell will then express the tripartite antigen fusion and usually one antibody scFv-BIP fusion.
- the reactivation of BLIP-inhibited ⁇ -lactamase by BIP when docked by the interaction of a model antibody scFv with a model antigen is illustrated using x-ray crystal structures in Figure 6. [144] With an scFv library of ⁇ 10 9 members it is desirable to transfect at least 10 u cells, so that at least 10 10 can be plated on each of several concentrations of antibiotic.
- the minimum antibiotic concentration for clean selection is established with a pilot transfection in which only a small aliquot of the library is transfected and plated on a series of antibiotic concentrations.
- the aliquot is small enough not to contain any antigen binders, but large enough to be statistically significant, e.g., 100-1000 clones.
- the antigen specificity of thus-selected antibody scFvs is then confirmed by rescuing the scFv- expressing phagemids, retransfecting each in the absence of antigen, and subjecting supernatants of overnight suspension cultures of cells expressing each antibody scFv clone to antibody capture ELISA using purified, immobilized antigen.
- the second round of selection is performed essentially as described for the first round, except that the number of transfectants should be increased if possible by a factor which reflects the multiplicity of epitopes represented by the original selectants.
- the newly-selected scFvs must be counter-screened to eliminate scFvs which activate ⁇ -lactamase by binding to something other than antigen such as the first scFv, BLIP, or ⁇ -lactamase itself. This can be readily accomplished by using an inducible promoter for the antigen, and restreaking each clone on antibiotic in the absence of inducer.
- Antigen-specific binders should not activate ⁇ -lactamase in the absence of antigen, and so should not restreak. Bona-fide antigen-binding scFvs are then verified as described above by antibody capture ELISA. For large antigens, the process may be repeated for a third round of selection, in the event that some epitopes are still excluded in the second round.
- examples of other binding molecules suitable for isolation and deployment suing the invention include other antibody fragments such as Fv, Fab, and variable region domains, as well as scaffolded peptides such as trxpeps.
- Isotropic binding molecules are obtained by presenting all potential binding surfaces of an ensemble member to other ensemble members or to a plurality of putative ensemble members.
- systems for isotropic selection/screening comprise a first binding ensemble member, a prior selected/screened binding ensemble member(s), and a plurality of putative binding ensemble members from which additional binding ensemble members will be selected/screened.
- the prior selected/screened binding ensemble member is either one that is known to interact with the first binding ensemble member or one that was obtained in a prior round of screening/selection using the first binding ensemble member against the plurality of putative binding ensemble members.
- the first binding ensemble member is subjected to additional round(s) of selection/screening in which the prior selected/screened binding ensemble members are present and the systems is placed under conditions where reactivation occurs if both the prior selected/screened binding ensemble member and a new member from the plurality of putative members bind to the first binding ensemble member.
- binding sites on the first binding ensemble member that did not screen/select for binding ensemble members in the prior rounds may do so in these subsequent rounds.
- the prior selected/screened ensemble members are obtained from previous round(s) of selection/screening against the plurality of putative ensemble members, the prior selected/screened ensemble members must be removed from their complexes for use in the subsequent round(s) of selection/screening against the plurality of putative ensemble members.
- the prior selected/screened ensemble member(s) replace the first binding ensemble member in its complex and the first binding ensemble member is free in solution.
- the prior selected/screened ensemble member is/are then used to present the first binding ensemble member to the plurality of putative binding ensemble members that are individually complexed to a plurality of the other complex.
- binding ensemble members that bind to different sites on the first binding ensemble member may be obtained from the plurality of putative binding ensemble members because the putative binding members "see" the first binding ensemble member in a different orientation from the earlier round(s) of selection/screening.
- an additional screening/selection step is added to the method where the prior selected/screened binding ensemble member(s) is free in solution and the system is placed under conditions where the responder is reactivated only when a member of the plurality of putative binding ensemble members can bind to a site of the first binding ensemble member that does not interfere with the binding of the prior selected/screened binding ensemble member(s).
- This embodiment is particularly useful for selections/screenings that are done in vitro.
- One important product of the above-described process is that for each such antigen, or target molecule, one or more bi-molecular detection reagents are obtained.
- These reagents comprise matched pairs of target molecule-binders which can bind to the target molecule simultaneously such that when used, respectively, in a responder complex and a reactivator complex, the responder is efficiently activated to produce the desired readout in proportional response to the amount of target molecule present. Because all of the components are stable, these reagents can be deployed in vitro, and may be used in humans for therapeutic or diagnostic applications.
- panels of these reagents can be deployed in 2-dimensional arrays in micro-well formats for automated high- throughput profiling of the expression levels of key gene products in healthy and diseased cells and tissues both for therapeutic target identification and validation, and for clinical diagnosis.
- detection arrays are severely limited by the requirement for target molecule labeling, and/or by throughput limitations on existing label-free detection systems.
- the reagents of the present invention can be robotically dispensed in solution directly into high-density 2-dimensional arrays of micro-wells along with specimen and substrate for a sensitive colorimetric or fluorometric readout on the presence of the target antigen within minutes. No other currently available detection system has such capability.
- Other in vitro applications of the invention include clinical diagnostics, environmental testing, quality assurance monitoring, food testing, and manufacturing process monitoring.
- Target-Mediated Reagent Activation can be directed to targets in vivo such as tumor markers to produce imageable signals at the site of an incipient tumor or other diseased tissue, or they can be used to activate cytotoxic prodrugs at such sites in the body.
- targets in vivo such as tumor markers to produce imageable signals at the site of an incipient tumor or other diseased tissue, or they can be used to activate cytotoxic prodrugs at such sites in the body.
- ⁇ -lactamases have been used in such applications to activate cephalosporin prodrugs of anti-tumor agents such as doxorubicin and nitrogen mustards (see, e.g., Jungheim and Shepherd (1994) Chem. Rev. 94: 1553-66; Francisco et al., ( 996) J Immunol, 157, 1652-8).
- ADPT Antibody-Directed Enzyme Prodrug Therapy
- a prodrug- activating enzyme such as a ⁇ -lactamase
- an inactive prodrug such as a cephalosporin derivative of doxorubicin
- ADEPT The main problem with ADEPT is that the unbound conjugate must clear the circulation before the prodrug can be administered in order to minimize systemic toxicity. However, by the time the conjugate has cleared the circulation >90% of the tumor bound enzyme has been lost (e.g., Bagshawe, 1995; Springer and Niculescu-Duvaz (1995) Anti-Cancer Drug Design 10: 361-72). In spite of this, ADEPT has been able to achieve higher active drug concentrations in the tumor than any other procedure (see, e.g., Sedlacek et al, in Contributions to Oncolosv. Huber and Queisser, eds. pp.
- prodrug activation can only occur at the target site, where the enzyme is activated.
- prodrug can be administered simultaneously with the tumor-targeting reagents to ensure the availability of maximum prodrug at the time of maximum target loading of the reagents, without regard for unbound reagents.
- enzyme reactivation systems of the present invention can be targeted for activation by surface markers on other types of cells and tissues, such as pathogen-infected cells, transplants, and sites of inflammation or atherogenesis.
- target-localized and activated enzymes can then be used to activate not just cytotoxins, but other types of therapeutic agents such as small molecule agonists or antagonists of biological response modifiers, as well as imaging reagents for precise localization of tissue with disease or other phenotype of interest.
- target- activated enzymes can be used to deliver: (1) immune stimulants to tumors, (2) immuno- suppressants to sites of chronic inflammation or to organ transplants to inhibit rejection, (3) antibiotics to specific pathogens, (4) cytotoxins and anti-virals to virus-infected cells, (5) hormones and other pleiotropic agents to specific cells and/or tissues, or (6) neuro- transmitters and other neuro-modulators to specific nerves or tissues.
- target-activated enzymes can be used to deliver to any tissue any small molecule cytotoxin, hormone, steroid, prostaglandin, neurotransmitter, or agonist/antagonist of peptide hormone, cytokine, or chemokine, etc., which can be inactivated by conjugation to the appropriate substrate.
- the antigen would be combined to one complex of the present system, i.e., a responder complex or a reactivator complex, and a library of test molecules, e.g., mutagenic variants of the antigen-binding molecule, would be combined to the other complex.
- a library of test molecules e.g., mutagenic variants of the antigen-binding molecule
- These complexes would then be co-expressed in appropriate host cells with the free parent antigen-binding molecule as the competitor, such that each cell would express the antigen fusion, the free parent binding molecule, and one of the test variants of the antigen-binding molecule fused to the other component.
- the parent and test binding molecules compete with each other for binding to the antigen such that the magnitude of the responder-conferred phenotype exhibited by a given cell is directly proportional to the affinity of the test variant expressed in that cell.
- those cells exhibiting the strongest phenotype should be expressing the highest-affinity test variants.
- any cell exhibiting a phenotype which is stronger than that which would result from competition of the parent antigen-binding molecule with itself, i.e., expressed as both competitor and test molecule should be expressing a higher-affinity test variant.
- the library of test variants of the parent binding molecule can be generated using a variety of mutagenesis methods including, for example, error-prone PCR (Cadwell and Joyce, in PCR Primer, A Laboratory Manual Dieffenbach and Dveksler, Eds. Cold Spring Harbor Press, Cold Spring Harbor NY, pp. 583-590, 1995), Parsimonious Mutagenesis (PM) (Balint and Larrick, Gene 137:109-118, 1993), DNA shuffling (Crameri et al, Nature Biotechnol. 14:315-19, 1996), random-priming recombination (RPR) (Shao et al., Nucleic Acids Res. 26:681-683, 1998), or the staggered extension process (StEO) (Zhao et al, Nature
- PM Biotechnol. 16:258-261, 1998.
- higher mutation rates can be used because only the antigen combining sites of the binding molecule are mutagenized. PM may therefore be advantageous for accessing larger affinity increments.
- PM has additional advantages in avoiding expression mutants, avoiding immunogenicity, and in ease of sequencing.
- FIG. 7 Affinity maturation of an antibody is illustrated in Figure 7 for an embodiment of the invention that uses E. coli cells expressing ⁇ -lactamase as the responder fused to BLIP, and expressing BIP as the reactivator.
- the antigen is fused, via (Gly Ser) 3 linker, to the amino terminus of BLIP, which is in turn fused via a similar linker to the amino terminus of ⁇ -lactamase.
- the ⁇ -lactamase - BLIP affinity is reduced by virtue of a D49A mutation in BLIP, and/or by an E104K, D, Q, or A mutation in ⁇ -lactamase, and/or by linker- induced strain, so that the enzyme is fully inhibited in cis, but can be readily reactivated when docked to BIP by the antigen-antibody interaction.
- the parent antibody is expressed free as the competitor, and a population of mutagenic variants of this antibody is expressed as the test molecule library fused to the amino-terminus of BIP, such that each cell expresses one test variant along with the competitor fusion and the tripartite antigen fusion.
- All three components also comprise amino terminal signal peptides for their translocation into the periplasmic space.
- Vectors for expression of these components can be constructed using standard art, and are illustrated in Figure 8. It is convenient to use a phagemid vector for the test variant library to allow facile recovery of selected variants for further testing.
- Selection of higher- affinity variants is accomplished by plating the cells onto solid medium containing a ⁇ -lactam antibiotic such as ampicillin at a concentration which is lethal for cells expressing the parent antibody competing against itself, i.e., as both competitor and test molecule.
- the optimal antibiotic concentration is typically one on which cells expressing the parent antibody as test molecule plate with ⁇ 1% efficiency, i.e., produce fewer than one colony per 100 cells plated.
- variants with higher plating efficiencies can be readily isolated by several rounds of replating at no more than about 10 cells per colony, in the course of which all unimproved test molecules are eliminated.
- Selection for ⁇ -lactamase-dependent antibiotic resistance has an advantage for affinity maturation in that the relationship between enzyme activity and antibiotic resistance is highly non-linear, such that small increments in affinity, leading to small increments in ⁇ -lactamase activity, can produce much larger increments in plating efficiency at the critical point in the antibiotic concentration curve.
- Some variants may be selected by virtue of higher stability or expression levels rather than by higher affinity.
- Expression variants can be detected by western blotting (an epitope tag is present on the carboxyl terminus of BIP for detection with anti-tag antibody), and then comparing the intensity of each selected variant-BlP fusion with that of the parent scFv-BIP fusion under the same conditions. Affinities of selected antibodies can be measured by established methods in the art, such as competition ELISA (e.g., Harlow and Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988) and surface plasmon resonance (e.g., Fagerstam et al.
- competition ELISA e.g., Harlow and Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988
- surface plasmon resonance e.g., Fagerstam et al.
- the systems and methods of the invention may also be used to identify natural interactions among the gene products of the human proteome, such as those interactions which comprise the operational circuitry of such fundamental cellular processes as signal transduction, the cell division cycle, gene expression, and the regulation of metabolism.
- the most successful current method for identifying natural ligands of proteins of interest from expressed sequence libraries is the yeast two-hybrid system (see, e.g., Fields and Song, Nature 340:245-247 (1989); Chien et al, Proc. Natl. Acad. Sci. (USA) 88:9578-9582 (1991)).
- the "bait" protein is fused to the DNA-binding domain of a transcription factor, and the expressed sequence library is fused to the transactivation domain of the same transcription factor.
- Both fusion proteins are expressed in yeast cells in which the expression of a responder gene is dependent on assembly of the transcription factor on upstream DNA, which is in turn dependent on an interaction between the bait protein and a product of the expressed sequence library.
- interactors are identified by responder signal generation.
- This method suffers from a number of limitations, including high false positive and false negative rates due to (1) the inherent variability of a multi-step signal generator, (2) variability due to the broad distribution of stabilities of expressed sequences fused to a meta-stable protein fragment, and (3) the need for heterologous proteins to translocate into and be stable in the alien environment of the yeast cell nucleus.
- the current invention circumvents most of the aforesaid limitations of the yeast two- hybrid system to greatly improve the efficiency of identification of natural protein-protein interactions in expressed sequence libraries.
- the invention allows signal generation directly from target interactions without intervening inter-molecular steps, the components of the invention are stable and therefore likely to have a stabilizing effect on unstable expressed sequence products, and finally the invention can be deployed in the natural environments of the target interactions.
- the bait protein is expressed as a fusion to the amino terminus of an inhibitor-responder fusion, e.g., BLIP - ⁇ - lactamase, and the expressed sequence library is expressed as a fusion to the amino terminus of a reactivator of the responder, which in the case of ⁇ -lactamase would be BIP.
- the ⁇ - lactamase - BLIP affinity is reduced by virtue of a D49A mutation in BLIP, and/or by an E104K, D, Q, or A mutation in ⁇ -lactamase, and/or by linker-induced strain, so that the enzyme is fully inhibited in cis, but can be readily reactivated when docked to BIP by an interaction of the bait protein with an expressed sequence product.
- This RAIR system can be expressed in a variety of cell types, including prokaryotic and eukaryotic cells.
- vectors analogous to those depicted in Figure 5 A can be used, except that the "antigen” would be replaced by the “bait” and the antibody scFv library would be replaced by an expressed sequence library.
- These vectors may be introduced into E. coli cells efficiently, for example, by chemical transformation or by high-voltage electroporation (Sambrook and Russell, supra).
- DNA libraries can be enriched for secreted protein sequences by templating from microsomal mRNAs. The isolation of rough microsomes and the preparation of mRNA therefrom (Gaetani et al,
- cytoplasm color screening or selection must be employed, using a chromogenic or fluorogenic substrate.
- the fluorogenic substrate CCF2/AM see, e.g., Zlokarnik et al., (1998) Science 279: 84-88) may be used, and expressed sequence products which interact with the bait protein may be recovered by subjecting the cells to fluorescence activated cell sorting (FACS, Wehrman et al, (2002) Proc Natl Acad Sci, in press).
- Vectors for expressing the components of these systems in eukaryotic cells are also known in the art (see, e.g., Sambrook and Russell, supra; and Current Protocols in Molecular Biology, supra), and ⁇ -lactamase is known to be stable and active in the mammalian cell cytoplasm (e.g., Moore et al. (1997) Analytical Biochem. 247: 203-209; Wehrman et al, supra).
- Vectors constructed to express the bait - BLIP - ⁇ - lactamase tri-partite fusion and the BIP- expressed sequence library fusions can be transfected into mammalian cell lines such as NIH 3T3 or Cos-7 cells with high efficiency by calcium phosphate co-precipitation or lipofection.
- Transformed cells will express the system components transiently at high levels, typically for up to 3 days, during which time the cells may be pulsed with fluorogenic substrate and subjected to FACS to enrich for cells expressing bait protein interactors.
- Stable transformants of the FACS-selected cells may be made by antibiotic selection, after which the putative bait protein interactors may be sequenced, identified, and subjected to verification tests such as co-immunoprecipitation with the bait protein.
- a general feature of such conditions is amplification of the MAPK pathway into a self-generating mitogenic signaling cycle in which the loop is closed by MAPK-induced autocrine growth factor production.
- One of the more common oncogenes is ErbB2, which is often over-expressed in adenocarcinomas of epithelial origin in breast, ovary, intestine, lung, and other tissues. ErbB2 is the most active of the EGFRs, and generally functions as a signal amplifier in hetero-dimers with other ligated receptors.
- a key player in signal transmission from activated ErbB2 to the MAPK pathway is growth factor receptor binding protein 2 (Grb2) which contains a src-homology phosphotyrosine binding domain (SH2) flanked by two polyproline-binding domains (SH3).
- the SH2 domain binds phosphotyrosine 1068 of the receptor C-terminal domain
- the SH3 domains bind SOS, a guanine nucleotide exchange factor, thereby docking SOS to the plasma membrane where it can activate Ras, a small GTPase, which in turn activates Raf-1, the first kinase in the MAPK pathway.
- Grb2 may be fused via flexible linker to the amino terminus of the BLIP - ⁇ -lactamase fusion, and BIP or BLIP-F142A may be similarly fused to the carboxyl terminus of ErbB2.
- a vector expressing these constructs may then be introduced, e.g., by lipofection, into an appropriate cancer cell line, such as the SK-BR3 human breast tumor line, in which autocrine signaling through the ErbB2 pathway is constitutive, and essential for tumor growth and survival.
- an appropriate cancer cell line such as the SK-BR3 human breast tumor line, in which autocrine signaling through the ErbB2 pathway is constitutive, and essential for tumor growth and survival.
- Tyr 1068 should be constitutively phosphorylated, and the interaction between the Grb2 SH2 domain and pTyrl068 therefore provides a pool of constitutively active ⁇ -lactamase, which can be readily detected using a fluorogenic substrate such as CCF2/AM.
- Antagonists of the pathway would have promising anti-cancer chemotherapeutic activities, and may be readily identified by their ability to extinguish fluorescence in these cells. Thus, these cells can be used in high- throughput screens for such antagonists.
- the systems of the invention can also be used to obtain molecules that inhibit the interaction between the binding ensemble members.
- the molecules disrupt the interaction of the binding ensemble members from the responder and reactivator complexes resulting in a loss of signal from the responder.
- U.S. Patent No. 6,294,330 discloses inhibitor selection/screens for a fragment based complementation systems that are adaptable to the systems of the invention. This patent is hereby inco ⁇ orated by reference.
- Example 1 Interaction-mediated reactivation of auto-inhibited ⁇ -lactamase.
- This example demonstrates the basic functionality of the RAIR methods and systems of the invention for detecting molecular interactions and inhibitors thereof.
- a model interaction was used to test the ability of reactivators, BIP ( ⁇ -lactamase- S70A) and BLIP-F142A, to reactivate a ⁇ -lactamase responder which was strongly inhibited in cis by fusion to BLIP-D49A, when the reactivator and the auto-inhibited ⁇ -lactamase were brought together by the model interaction in E. coli cells.
- the RAIR system was tested for competitive inhibition of the reactivation when the reactivating interaction was inhibited by one of the model interactors expressed from a second gene without a fusion partner.
- the model interaction was comprised of the leucine zipper helices of the c-fos and c-jun subunits of the AP-1 transcription factor (Karin et al, (1997) Curr Opin Cell Biol 9, 240-6).
- the c-fos helix was fused via a flexible linker to the amino terminus of the auto- inhibited responder, comprised of BLIP-D49A fused via similar linker to the amino terminus of ⁇ -lactamase.
- the c-jun helix was fused via a flexible linker to the amino terminus of the reactivator, either BIP or BLIP-F142A.
- the reactivation inhibitor was comprised of the c-jun helix fused to a chaperone, thioredoxin.
- the expression vectors for the tri-partite fusion, the reactivator fusion, and the reactivation inhibitor fusion are illustrated in Figure 10.
- the tri-partite fusion expression cassette was comprised of a constitutive mutant of the / ⁇ cUV5 promoter, followed by the coding sequence for the tri-partite fusion protein and a transcription termination sequence.
- the tri-partite fusion gene comprised coding sequences for a signal peptide for translocation of the fusion protein into the periplasmic space of the bacterial cell, followed by the c-fos helix fused via a (Gly 4 Ser) linker to BLIP-D49A, followed via a similar linker by ⁇ -lactamase.
- This cassette was assembled in a plasmid based on the pl5A replicon (Rose, Nucleic Acids Res. (1988) 16:355-356) containing a kanamycin resistance gene (ka ⁇ ) for plasmid maintenance.
- the reactivator fusion expression cassette was comprised of the lacUV5 promoter, followed by the coding sequence for a signal peptide, followed by the c-jun helix fused to BIP or BLIP-F142A via a (Gly 4 Ser) 3 linker.
- This cassette was assembled in plasmid pBR322 in which the ⁇ -lactamase gene was replaced with the gene for chloramphenicol resistance (cam) for plasmid maintenance.
- Negative control cassettes comprised of the reactivators without amino-terminal c-jun helices were constructed similarly. For reactivation inhibition tests, an interaction inhibitor expression cassette was inserted into the reactivator fusion vector, as illustrated in Figure 10.
- This cassette was comprised of a / ⁇ cUV5 promoter, and the coding sequences for a signal peptide, followed by the c-jun helix fused to thioredoxin via a (Gly Ser) 3 linker. All expression vectors were assembled using standard recombinant DNA methods (e.g., Sambrook and Russell, eds, Molecular Cloning: A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001 ; and Current Protocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc. New York, 1997).
- E. coli cells e.g., TOPI OF'
- TOPI OF' high- voltage electroporation
- E coli cells were transformed with the constructs of Figure 10 to express the c-fos leucine zipper helix fused to the BLIP D49A - ⁇ -lactamase fusion, BIP or BLIP F142A with or without the amino terminal c-jun helix, and, where indicated, further expressing the c-jun helix fused to thioredoxin (trx) as a competitive inhibitor of the reactivating fos-jun helix interaction.
- the transformed cells were plated on solid medium containing 2% glucose, increasing amounts of ampicillin, and, where indicated, IPTG, which is required for expression of the reactivator fusions and the interaction inhibitor. The data are expressed as % plating efficiency, I e., the percent of transformed cells plated forming colonies after overnight growth at 33°C.
- Free BIP produced little reactivation by itself, but when fused to the c-jun helix, so that BIP could be docked to the auto-inhibited ⁇ -lactamase by the fos-jun helix interaction, ⁇ -lactamase was fully reactivated, i.e., every plated cell produced a colony on each concentration of ampicillin up to lOO ⁇ g/ml. [177] At lOO ⁇ g/ml ampicillin, IO 6 cells had to be plated in a separate experiment before any colonies appeared.
- bona-fide interactors such as antigen-specific antibodies or natural ligands, can be selected for a protein of interest from a library of at least IO 5 non-interactors in a single plating. Even lower frequency interactors can readily be isolated by replating the colonies one or more times at only a few cells per colony, so that low-efficiency platers would be partitioned out. This demonstrates the utility of the system for sensitive detection of specific protein-protein interactions.
- This example demonstrates the utility of the invention for detection of antigen- antibody interactions, selection of antibodies against specific antigens, and affinity maturation of antibodies. Also, the ability of the system to detect free antigen molecules via a tri-molecular interaction is demonstrated. Finally, reactivation of ⁇ -lactamase by antigen- antibody interaction when the responder complex comprises non-covalent linkages is demonstrated.
- One of the antibodies used in the example was a mouse monoclonal raised against the extra-cellular domain of the human B-cell activation antigen CD40, and isolated by hybridoma technology.
- This antibody designated HB15, had a K d for CD40 of 7.6 nM, as determined by surface plasmon resonance (Fagerstam et al, (1992) J Chromatog 597: 397- 410).
- a higher-affinity variant of this antibody was subsequently identified which contained two mutations in the third complementarity-determining region (CDR3) of the heavy chain variable region (VH), which conferred a 12-fold increase in the affinity of the antibody.
- This variant was designated HB 15 Y.
- the vectors for expression of the system components for CD40-HB15 interaction- mediated reactivation of auto-inhibited ⁇ -lactamase, and for inhibition of that reactivation by a competitor antibody are depicted in Figure 11.
- the CD40 - BL1P-D49A - ⁇ -lactamase fusion contained (Gly 4 Ser) 3 linkers and was expressed from a constitutive mutant of the / ⁇ cUV5 promoter in a pi 5 A plasmid containing a kanamycin resistance gene (kan) for plasmid maintenance.
- the HB15 antibodies were expressed in chimeric Fab form, i.e., VH- CH1 (Fd) with full-length light chain (LC), in which both constant regions were human in origin.
- the Fabs were expressed from dicistronic transcripts driven by the / ⁇ cUV5 promoter.
- the upstream cistron encoded the LC, followed by an internal ribosome entry site (IRES) to allow translation to re-initiate on the downstream cistron, which encoded BL1P-F142A fused (Gly 4 Ser) 3 linker to the amino terminus of the Fd fragment of either HB15 or HB15Y.
- This reactivator/Fab fusion cassette was inserted into plasmid pBR322 in which the ⁇ -lactamase gene had been replaced by the gene for chloramphenicol resistance (cat) for plasmid maintenance.
- the reference antibody HB15 Fab
- a Fab specific for an irrelevant antigen, glutathione S-transferase (GST) was used as a negative control.
- Table II presents data showing the ability of the interaction between CD40 and anti- CD40 Fabs to facilitate strong reactivation of the auto-inhibited ⁇ -lactamase/BLIP-D49A fusion by the BLIP-F142A reactivator when antigen and antibody are fused to the inhibited responder and reactivator, respectively.
- host cells expressing the anti-GST Fab as the test Fab fused to the reactivator produced less than one colony per 10,000 cells plated on 25 ⁇ g/ml ampicillin and above, both the HB15 and HB15Y Fabs plated at 100% efficiency up to at least 200 ⁇ g/ml ampicillin.
- antigen-specific antibodies can be selected from a library of at least IO 6 non-binding antibodies in a single plating. Even lower frequency binders could readily be isolated by replating the colonies one or more times at only a few cells per colony, so that low-efficiency platers would be partitioned out.
- E. coli cells were transformed with the constructs of Figure 11 to express CD40 fused to auto-inhibited ⁇ -lactamase, the indicated test Fabs fused to BLIP-F142A as the reactivator, and, where indicated, HB15 as the competitor.
- Transformed cells were plated on solid medium containing 2% glucose, lOO ⁇ M IPTG, and the indicated concentrations of ampicillin. The data are expressed as plating efficiencies, i.e., the percent of transformed cells plated which formed colonies after overnight growth at 33°C.
- CD40 - anti-CD40 interaction was further used to test the invention for its ability to detect a free binding ensemble member in a tri-molecular interaction.
- one binding ensemble member (CD40 antigen) is linked non-covalently in the responder complex.
- CD40 was instead fused directly to the amino terminus of the inhibitor, BLJP-D49A, in the auto-inhibited ⁇ -lactamase complex, CD40 was instead fused to the c-jun leucine zipper helix, and the c-fos helix was fused to the amino terminus of the BLIP-D49A - ⁇ -lactamase fusion.
- the high affinity fos-jun helix interaction linksthe antigen non-covalently to the auto-inhibited responder complex, so that antibody binding to the antigen can dock the reactivator to the responder.
- This and similar configurations have the advantage that binding ensemble members which do not tolerate fusion partners such as the components of the system, will nevertheless usually tolerate the addition of much smaller fusion partners such as high-affinity leucine zipper helixes, which can then be used to non-covalently dock the binding ensemble members to the components of the system.
- the fos and jun fusions were each expressed from constitutive / ⁇ cUV5 promoters in separate cassettes in the auto-inhibited responder vector.
- the anti-GST Fab served as the negative control, fused to the reactivator.
- the fos and jun fusions were co-expressed in E. coli cells with each of the Fabs, anti- GST, HB15, and HB15Y fused to the BLIP-F142A reactivator, and scored for plating efficiency on increasing amounts of ampicillin.
- the plating efficiency data are shown in Table III.
- non-covalent linkages can replace covalent linkages in the responder complex, and still allow responder reactivation by interaction of binding ensemble members, at least one of which is not covalently linked to either the auto-inhibited responder or the reactivator.
- this example demonstrates by equivalence that auto-inhibited responders can be reactivated efficiently by tri-molecular interactions in which two non-competing antigen binders (antibody and helix) engage free antigen (CD40-helix fusion) to dock the reactivator to the auto-inhibited responder, activating the latter.
- Table III Reactivation of Auto-Inhibited ⁇ -lactamase by Free Antigen in a Tri- molecular complex with Antibody and Leucine Zipper 3 '
- a ' E. coli cells were transformed with the constructs of Figure 1 IB to express CD40 fused to a c-jun helix, a c-fos helix fused to auto-inhibited ⁇ -lactamase, the indicated test Fabs fused to BLIP-F142A as the reactivator.
- Transformed cells were plated on solid medium containing 2% glucose, lOO ⁇ M IPTG, and the indicated concentrations of ampicillin. The data are expressed as plating efficiencies, i.e., the percent of transformed cells plated which formed colonies after overnight growth at 33°C.
- the responder and the inhibitor are non-covalently linked in the responder complex, and the binding ensemble member is covalently linked to the inhibitor. This is accomplished by expressing the CD40-BLIP fusion and ⁇ -lactamase from separate cistrons in the responder expression vector. To ensure constitutive inhibition of ⁇ -lactamase, wild-type BLIP may be used instead of the D49A mutant.
- the responder expression vector shown in Figure 11 A is modified by replacing the coding sequence for the (G 4 S) linker between BLIP and ⁇ -lactamase with a DNA sequence encoding a translation stop codon at the end of BLIP, followed by an internal ribosome entry site (IRES) for translation of ⁇ -lactamase, followed by a translation start codon and signal peptide for production and secretion of free ⁇ -lactamase.
- IRES internal ribosome entry site
- the CD40-BLIP fusion and ⁇ -lactamase were co-expressed in E. coli cells with each of the Fabs, anti-GST, HB15, and HB15Y fused to the BIP- ⁇ 104Q reactivator, and scored for plating efficiency on increasing amounts of ampicillin.
- the Reactivator Fusion Expression Vector is the same as shown in Figure 11A, except that BIP-E104Q replaces BLIP-F142A as the reactivator.
- the plating efficiency data are shown in Table IV. As expected, with the anti-GST Fab, no colonies appeared above lO ⁇ g/ml ampicillin.
- HB15 and HB15Y anti-CD40 Fabs plating was quantitative on at least 50 ⁇ g/ml ampicillin for HB15, and on at least lOO ⁇ g/ml for the higher-affinity HB15Y. These levels of activation compare favorably with reactivation when the inhibitor and responder are covalently linked. From this, it may be concluded that non-covalent linkages can replace covalent linkages in the responder complex, and still allow efficient responder activation by interaction of binding ensemble members when inhibitor and responder are not covalently linked.
- E. coli cells were transformed with a responder complex construct expressing ⁇ -lactamase and CD40 fused to the BLIP inhibitor, and a reactivator complex construct expressing the BIP-E104Q reactivator fused to each of the indicated test Fabs.
- Transformed cells were plated on solid medium containing 2% glucose, 1 OO ⁇ M IPTG, and the indicated concentrations of ampicillin. The data are expressed as plating efficiencies, i.e., the percent of transformed cells plated which formed colonies after overnight growth at 33°C.
- ⁇ -lactamase activation by antigen-antibody interaction with non-covalent linkage of responder and inhibitor in the responder complex could have been accomplished with equal efficiency by covalently linking the binding ensemble member (CD40) to the responder instead of to the inhibitor.
- CD40 binding ensemble member
- BLIP-F142A would be used as the reactivator fused to the Fabs, such that a CD40-Fab interaction would dock BLIP- F142A to ⁇ -lactamase, thereby protecting it from inhibition by BLIP.
- Example 3 Re-activation of auto-inhibited ⁇ -lactamase in vitro by antigen-antibody interaction.
- fusion protein [5 supplemented with antibiotics (either kanamycin or chloramphenicol) for plasmid maintenance. Expression of the fusion protein was then induced for 4 hours with lmM IPTG, and the cells were collected and lysed in PBS supplemented with 150mM NaCl, 1% Triton XI 00, 5% Glycerol, lOmM Benzamidine, (Sigma Chemical Co., St. Louis, MO) E. coli- specific protease inhibitor cocktail and lmM PMSF. The fusion proteins were batch-
- both the HB15 and HB15Y Fabs produced a steady increase in ⁇ -lactamase activity throughout the 30' incubation period. In both cases the reaction product appeared to accumulate exponentially for -15-20 minutes before equilibrium was reached, after which the product appeared to accumulate more or less linearly.
- the HB15Y Fab consistently outpaced the lower affinity HB15 antibody, as might be expected from its higher affinity. Since the dissociation rates for both Fabs are expected to be low compared to the time scale of this reaction, the higher reaction rate for the HB15Y Fab specifically implies a higher association rate constant for this antibody.
- Example 4 An antibody-antigen interaction-mediated ⁇ -lactamase reactivation system for target-activated enzyme prodrug therapy (TAcEPT)
- ADPT Antibody-directed enzyme prodrug therapy
- Enzymes such as ⁇ -lactamase have been chemically or genetically conjugated to tumor-targeting antibodies and used with ⁇ -lactam derivatives of anti-tumor drugs such as cephalosporin mustards and anthracyclines to achieve promising anti-tumor effects in animals.
- the efficacy of ADEPT is limited, however, by the need for unbound conjugate to clear the circulation before the prodrug can be administered. By the time the circulating conjugate is depleted to the threshold below which systemic activation of the prodrug would produce acceptable levels of toxicity, so much of the conjugate has been lost from the tumor that efficacy is often seriously compromised.
- This problem can be overcome by using an interaction-dependent ⁇ -lactamase reactivation system with tumor targeting antibodies.
- scFv single-chain antibody fragments
- the auto- inhibited ⁇ -lactamase and reactivator can localize to the tumor and reconstitute sufficient ⁇ - lactamase activity on the tumor cell surface to produce high levels of tumor-localized cytotoxicity from ⁇ -lactam prodrugs.
- the resulting Tumor-Activated Enzyme Prodrug Therapy (TAcEPT) system is then tested for its ability to ablate SKBR3 and other Her-2/neu-expressing human tumors in severe combined immuno-deficient (scid) mice. Once the efficacy and safety of the system has been demonstrated in animal models, toxicity and efficacy trials are initiated in human breast cancer subjects.
- TcEPT Tumor-Activated Enzyme Prodrug Therapy
- the tumor activation mechanism for these components employ two scFvs such as those described by Schier et al. (1996, Gene 169: 147-155), which were derived from a phage display library of a human non-immune repertoire (Marks et al. (1991) J. Mol. Biol. 222: 581-597) by panning against a recombinant fragment comprising the extra-cellular domain (ED) of Her-2/neu. These two scFv appear to recognize non-overlapping epitopes, since they do not compete for binding to the Her-2/neuED by ELISA. The affinity of one of these scFv was improved to sub-nM K d in vitro (Schier et al.
- the coding sequences for the scFv are subcloned for expression of the scFv as fusions via (Gly 4 Ser) linkers to the amino termini of the BLIP-D49A - ⁇ -lactamase fusion, and BLIP-F142A, respectively.
- Appropriate vectors are derived from pET26b (Novagen, Inc., Madison, WI), and have convenient restriction sites for insertion of scFv, ⁇ - lactamase, and the BLIP coding sequences.
- each fusion protein is fully induced with lmM IPTG from the bacteriophage T7 promoter (Moffatt and Studier (1986) J. Mol Biol 189: 113-130) under the control of the lac repressor.
- Each primary translation product contains a pelB signal peptide for secretion into the bacterial periplasm and a carboxyl-terminal His 6 tag for one-step purification from non-denatured lysates (e.g., osmotic shock; (Neu and Heppel (1965) JBiol Chem 240: 3685-92) by IMAC.
- the yield of each fusion protein can be optimized primarily by manipulation of the inducer concentration and the growth temperature.
- the purified fusion proteins are tested for binding to an immobilized recombinant fusion of the Her-2/neu extra-cellular domain (ED) to a stabilizing immunoglobulin domain (Ig) by ELISA using an anti-His 6 tag antibody (Qiagen). His 6 -tagged Her-2/neu - Ig fusion may, like the fusion proteins, be purified by IMAC, and immobilized on the surface of microtiter plate wells.
- the wells of microtiter plates are coated with antigen, and exposed to increasing amounts of the first scFv fusion until the ELISA signal plateaus. At this level the antigen is saturated with the first scFv fusion protein, and increasing amounts of the second scFv fusion are added.
- Immobilized BSA may be used as the negative control.
- V max is a more or less linear function of the concentration of the second scFv fusion. As the amount of second scFv fusion is increased, at some point V max should plateau.
- the amount of the second fusion bound can be determined by ELISA, and a relative specific activity (k cat rel ) may be computed for the reactivated ⁇ -lactamase.
- the K M may be estimated in solution with saturating antigen and saturating first scFv fusion and limiting amounts of the second scFv fusion. A range of nitrocefin concentrations is added and the initial rates of change of absorbance at 485 nm is measured as a function of second scFv fusion concentration. The K M is then computed from data by standard regression analysis.
- V max should be a more or less linear function of the amount of intact ⁇ -lactamase fusion and should plateau at saturation.
- the amount of intact ⁇ -lactamase fusion bound, as determined by ELISA should be comparable to the amount of the second fragment fusion bound, and the ratio of V max should reflect the ratio of specific activities of the intact and fragment- reconstituted ⁇ -lactamases.
- the K M should be estimated as described above for the reactivated enzyme.
- the reactivated ⁇ -lactamase is expected to have a maximum activity (k cnl ) near that of the free enzyme because the BLIP-D49A - ⁇ -lactamase complex is under strain with the 15-mer linker, and the affinity of the complex is at least 10- fold lower than that of BLIP-F142A for ⁇ -lactamase.
- the optimized fusions of anti-Her-2/new scFv with ⁇ -lactamase reactivation components may then be tested for activation of ⁇ -lactamase activity in the presence of human tumor cells expressing the Her-2/neu antigen.
- Cell killing may be assayed using any of the three cephalosporin prodrugs shown in Figure 14.
- the reactivated ⁇ -lactamase activity may again be compared with the free ⁇ -lactamase activity, this time with respect to tumor cell killing.
- Such results should indicate the dose range which may be required to show a significant anti-tumor effect in animals, which will be the next step in preclinical evaluation of the tumor-targeted ⁇ -lactamase.
- the SK-BR-3 line of human breast adenocarcinoma cells may be seeded in 6-well tissue culture plates at 3xl0 5 cells per well in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal calf serum (FCS), and allowed to grow to confluency at 37°C in 10% CO 2 .
- DMEM Dulbecco's Minimum Essential Medium
- FCS fetal calf serum
- the saturability of both Her-2/neu epitopes on the cells may be determined with increasing amounts of intact ⁇ -lactamase fused to each scFv, as determined spectrophotometrically after nitrocefin hydrolysis.
- V max of the reactivated, auto-inhibited enzyme may then be determined on the cells with saturating concentrations of both scFv fusions and nitrocefin.
- V max is expected to conform to the predicted activity based on the maximum uninhibited ⁇ -lactamase activity and the ratio of V max observed on the immobilized recombinant antigen.
- the sensitivity of the cells to any of the three prodrugs shown in Figure 14 may be determined essentially as described by Marais et al.
- prodrugs are dissolved in DMSO and diluted into DMEM/FCS to a range of concentrations immediately prior to use. One ml is added to each well and the cells are incubated overnight. The cells are then washed, trypsinized, and viability is determined by dye exclusion. Aliquots are then seeded into fresh dishes.
- Example 5 Affinity Maturation of an antigen-specific antibody [203]
- the auto-inhibited reporter is comprised of a responder protein (also referred to in this example as a reporter protein) and an inhibitor of the reporter that are linked genetically so that the reporter is in a constitutively inhibited state.
- the system further comprises a reactivator protein that binds to the inhibitor competitively with the reporter and with an affinity that is at least comparable to that of the reporter, but which is not high enough to displace the inhibitor from the reporter when the reactivator and the auto-inhibited reporter are not physically linked.
- the auto-inhibited reporter and the reactivator are each linked to molecules that interact with one another, such as an antibody and an antigen, that interaction brings the auto-inhibited reporter and the reactivator into close proximity, whereupon the inhibitor re-equilibrates between the reporter and the reactivator, causing the detectable activation of the reporter.
- the auto-inhibited reporter fusion and the reactivator fusion are co-expressed in cells, and when the reporter confers a selectable phenotype on the cells, such as antibiotic resistance, the antigen-antibody interaction can be monitored by the phenotype of the cells.
- the antibody is a library of candidate antigen-binders
- the selectable phenotype can be used to identify bona fide antigen-binders in the library. This is illustrated in Figure 15.
- K d S in the sub-nanomolar range are typically desired for antibodies, but such concentrations of signaling molecules in cells are usually not sufficient to generate robust phenotypes.
- the antibody library is a library of candidate higher-affinity variants of the antibody, and the goal is to find the highest affinity variants, then it is necessary to disengage affinity from expression levels, so that reporter activation remains proportional to affinity at expression levels which are higher than the desired K d values.
- affinity maturation the reference antibody is expressed as an unlinked "competitor" in each cell, along with the fusions of the library members and the antigen to the auto-inhibited reporter and reactivator.
- Each cell expresses a single library member that competes one-on-one in the cell with the reference antibody for binding to the antigen, so that the higher the affinity of a given library member, the more reactivator is docked to the auto-inhibited reporter, and the higher the reporter activity.
- the strength of the phenotype conferred by the reporter is proportional to the affinity of the library member.
- CD40ED human CD40 extra-cellular domain
- KB2F10 is a humanized murine antibody that binds the extra-cellular domain of human CD40 antigen.
- the ability of KB2F10 to compete with itself for reactivation of ⁇ - lactamase in the RAIR system was determined. This establishes a threshold above which the stringency of selection has to be set in order to restrict the selection to only higher affinity variants.
- the variable region coding sequences of KB2F10 were subcloned into the reactivator fusion vector and into the competitor cassette in the reporter/competitor vector shown in Figure 16 for expression as Fab fragments with human CHI and CL constant regions to make the light chain and Fd chain.
- the Fabs in the two vectors are expressed from dicistronic transcripts.
- the order of cistrons is determined by which V-region is to be mutagenized.
- the chain which is common to the competitor and the library is expressed from the upstream cistron to ensure that it is in excess for the competing chains, so that the competing chains are competing for antigen and not for the common chain.
- Any of the six CDRs of the antibody may be affinity matured in any order.
- VH generally bears most of the binding affinity of an antibody, VH is generally mutagenizd first.
- the light chain is expressed from the upstream cistron and the Fd chain from the downstream cistron.
- a non-binding Fab sequence was also inserted into the competitor cassette of the reporter/competitor vector, and for a negative test antibody control, the non-binding Fab sequence was inserted into the reactivator fusion cassette.
- Plasmid DNA of the Reference-Reactivator Fusion vector and the Reporter/Competitor vector (or each vector with a negative control vector of the other) were introduced simultaneously into E. Coli strain TOPI OF' by high- voltage electroporation, and after a one-hour recovery, the cells were plated on solid rich medium (2xYT) containing increasing concentrations of the ⁇ -lactam antibiotic ampicillin which is hydrolyzed by ⁇ - lactamase.
- test antibody-reporter fusion is expressed from an inducible promoter, which allows the expression of the reference antibody to be tuned to roughly the mid-point of the dynamic range. Since the relationship of test antibody affinity to PE is sigmoidal, this is the area of greatest sensitivity of the selection system, where the increase in PE per unit increase in affinity of the test antibody is maximal. To achieve this condition, the cells expressing the KB2F10 reference antibody as both competitor and test antibody were plated on several concentrations of isopropyl thiogalactoside (IPTG), an inducer of the lactose operon promoter. At 50 ⁇ M IPTG the cells plated quantitatively out to 75 ⁇ g/ml ampicillin, above which they declined rapidly.
- IPTG isopropyl thiogalactoside
- the background PE due to reactivation by the KB2F10 reference antibody and unimproved variants will be between 0.4 and 0.01%, or 250-1000-fold lower than the PEs of variants with affinities less than 10-fold higher than that of the reference antibody.
- These background clones can then be eliminated by replating the selected clones 2-3 times under the same conditions, where with each replating the enrichment of higher-affinity variants over background is 250- 1000-fold.
- the oligonucleotides are then used to prime amplification of the intervening V-region sequences, which are then ligated into the expression cassette in place of the reference V-region sequences.
- Most of the diversity of such libraries covering e.g., all three CDRs in VH simultaneously, can be accessed in libraries of -10 9 clones.
- the first round of selection is performed by plating at least IO 9 double transformants, expressing the CD40-BLIP- ⁇ -lactamase fusion, the KB2F10 Fab competitor, and the BIP-KB2F10 Fab fusion with PM library covering all three VH CDRs on 100 and 125 ⁇ g/ml ampicillin with 50 ⁇ M IPTG. Colonies appearing after overnight growth are pooled and the BlP-library member fusion plasmids purified and retransformed with the CD40-reporter/competitor plasmid. -10 transformed cells per original colony are plated again on 100 and 125 ⁇ g/ml ampicillin with 50 ⁇ M IPTG.
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US10/076,845 US20030157579A1 (en) | 2002-02-14 | 2002-02-14 | Molecular sensors activated by disinhibition |
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US37380202P | 2002-04-18 | 2002-04-18 | |
US373802P | 2002-04-18 | ||
US373765P | 2002-04-18 | ||
US37971802P | 2002-05-10 | 2002-05-10 | |
US379718P | 2002-05-10 | ||
US208730 | 2002-07-29 | ||
US10/208,730 US7335478B2 (en) | 2002-04-18 | 2002-07-29 | Reactivation-based molecular interaction sensors |
PCT/US2003/004925 WO2003068945A2 (en) | 2002-02-14 | 2003-02-14 | Detection of molecular interaction by reactivation of an auto-inhibited responder (rair) |
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WEHRMAN T ET AL: "Protein-protein interactions monitored in mammalian cells via complementation of beta-lactamase enzyme fragments" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 99, no. 6, 19 March 2002 (2002-03-19), pages 3469-3474, XP002254024 ISSN: 0027-8424 * |
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