WO2000071702A1 - Interaction-activated proteins - Google Patents
Interaction-activated proteins Download PDFInfo
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- WO2000071702A1 WO2000071702A1 PCT/US2000/007108 US0007108W WO0071702A1 WO 2000071702 A1 WO2000071702 A1 WO 2000071702A1 US 0007108 W US0007108 W US 0007108W WO 0071702 A1 WO0071702 A1 WO 0071702A1
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- fragment
- protein
- oligopeptide
- lactamase
- interaction
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
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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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- G01N2333/986—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)
Definitions
- the present invention is concerned with detecting interactions between proteins by expressing them as part of a fusion sequence that also encodes for one fragment of a fragment pair that reassembles into a directly detectable protein.
- the interaction-dependent enzyme association (IdEA) systems of the present invention are exemplified by the bacterial ⁇ -lactamases, a large group of structurally-related enzymes which segregate into several groups on the basis of structural homologies and substrate specificities.
- the domains can function independently, and in fact are often in separate, interacting subunits.
- the "bait" protein is expressed as a fusion with a cw-element sequence-specific DNA-binding domain, and cDNAs are expressed as fusions with a transactivation domain.
- reporter gene can be expressed, since its transcription is dependent on transactivation from the cz ' s-element.
- Reporters can be either screenable (e.g., ⁇ -galactosidase for color) or selectable (e.g., HIS 3 for growth in the absence of histidine).
- the fundamental limitation with this system is that many steps are required between the test interaction and the generation of a selectable phenotype. Each such step presents an opportunity for non-specific interaction to raise the false positive background, and for dissociation to allow bona fide interactors to be missed.
- the false positive problem is exacerbated by the highly combinatorial nature of the transcription machinery and the abundance of protein domains encoded in cDNA libraries which can interact with one or more components of the transcription initiation complex, including transactivator-bound promoter DNA (Bartel et al., BioTechniques (1993) 14:920).
- Another limitation of the original two-hybrid system is that it generally cannot accommodate secreted or membrane proteins and cytoplasmic proteins must be stable in the yeast nucleus.
- the interaction is then monitored by its ability to allow phage encoding the interactors to transfer a selectable phenotype to susceptible cells by infection.
- this method also suffers from requiring many low-efficiency steps between the target interaction and the expression of the selectable phenotype by the recipient cell.
- the efficiency of this system suffers from the fact that most natural protein-protein interactions have affinities in the micromolar range, with half-lifes on the order of seconds. When the time delay between interaction and signal generation exceeds this half-life, which it does in these systems, the efficiency of interaction detection declines sharply.
- interactors can be fused to variants of the Green Fluorescent Protein of Aequorea victoria (GFP), which are capable of detectable fluorescence resonance energy transfer (FRET) when brought into close proximity by the interactors (Cubitt et al., Trends Biochem (1995) 20:448).
- GFP Green Fluorescent Protein of Aequorea victoria
- FRET fluorescence resonance energy transfer
- Some enzymes which confer selectable or screenable phenotypes on cells can also be adapted for two-hybrid type protein-protein interaction detection (Rossi et al.
- the selectable signal is produced directly from the desired interaction, without any intervening steps which are the main sources of inefficiency in the earlier systems.
- improvements in efficiency and background should make these methods more amenable to high-throughput applications.
- IdEA and GFP FRET systems in theory can be set up in both prokaryotic and eukaryotic cells, and either in the cytoplasm or in a secretory pathway to allow interactions to be monitored in natural milieus, they have not.
- the ecto-domain of the bait receptor is fused to the transmembrane domain and endo-domain of an E. coli receptor.
- this fusion protein is co-expressed with an expression library in the bacterial periplasm, ligands for the receptor can be identified by their ability to dimerize the receptor and induce expression of a selectable phenotype.
- this system suffers from the same limitation as the yeast two-hybrid and SIP systems, namely, that multiple steps between interaction and phenotype cause severe loss of efficiency due to high false positive and false negative rates.
- IdEA systems capable of simultaneous detection of multiple interactions between extra-cellular as well as intracellular proteins in a high throughput format.
- USPN 5,585,245 discloses a ubiquitin-based protein sensor complementation system where binding of two predetermined proteins of a binding pair is detected as specific proteolysis of ubiquitin by ubiquitinases.
- PCT publication WO 98/44350 discloses a reporter subunit complementation system employing fusion proteins of ⁇ -galactosidase subunits.
- PCT publication WO 98/34120 discloses a protein fragment complementation system employing dihydrofolate reductase.
- SUMMARY Compositions and methods are provided for identifying interactions between polypeptides using an interaction-dependent protein association system.
- the system is characterized by using fragment pairs comprised of a first and a second member that functionally reassemble into a marker protein having a directly detectable signal, such as a visible phenotypic change or antibiotic resistance.
- the fragment complementation system of the present invention involves co-expression in a host cell of a first and a second oligopeptide, where each is a fusion protein separated by a flexible polypeptide linker with a member of the marker protein fragment pair.
- Binding of the first oligopeptide to the second oligopeptide results in the functional reconstitution of the fragment pair into a marker protein, and the interacting first and second oligopeptides are identified by isolating and sequencing plasmids from a host cell that displays a directly detectable signal indicative of the marker protein.
- Functional reconstitution of the fragment pairs into a marker protein can be enhanced by including elements such as a cysteine residue or a randomly encoded peptide of from 3-12 amino acids at or near the break-point termini of the fragment pair member, or by introducing 1-3 codon changes within the nucleotide sequence encoding for a member of a fragment pair.
- the invention also provides for efficient methods of finding functional fragment pairs of a marker protein that involve identifying functional break- points within flexible loops using tertiary or secondary structural information.
- the interaction-dependent protein activation systems of the present invention find particular use in identifying immunoglobulin epitopes, polypeptide sequences that bind to extracellular proteins, and inhibitors of phophorylati on-regulated signal transducer proteins.
- the systems also find use in allowing single antibiotic selection of cells transformed to express genes for multiple traits and for targeted and localized activation of derivitized anti-tumor prodrugs.
- Figure 1 Mechanism for Interaction-dependent Enzyme Activation (IdEA). Interaction-dependent fragment complementation requires enzyme ⁇ and co fragments which can refold to form active enzyme when and only when they are brought together by an interaction of heterologous domains fused to their termini.
- IdEA Interaction-dependent Enzyme Activation
- FIG. 1 Nucleotide coding sequence for the mature form of TEM-1 ⁇ -lactamase and the encoded amino acid sequence (Sutcliffe, Proc Natl Acad Sci (1978) 75:3737). From the sequence for plasmid pBR322 (SYNPBR322), Genbank accession no. JO 1749. The breakpoints between the ⁇ and ⁇ fragments at residues Asn52/Ser53, Glu63/Glu64, Gln99/Asnl00, Prol74/Asnl75, Glul97/Leul98, Lys215/Val216, Ala227/Gly228 and Gly253/Lys254 are indicated.
- Figure 3 Three-dimensional structure of mature TEM-1 ⁇ -lactamase. Rendering of the x-ray crystal structure of Jelsch et al. (Proteins Struct Fund (1993) 76:364ff), using red and blue solid ribbons to show ⁇ -helix and ⁇ -sheet, respectively. The molecule is oriented to emphasize the two-domain structure ( ⁇ - ⁇ and / ⁇ ). The active site nucleophile, Ser70, is shown as a ball-and-stick model.
- Figure 4 Three-dimensional representation of interaction-dependent activation of ⁇ -lactamase by fragment complementation. Docking of TEM-1 ⁇ l97 and ⁇ l98 fragments by the interaction of the hetero-dimerizing helixes from the fos and jun subunits of the AP-1 transcription activator allows re-folding of the fragments into the active conformation of the enzyme (compare with Figure 3).
- YW-200 and YW-285 are a DNA-binding tri-indole and its cephalosporin prodrug (Wang et al. , 1998, US Patent 5,843,937)
- Vectors and strategy for the expression of heterologous proteins as fusions to the ⁇ l97 and ⁇ l98 fragments of TEM-1 ⁇ -lactamase for interaction-dependent ⁇ -lactamase activation by fragment complementation are a high-copy pUC119-based phagemid for expression of ⁇ 198 fusions and free ligands from dicistronic transcripts, which can be rescued as phage for quantitative introduction into host cells by high- multiplicity infection.
- Vector pAEl is a low-copy pl5A replicon with a strong promoter for expression of ⁇ 197 fusions at comparable or higher levels than expression from the pAOl vector.
- Trxpeps are 12-mer peptides inserted into the active site of thioredoxin.
- Tripep-trx libraries are random tri-peptides at the N-terminus of thioredoxin with an intervening Gly 4 Ser linker.
- ScFv single-chain antibody Fv fragment.
- LC-CH1 antibody fragment composed of light chain and first constant region of heavy chain.
- VL antibody light chain variable region, lac prom, lactose operon promoter.
- SP signal peptide. (Gly 4 Ser) 3 , flexible 15-mer linker.
- m.o. multiplicity of infection, trc prom, fusion promoter from tryptophan and lactose operons.
- tt transcription terminator, kan, kanamycin resistance gene.
- FIG 7. TEM-1 ⁇ -lactamase fragment complementation by interaction between representative single-chain antibody Fv fragment (scFv) and thioredoxin-scaffolded peptide (Trx).
- the N-terminal ⁇ -lactamase fragment, ⁇ l97 ( ⁇ ) is colored red.
- the C-terminal fragment, ⁇ l98 ( ⁇ ) is colored blue.
- TEM-1, thioredoxin, and the scFv were rendered from published structures. The peptide and the linkers were drawn in.
- Figure 8. TEM-1 ⁇ -lactamase fragment complementation by interaction between the CD40 extra-cellular domain (CD40) and a thioredoxin-scaffolded peptide (Trx).
- the N-terminal ⁇ -lactamase fragment, ⁇ l97 ( ⁇ ), is colored red.
- the C-terminal fragment, ⁇ l98 ( ⁇ ), is colored blue.
- TEM-1, thioredoxin, and the scFv were rendered from published structures.
- the peptide and the linkers were drawn in.
- Figure 9 Vectors and protocol for construction of a multiplex protein-protein interaction library using interaction-dependent ⁇ -lactamase fragment complementation systems.
- Expressed sequence (ES) i.e., random-primed cDNA libraries, are subcloned into phagemid vectors for expression as fusions to the ⁇ -lactamase ⁇ and ⁇ fragments, via the flexible linker (Gly 4 Ser) 3 .
- the vectors encode a peptide epitope tag, such as the 12-residue Myc tag, at the C-terminus of the ES.
- the ES libraries When co-expressed with anti-Tag scFv, such as anti- myc 9E10, fused to the other fragment, the ES libraries can be selected for ⁇ -lactamase activity driven by the Tag-anti-Tag interaction, which will require stable expression of the
- the resultant libraries may then be rescued as phage and co-infected into male cells for selection of interacting AFD pairs (Multiplex Interaction Library).
- the AFD libraries can also be co-infected with scFv libraries, antibody light chain variable region libraries (VL), or peptide libraries displayed on thioredoxin (trx-peptide) for simultaneous selection of binding proteins for each AFD (Multiplex Antibody /Peptide Binder Selection). See legends to Figures 6 and 10 for identification of other abbreviations.
- Figure 10 Abbreviated output of the PredictProtein Program for prediction of secondary structure and solvent exposure for NPTII (Rost and Sander, 1993, 1994).
- the top line shows the amino acid sequence in single letter code.
- the second and third lines show secondary structure prediction. H, helix; E, strand; L, loop.
- the fourth line shows a measure of reliability on a scale from 1 to 10, with 10 being highest.
- the fifth line shows solvent accessibility - e, exposed; b, buried.
- the bottom line shows a measure of reliability for solvent accessibility on a scale of 1 to 10, with 10 being highest.
- Ten regions of the sequence predicted to have little secondary structure and to be exposed to solvent are indicated by underlining as potential sites for productive fragmentation.
- T7 prom, bacteriophage T7 promoter; SP, pelB signal peptide; scFv is comprised of VH (antibody heavy chain variable region), (Gly 4 Ser) 3 (15-mer flexible linker), and VL (antibody light chain variable region); kan, kanamycin resistance; His 6 , hexa-histidine tag for metal ion affinity purification; lacF, high-affinity lac operon repressor mutant; f 1 ori, phage origin of replication.
- an interaction-dependent protein activation system useful in detecting an interaction between a first protein and a second target protein.
- the method detects the interaction of a first known or unknown interactor domain with a second unknown interactor domain by bringing into close proximity members of a fragment pair of a marker protein, such that the parent marker protein is reassembled to its original functionality, and such that reassembly requires the prior interaction of the heterologous interactor domains.
- the system is characterized by N- terminal and C-terminal fragment members that comprise fragment pairs which are derived from, and can functionally reassemble into a marker protein that provides for a directly detectable signal that does not involve downstream steps necessary for recognition.
- a marker protein of interest for the instant invention functions of itself to produce a selectable signal such as a visible phenotypic change or antibiotic resistance.
- the fragment pairs are used in methods that involve the co-expression of a first and a second oligopeptide sequence, in which the first oligopeptide sequence is a fusion protein comprised of in the direction of translation, an N-terminal fragment fused through a break- point terminus to a flexible polypeptide linker and a first interactor domain, and the second oligopeptide sequence is a fusion protein comprised of in the direction of translation, a second interactor domain and a flexible polypeptide linker fused through a break-point terminus to a C-terminal fragment.
- the flexible polypeptide linker separates the fragment domain from the interactor domain and allows for their independent folding.
- the linker is optimally 15 amino acids or 60 A in length ( ⁇ 4 A per residue) but may be as long as 30 amino acids but preferably not more than 20 amino acids in length. It may be as short as 3 amino acids in length, but more preferably is at least 6 amino acids in length.
- the linker should be comprised of small, preferably neutral residues such as Gly, Ala and Val, but also may include polar residues that have heteroatoms such as Ser and Met, and may also contain charged residues.
- the first interactor domain is a known or unknown protein or protein fragment that binds directly or indirectly to a second target interactor domain that is an unknown protein or protein fragment and either or both the first and second interactor domain can be a member of a library.
- the interactor domain libraries are preferably constructed from cDNA, but may also be constructed from, for example, synthetic DNA, RNA and genomic
- Bound interactor domains are identified by expressing a functionally reconstituted marker protein, and then the nucleotide sequences encoding for bound interactor domains or the bound interactor domains themselves are characterized by methods including electrophoresis, polymerase chain reaction (PCR), nucleotide and amino acid sequencing and the like.
- Advantages of the present invention over previously disclosed fragment complementation systems include a reporter protein that provides for a directly detectable signal upon reassembly, and background levels of 1 in 10 6 or less. Additionally, the invention provides for rationally incorporated enhancement modifications to the fusion oligopeptides that increase the functional activity of the reconstituted protein to wild-type levels by improving folding and reassembly of the fragments into the parent protein, while at the same time maintaining dependence on the interactor domains for reassembly.
- the interaction-dependent enzyme activation system of the subject invention may be used to detect in vitro protein interactions, such as in cell ly sates, or the interactions of intracellular or extracellular proteins of a host cell.
- the first and second fusion oligopeptides can be expressed with a signal peptide.
- an N-terminal signal peptide can provide for translocation of the fusion oligopeptides to the periplasm.
- the combined lengths of the N-terminal fragment and the C-terminal fragment may be discontinuous with residues around the break-point deleted, contiguous, or overlapping with residues around the break-point repeated, thereby comprising from 90% to 110% of the total length of the parent protein. Break-point termini are herein defined as the C-terminus of the N-terminal fragment and the N-terminus of the C-terminal fragment.
- the subject invention provides for enhancing the performance of the reassembled parent protein by introducing at least one of the following modifications, including: i) a randomly-encoded peptide of 3-12 amino acids between the break-point terminus of each fragment and the flexible polypeptide linker, ii) a randomly-encoded peptide of 3-12 amino acids expressed separately as a fusion to the N-terminus of a thioredoxin with an intervening flexible linker, iii) a cysteine residue encoded at or within 5 amino acid positions of the break-point and between the break-point terminus of each fragment and the flexible polypeptide linker so that a disulfide bond can form between the members of a fragment pair, and iv) 1-3 codon changes within a member of a fragment pair introduced, for example, by PCR amplification of a nucleotide sequence encoding for a member of a fragment pair under error-prone conditions, to enhance the folding stability of a functionally reconstituted marker protein.
- the invention is also directed to plasmids containing expression cassettes constructed to express fusion oligopeptides comprised of a fragment domain and an interactor domain.
- the expression cassettes for the N-terminal and C-terminal fragment pair members are designed with their components in different sequential orders.
- the expression cassette will comprise as operably linked components in the direction of transcription nucleotide sequences encoding for (i) a promoter functional in a host cell, (ii) a polypeptide interactor domain, (iii) a flexible polypeptide linker and (iv) a C-terminal fragment of a marker protein that provides for a directly selectable phenotype.
- the expression cassette for the N-terminal fragment pair member will comprise as operably linked components in the direction of transcription nucleotide sequences encoding for (i) a promoter functional in a host cell, (ii) an N-terminal fragment of a marker protein that provides for a directly selectable phenotype, (iii) a flexible polypeptide linker and (iv) a polypeptide interactor domain.
- the invention is also concerned with host cells that contain plasmids having the sequences of the above- described expression cassettes.
- Appropriate host cells for application of the subject invention include both eukaryotic cells, such as mammalian, yeast and plant cells, and prokaryotic cells, such as bacterial cells.
- eukaryotic cells such as mammalian, yeast and plant cells
- prokaryotic cells such as bacterial cells.
- a variety of prokaryotic expression systems can be used to express the fusion oligopeptides of the subject invention.
- Expression vectors can be constructed which contain a promoter to direct transcription, a ribosome binding site, and a transcriptional terminator. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky (1984) J. Bacteriol.
- Plasmids useful for transforming bacteria include pBR322 (Bolivar, et al, (1977) Gene 2:95-113), the pUC plasmids
- Plasmids may contain both viral and bacterial elements. Methods for the recovery of the proteins in biologically active form are discussed in U.S. Patent Nos. 4,966,963 and 4,999,422, which are inco ⁇ orated herein by reference. See Sambrook, et al (In Molecular Cloning: A Laboratory Manual, 2 nd Ed.,
- host cells for use in practicing the present invention include mammalian, avian, plant, insect, and fungal cells.
- a promoter will depend in part upon whether constitutive or inducible expression is desired and whether it is desirable to produce the fusion oligopeptides at a particular stage of plant development and/or in a particular tissue.
- Expression can be targeted to a particular location within a host plant such as seed, leaves, fruits, flowers, and roots, by using specific regulatory sequences, such as those described in USPN 5,463, 174, USPN 4,943,674, USPN 5,106,739, USPN 5, 175,095, USPN 5,420,034, USPN 5,188,958, and
- transcription and translational regions functional in yeast cells are provided, particularly from the host species.
- the transcriptional initiation regulatory regions can be obtained, for example from genes in the glycolytic pathway, such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglucoisomerase, phosphoglycerate kinase, etc. or regulatable genes such as acid phosphatase, lactase, metallothionein, glucoamylase, etc.
- Any one of a number of regulatory sequences can be used in a particular situation, depending upon whether constitutive or induced transcription is desired, the particular efficiency of the promoter in conjunction with the open-reading frame of interest, the ability to join a strong promoter with a control region from a different promoter which allows for inducible transcription, ease of construction, and the like.
- promoters which are activated in the presence of galactose.
- Galactose-inducible promoters (GAL1, GAL7, and GAL 10) have been extensively utilized for high level and regulated expression of protein in yeast (Lue et al, (1987) Mol. Cell. Biol. 7:3446; Johnston, (1987) Microbiol. Rev. 51 :458).
- the invention also provides for efficient methods of identifying functional fragment pairs of a marker protein of interest that involves preparing a multiplicity of fragment pair members with break-point termini within a solvent exposed loop or a flexible loop defined by tertiary or secondary strucmre analysis to obtain a fragment pair library.
- the fragment pair members are expressed in a multiplicity of host cells, and the host cells exhibiting the directly detectable signal associated with the marker protein of interest are isolated as indicative of containing fragment pair members that functionally reconstitute the marker protein. Plasmids containing expression cassettes coding for the fragment pair members are then sequenced to identify functional fragment pairs.
- the fragment pair members can be expressed as fusion proteins with interactor domains known to bind to each other, such as the fos and jun transcription factors that associate through a leucine zipper interaction.
- interactor domains known to bind to each other, such as the fos and jun transcription factors that associate through a leucine zipper interaction.
- the sequences encoding the hetero-dimerizing helices of the fos and jun transcription factors are sufficient to use as effective interactor domain for this purpose.
- the systems and methods of the subject invention find particular use in identifying epitopes recognized by immunoglobulin molecules, polypeptide sequences that bind to extracellular domains of a transmembrane protein, inhibitors of phosphorylation-regulated signal transducer proteins, and interaction between oligopeptides of two different proteomes.
- first and second fusion oligopeptides comprised of a fragment domain and an interactor domain are expressed in a host cell where the first fusion oligopeptide has an interactor domain comprised of a randomly encoded peptide inserted into the active site of a thioredoxin protein and the interactor domain of the second fusion oligopeptide is comprised of a single-chain variable region (scFv) or antibody light chain variable region (VL).
- scFv single-chain variable region
- VL antibody light chain variable region
- a similar strategy is followed for identifying polypeptide sequences that interact with the extracellular domain of a transmembrane protein, where the first interactor domain is comprised of a randomly encoded peptide inserted into the active site of a thioredoxin protein and the second interactor domain is comprised of a transmembrane protein.
- Identification of inhibitors of a phosphorylation-regulated signal transduction protein involves expressing a first fusion oligopeptide with a first interactor domain comprised of a phosphorylation-regulated signal transduction protein, such as Her-2/neu, and a second fusion oligopeptide with a second interactor domain comprised of a scFv or antibody light chain variable region that only binds to the unphosphorylated signal transduction protein.
- Inhibitory compounds are identified from host cells that change color in the presence of a chromogenic ⁇ -lactamase substrate.
- members of a first and second cellular expression library comprise the first and second interactor domain, respectively, of a fusion oligopeptide.
- the expression library is preferably a cDNA library, but may also be constructed from synthetic nucleotides to screen randomly generated polypeptides.
- a library of particular application for the present invention should represent all the protein members of a proteome of interest. Libraries derived from nucleotide sequences that all members of a total protein population (i.e. a proteome) of interest may be isolated from a host cell such as a prokaryotic or a eukaryotic cell, or also from a viral host. Viral hosts that encode for oncogenes are of particular interest. Mammalian tumor cells, immune cells and endothelial cells also provide proteomes of particular interest for the subject invention.
- the invention also finds use in selecting with a single marker protein the incorporation of multiple genetic traits in a host cell, where detectable expression of a functionally reassembled marker protein is indicative of co-expression of multiple genes that encode for individual traits in a host.
- the invention provides therapeutic utility in a method for specifically activating derivitized prodrugs in the vicinity of a target organ in a host, where each member of a marker protein fragment pair is expressed as a fusion protein with individual immunoglobulin molecules that recognize neighboring but non- overlapping epitopes on a target protein. Binding of both antibodies to the target protein allows functional reconstitution of the marker protein which then activates subsequently administered prodrug only in the vicinity of a target organ.
- the invention is exemplified by the antibiotic resistance enzyme, TEM-1 ⁇ - lactamase, although fragment pairs of other enzymes that provide for antibiotic resistance are included in the present invention, including: aminoglycoside phosphotransferases, particularly neomycin phosphotransferase, chloramphenicol acetyl transferase, and the tetracycline resistance protein described by Backman and Boyer (Gene (1983) 26: 197).
- Other proteins that can directly elicit a visible phenotypic change such as a color change or fluorescence emission also are applicable to the subject invention. Examples of such proteins include ⁇ -galactosidase and green fluorescent protein (GFP) or other related fluorescent proteins.
- TEM-1 ⁇ -lactamase of E. coli is the 264 amino acid product of the ampicillin resistance gene of plasmid pBR322 (Sutcliffe, 1978, supra), the nucleotide sequence of which is shown in Figure 2 along with the encoded amino acid sequence.
- TEM-1 is the archetype member of the homologous Class A ⁇ -lactamases, or penicillinases. Its three- dimensional structure is shown in Figure 3 (Jelsch et al., Proteins Struct Funct (1993) 6:364ff).
- the Class A ⁇ -lactamases are comprised of two domains.
- One domain, ⁇ - ⁇ is made up of N-terminal and C-terminal sequences, which form an anti-parallel two-helix bundle packed against a flat 5-stranded ⁇ -sheet.
- the inner face of the sheet packs against the other domain ( ⁇ ), a seven helix bundle with two extended loops and two small ⁇ - structures.
- An outside strand of the ⁇ -sheet borders the substrate binding pocket, opposite the catalytic nucleophile, Ser70, and contributes substrate-binding residues.
- the remainder of the active site residues, including Ser70, are contributed by the ⁇ domain.
- the two domains are connected by two loops: R61-R65 and D214-W229.
- the subject invention also provides a method of identifying optimal break-points in a parent protein that provides for a directly detectable signal.
- a search of the "fragment space" of TEM-1 ⁇ -lactamase was conducted to identify fragment pairs which could complement for activity only when the break-point termini of the fragments were genetically fused to hetero-dimerizing helixes from the c-fos and c-jun subunits of the AP-1 transcription factor (Karin et al., Curr Opin Cell Biol (1997) 9:240. To do this, libraries of all possible N- and C- terminal fragments of the enzyme were generated by progressive exonucleolytic digestion of the full coding sequence from both termini.
- Fragments of less than 25 amino acids were considered non-viable.
- the fragment sequences co-expressed in the same E. coli cells so that each cell expressed a single pair of N- and C- terminal fragments and every possible pair may be represented.
- N- and C-terminal fragment pairs For example, for a 100 kDa enzyme there are only 10 6 possible N- and C-terminal fragment pairs, so an exhaustive search of the fragment space of most enzymes could be conducted with libraries of a manageable size. An exposed loop was identified by this method between two ⁇ -helixes of E.
- coli T ⁇ M-1 ⁇ -lactamase (approximately Thrl95 to Ala202, between helixes 7 and 8) within which the chain could be broken to produce fragments which could only complement for activity when fused to X efos and jun helixes.
- Representative fragments with contiguous break point termini at Glul97 and Leu 198 were designated ⁇ l97 (N-terminal fragment) and ⁇ l98 (C-terminal fragment), and subsequently shown to produce selectable activity in the E.
- coli periplasm with interactions between a variety of heterologous domains fused to the break-point termini, including single-chain antibody Fv fragments (scFv), antibody light chains (LC), thioredoxin with 12-mer peptides inserted into the active site (trxpeps), and the extracellular domain of the B-cell activation antigen CD40 (CD40ED).
- scFv single-chain antibody Fv fragments
- LC antibody light chains
- thioredoxin with 12-mer peptides inserted into the active site trxpeps
- CD40ED B-cell activation antigen CD40
- Activation by complementation of ⁇ 197 and ⁇ l98 could also be driven by interaction of the heterologous domains with a third polypeptide, such as a receptor.
- a third polypeptide such as a receptor.
- coli TEM-1 ⁇ -lactamase in addition to E197/L198 include amide-bond junctions between amino acid residues N52/S53, E63/E64, Q99/N100, P174/N175, K215/V216, A227/G228, and G253/K254.
- the combined lengths of the fragment pairs may be discontinuous or overlapping, however, comprising from 90% to 110% of the total length of the parent protein, and the actual break-point could be within ten amino acid residues in either direction from an identified functional contiguous break-point junction.
- the specific activity of the reconstituted enzyme can be enhanced to near wild-type levels by the interaction-driven formation of a disulfide at the break-point, which restores the integrity of the native polypeptide backbone (see Figure 4).
- the ⁇ -lactamase l97 and ⁇ l98 fragments cooperatively produce selectable activity in the bacterial periplasm in a manner that is strictly dependent on specific interaction between heterologous domains fused to the break-point termini of the fragments is an example of an enzyme-based molecular interaction sensor that can undergo secretory translocation across a plasma membrane into an extra-cellular compartment, and therefore can reliably detect interactions between and among extra-cellular proteins.
- the interaction-dependent enzyme association systems of the present invention finds use in many applications in human therapeutics, diagnostics, and prognostics, as well as in high-throughput screening systems for the discovery and validation of pharmaceutical targets and drugs.
- One particular application is concerned with the localized and controlled activation of inactive or weakly active compounds.
- useful compounds such as drugs, chromophores, and fluorophores
- Such conjugates can then be activated by the appropriate hydrolytic enzymes such as esterases, carboxypeptidases, alkaline phosphatases, glycosidases, glucuronidases, ⁇ -lactamases, and Penicillin-amidases.
- hydrolytic enzymes such as esterases, carboxypeptidases, alkaline phosphatases, glycosidases, glucuronidases, ⁇ -lactamases, and Penicillin-amidases.
- cephalosporins may be conjugated at the 3' position via a variety of different leaving groups to a variety of anti-cancer drugs, such as nitrogen mustards, methotrexate, anthracyclines, and vinca alkaloids (Svensson et al., J Med Chem (1998) 41: 1507; Vrudhula et al., J Med Chem (1995) 5S:1380; Jungheim and Shepherd, 1994, supra; Alexander et al. Tetrahedron Lett (1991) 52:3269;, see also Figure 5). All of these are good substrates for broad spectrum ⁇ -lactamases, and most are much less active than their parent drugs.
- anti-cancer drugs such as nitrogen mustards, methotrexate, anthracyclines, and vinca alkaloids (Svensson et al., J Med Chem (1998) 41: 1507; Vrudhula et al., J Med Chem (1995) 5S:1380; Jungheim and Shepherd, 1994, supra
- enzymes which could be activated to hydrolyze chromogenic substrates only upon binding to target analytes could form the basis of assays for those analytes of unparalleled sensitivity and convenience.
- assays would be homogeneous, requiring no manipulations other than the mixing of two components, namely the enzyme and substrate, with a biological specimen, in which the presence of the analyte is then quantitatively indicated by the rapid development of color.
- K d concentrations of reagents so that typically only a fraction of analyte molecules participates in signal generation, and equilibration is often slow or does not even reach completion.
- an enzyme 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.
- interaction-activated enzymes could be adapted for activation by binding to specific cell surface molecules. This would allow the enzyme 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 (ADEPT; Bagshawe, 1995, supra) 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.
- an inactive prodrug such as an anthracycline cephalosporin
- an inactive prodrug such as an anthracycline cephalosporin
- the unbound conjugate problem could be completely obviated by a prodrug- activating enzyme which would be active only when bound to the tumor, so that the prodrug could be administered simultaneously with the enzyme or at the point of peak tumor loading without regard for unbound enzyme which would be inactive.
- interaction-activated enzymes could be targeted for activation by surface markers on the cells of other types of diseased tissues, such as sites of inflammation or atherogenesis, or even healthy tissues.
- the target-localized and activated enzymes could 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-activatable enzymes could be used to deliver: (1) immune stimulants to tumors, (2) immuno-suppressants to sites of chronic inflammation or to organ transplants, (3) antibiotics to specific pathogens, (4) cytotoxins and anti-virals to virus-infected cells,
- interaction- activated enzymes could 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 could be inactivated by conjugation to the appropriate substrate.
- interaction- activated enzymes could be adapted for efficient simultaneous detection of multitudes of interactions among proteins within cells, including expressed sequence libraries, single-chain antibody fragment (scFv) libraries, and scaffolded peptide libraries.
- enzyme-based interaction traps could enable the comprehensive mapping of pairwise protein-protein interactions within and between the proteomes of human cells, tissues, and pathogens for the rapid identification and validation of new pharmaceutical targets. They could also be used for rapid selection of binding molecules from single-chain antibody fragment (scFv) libraries, or from scaffolded peptide libraries for use as reagents in functional genomics studies, or for identification of natural ligands and epitopes by homology.
- interaction-dependent ⁇ -lactamases could be used immediately to screen for inhibitors of the interaction by exploiting the great substrate diversity of these enzymes to reverse the polarity of selection.
- interaction-dependent activation of ⁇ -lactamase could be used to confer selective growth on host cells in the presence of ⁇ -lactam antibiotics, it could also be used to confer selective cytotoxicity on the cells in the presence of ⁇ -lactam pro-antibiotics
- the latter substrates would only become cytotoxic upon hydrolysis of the ⁇ -lactam moiety by the interaction-activated enzyme, and so could be used to select inhibitors of the interaction by their ability to confer selective growth on host cells.
- enzyme-based interaction sensors could be used for rapid detection of the activation or inhibition of key molecular interactions in signal transduction pathways, enabling high-throughput cellular screens for inhibitors or activators of those pathways.
- screening for agonists or antagonists of receptor tyrosine kinases 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, like the yeast two-hybrid system, and are therefore poorly suited to high- throughput screening.
- interaction-dependent ⁇ -lactamases could be set up for activation by phospho-tyrosine sensitive interactions, so that a selectable phenotype would be generated just downstream from receptor ligation.
- Interaction between the receptor tyrosine kinase substrate and a binder peptide could be designed to be either dependent on, or inhibited by phosphorylation, so that either receptor agonists or receptor antagonsists could be selected.
- the present invention provides for general strategies for the use of heterologous interactors, break-point disulfides. random tri-peptide libraries, and mutagenesis to obtain stable enzyme fragments which are capable of forming of catalytically robust complexes. It has been suggested that it might be possible to identify such fragment pairs for any enzyme simply by conducting thorough searches of all possible fragment pairs for the enzymes in question (Ostermeier et al. , Proc Natl Acad Sci (1999) 96:3562). In practice, however, the success of such endeavors is strongly dependent on the stringency of selection, that is, how much functional enzyme must be produced by the expressed fragments to produce an efficiently selectable phenotype. An efficiently selectable phenotype is one in which the background frequency, or false positive rate, is not appreciably higher than the frequencies of the desired fragments in the fragment libraries.
- the most useful fragment complementation systems for a given enzyme are not necessarily those fragments of wild-type sequence which are most capable of unassisted complementation, but rather the most useful fragment complementation systems comprise those fragments which, when using the engineering techniques described, can be made to meet more specific performance requirements.
- naturally evolved proteins are generally expected to exhibit a roughly inverse correlation between fragment stability and complex stability. This is due to the energy cost of inter-conversion. The more stable the fragments are, the more energy is required to form the complex and vice versa. As a result, those fragments capable of producing the highest specific activities might be missed or dismissed because fragment instability may prevent them from producing selectable levels of activity.
- libraries of fragment pairs can be simultaneously expressed with libraries of random tri-peptides to insure that every fragment pair has a chance to perform in the presence of fragment-stabilizing tri-peptides, thereby minimizing the dependence of the phenotype on fragment stability.
- This strategy is especially useful if dependence of activation on the interaction of heterologous domains fused to the fragments is desired. If constitutive activation is desired, the fragment libraries could also be amplified by error-prone PCR to introduce fold-accelerating mutations which could mitigate both fragment instability and complex instability, as was found for ⁇ -lactamase.
- fragment stability is especially important, but the most stable fragments might not be selectable if they cannot produce stable complexes without assistance, as would be predicted by the inverse correlation of fragment stability and complex stability.
- fragment libraries could be expressed in the E. coli periplasm with a disulfide at the break-points and heterologous interactors fused to the break-point termini. These tools provide mechanisms for docking the fragments, accelerating folding, and stabilizing the active complex. As was shown with ⁇ -lactamase, a substantial fraction of fragment pairs can be made to produce robust selectable activity in the bacterial periplasm with such molecular prostheses.
- Each of the four tools described for enhancement of functional reconstitution of the parent protein of the fragment pairs i.e., heterologous interaction, break-point disulfide, tri-peptide stabilizers, and mutagenesis, can be used alone or in combination to insure selection of the best fragments for the desired application, and also to improve and optimize the performance of selected fragment pairs for a desired application.
- each tool enhances performance by a different mechanism, so that the effects of multiple tools are generally additive.
- Heterologous interactors bring and hold the fragments together to facilitate re-folding into the active complex.
- Break-point disulfides can stabilize the active fold by restoring the integrity of the polypeptide backbone at the break-point.
- Tethered or free tri-peptides can protect the fragments from aggregation without interfering with folding into the active complex.
- Mutagenesis can protect the fragments by accelerating folding into the active complex.
- the first step in the development of high-performance enzyme fragment complementation systems is to construct vectors to express each fragment in the fragment pair library.
- a convenient system for selective fragment library expression may be derived from the expression system illustrated in Figure 6. All fragment pairs regardless of the intended application can potentially benefit from and would not be impaired by the docking function provided by interactors such as the fos and jun helixes fused to the break-point termini.
- the C-terminal, or ⁇ fragment library would be expressed as N-terminal fusions via a flexible polypeptide linker such as a (Gly 4 Ser) 3 linker to the fos helix (Interactor 2 in Figure 6) from the lac promoter in the phagemid vector pAOl (the upstream cistron could be removed if desired).
- a flexible polypeptide linker such as a (Gly 4 Ser) 3 linker to the fos helix (Interactor 2 in Figure 6) from the lac promoter in the phagemid vector pAOl (the upstream cistron could be removed if desired).
- the amino acid sequence of the flexible polypeptide linker is not critical, however, it must be of a sufficient length and flexibility such that the fragment domain and heterologous interactor domain fold independently and unhindered.
- N-terminal, or ⁇ fragment library would be expressed as C-terminal fusions via a flexible polypeptide linker such as a (Gly 4 Ser) 3 linker to the jun helix (Interactor 1 in Figure 6) from the trc promoter in the compatible pAEl vector. Coding sequences for signal peptides would be included if translocation to the periplasm were desired.
- a flexible polypeptide linker such as a (Gly 4 Ser) 3 linker to the jun helix (Interactor 1 in Figure 6) from the trc promoter in the compatible pAEl vector. Coding sequences for signal peptides would be included if translocation to the periplasm were desired.
- one or more of the performance-enhancing tools may be inco ⁇ orated into the expression vectors to maximize the probability of selecting the best fragment pair for the intended application(s).
- cysteines should be encoded at the break-point termini to allow disulfide formation. If the enzyme contains other cysteines, at least 1 mM and not more than 5 mM of a reducing agent such as GSH or DTT should be included in the growth medium to inhibit the formation of mixed disulfides.
- a random or VRK tri- peptide library may be encoded in frame with each fragment fusion between the break-point terminus and the flexible polypeptide linker. If VRK libraries were used for each fragment in a 50-fragment pair library, every possible tri-peptide-fragment combination would be contained in a combined library of ⁇ 10 8 . Alternatively, a single tri-peptide library could be used for each fragment pair in trans, as was described above. The tri-peptide library would be fused operably in frame via the flexible polypeptide linker to the N-terminus of thioredoxin and expressed from the upstream cistron in the pAOl phagemid vector (see Figure 6).
- the second step in the development of high-performance enzyme fragment complementation systems is to construct an expression library of candidate enzyme fragment pairs.
- Methods for generating libraries of random fragment pairs have been described (Ostermeier et al., 1999, supra). However, such libraries are quite inefficient as the vast majority of fragment pairs will be dysfunctional.
- For combinatorial screening of fragment pair libraries with mutagenic or random tri-peptide libraries much more efficient fragment pair libraries will be necessary. For a variety of reasons it may be assumed that the most functional fragment pairs will correspond to scission of the polypeptide chain in exposed regions between elements of secondary strucmre.
- Exposed break-points will be required for use of tethered heterologous interactors and tri-peptides, and scission within secondary structure elements can irreversibly destabilize such elements. If a 3-dimensional strucmre is available for the enzyme of interest, or for a homolog, it can be used to identify exposed loops as candidate sites for chain scission. Typical globular proteins will not have more than 20-25 such sites that are far enough from the ends so that the larger fragment is not independently active. This is a manageable number for construction of coding sequences for each fragment pair by PCR. Two end-specific primers would be required, plus a head-to-head pair of primers for each break-point, which should be located more or less in the center of the exposed loop.
- mutagenesis can be limited to 1-3 unbiased coding changes per molecule (Cadwell and Joyce, 1995, in PCR Primer-A Laboratory Manual C. Dieffenbach and G. Dveksler, Eds. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 583-590). Since most mutations would be non-phenotypic, this could easily be combined with the other performance-enhancing tools without compromising the selectability of optimal fragment-tri-peptide combinations.
- the fragment coding sequences Once the fragment coding sequences have been amplified, gel-purified, and ligated into the vectors, the ligation products may be desalted and concentrated to allow efficient co- transformation of E.
- fragment complementation is to be dependent on the direct or ligand-mediated interaction of heterologous domains fused to the break-point termini
- mutagenesis should not be used because folding acceleration usually eliminates the need for docking assistance.
- selected fragment pairs must be counter-screened for loss of activity in the absence of the fos-jun interaction and activation indexes must be determined as the ratio of interaction-dependent activity to interaction-independent activity.
- activation indexes of the order of at least 10 6 are preferred since rare genes are expected to have frequencies in that range.
- lower activation indexes are usually acceptable.
- affinities K d
- the fragment binder fusions need only to be used at 100 nM concentrations to saturate the ligand. Under these conditions — 90% of the fragment-binder fusions will be unbound. If the activation index is > 100, the background will be ⁇ 10% of the signal.
- Selected fragment pairs can be optimized for maximum activity and/or maximum activation index.
- break-point disulfides produce the highest specific activities because they allow the greatest amount of native strucmre in the fragment complex. However, they also may in the background so that activation indexes are often lower.
- Two parameters may be adjusted to control the formation of break-point disulfides. (1) The proximity of the disulfide-forming cysteines to the break-point may be adjusted to place greater orientational stringency on disulfide formation. (2) The concentration of reducing agent in the medium may be increased to reduce the effective concentration of
- DsbA the principle disulfide-forming oxidase in the periplasm.
- a fragment pair library of a non-phenotypic protein is expressed as fusions to the interaction-dependent T ⁇ M-1 ⁇ -lactamase fragments, it is expected that only those fragment pairs which associate and fold into the native conformation will provide sufficient docking function to facilitate selectable ⁇ -lactamase activation.
- the subject fragments serve the pu ⁇ ose of the heterologous interactors in facilitating complementation of ⁇ -lactamase fragments.
- fragment/heterologous interactor fusion sequences could be encoded into the fragment/heterologous interactor fusion sequences to enhance functional reassociation of the ⁇ -lactamase fragments, including a break-point disulfide, a randomly-encoded peptide of from 3-12 amino acids, and mutagenesis of several amino acids within the fragment domain. All of these tools would specifically impact only complementation of the subject fragments by stabilizing the fragments, accelerating folding, and/or stabilizing the active fragment complex. Selected fragment pairs could then be tested individually for reconstitution of enzymatic activity or other function of the parental protein. In this way many useful fragment complementation systems could be developed for proteins which are active in eukaryotic cells, such as kinases or herbicide-resistance proteins.
- interaction-activated enzyme association systems of the subject invention find use in many applications as summarized below.
- Simplex refers to the use of single bait proteins to fish natural interactors out of expressed sequence libraries.
- Multiplex refers to the combinatorial pair-wise interaction of two expressed sequence libraries for the pu ⁇ ose of simultaneously isolating as many natural interactions as possible. Individual interactors can be readily identified by nucleic acid hybridization.
- Interaction-dependent ⁇ -lactamase systems may also be used to enrich randomly- primed expressed sequence libraries for fragments which encode autonomously- folding domains (AFD). Interference with folding by the fusion partner is avoided by using epitope tags and hetero-dimerizing helixes only at the N- and C-termini of the expressed sequence, respectively. The fragments would have N- and C-terminal anti- tag binder and the partner hetero-dimerizing helix. The disulfide switch can accommodate diverse interaction geometries. (3) Simplex and multiplex selection of binding molecules such as single chain antibody fragments (scFv) and antibody light chain variable regions (VL).
- scFv single chain antibody fragments
- VL antibody light chain variable regions
- Non-immune human scFv repertoire libraries can be used with TEM-1 ⁇ -lactamase interaction- dependent activation systems to isolate scFv to single baits or simultaneously to expressed sequence libraries. In the latter case scFv specific for individual targets can be readily identified by nucleic acid hybridization. (4) Interface mapping and ligand identification by mimotope homology. Constrained peptide libraries displayed on the surface of a carrier or "scaffold" protein may be used with ⁇ -lactamase interaction-dependent activation systems to isolate surrogate ligands for proteins or AFDs of interest.
- Consensus sequences from panels of such surrogate ligands for a given polypeptide may then be used to identify natural ligands of the polypeptide or interaction surfaces on natural ligands of the polypeptide.
- a common application of interface mapping is epitope mapping for antibodies, whereby the specific region to which an antibody binds on the surface of its antigen is identified.
- Bio- Action Sensors The efficiencies of most screening systems for signal transduction agonists and antagonists are compromised by the need for multiple steps between receptor ligation and selectable phenotype generation, which usually requires de novo gene expression.
- Interaction-activated ⁇ -lactamases can be tailored for activation or inhibition by any component of a target signal transduction pathway to allow selection of agonists or antagonists of the pathway in any appropriate cell type without the need to wait for gene expression to generate a selectable phenotype.
- Interaction-dependent complementing fragments can be fused to two scFv or other binding molecules which bind non-overlapping epitopes on target molecules, so that ⁇ -lactamase activation becomes dependent on binding to the target ligand.
- the use of ligand-dependent ⁇ -lactamases in homogeneous assays for two-epitope analytes from proteins to pathogens affords unparalleled sensitivity because saturation kinetics can be used instead of the equilibrium kinetics required by most assays.
- the binding molecules could also be oligonucleotides which anneal to contiguous sequences in the genome of a target pathogen.
- Target-Activated Enzyme Prodrug Therapy T AcEPT
- T AcEI Target- Activated Enzyme Imaging
- Antibody-directed enzyme prodrug therapy is a promising chemo- therapeutic strategy in which patients are treated with prodrug-activating enzymes such as ⁇ -lactamase conjugated to tumor-targeting antibodies (Bagshawe, 1995, supra). When unbound antibody-enzyme conjugate has cleared the circulation, prodrugs can be administered which are preferentially activated at the site of the tumor.
- the efficacy of this therapy is severely limited by the need for unbound conjugate to clear the circulation before the prodrug can be administered in order to avoid excessive toxicity, during which time most of the bound enzyme is lost from the tumor.
- the use of tumor-activated ⁇ -lactamases allows the prodrug to be administered at peak tumor loading of the enzyme since the latter is inactive in the circulation, and can only activate the prodrug when bound to the tumor.
- the same strategy can be used for antibody-directed site-specific activation of reagents for imaging of tumors or other tissue pathologies, or for other therapeutic indications such as inflammation or transplant rejection.
- ScFv single-chain antibody Fv fragments
- 12-amino acid peptides by inserted into the active site of E. coli thioredoxin (trxpeps, Colas et al., Nature (1996) 380: 548).
- ScFv are comprised of antibody heavy chain and light chain variable regions (VH and VL) tethered into a continuous polypeptide by most commonly a (Gly 4 Ser) 3 linker encoded between most commonly the C-terminus of VH and the N- terminus of VL.
- ScFv from a human non-immune antibody repertoire were amplified by PCR using a consensus primer mix (Marks et al., Eur J Immunol (1991) 27:985), and subcloned into a pUC119-based phagemid vector (Sambrook et al., supra) for expression of the scFv as fusions to the N-terminus of the col 98 fragment with an intervening (Gly 4 Ser) 3 linker (pAOl; see Figure 6A).
- An N-terminal signal peptide was provided for translocation to the bacterial periplasm.
- a commercial trxpep library was obtained and amplified by PCR using primers specific for the N- and C-termini of E. coli thioredoxin (Genbank accession no. M54881 ). This product was subcloned into a pl5A replicon (Rose, Nuc Acids Res
- FIG. 7 illustrates the activation of T ⁇ M-1 by complementation of ⁇ 197 and col 98, mediated by interaction between an scFv and a trxpep.
- the false positive rate was exceedingly low, much lower than has been reported for other intra-cellular interaction sensors such as the yeast two-hybrid system (Barrel et al., 1993, supra; Bartel et al., 1996, supra). This property is essential for high-throughput applications.
- the false negative rate with respect to the scFv was immeasurably low, as trxpeps were recovered for all functional scFv, and this too is essential for high-throughput applications.
- mimotopes were recovered for all scFv enables the system for high-throughput multiplex epitope mapping for scFv.
- the system is capable of efficient recovery of multiple interactions between two diverse populations of proteins simultaneously.
- the throughput of the system should be limited only by the sizes of the interacting libraries, and/or the number of co-transformants which can be handled conveniently.
- construction of recombinant protein libraries in the 10 9 -10 10 range is routinely possible for scFv, trxpeps, or cDNAs (Hoogenboom et al., Immunotech (1998) 4:1).
- VL antibody light chain V-regions
- This example demonstrates the ability of the system to work with larger antibody fragments, such as Fab, which are comprised of entire light chains disulfide-bonded to Fd fragments which contain VL plus the first heavy chain constant region.
- Fabs fragments from a human repertoire library was subcloned for expression as C-terminal col 98 fusions from a dicistronic transcript from the lac promoter in the pAOl vector (see Figure 6A).
- the first cistron encoded the light chain with a signal peptide for translocation to the periplasm.
- the light chain termination codon was followed by a short spacer sequence and then a ribosome binding site approximately 10 bp upstream from the start of translation for the signal peptide of the Fd fragment, which was followed by ⁇ 198 with an intervening (Gly 4 Ser) 3 linker.
- This construct was then co-expressed with the ⁇ l97-trxpep library in the pAEl vector in strains DH5 ⁇ and TGI . Spontaneous association of the light chain with the Fd-c l98 fusion protein in the periplasm was expected to produce a functional Fab fragment. Binding of the latter to the peptide on a ⁇ l97-trxpep fusion was then expected to facilitate assembly of the functional TEM-1 ⁇ -lactamase in amounts sufficient to confer selectable resistance to ampicillin on the host cells.
- Plasmid DNA was then purified from this light chain library and used as the recipient for insertion of the VH repertoire to complete the Fab library.
- the resulting library was contaminated with approximately 15% of clones which contained the intermediate vector. Only these LC-CH1 complexes were capable of driving ⁇ l97-col98 complementation by binding of the VL combining site with the peptide on the appropriate trxpep. It is not known why full-length Fabs were not selected, however, the larger size and rigidity of the Fab-trxpep complex ( ⁇ 67 kDa) may have sterically inhibited fragment complementation, whereas the smaller size and flexibility of the LC-CHl complex did not.
- This example demonstrates the ability of the present system to isolate panels of trxpeps that bind to a given protein of interest, and which could be used to map interaction surfaces on the protein, and which could also assist in the identification of new ligands by homology.
- the extra-cellular domain of the human B-cell activation antigen CD40 is known to reliably express in the E. coli periplasm (Noelle et al., Immunol Today (1992) 75:431; Bajorath and Aruffo, Proteins: Struct, Fund, Genet (1997) 27:59).
- a T-cell surface molecule, CD40 ligand (CD40L) is known to co-activate B-cells by ligation to CD40, but there may be other ligands.
- T ⁇ M-1 ⁇ l97/ ⁇ l98 fragment complementation was used to select a panel of CD40-binding trxpeps.
- the sequences of these peptides would then be examined for homology to the known ligand and other potential ligands.
- CD40ED was amplified by PCR using primers homologous to the N-terminus of the mamre protein and to the C-terminus of the ⁇ 190-residue extra-cellular domain (Genbank accession no. X60592).
- the PCR product was then subcloned into the pAOl phagemid vector ( Figure 6A) for expression from the lac promoter as a C-terminal fusion to the TEM-1 co 198 fragment with an intervening (Gly 4 Ser) 3 linker. Expression of the correct product was confirmed by PAGE, and the CD40 fusion vector was then rescued as phage and transfected into TG-1 cells bearing the same trxpep library construct as described above.
- Ampicillin-resistant clones encoding thirteen unique trxpeps were recovered. In all cases amp resistance was strictly dependent on the presence of CD40ED and the peptide portion of the trxpep. No activity was seen if CD40ED was replaced with an irrelevant protein or if the trxpep was replaced by wild-type thioredoxin.
- the sequences of the selected CD40-binding peptides are shown in Table 2 below along with their homologies to each other and to CD40L.
- the thirteen peptides sort into eight homology groups: two groups with three each (1 and 2), one with two (3), and five with one each. Groups 1 and 2 are defined by homology of three peptides in each group to the same region of CD40L.
- Group 1 is homologous to the region of CD40L from Pro217 to Gly234, and Group 2 is homologous to the region from Glyl58 to Leu 168.
- Group 3 is defined only by inter- pep tide homology and has no detectable homology to CD40L.
- Group 4 is homologous to CD40L from Serl lO to Pro 120, and Group 5 is homologous to CD40L from Pro244 to Gly257. Groups 6-8 have no discernable homologies.
- CTLA-2A a matrix metalloproteinase
- VEGI vascular endothelial cell growth inhibitor
- CD3 ⁇ bone mo ⁇ hogenetic protein 3B
- SB may define an interaction motif or motifs, which have been used repeatedly for extra-cellular protein-protein interactions. They may also indicate multiple interaction sites on CD40.
- Inter-trxpep competition was tested by expressing each of five selected CD40- binding trxpeps from a second cistron in the pAOl phagemid vector, downstream from the
- CD40 - co 198 fusion Each of these constructs was then co-expressed with each of the same five plus three additional selected ⁇ l97-trxpep fusion constructs in strain TGI and scored for growth on 25 ⁇ g/ml ampicillin. The results are shown in Table 3 below.
- p44-4-2Al and p45-7-2A3 compete strongly and have similar competition profiles. They compete with BW10-1 and nothing else except BW10-8 slightly. BW10-9 competes slightly with BW10-8 and p58-12-9Al . p65-2-9Al is inhibited by nothing.
- Group 2 P58-12-9A1, BlO-8, BlO-4
- the competition data is consistent with the homology data with the caveat that simultaneous binding to non-overlapping epitopes is sometimes not tolerated.
- This allows unrelated sequences like p58-12-9Al and BW10-8 to compete strongly with one another and have similar competition profiles.
- This is probably due to steric interference with enzyme reassembly, and may account for the discordance between homology and competition data for BWlO-l and p58-12-9Al in particular. These two probably bind near the same CD40 interaction epitope, which may sterically inhibit fragment complementation for many (but not all) other trxpeps.
- ⁇ -lactamase activation it will be useful for ⁇ -lactamase activation to be mediated by simultaneous binding of both ⁇ l97 and ⁇ 198 to non-overlapping epitopes on a separate molecule, either a free ligand or cell surface receptor.
- Two CD40-binding trxpeps which had been identified as non-competing by the competition tests, were used to test this utility.
- One of the two trxpeps was subcloned for expression as the C-terminal ⁇ l98 fusion from the pAOl vector (see Figure 6).
- the other trxpep was expressed as the ⁇ l97 fusion from the pAEl vector as before. Co-expression of these two constructs was used as the negative control.
- CD40ED coding sequence (including signal peptide) was subcloned into the trxpep- ⁇ l98 expression cassette between the promoter and the trxpep- ⁇ l98 sequence.
- An additional 20 bp containing a ribosome binding site was included downstream from the CD40 stop codon to allow expression of both CD40 and trxpep-col98 from the same dicistronic transcript, as was described above for the Fab.
- Table 4 shows that CD40 expression induced resistance to 50 ⁇ g/ml ampicillin, whereas without CD40 the cells expressing the control constructs produced fewer than 10 "6 colonies per cell on 25 ⁇ g/ml ampicillin.
- ⁇ -lactamase fragment complementation can be efficiently induced by a tri-molecular protein-protein-protein interaction.
- ⁇ -lactamase activation by ⁇ l97- ⁇ l98 fragment complementation could be driven efficiently by interaction between scFv and trxpeps, it was important to show that it could also be driven by interaction between scFv and a bona fide protein antigen, preferably a cell surface receptor. This was especially important because the ligand- binding domains for type 1 trans-membrane receptors are N-terminal, therefore their expression as C-terminal fusions is preferred. However, the preferred orientation for scFv expression is also N-terminal.
- the CD40-fos fusion and the scFvcol98 fusion were expressed from a dicistronic transcript in the pAOl vector, and ⁇ l97-jun fusion was expressed from the pAEl vector.
- the fos-jun interaction has a K d in the 10 "8 M range, so it should quantitatively ligate CD40 with ⁇ l97, which are much more abundant than this in the periplasm. Binding of the scFv to CD40 should then dock ⁇ l98 with the complex to facilitate fragment complementation.
- CD40-fos expression induced resistance to up to lOO ⁇ g/ml ampicillin, whereas cells expressing only the control constructs without CD40-fos again produced fewer than 10 "6 colonies per cell on 25 ⁇ g/ml ampicillin.
- ⁇ -lactamase fragment complementation can be efficiently induced by a tri-molecular interaction of two extra-cellular proteins in preferred C-terminal fusions.
- the ⁇ -lactamase activity produced by interaction-dependent complementation of the ⁇ l97 and ⁇ l98 fragments is substantially less than that of the wild-type enzyme under the same expression conditions.
- This loss of activity could be due to a tendency of the fragments to aggregate or turnover when they are not folded into the native conformation, and it could also reflect a loss of specific activity due to the reduced ability of the loosely tethered heterologous interaction to stabilize the native conformation. It was reasoned that both folding kinetics and stability could be enhanced by the introduction of a disulfide at the break-point, and this could lead to a substantial increase in interaction-dependent activity.
- Cysteines were added to the sequences of 197 and col 98, between the break-point termini and the linkers leading to the heterologous interactors.
- quantitative ampicillin resistance > 10% plating efficiency
- the plating efficiency on 25 ⁇ g/ml ampicillin increased at least 2-fold.
- disulfide formation must be accelerating folding and/or stabilizing the active conformation.
- the disulfide produced nearly as much activity without the interactors.
- Disulfide formation was made to be more dependent on the heterologous interaction by two modifications.
- disulfide formation could be inhibited by inclusion of a reducing agent in the growth medium.
- Dithiothreitol (DTT) at 10 mM reduced the plating efficiency of the disulfide-assisted fragments on 100 ⁇ g/ml ampicillin to ⁇ 10 ⁇ colonies per cell in the absence of an interaction, whereas with the fos-jun interaction the activity of the same fragments was little affected by DTT, so that the activation index was increased to
- the 8 A increase in thiol separation alone increased the activation increment substantially over that of the fos-jun interaction without disulfide.
- the enhancement of interaction-dependent specific activity provided by the disulfide should allow weak interactions and/or poor expressors to produce selectable ⁇ -lactamase activity with fewer than 10 molecules per cell of the activated enzyme.
- break-point disulfide to enhance activation of TEM-1 ⁇ l97/col98 fragment complementation, suggests that break-point disulfides might be able to activate many enzyme fragment pairs which produce weak or no selectable activity with a heterologous interaction alone.
- the heterologous interaction may be essential for fragment docking, but since it is tethered with — 60 A linkers it cannot restore the tight junction of the polypeptide backbone at the break-point.
- formation of a disulfide across the break-point should restore the integrity of the backbone, and should thereby help stabilize the active site of the complex. This idea was tested by screening nine additional pairs of TEM-1 ⁇ -lactamase fragments, corresponding to scission in nine exposed loops of the polypeptide chain. The nine fragment pairs were screened for selectable activity with the break-point disulfide alone, the fos-jun interaction alone, and with both together. The results are summarized in Table 5.
- the ten fragment pairs may be sorted into three groups. One group comprises the two negative pairs. The second group comprises three pairs which can only be activated by disulfide and fos-jun interaction together. In each case, the plating efficiency is at least 10% on 25 ⁇ g/ml ampicillin, with an activation index of at least 1000.
- the third group comprises five pairs, all from break-points in the C-terminal third of the molecule, which produce modest-to-robust activity with fos-jun alone, but potent activity with both fos-jun and the disulfide together.
- G253/K254 ++++ 400 +++ 50 a' Fragment pairs were expressed in TGI cells and plated onto ampicillin in the presence of ImM IPTG. Fragments were expressed with or without break-point terminal thiols (S-S) and with or without break-point terminal fos ( ⁇ ) or jun ( ⁇ ) helixes.
- S-S break-point terminal thiols
- ⁇ break-point terminal fos
- jun ( ⁇ ) helixes b' Activities are expressed as plating efficiencies (colonies per cell) on 25 ⁇ g/ml ampicillin (amp25). -, ⁇ 10 "4 ; +/-, 0.01 ; +,
- HiAmp refers to the maximum ampicillin concentration in ⁇ g/ml on which fragment-expressing cells plate with >10% efficiency.
- fragment pairs which produced the highest activities are not the same as those with the highest activation indexes and vice versa, indicates that different fragment pairs may be optimally suited for different applications.
- the activation index is more important than maximum activity for intra-cellular interaction mapping, where natural interactions must be identified against backgrounds of 10 6 or more non-interacting pairs.
- P174/N175 may be the best fragment pair for intra-cellular interaction mapping.
- maximum activity is more important than the activation index for in vitro applications because the activating target ligands will always be limiting in such applications.
- G253/K254 may be the best fragment pair for in vitro applications such as biosensors or homogeneous assays.
- the break-point disulfide overcomes a significant shortcoming of interaction- dependent enzyme fragment complementation systems. It is essential for high-throughput applications that such systems be capable of efficient activation by a wide range of heterologous protein-protein interactions. In other words, to minimize the false negative rate, the system must be activatable by any interaction between two proteins or fragments within the size range of single, naturally evolved protein domains, i.e., between — 100 and 300 amino acids in length. Globular proteins in this size range have radii in the range — 30-50A. This means that the points of attachment for the linkers could be up to lOOA apart, and this distance must be spanned by the linkers in order for the break-points of the fragments to be able to come together.
- the (Gly 4 Ser) 3 linker was selected, which is expected to be fully extended and flexible, and to have a length of ⁇ 6 ⁇ A, thereby providing a combined length of up to 12 ⁇ A to allow close approach of the break-point termini during folding. Nevertheless, it is reasonable to expect the stability of the active conformation to be quite sensitive, and generally inversely proportional to the dimensions of the heterologous interaction. Thus, for all such systems described to date it may be assumed that the longer the linkers, the larger the proportion of possible interactions that can accommodate refolding, but the less the interaction can contribute to stabilization of the active conformation.
- break-point disulfide overcomes this limitation because, if the linkers are long enough, it will form readily during re-folding, and once the break-point disulfide is formed the specific activity of the reconstimted enzyme should be independent of the dimensions of the heterologous interaction, and in fact should not even require the continued integrity of the interaction.
- the break-point disulfide acts as a one-way switch, with an activation energy which can be supplied by a broad range of heterologous interactions, limited only by the ability of the interactors to fold properly, and by the length of the linkers to allow close approach of the break-point cysteines. This has two important consequences which allow a larger proportion of natural interactions to produce selectable activity. Longer linkers can be used, and interactions which are too weak to sustain selectable enzyme activity by themselves should still be able to "throw the disulfide switch" to produce selectable activity.
- Another way to enhance interaction-dependent enzyme fragment complementation is to introduce short, random peptide sequences at the break-points, and then to select for increased activity with a model interaction.
- pep tide-dependent enhancements could occur by any of several mechanisms.
- the peptides could stabilize the active conformation of the reconstimted enzyme by interacting with each other or with the enzyme itself, or the peptides could stabilize one or both of the fragments, thereby increasing steady-state activity by increasing fragment concentration.
- Synthetic oligonucleotides were used to add three randomized residues to each fragment between the break-point residue and the linker for the heterologous domain.
- the c-fos helix at the N-terminus of co 198 and the c-jun helix at the C-terminus of ⁇ 197 was used.
- a degenerate codon was used, which encoded a subset of amino acids which was biased toward charged residues to favor charge-charge interactions, which are the strongest.
- the VRK codon places c, a, or g in the first position, a or g in the second position, and t or g in the third position.
- the jun helix was removed from ⁇ l97 in the same starting 10 4 clones of the library, and when these clones were plated onto the same concentrations of ampicillin, only a few colonies grew on 200 ⁇ g/ml ampicillin, and no colonies appeared on higher concentrations. This level of ampicillin resistance is comparable to that produced by the fos-jun interaction alone.
- the most likely effect of the GRE tri-peptide is to stabilize the ⁇ l97 fragment by interfering with loss of the fragment by amo ⁇ hous aggregation. Since the ⁇ l98 fragment is quite stable, but the ⁇ l97 fragment is somewhat less so, the latter is expected to be limiting for fragment complementation, and any stabilization of ⁇ 197 leading to an increase in its concentration would increase the steady state activity of the interaction-activated enzyme accordingly. Though the GRE tri-peptide could inhibit aggregation of ⁇ 197, it apparently did not interfere with re-folding of the fragment complex. Since aggregate formation proceeds exponentially, it isakily sensitive to small shifts in the inter- molecular association rate constants (Dobson, Trends Biochem Sci (1999) 24:329).
- FHT400-1A1, -1B1 HSE cat agt gag
- REQ egg gag cag
- GRE was selected again from the ⁇ tri-peptide library.
- NGR was selected twice from the ⁇ tri-peptide library, with two different co tri-peptides. In all cases, activation continued to be dependent on the fos-jun interaction. However, in contrast to the original GRE tri- peptide, activity was enhanced in all cases by the presence of the both the ⁇ and ⁇ tri- peptides. Even the activity of the GRE tri-peptide was enhanced by the DGR tri-peptide on the ⁇ fragment. Also, the fragments were interchangeable to some extent. Different ⁇ tri- peptides could be paired with different ⁇ tri-peptides.
- the GRE tri-peptide was also found to stabilize ⁇ 197 in trans.
- the ⁇ l97-fos and jun-col98 fusions were co-expressed in the E. coli periplasm with the GRE tri-peptide fused to the N-terminus of thioredoxin via a Gly 4 Ser linker, the cells plated with 100% efficiency on 50 ⁇ g/ml ampicillin, whereas cells expressing the ⁇ l97-fos and jun-col98 fusions either alone, without the GRE-t ⁇ x fusion, or with a different tri-peptide-ttx4 fusion, plated with only — 1 % efficiency on 50 ⁇ g/ml ampicillin.
- the GRE-tr fusion conferred no resistance to ampicillin in the absence of the interacting helixes, thus it does not stabilize the re-folded fragment complex, but rather it must stabilize the ⁇ l97 fragment since activity is limited by the amount of soluble ⁇ l97. Since the GRE tri-peptide had the same stabilizing effect on ⁇ 197 fragment when a different carrier was used, its activity must be context independent. Thus, an 18 kDa enzyme fragment could be stabilized at least 100-fold by a tri-peptide selected from a random sequence library. As with the tethered tri-peptide, the free GRE tri-peptide could inhibit aggregation of ⁇ 197 without apparently interfering with re-folding of the fragment complex.
- ⁇ l97 coding sequence is actually about 520 nucleotides in length, and —75% of mutations change the encoded amino acids, less than three coding changes per molecule should be produced.
- About 10 8 clones of the ⁇ l97 mutant library were collected and co-expressed as the jun helix fusion with the fos helix fusion of wild-type col 98.
- the mutagenized ⁇ l97-jun fusion was expressed from the pAEl vector and the fos- ⁇ l98 fusion was expressed from the pAOl phagemid vector (see Figure 6). When both constructs were co-expressed in strain DH5 ⁇ colonies were recovered in the presence of 600 ⁇ g/ml ampicillin.
- FI600-1 and -3 Upon sequencing, two of three clones recovered (FI600-1 and -3) had the same sequence with two coding mutations, K55E (aag ⁇ gag) and M182T (atg ⁇ acg).
- the third clone (FI600-4) also had two coding mutations, one of which was shared with the other two (M182T), and the other of which, P62S (ccc-»tcc), was proximal to the other mutation of the other clones.
- the tri-peptides stabilized the fragments by reversibly interfering with aggregation. Reversibility allows them to inhibit aggregation without interfering with folding.
- mutations are not reversible in this sense. If aggregation is caused primarily by the inter-molecular formation of native folding contacts, disruption of these by mutation might be expected to interfere with folding. In fact, it may be thermodynamically impossible to stabilize the fragments by mutation without inhibiting the re-folding process required to form the active fragment complex. This is because the native folds of the fragments have too much exposed hydrophobic surface to be stable. Thus, mutations can only stabilize the fragments by stabilizing alternative folds, which minimize exposed hydrophobic surface. However, these alternative folds must be unfolded before the native folding pathway can proceed to the active complex, and the energy required for this process may be prohibitive.
- the rate of aggregation is proportional to the lifetimes of such species.
- the effects of the break-point disulfide described above indicated that the fragments are capable of association and initiation of folding in the absence of the heterologous interaction, but that the folding process is aborted when the fragments are not held together in some way, such as by the heterologous interaction or by the formation of a disulfide at the break-point. In the absence of either of these the probability that the fragments will dissociate before folding is complete is proportional to the folding rate, which in turn is proportional to the lifetimes of the folding intermediates.
- Ligand-activated or interaction- activated ⁇ -lactamases can be activated in multiple locations, including the bacterial periplasm, bacterial cytoplasm, eukaryotic cell cytoplasm, or in vitro.
- ⁇ -lactamase fragment complementation systems have been demonstrated for monitoring interactions between and among cell-surface receptors, antibodies, and random peptide libraries displayed on the surface of a natural protein.
- the activation index is the most important parameter of the interaction-dependent fragment complementation system for cleanly discriminating bona fide interactions from large pools of non- interacting protein pairs.
- the P174/N175 fragment pair of TEM-1 ⁇ -lactamase ( ⁇ l74 and co 175) because with the break-point disulfide this pair has the largest activation index, — 10 7 .
- fragment- stabilizing tri-peptides should allow weaker bona fide interactions in the expressed sequence libraries to confer selectable activity.
- Rough microsomes which are derived from membranes of rough ER and are therefore enriched in mRNA for secreted and membrane proteins, may be isolated from unfractionated lymphocytes from pooled human blood by sedimentation velocity in sucrose density gradients (Gaetani et al., Methods in Enzymology (1983) 96:3; Natzle et al., J Biol
- RNA may then be purified from the rough microsomes using a commercially available kit (e.g., Poly(A) Select, Promega, Inc., Madison, WI).
- a randomly-primed cDNA library is then made from the RNA template and cloned directionally.
- First-strand cDNA is made with AMV reverse transcriptase (RT) and random hexamer primers (Sambrook et al., 1989, pp. 8.11-8.21).
- the primers contain a unique 5' extension with convenient restriction sites for ligation into the ⁇ -lactamase ⁇ and ⁇ fusion expression vectors.
- the template is destroyed by the RNAseH activity of AMV RT and the unused primers are removed using a spun column.
- the second strand is then made with the Klenow fragment of DNA polymerase I and random hexamer primers containing a different unique 5' extension with a different restriction site for insertion into the expression vectors.
- the cDNA is PCR- amplified with primers corresponding to only the unique sequence on each original primer (Dieffenbach and Dveksler, in PCR Primer: A Laboratory Manual, Cold Spring Harbor Press, cold Spring Harbor, NY, 1995), so that the majority of amplified fragments have the correct orientation for expression in E. coli.
- the product is then normalized by exhaustive hybridization to a limiting amount of human genomic DNA immobilized on magnetic beads (Kopczynski et al., 1998, supra). Since coding sequences are naturally normalized in genomic DNA, cDNA recovered from the genomic DNA hybrids should be normalized.
- the PCR product is size selected by centrifugal gel filtration on Sephacryl S- 400 spun columns for fragments > — 200 bp.
- the cDNA is then digested with appropriate restriction enzymes and ligated into the interaction-dependent ⁇ -lactamase ⁇ l74 and c l 75 fusion expression vectors, which are essentially the same as those shown in Figure 6, except for some modifications required for fold selection.
- the vectors and protocol for fold selection and interaction mapping of the cDNA library are illustrated in Figure 9.
- both vectors for expression of the library as ⁇ and ⁇ fusions are compatible phagemids.
- a peptide epitope tag such as the well- known 12-mer derived from the c-myc oncogene (Hoogenboom et al., 1998, supra) is encoded at the C-terminus of the cDNA, or expressed sequence ( ⁇ S) library in the ⁇ -fusion vector, and at the N-terminus of the ⁇ S library in the co-fusion vector.
- each fusion library When co-expressed with an anti-tag scFv, such as the anti-myc 9 ⁇ 10 scFv (Hoogenboom et al., 1998, supra) fused to the other ⁇ -lactamase fragment, each fusion library can be enriched for clones which express autonomously folding domains (AFD) in the correct reading frame.
- AFD autonomously folding domains
- the normalized cDNA library-vector ligation products are transduced into E. coli strain TG-1 by high-voltage electroporation (Dower et al., Nucleic Acids Res (1988) 76:6127), and plated onto the minimum ampicillin concentration on which non-interactors are known to plate with efficiencies of ⁇ 10 "3 since at least a 100-fold excess of non-AFD- encoding fragments is expected in the libraries.
- the recommended ampicillin concentration would be —25 ⁇ g/ml.
- the libraries can be rescued as filamentous phage by high-multiplicity super-infection of at least 10 8 cells of each library with the helper phage M13K07 (Sambrook et al. , 1989, pp. 4.17-
- the library phage are recovered from the culture supernatant by precipitation with polyethylene glycol, and reconstimted in phosphate-buffered saline.
- the library phage stocks may be stored frozen in 15% glycerol.
- Fresh E. coli TG-1 cells may then be co-infected with a high-multiplicity of each phage library and plated onto a concentration of ampicillin on which the activation index of the system is known to be maximal.
- 100 ⁇ g/ml ampicillin is optimal, since the activation index is at least 10 7 and the fos-jun interaction-mediated plating efficiency is at least 50% .
- At least 10 14 transforming units of each fusion library phage should be used to infect at least 10 12 log phase TG-1 cells to insure that most of the possible pair-wise combinations of 10 6 clones of each AFD library are present in the doubly infected cell population before selection.
- the cells After a one-hour adso ⁇ tion at 10 9 cells per ml, the cells are washed, resuspended in fresh medium, and incubated for another hour with gentle shaking to allow the phagemid genes to express.
- the cells are then concentrated and plated on 100 large petri dishes (150 mm dia.) containing solid LB medium containing 1 mM IPTG and 100 ⁇ g/ml ampicillin. A small aliquot is plated on chloramphenicol and kanamycin to determine the number of co-transformants.
- Labeled oligonucleotide hybridization probes may be prepared for these known interactions, and colony lifts of the entire interaction library may be probed to see what fraction of expected interactors are actually represented in the library. Interaction partner sequences from positive clones may be recovered, and homology searched to determine if known or new interactors have been identified. Colonies expressing bona fide interactions may be grown up and stored indefinitely in 15% glycerol at -70°C, pending further characterization or use for e.g., drug screening.
- Interaction-dependent ⁇ -lactamase fragment complementation systems can be adapted for activation or inactivation by virtually any post-translational modification that occurs namrally in cells. As a result they may be deployed intra-cellularly as biosensors to monitor the activity of any process which is regulated by post-translational modification.
- a major class of such processes is phosphorylation-regulated signal transduction pathways.
- Phosphorylation-regulated intermediates are obligatory components of most processes by which cells respond to extra-cellular conditions or messenger molecules by altering gene expression.
- Cellular responses to extra-cellular signals may be fall into three general categories, growth, survival, and differentiation.
- a ubiquitous component of neoplastic transformation is the deregulation of growth control signaling, often accompanied by the deregulation of survival signalling as well.
- oncogenes are phosphorylation- regulated growth signal transducers, which become over-expressed or mutated to constimtive activity in cancer cells.
- the Her-2/neu oncogene is a 185 kDa Type I transmembrane receptor tyrosine kinase, which is a member of the epidermal growth factor receptor (EGFR) family. This growth factor receptor is over-expressed in particularly aggressive adenocarcinomas of epithelial origin in a number of tissues, notably breast.
- Her- 2/neu When normally expressed, Her- 2/neu hetero-dimerizes with other EGF-family receptors when they are ligated by growth factor. This leads to cross phosphorylation of multiple tyrosines on the cytoplasmic domains of the receptors. Phosphorylation of tyrosine 1068 (Tyrl068) on Her-2/neu leads via phospho-tyrosine-binding accessory proteins and guanosine nucleotide exchange factors to activation of p21TM ⁇ and thence to activation of cell division via the MAP kinase cascade.
- a cell-based biosensor which produces a readily detectable and quantifiable signal when Her-2/neu activation is blocked, would be particularly useful for high- throughput screening of chemical libraries for compounds with anti-breast mmor potential.
- Such a biosensor may be set up with a ⁇ -lactamase fragment complementation system as follows.
- the ⁇ fragment could be fused via flexible linker to the C-terminus of Her-2/neu, which is proximal to the Tyrl068 substrate of the receptor kinase.
- the ⁇ fragment could then be fused to a binding protein, such as a scFv or VL, which binds to the Tyrl068 region of the receptor only when Tyrl068 is unphosphorylated.
- Tyrl068 is mostly phosphorylated in Her-2/neu over-expressing cells, especially in the presence of EGF, ⁇ -lactamase activation would be minimal. However, in the presence of an inhibitor of Her-2/neu activation, the proportion of unphosphorylated Tyrl068 would rise, recruiting the ⁇ - Tyrl068 binder fusion to the receptor where ⁇ - ⁇ complementation would increase ⁇ - lactamase activity in the cells. In the presence of a fluorogenic ⁇ -lactamase substrate, inhibitors of Her-2/neu activation could be readily identified by increasing fluorescence in a matter of minutes, since dephosphorylation of Tyr 1068 occurs rapidly upon inhibition of the Her-2/neu kinase activity.
- the first step in developing the Her-2/neu inactivation biosensor would be to obtain a Tyrl068-binding protein. This could be accomplished by inserting the coding sequence for the substrate peptide, PVPEYINQS, into the active site of thioredoxin, between G33 and P34, flanked by short flexible linkers such as PGSGG to minimize structural constraints on the peptide, which does not require a rigid strucmre for binding to its namral ligand, the Grb2 SH2 domain.
- This Tyrl068 trxpep can then be fused via a (Gly 4 Ser) 3 linker to the N-terminus of co254, and co-expressed in E.
- coli TG-1 cells with a scFv library of at least 10 8 clones, or a VL library of at least 10 6 clones fused to the C-terminus of ⁇ 253 via the (Gly 4 Ser) 3 linker. Since the Tyrl068-binder is being selected for deployment in the mammalian cell cytoplasm, it might be prudent to perform the selections in the E. coli cytoplasm. For this pu ⁇ ose the vectors in Figure 6 could be used with the signal peptides removed.
- binders with sub-micromolar affinities since Tyr is the most common amino acid in high-affinity protein
- the entire coding sequence for the ⁇ 253 - Tyrl068-binder fusion may be subcloned into a mammalian expression vector, such as the pCMV-Tag vectors (TKX1 cells, Stratagene,
- the co254 fragment must be expressed as a fusion to the C-terminus of the Her-2/neu cytoplasmic domain, which contains Tyrl068.
- the coding sequence of the 1210-residue EGF receptor (Genbank accession no. X00588; Ullrich et al., Nature (1984) 509:418) may be used as it is operationally identical to Her-2/neu, and its Tyrl068 will become phosphorylated under the same conditions of over-expression and/or growth factor ligation in mmor cells.
- the 35- residue co254 ⁇ -lactamase fragments will be only 152 residues away from Tyrl068.
- Both the EGFR- ⁇ 254 fusion and the ⁇ 253-Tyrl068-binder fusion may be expressed from the same vector from a dicistronic mRNA. This is accomplished by inserting an internal ribosome entry site (IRES; Martinez-Salas, Curr Opin Biotechnol (1999) 70:458) between the termination codon of the upstream cistron and the initiation codon of the downstream cistron. This will allow both proteins to be made simultaneously from the same mRNA.
- the vector may be introduced into the mmor cell line by cationic liposome-mediated transfection, using e.g., lipofectamine (Gibco-BRL, Gaithersburg, MD) according to the protocol in the product literature.
- Operation of the biosensor may be tested in transiently transfected cells, and if operational, stable transformants may then be isolated by selection for long term antibiotic resistance. Multiple free-diffusible chromogenic and fluorogenic substrates are available for continuous monitoring of ⁇ -lactamase activity. Operationally, the co254 fragment will be anchored to the plasma membrane at the C-terminus of the cytoplasmic domain of the receptor near Tyr 1068, and the ⁇ 253 fragment will be free in the cytoplasm as the Tyrl068-binder fusion. ATP-analog tyrosine kinase inhibitors are available commercially and can be used as positive controls for inhibitor selection, and to determine the signal increment from fully-activated to fully-inhibited EGFR.
- Example 10 A Fragment Complementation System for Neomycin Phosphotransferase.
- Enzyme fragment complementation systems may also be useful for selection for the simultaneous inco ⁇ oration of multiple genetic elements into the same cell or organism.
- the production of secretory IgA antibodies in plants requires the introduction of four different genes into the same plant. For practical reasons this requires the introduction of at least two and preferably three different DNA molecules.
- each DNA molecule must carry its own selectable marker.
- the use of multiple antibiotic selection systems on the same transformants is cumbersome and inefficient, as the overall false positive and false negative rates tend to scale as the product of the rates for the individual antibiotics.
- two- or three-piece fragment complementation systems for a single antibiotic offer a distinct advantage over multiple antibiotic selection.
- the fos and jun interactors should be used with tri-peptide libraries between the break- points and the (Gly 4 Ser) 3 linkers.
- the tri-peptide libraries will provide stabilizers for each fragment so that the selection will be biased toward the fragments producing the highest specific activities.
- two-trait selection applications i.e., bi-molecular selections, where a heterologous interaction is not required
- specific activity may be increased further by mutagenesis and selection for fold accelerating mutations.
- selected fragment pairs will have to be tested for dependence on the heterologous interaction. In this case, the activation index will be of some importance, but as with in vitro applications a modest index of 1000 will be more than adequate for clean selections.
- Neomycin phosphotransferase II (NPTII; Genbank accession no. M77786) is a 267- amino acid enzyme from E. coli which inactivates aminoglycoside antibiotics such as neomycin and kanamycin by phosphorylation from ATP.
- NPTII is widely used as a selectable marker for plant and animal cell transformation.
- fragment complementation systems for NPTII would be particularly useful for facile generation of multiple-trait plant and animal transgenics.
- the three-dimensional strucmre of NPTII is not known, and its homology to known structures is too low for reliable prediction. However, as described above, empirically-derived neural net algorithms are available which allow fairly accurate prediction of secondary strucmre and solvent exposure for any protein sequence.
- the vectors would differ from those in Figure 6 in not encoding signal peptides, and the pAOl vector would have ampicillin resistance instead of kanamycin resistance. Also, the vectors should contain VRK or NNK random tri-peptide-encoding sequences between the cloning sites for the enzyme fragments and the (Gly 4 Ser) 3 linkers.
- the PCR product for each fragment is restriction digested and ligated into the appropriate vector, ⁇ fragments into the pA ⁇ l-type vector and ⁇ fragments into the pAOl- type vector.
- the ligation products are then introduced into TG-1 cells by high-voltage electroporation, and plated onto chloramphenicol or ampicillin. At least 10 4 transformants should be collected for each fragment.
- kanamycin sensitivity should be determined for each fragment library, both to prevent false positives and to determine the minimum quantitatively selective kanamycin concentration. This should be the concentration on which single fragment plating efficiencies are ⁇ 10 "6 , since the frequencies of the fragment- stabilizing peptides could be this low.
- the cells of each fragment pair are centrifuged, washed, and plated onto ten 150-mm dishes containing solid LB medium with the minimum quantitatively selective concentration of kanamycin.
- two hetero-dimerizing helix pairs may conveniently be used, such as the parallel -binding helixes from fos and jun as described above, and the anti- parallel-binding helixes from yeast DNA topoisomerase II (TopII; Berger et al. , Nature (1996) 579:225).
- One of each helix pair would be fused to an NPTII fragment, and the other two helixes would be fused to each other, so that the NPTII fragments would only come together when the 2-helix fusion was present to form the tri-molecular complex.
- an ⁇ -TopIIN fusion and a fos-co fusion could only be brought together and activated by a jun-TopIIC fusion.
- Genes encoding each of the three fusions could then be distributed among three different DNA constructs which also encode genes of interest.
- eukaryotic cells could be transformed with a mixture of the three different constructs and selected for the simultaneous presence of all three genes in the same cell simply by selection for growth on a single antibiotic.
- Example 11 Tar get- Activated Enzyme Prodrug Therapy.
- 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 mmor that efficacy is often seriously compromised.
- This problem may be overcome by using an interaction-dependent ⁇ -lactamase fragment complementation system with mmor targeting antibodies.
- a-lactamase fragment complementation system with mmor targeting antibodies.
- scFv single- chain antibody fragments
- the ⁇ -lactamase fragments can localize to the mmor and reconstitute sufficient ⁇ - lactamase activity on the mmor cell surface to produce high levels of tumor-localized cytotoxicity from ⁇ -lactam prodrugs.
- the great advantage of such a system is that prodrug activation cannot occur in the general circulation or anywhere the tumor marker is not encountered, so that the prodrug may be administered either simultaneously with high doses of the scFv-fragment fusions, or at the point of highest mmor load of the fragments, without regard for the circulating levels of the fragments which would be completely inactive.
- the construction and purification of fusions of interaction-dependent ⁇ -lactamase fragments with scFv which bind non-overlapping epitopes on the human breast mmor marker Her-2/neu is described.
- One may then determine the kinetics of reconstitution of ⁇ -lactamase activity on the surface of Her-2/neu - expressing SKOV3 human ovarian cancer cells.
- TcEPT Tumor- Activated Enzyme Prodrug Therapy
- ⁇ -lactamase fragment complementation systems are similar to those for in vitro use in general.
- the most important parameters are specific activity and fragment stability, while activation indexes above 1000 confer little additional efficacy.
- the ⁇ 253/co254 would be the recommended fragment pair for this application because it has the highest interaction-dependent specific activity, the fragments are moderately stable, and its activation index is more than adequate.
- the stability of the ⁇ 253 fragment could probably be improved by a custom fragment- stabilizing tri-peptide.
- ⁇ 253-stabilizing tri-peptides would then be selected by plating at least 10 4 library transformants on increasing ampicillin from 400 to 1000 ⁇ g/ml, since ⁇ 253/co254 plates quantitatively on 400 ⁇ g/ml even without a stabilizing peptide, and wild-type TEM-1 ⁇ -lactamase does not plate on more than 1000 ⁇ g/ml when expressed under these conditions.
- the mmor activation mechanism for these fragments may employ two scFvs such as those described by Schier et al. (Gene (1996) 769: 147), which were derived from a phage display library of a human non-immune repertoire (Marks et al., 1991) by panning against a recombinant fragment comprising the extra-cellular domain (ED) of Her-2/neu.
- scFvs such as those described by Schier et al. (Gene (1996) 769: 147), which were derived from a phage display library of a human non-immune repertoire (Marks et al., 1991) by panning against a recombinant fragment comprising the extra-cellular domain (ED) of Her-2/neu.
- ED extra-cellular domain
- the coding sequences for the scFv may be subcloned into the ⁇ -lactamase ⁇ and ⁇ fusion production vectors, p ⁇ lac ⁇ and p ⁇ lac ⁇ , shown in Figure 11. These vectors are derived from pET26b (Novagen), and have convenient restriction sites for insertion of both scFv and ⁇ -lactamase fragment sequences. Each fusion protein is inducibly expressed (IPTG) from the strong phage T7 promoter under the control of the lac repressor.
- IPTG inducibly expressed
- Each primary translation product contains a pelB signal peptide for secretion into the bacterial periplasm and a C-terminal His 6 tag for one-step purification from osmotic shock extracts by immobilized metal ion affinity chromatography (IMAC, Janknecht et al., Proc Natl Acad Sci (1991) SS:8972).
- the yield of each fusion protein can be optimized primarily by manipulation of the inducer concentration and the growth temperature.
- Each scFv may be expressed as both ⁇ and ⁇ fusions to determine which arrangement(s) (1) support the highest binding activity, (2) support the highest enzymatic activity, and (3) support the highest yields.
- expression may be optimized by the criterion of silver-stained PAGE.
- fusion proteins should be purified from osmotic shock extracts (Neu and Heppel, 1965, supra) by IMAC. The purified fusion proteins may be 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).
- Immobilized BSA may be used as the negative control.
- an established vehicle i.e., an antibody- ⁇ -lactamase conjugate.
- conditions are established for samrating the antigen with one of the scFv- ⁇ -lac fragment fusion proteins.
- the wells of microtiter plates are coated with antigen, and exposed to increasing amounts of the first scFv-fragment fusion until the ELISA signal plateaus.
- V max should be a more or less linear function of the concentration of the second fusion. As the amount of second fusion is increased, at some point V ⁇ nax 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 fragment- reconstituted ⁇ -lactamase.
- the K M may be estimated in solution with samrating antigen and samrating first fusion and limiting amounts of the second 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 fusion concentration. The K M is then computed from standard regression analysis.
- a fusion of intact ⁇ -lactamase to the second scFv may be prepared. This is then added in increasing amounts to antigen-coated wells which had been saturated with the first fusion as had been done before.
- 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 fragment-reconstituted enzyme.
- the TEM-1 ⁇ 253/ ⁇ 254 fragment complex is expected to have a maximum activity (k ca near that of the intact enzyme. If the
- K M are also comparable, activities on a mmor up to 100-fold higher at the peak of prodrug activation than with the conventional antibody- ⁇ -lactamase fusion might be expected, which may have 1 % or less of its peak activity left when the unbound fusion has cleared the circulation enough to allow prodrug administration.
- the fragment-reconstituted activity may again be compared with the intact ⁇ -lactamase activity, this time with respect to mmor 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-OV-3 line of human ovarian 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.
- the V ⁇ mx of the fragment- reconstituted enzyme may then be determined on the cells with samrating concentrations of both fusions and nitrocefin. It would be expected to conform to the predicted activity based on the maximum intact ⁇ -lactamase activity and the ratio of V ⁇ ma observed on the immobilized recombinant antigen.
- the sensitivity of the cells to any of the three prodrugs shown in Figure 5 may be determined essentially as described by Marais et al. (Cancer
- prodrugs are dissolved in DMSO and diluted into DMEM/FCS to a range of concentrations immediately prior to use.
- DMSO DMEM/FCS
- 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. After four days of growth, cell viability is assessed by inco ⁇ oration of [ 3 H] thymidine as determined by liquid scintillation counting of acid insoluble material.
- results are expressed as percentage of untreated control cells.
- the relative cytotoxicities of the prodrugs with the ⁇ -lactamase fragment system may be compared to those of the intact ⁇ -lactamase fusions, particularly at the lower prodrug concentrations where second order rate constants (k cat /K M ) may be important, to give an indication of the potential increase in efficacy of TAcEPT over conventional ADEPT in vivo.
- All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein .inco ⁇ orated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be inco ⁇ orate by reference.
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AU60459/00A AU6045900A (en) | 1999-05-25 | 2000-03-16 | Interaction-activated proteins |
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WO2002036738A2 (en) * | 2000-10-30 | 2002-05-10 | Kalobios, Inc. | Affinity maturation by competitive selection |
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Also Published As
Publication number | Publication date |
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CA2374476A1 (en) | 2000-11-30 |
AU6045900A (en) | 2000-12-12 |
JP2003500051A (en) | 2003-01-07 |
EP1183347A1 (en) | 2002-03-06 |
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