WO2009079618A1 - Systèmes et procédés de génie enzymatique - Google Patents

Systèmes et procédés de génie enzymatique Download PDF

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WO2009079618A1
WO2009079618A1 PCT/US2008/087342 US2008087342W WO2009079618A1 WO 2009079618 A1 WO2009079618 A1 WO 2009079618A1 US 2008087342 W US2008087342 W US 2008087342W WO 2009079618 A1 WO2009079618 A1 WO 2009079618A1
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proteolytic
reporter
target
activity
library
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PCT/US2008/087342
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Robert F. Balint
Jeng-Horng Her
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Cytodesign, Inc.
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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
    • 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
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)

Definitions

  • Target-specific proteases have many important industrial, pharmaceutical, and environmental applications. For example, most human diseases are dependent in one way or another on specific proteins, whether they are the proteins that mediate the entry of virus particles into host cells, or cell surface receptors and other proteins that mediate signal transduction essential for cell survival, or soluble toxins, growth factors, or other biological response modifiers. Thus, most diseases have one or more protein "Achilles' heels" by which, if they can be targeted efficiently for neutralization and/or elimination, the disease can be mitigated. For example, chronic inflammatory diseases such as rheumatoid arthritis can be mitigated by treatment with antibodies against inflammatory cytokines such as TNF ⁇ or IFN ⁇ . Many cancer cells depend on continuous autocrine or paracrine growth factor stimulation for survival.
  • Antibodies have shown great promise in the clinic for neutralizing a wide variety of target antigens.
  • target neutralization by binding is a stoichiometric phenomenon, and large doses of antibody are generally required for quantitative neutralization of target antigens.
  • Large antibody doses are costly and can be sufficiently immunogenic to cause significant loss of efficacy during treatment.
  • Enzyme therapeutics are mainstays of treatment for certain diseases.
  • DNase proteozyme
  • tPA tissue plasminogen activator
  • glucocerebrosidase Currative X-ray plasminogen activator
  • mAb monoclonal antibodies
  • enzyme therapeutics have the potential to be more effective at lower doses than binding reagents, provided, of course, they possess at least comparable, if not greater, specificities for their targets.
  • the human genome encodes a large number of proteolytic activities, which fall into two broad categories: digestive or degradative enzymes, and maturation enzymes that process specific gene products into functional polypeptides.
  • digestive or degradative enzymes and maturation enzymes that process specific gene products into functional polypeptides.
  • maturation enzymes that process specific gene products into functional polypeptides.
  • the engineering of alterations in protease specificity has been reported (Bone and Agard, 1991; Hopfner et al., 1998; Butler et al., 1999; McGinness et al., 2006).
  • human proteases could be engineered to cleave desired substrates with sufficient efficiency and specificity for therapeutic use, particularly if a high- throughput screening or selection system were available that could accommodate large numbers of mutants.
  • antibodies are uniquely adapted for rapid optimization for a wide variety of activities.
  • certain disease states provoke the formation of antibodies with catalytic activities (Paul, 1998).
  • hydrolytic anti- DNA antibodies can be found in patients with systemic lupus erythematosus.
  • the first proteolytic antibody was found in an asthma patient, and could specifically cleave vasoactive intestinal polypeptide (VIP) (Paul et al., 1989).
  • VIP vasoactive intestinal polypeptide
  • Antibodies which cleave thyroglobulin were identified in autoimmune thyroid disease (Li et al., 1995), and Bence- Jones proteins from multiple myeloma patients exhibited catalytic activity (Paul et al., 1995). Some 20% of hemophiliacs become resistant to factor VIII, and antibodies capable of hydrolyzing factor VIII in serum are associated with this resistance (Lacroix-Desmazes et al., 2002). Besides autoimmune disease patients, normal donors have also been found to possess IgG which contain proteolytic activities against model peptide substrates (Kalaga et al., 1995; Li et al, 2000).
  • Serine proteases such as trypsin and subtilisin contain a conserved catalytic triad comprised of Asp, Ser, and His residues that are arranged in space such that the Asp and His provide an electron shuttle that partially abstracts a proton from the Ser, making it highly nucleophilic.
  • This active site nucleophile is highly attractive to the peptide carbons of polypeptide chains, and hydrolyzes the peptide bond by forming an acyl-enzyme intermediate, which is then hydrolyzed by water to regenerate the active apo-enzyme.
  • the proteolytic antibodies identified to date appear to utilize a similar mechanism.
  • HIV gp41 antibody was identified serendipitously because of its ability to degrade the gp41 antigen (Hifumi et al., 1999). Further studies indicated that the proteolytic function was due exclusively to the light chain, and molecular modeling predicted possible catalytic triads consisting of Ser-Asp-His (Hifumi et al., 2000). The most thoroughly studied proteolytic antibody is anti-VIP, which was produced in mice, and whose protease function has also been found to reside in the light chain (Sun et al., 1997; Sun et al., 1994). The catalytic activity requires a triad consisting of Asp 1, Ser27a, and His93.
  • Figure 1 shows the human germline repertoire of 40 kappa light chain V-regions (VK), and indicates the 13 VK that contain potential catalytic triads.
  • proteolytic antibodies in hemophiliacs demonstrate the potential efficacy of proteolytic antibodies in the clinic.
  • the ability to harness proteolytic antibodies for therapeutic uses would provide more potent pharmaceuticals that could be given at lower doses. Indeed, Zhou, et.al. (2002) have calculated that an antibody with a modest turnover number (0.1/min) could inactivate over two-thousand times as many antigen molecules as an antibody that binds stoichiometrically.
  • Several mechanisms have been employed to identify antigen-specific proteolytic antibodies. Some have been found by chance, others have used transition-state analogs, which utilize phosphonate esters to mimic the tetrahedral intermediate of target peptide bond hydrolysis.
  • Such methods have used phage display libraries to identify antibody single-chain Fv fragments (scFv) capable of covalently binding the intermediate (Paul et al., 2001).
  • Haptenized phosphonate diesters in the context of target sequence have been used to immunize animals from which a proteolytic antibody specific for HIV gpl20 was obtained (Paul et al., 2003).
  • Robust methods for screening large numbers of variants e.g., 10 8 -10 9 ), which are not currently available, could greatly improve the ability to identify such antibodies.
  • the invention is based on the discovery of methods and systems for detecting a protease activity using a proteolytic sensor complex.
  • the invention provides a method for detection of proteolytic activity comprising the steps of: providing a reporter complex comprising a reporter molecule, an inhibitor of the reporter molecule, and a target polypeptide of interest where the inhibitor inhibits the reporter molecule; providing a proteolytic molecule with a proteolytic activity for the target polypeptide, combining the reporter complex and the proteolytic molecule whereupon cleavage of the target polypeptide causes dissociation of the reporter and inhibitor; and detecting an activity of the reporter molecule, thereby detecting proteolytic cleavage of the target polypeptide.
  • the target polypeptide is covalently linked to the reporter and the inhibitor of the reporter. In some embodiments, the target polypeptide and the inhibitor are covalently linked and the proteolytic cleavage of the target polypeptide inactivates the inhibitor.
  • the reporter may be covalently linked to the inhibitor, or may be non-covalently associated with the inhibitor.
  • the proteolytic molecule has a serine protease activity.
  • the reporter is TEM-I ⁇ -lactamase or ⁇ - lactamaseE104K, and the inhibitor is BLIP.
  • the method of the invention can be performed where the proteolytic molecule is a member of a library of candidate proteolytic polypeptides, e.g., a library of candidate proteolytic antibody molecules, that are being screened for proteolytic activity.
  • the methods of the invention can also be performed where the target polypeptide is a member of a library of candidate target polypeptides.
  • the reporter complex and proteolytic molecule can be expressed using any number of expression systems, including eukaryotic expression systems, e.g., yeast or mammalian systems, or bacterial expression systems.
  • the expression system is one in which the expressed reporter complex and proteolytic molecule are secreted into the periplasmic space, e.g., an E. coli expression system.
  • the invention provides a system for selecting a proteolytic molecule that cleaves a target polypeptide from a library of candidate proteolytic molecules, said system comprising a population of cells in each of which a reporter complex is co- expressed with at least one member of a library of candidate proteolytic molecules, where the reporter confers a selectable phenotype when the target polypeptide is cleaved by a candidate proteolytic molecule.
  • the cells are bacterial cells such as E. coli.
  • the reporter molecule is TEM-I ⁇ -lactamase or ⁇ -lactamaseE104K and the inhibitor is BLEP.
  • the candidate proteolytic molecules are antibodies or antibody fragments, e.g., VK domains, kappa light chains, Fab fragments, single-chain Fv fragments, or VH domains.
  • the candidate proteolytic molecules are proteases diversely mutated to alter substrate specificity.
  • the candidate proteolytic molecules are polypeptide domains representing structural classes known to provide scaffolds for containing polypeptides with diverse activity. Such scaffold include, but are not limited to, immunoglobulin domains and ⁇ -barrels.
  • FIG. 1 The human germline kappa V-region repertoire.
  • the kappa germline repertoire is grouped into six families based on sequence homology (labeled at the left).
  • the complementarity-determining regions (CDRs) and framework regions (FR) are designated above.
  • Putative proteolytic light chains are identified by those V-regions (shaded in cyan) that contain Asp or GIu at the N-terminus, and Ser and His in the CDRs (shaded in magenta). 13 potential serine protease V regions are identified out of 40 human germline VK genes (33%).
  • a hydrolytic activity sensor is comprised of a Reporter and a low-affinity inhibitor of the Reporter, each linked to the same Target molecule in such a way that if the Target is cleaved by an Enzyme, the Reporter and Inhibitor separate, activating the Reporter to produce a detectable signal.
  • the hydrolytic activity sensor is comprised of a Reporter and a Target molecule inserted into a high-affinity Inhibitor of the Reporter in such a way that cleavage of the Target causes the inactivation of the Inhibitor, thereby activating the Reporter to produce a detectable signal.
  • Target cleavage leads to Reporter activation by inactivation of an inhibitor, in one case by separation from the Reporter, and in the other case by fragmentation of the Inhibitor.
  • the Target can be a protein or any other hydrolysable molecule.
  • the enzyme can be a protease, proteolytic antibody, or any other Enzyme with the ability to hydrolyze the Target. If the Enzyme and hydrolytic activity sensor are expressed in a cell, the activated Reporter may confer a selectable phenotype on the cell. In this way the sensor can be used to isolate proteins with desired hydrolytic activities from populations of candidate proteins.
  • FIG. 3 Reactivation of Auto-Inhibited ⁇ -lactamase in the E. coli periplasm by cleavage of Botulinum neurotoxin (BoNT) light chain by a proteolytic antibody VK domain derived from a VK library.
  • the VK library is based on the human Al 8 germline VKII domain with catalytic triad comprised of Dl, S27a, and H93, plus 11 other positions in CDRl and CDR3 diversified by Parsimonious Mutagenesis (Balint and Larrick, 1993).
  • BoNT Light chain (Segelke et al, 2004), BLIP (Lim et al., 2000), ⁇ -lactamaseE104K (Jelsch et al., 1993), and the VK domains (Yin et al., 2001) are shown as ribbon structures rendered from x-ray coordinates using DS ViewerPro 5.0 (Accelrys, San Diego).
  • the catalytic triad of the VK domain (Dl, H93, and S27a) is depicted in CPK, as are the sites which are diversified in the library and the active site nucleophile, S70, of ⁇ - lactamaseE104K ( ⁇ -lacE104K).
  • the VK domain may also be expressed as a full-length light chain with a C-terminal kappa chain constant region domain (CK).
  • BONT light chain is shown being cleaved in an exposed loop at Y249, which remains attached by ester linkage to S27a in an Acyl Adduct of the VK domain.
  • the proteolytic VK domain is regenerated by hydrolysis of the Acyl Adduct, which is the rate-limiting step in the catalytic cycle.
  • cleavage of BoNT Light Chain confers ampicillin resistance on the cells in proportion to the proteolytic activity of the VK domain.
  • increasing ampicillin concentrations can be used to identify the highest proteolytic activities in the VK library.
  • FIG. 1 A. TFS expression vector.
  • the BLIP - Target - BLAC ( ⁇ -lactamaseE104K) triple fusion proteolytic activity sensor protein is expressed with an N-terminal signal peptide (SP) for efficient translocation to the bacterial periplasm, from the inducible lactose operon promoter (P LAC )-
  • SP N-terminal signal peptide
  • P LAC inducible lactose operon promoter
  • the (Gly 4 Ser) 3 linkers are ⁇ 6 ⁇ A in length to allow fully-relaxed BLIP-BLAC complex formation from the termini of most targets of up to 5OkDa in size regardless of where the termini are located on the target protein.
  • B IIS expression vector. Similar to A, except that the Target is inserted into an exposed loop on the surface of BLIP between Ala77 and Pro78 instead of between BLIP and BLAC, and free wild-type BLAC is expressed from a separate promoter, cPuc, a constitutive mutant of the lactose operon promoter.
  • Monomelic candidates such as human antibody VK domains or full-length light chains, may be expressed from the arabinose operon promoter ⁇ P BAD ) which is activated by arabinose via AraC, a suppressor/activator of the promoter.
  • D Proteolytic antibody Fab fragment expression vector. Essentially the same as C, except that in the preferred embodiment, the Fab Fd and light chains are expressed from a dicistronic transcript, in which translation of the downstream cistron is initiated internally via a ribosome binding site (RBS) inserted between the termination codon of the upstream cistron and the initiation codon of the downstream cistron. Arrows indicate direction of transcription or replication.
  • RBS ribosome binding site
  • HiS 6 hexa-histidine tag for metal-ion affinity purification
  • Flag tag epitope tag for expression analysis
  • tt transcription termination signal
  • pBR322 ori pl5A ori, mutually compatible plasmid origins of replication
  • flori phagemid origin of replication for phage rescue
  • cat chloramphenicol acetyl transferase
  • kan kanamycin resistance.
  • FIG. 5 Mechanism of action of serine proteases.
  • a serine hydroxyl in the enzyme active site Serl57 in Trypsin
  • Histidine-aspartate electron shuttle His57- AsplO2 in Trypsin
  • the activated ⁇ O of the serine then attacks the peptide carbonyl of the substrate.
  • the peptide bond breaks, and the carboxyl terminal fragment of the substrate is released, leaving the acyl-enzyme intermediate.
  • water is activated by withdrawal of a proton by the His-Asp shuttle, and attacks the carbonyl of the acyl-enzyme intermediate.
  • step 4 the amino-terminal fragment of the substrate is released, and the apo-enzyme is regenerated.
  • Figure 6 Location of the catalytic triad in a proteolytic VK domain, and the location of the sites to be diversified by PM in a library based on the human germline VK domain Al 8. The Al 8 sequence is shown at top with catalytic triad in bold-face underlined and PM sites in bold-face.
  • B 3-d location of PM sites surrounding catalytic triad.
  • the VK domain is shown as a ribbon structure rendered from x-ray coordinates using DS ViewerPro 5.0 (Accelrys, San Diego).
  • the catalytic triad of the VK domain (Dl, H93, and S27a) is depicted in CPK, as are the sites which are diversified by PM in the library.
  • the VK domain is normally expressed as a full-length light chain with a C-terminal kappa constant region (not depicted).
  • This rate was selected to maximize the diversity of a library of 10 9 independent clones with respect to clones bearing 3-5 coding changes among the 11 selected positions, which comprise -31% of the library.
  • each position will be altered in 57% of clones in the library.
  • FIG. 9 Plating efficiencies (PE) on increasing ampicillin of E. coli cells co- expressing antibody light chains with the Al 8 human germline proteolytic VK domain or a negative control VK (Neg Con) and BLIP-BLAC(El 04K) fusion containing the Al 8 substrate tripeptide PFR in the linker.
  • the light chains were expressed from an arabinose operon promoter induced by the indicated arabinose (ara) concentrations. Cleavage of the linker between BLAC and BLIP leads to the activation of BLAC and ampicillin resistance in proportion to Al 8 light chain expression. No BLAC activation above background was produced by full induction of the negative control light chain.
  • Figure 10 Figure 10.
  • PE Plating efficiencies
  • E. coli cells co-expressing antibody light chains with the Al 8 human germline proteolytic VK domain or a negative control VK (Neg Con), wild-type ⁇ -lactamase, and the Inhibitor Insertion Sensor (IIS), in which a target sequence containing the Al 8 substrate tripeptide PFR has been inserted between Ala77 and Pro78 in the center of the inter-subdomain loop of BLIP.
  • the light chains were expressed from an arabinose operon promoter induced by the indicated arabinose (ara) concentrations. Cleavage of the target sequence leads to fragmentation of BLIP and the activation of BLAC and ampicillin resistance in proportion to Al 8 light chain expression. No BLAC activation above background was produced by full induction of the negative control light chain.
  • the invention provides systems and methods for the detection of proteolytic activities.
  • the desired proteolytic activities are selected from populations of antibody fragments that contain protease-like catalytic residues in the complementarity determining regions (CDR) of their variable domains.
  • the libraries of the invention may comprise non-antibody proteins that contain protease-like catalytic residues on their surfaces.
  • the present invention allows the determination of substrate specificities for proteases of interest, selection of altered substrates for proteases of interest, and selection of inhibitors of proteases of interest.
  • proteolytic activity or protease is meant a polypeptide that is capable of catalyzing the hydrolysis of a peptide bond, i.e., an amide bond in the peptide backbone of at least one polypeptide.
  • Natural proteases are classified according to the catalytic mechanism (Handbook of Proteolytic Enzymes, 2" Ed.; Barrett, Rawlings and Woessner, eds., Elsevier, 2004).
  • Serine proteases comprise the largest class, in which the peptidolytic nucleophile is an activated serine residue.
  • the other major classes include cysteine, aspartic, and metallo-proteases.
  • Lesser classes include glutamic, threonine, and proteases of unknown mechanisms.
  • serine proteases there is a general, but by no means exclusive, preference for cleavage on the carboxyl side of basic residues, and a similar preference for hydrophobic residues on the amino side of the scissile residue.
  • target, or substrate sequence specificity is weak, especially for digestive enzymes.
  • target specificity is conferred by proximal structures which limit access to the active site and/or increase affinity for the target.
  • Bacterial cells are eminently suited to provide such reaction vessels, since they can be genetically engineered to make candidate proteolytic abzymes, and to exhibit a variety of selectable phenotypes in response to the desired reaction, such as color, fluorescence, luminescence, or the ability to grow under non-permissive conditions.
  • the present invention is comprised of a proteolytic activity sensor, comprising a reporter, an inhibitor of the reporter, and a target substrate of the desired proteolytic activity, all present in a single cell.
  • the target and inhibitor are covalently linked to each other, and the reporter is physically linked to the inhibitor, either covalently (via the target) or non-covalently.
  • the reporter is constitutively inhibited.
  • the inhibitor is caused to dissociate from the reporter, allowing the latter to confer a selectable phenotype on the cells expressing the target-cleaving activity. Either of two mechanisms may be employed whereby target cleavage is causally linked to dissociation of the inhibitor from the reporter. These are illustrated in Figure 2.
  • the "Triple Fusion Sensor” (TFS), reporter, target, and inhibitor are expressed as a single continuous polypeptide chain in which the target is situated between the reporter and inhibitor and separated from them by flexible polypeptide linkers.
  • the reporter is constitutively inhibited by forming a complex with the inhibitor, and therefore no selectable phenotype is conferred on the cells.
  • the affinity between reporter and inhibitor is sufficiently low that when the proteolytic target is cleaved, the inhibitor and reporter are no longer held together, but dissociate and diffuse apart, thereby activating the reporter to confer the selectable phenotype on the cells.
  • the TFS when the TFS is co-expressed in cells with a library of candidate target-cleaving proteins, such that each library member is expressed in a different cell, candidates which cleave the target protein cause the activation of the reporter, and are identified by the selectable phenotype conferred thereby on the cells.
  • the proteolytic target can be a variety of sizes, e.g., depending on the expression system employed.
  • the target protein is inserted with flanking linkers into a specific location on the surface of the inhibitor to form a stable, soluble fusion protein in which the inhibitor remains fully functional.
  • the affinity of the inhibitor for the reporter may be sufficiently high that covalent linkage is not required for quantitative constitutive inhibition of the reporter when both reporter and target-inhibitor fusion are co-expressed in the same cells.
  • the affinity of the reporter and inhibitor-with-target-insertion is too low for quantitative inhibition at operating expression levels, they may be covalently linked via flexible peptide linker of sufficient length to allow relaxed complex formation, preferably at least 30 residues ( ⁇ 12 ⁇ A).
  • candidates which cleave the target protein cause the fragmentation of the inhibitor, thereby causing the activation of the reporter, and are identified by the selectable phenotype conferred thereby on the cells.
  • the reporter is the TEM-I ⁇ - lactamase of E. coli (BLAC; Jelsch et al., 1993)
  • the inhibitor is the ⁇ -lactamase inhibitor protein of Streptomyces clavuligerus (BLIP; Lim et al., 2001)
  • the sensor is expressed in the periplasmic space, i.e., the secretory compartment, of E. coli cells.
  • the preferred site of target insertion into BLIP is between Ala77 and Pro78 or between Pro78 and Ser79 of the mature sequence, though other sites are also possible.
  • the preferred site is in a loop which connects the two subdomains of the protein, such that cleavage of the loop, or of a target sequence in the loop, leads to dissociation of the subdomains, and inactivation of the inhibitor.
  • the periplasmic BLAC/BLIP system has the advantage that most proteolytic targets and candidate proteases will be extra-cellular proteins, and therefore more at home in this compartment. Another advantage of these systems is that BLAC confers antibiotic resistance on the cells, and this allows selection from much larger libraries than could be screened using a color or fluorescence phenotype, though substrates are also available for these phenotypes (McManus-Munoz and Crowder, 1999; Campbell, 2004; Zlokarnik et al., 1998).
  • any reporter enzyme that confers a selectable phenotype on host cells could be used in the proteolytic activity sensor.
  • chromogenic enzymes such as ⁇ -galactosidase, or other antibiotic resistance-conferring enzymes such as kanamycin phosphotransferase or chloramphenicol acetyl transferase could also be used.
  • Proteolytic activity sensors with appropriate reporters can also be deployed in other compartments of the cell, such as the cytoplasm, and in eukaryotic cells, such as mammalian cells.
  • luciferase or neomycin phosphotransferase could be used as reporter along with appropriate targets in a TFS or IIS system co-expressed in the cytoplasm of mammalian cells with a library of candidate proteases, wherein target-cleaving activity would confer a chromogenic (luciferase) or antibiotic-resistance (NPT) phenotype on the cells.
  • a suitable polypeptide inhibitor of the reporter is not available, one with suitable affinity can readily be obtained from a panel of anti-reporter antibodies.
  • a panel can be obtained by a variety of methods, such as by hybridoma production from reporter-immunized mice (K ⁇ hler and Milstein, 1975, Nature 256:495-7), or by biopanning from a bacteriophage-displayed or yeast cell-displayed antibody fragment library (e.g., McCafferty et al, 1990, Nature 348:552-554).
  • a substantial fraction of antibodies which bind an enzyme will have usable inhibitory activity.
  • Antibody inhibitors may be used in standard antibody fragment formats, such as single-chain variable domain fragments (scFv) or light chain with heavy chain ab fragments (Fab).
  • scFv inhibitors are convenient because targets can be inserted into the linker between the heavy and light chain variable domains, such that cleavage of the target leads to dissociation of the variable domains and inactivation of the inhibitor.
  • Any polypeptide target may be deployed in a TFS or IIS system for selection of proteolytic activities.
  • Particularly attractive therapeutic targets include autocrine/paracrine growth factor receptors which are over-expressed on cancer cells, such as the epidermal growth factor receptors (EGFR), or cytokines involved in chronic inflammatory disease, such as TNF ⁇ , or bacterial exotoxins, such as Botulinum neurotoxin or anthrax toxin.
  • Attractive targets are often multi-subunit proteins, of which one subunit in particular is to be targeted.
  • a target polypeptide may be too large, or otherwise unable to be stably expressed in the sensor, or cleavage may not lead to efficient dissociation of reporter and inhibitor.
  • a fragment of the target that serves as a target often contains a surface loop, whose cleavage would lead to inactivation of the target.
  • receptor targets typically an extra-cellular domain is to be targeted, which is preferably deployed as a fragment because membrane receptors are usually not soluble.
  • targets or target fragments initially do not express well in a TFS or IIS system stable expression of the sensor can often be achieved using standard tools for optimization of recombinant protein expression in microbial systems ⁇ see, e.g., Sambrook and Russell, eds, Molecular Cloning: A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001; and Current Protocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc. New York, 1997), including, for example, codon optimization, linker optimization, growth at reduced temperatures, etc.
  • codon optimization e. John Wiley & Sons, Inc. New York, 1997)
  • codon optimization linker optimization
  • growth at reduced temperatures etc.
  • efficient cleavage of an exposed site on a target polypeptide will lead to inactivation of the target. This can be confirmed for selected target sequence-cleaving molecules, and multiple examples may need to be tested to obtain the desired activity.
  • TFS systems employing BLAC and BLIP
  • An example of a TFS system for selection of proteolytic antibody kappa light chain variable region domains (VK) against the Botulinum neurotoxin (BoNT) light chain from a human proteolytic VK library using BLAC and BLIP is illustrated in Figure 3.
  • BLAC and BLIP are separated from the target protein by flexible linkers, e.g., (Gly 4 Ser) 3 , which at 6 ⁇ A in length, should allow relaxed BLIP-BLAC complex formation from the termini of most targets of up to 5OkDa in size regardless of where the termini are located on the target protein.
  • BLAC has a mutation, GlulO4Lys (E104K), which reduces its affinity for BLIP by -1000-fold ⁇ K d >10 "7 ; Petrosino et al, 1999, J Biol Chem 274:2394-2400; Huang et al, 1996, J MoI Biol. 258:688-703) without appreciably affecting its enzymatic activity, such that at working expression levels in the cells, BLAC(El 04K) is only inhibited by BLIP when they are physically linked to each other. Wild-type BLAC is used in the IIS system.
  • BLAC(E 104K) Separation of BLAC(E 104K) from BLIP upon target cleavage is based on the expectation that many locations on the surface of any given target protein will be vulnerable to destabilization if the backbone is cleaved, so that at least some unfolding of the protein occurs in the vicinity of the cleavage site, exposing hydrophobic sequence to the endo- and exo-proteolytic activities of the periplasm.
  • cleaved proteins generally turn over rapidly, with exo-proteolytic digestion proceeding with varying efficiencies into the flanking linkers, causing the complete separation of the reporter from the inhibitor.
  • the periplasm appears to have no unfolding mechanism for unstable proteins.
  • coli expression vector (Sambrook and Russell, 2001), e.g., the inducible lactose operon promoter in a pl5A replicon such as pACYC184 (Rose, 1988) from which the resident BLAC gene has been removed.
  • the sensor need only be expressed at relatively low levels, since very little active BLAC is required to confer selectable levels of antibiotic resistance on the cells.
  • Low-level expression has other advantages as well. For example, many heterologous target proteins will only tolerate low levels of expression, especially as triple fusion proteins. Also, low expression levels typically favor selection of variants with higher turnover rates on higher concentrations of antibiotic.
  • candidate proteolytic proteins such as nucleophilic antibody light chain variable region domains, antibody Fv or Fab fragments containing such domains, or variants of a mutagenized protease
  • a second standard expression vector such as a modified pBAD vector (Guzman et al., 1995) based on the compatible pBR322 replicon with the tunable arabinose operon promoter (P BAD ) for precise control of proteolytic candidate expression, and kanamycin resistance for plasmid maintenance.
  • Vectors for the expression of the TFS and IIS configurations of the proteolytic activity sensor and for the expression of the library of candidate target-cleaving proteins are illustrated in Figure 4.
  • An initial enrichment of cells expressing target-cleaving activities can be accomplished by growing the library in suspension cultures.
  • Large proteolytic candidate libraries e.g., >10 9 members, with each cell expressing the BLAC-target-BLIP (or BLIP- target-BLAC) sensor and one member of the library, can be inoculated at e.g., 10 8 cells per ml into rich medium such as 2xYT broth (Sambrook and Russell, 2001) containing low levels of the transcription inducers and concentrations of a ⁇ -lactam antibiotic such as ampicillin or carbenicillin which are lethal for cells not containing a desired level of target- cleaving activity, e.g., cells expressing the sensor and a non-cleaver, or the parent cleaver of a mutagenic library.
  • rich medium such as 2xYT broth (Sambrook and Russell, 2001) containing low levels of the transcription inducers and concentrations of a ⁇ -lactam antibiotic such as ampicillin or carb
  • Antibiotic resistance generally correlates quantitatively with BLAC activation, so that clones which grow on the higher antibiotic concentrations will exhibit higher proteolytic activities and/or higher expression levels. If such clones are subjected to additional rounds of low-level mutagenesis and selection under expression conditions in which the sensor is generally present in excess over the candidate target-cleavers, further gains in antibiotic resistance will be biased toward increased catalytic activity of the target cutters.
  • nucleophilic antibodies of the pre-immune repertoire form stable adducts with phosphonate diesters, which are irreversible inhibitors of serine proteases, and they also attack synthetic peptides with basic residues at the cleavage site.
  • the nucleophilic activities of IgM are considerably higher than those of IgG, suggesting that this activity might be diminished during the affinity maturation process. This is understandable if such nucleophilic activities are accompanied by significant turnover, which would tend to reduce B-cell receptor occupancy and, therefore, inhibit affinity-dependent B-cell activation. Thus, rapid dissociation of the antibody from the product would be disfavored by the affinity maturation process, and high catalytic efficiencies would actually be selected against.
  • mice are immunized with antigens haptenized with phosphonate diesters
  • the resulting antigen-binding polyclonal antibody repertoires are enriched for nucleophilic activity relative to non-immune repertoires (Paul et al, 2003, J Biol Chem 278:20429-35). This is consistent with the ability of these antibodies to form stable covalent adducts of the phosphonate antigen, which might trigger B-cell clonal selection and proliferation in the same way that high non-covalent affinities would.
  • nucleophilic activities of such antibodies can exceed those of serine proteases, and they are active against the peptide analogs of the antigens as well, indicating a considerable flexibility in the VH-VK complex that would allow nucleophilic attack at different positions separated by as much as 5 ⁇ A.
  • Such antibodies against peptide substrates still do not have mechanisms to accelerate hydrolysis of the acyl adduct, and so their turnover numbers are still low, precluding the likelihood of obtaining catalytically efficient antibodies by immunization with phosphonate diesters.
  • proteolytic VK Libraries [0049] From the prevalence of natural proteolytic antibodies it may be concluded that the stable structure/activity space defined by the nucleophilic antibody light chain VK domain repertoire combined with diversification of the positions surrounding the catalytic triad should contain useful proteolytic activities for a broad spectrum of important protein and peptide targets.
  • the present invention provides an efficient means to isolate desired proteolytic activities from VK libraries that would encompass such a structure/activity space.
  • antibodies with antigen-specific nucleophilic activities may be expected in the repertoires of immunized animals, as discussed above, antibodies with significant turnover rates tend to be counter-selected by the affinity maturation process.
  • recombinant methods are employed to obtain antibody libraries enriched for high catalytic efficiencies.
  • Some of the human germline VK domains that contain potential catalytic triads already display detectable proteolytic activity for peptides with basic residues at the cleavage site, and they represent much of the structural diversity of the human VK repertoire. Also, they are active as free light chains, which tend to form dimers.
  • the human proteolytic VK repertoire can be used to construct large mutagenic libraries containing diverse proteolytic activities.
  • the human proteolytic light chain libraries of the present invention are based on representative sequences from each of the six VK domain structural groups, each containing a potential catalytic triad comprised of an N-terminal Asp or GIu, one or more Ser in CDRl , and at least one His in CDR3.
  • Preferred representative sequences include A30 (VKI), Al 8 VKII), L20 (VKIII), B3 (VKIV), B2 (VKV), and A26/10 (VKVI). All but B3 already contain potential catalytic triads, and B3 requires only the addition of a His in CDR3.
  • Each structural class is further diversified by random mutagenesis in CDRl and CDR3.
  • PM Parsimonious Mutagenesis
  • PM involves doping the sequence of each CDR with degenerate codons, which are designed to encode selected sets of amino acids at selected positions with predetermined frequencies. This is accomplished using mutagenic oligonucleotide primers to amplify the sequence between the N-terminus of CDRl and the C-terminus of the VK just downstream from CDR3. The resulting PCR product is then ligated into an expression vector which already contains the N-terminal fragment of the VK and the downstream CK domain in the same reading frame.
  • the PM library for each human proteolytic VK structural class is constructed from two degenerate oligonucleotide primers, which are used to amplify the sequence from -20 bp upstream from the C-terminus of Framework 1 (FRl) through the end of FR4.
  • This product is then be amplified a second time by overlap extension PCR with a fragment containing the coding sequence for all of FRl, so that the two fragments overlap in the C -terminal region of FRl .
  • the final product contains unique restriction sites outside the coding sequence for ligation into the expression vector in frame with the N-terminal signal peptide and C-terminal kappa constant region (CK).
  • the expression vector is a modified pBAD vector (Guzman et al., 1995) based on pBR322 with the tunable arabinose operon promoter (P BAD ) for precise control of light chain expression, and kanamycin resistance for plasmid maintenance.
  • P BAD tunable arabinose operon promoter
  • the pelB signal peptide is appended to the N-terminus of the VK for efficient translocation to the periplasm, and a HiS 6 tag is present at the C- terminus of CK for expression analysis and purification (Janknecht et al., 1991). Standard procedures are used for all cloning and analyses of structure and expression (Sambrook and Russell, 2001).
  • the mutagenic oligonucleotide primers for PM library construction are designed with the aid of a software tool (Balint and Larrick, 1993). Coding change frequencies are selected on the basis of the expected number of adaptive coding changes in optimal variants (typically a small number), the maximum library size that can be screened with available methods, and the number of amino acids to be sampled at each position. The positions to be doped and the set of amino acids to be sampled at each position are selected on the basis of available structure-activity information. The operator specifies the number of sites to be diversified, the coding change frequency, and the desired library size.
  • the coding change frequency is then adjusted to maximize diversity, and the program computes the binomial distribution of n coding changes among the diversified sites, and the number of permutations and clones per permutation in the library for each value of n. Then the sets of amino acids to be sampled at each position are selected, and the program computes the required doping codon for each position. With this information, the program then computes the precise composition of the nucleotide mixtures required at each position of the coding sequence during primer synthesis.
  • the acyl adduct hydrolysis rate can be accelerated by several mechanisms, including (1) electrophilic activation of the ester linkage toward hydrolysis, (2) nucleophilic activation of water for attack at the ester carboxyl, (3) destabilization of the acyl adduct by repulsive contact, and (4) increased exposure of the ester linkage to the aqueous environment. Strongly conserved positions in this region were avoided since they may play essential structural roles.
  • Figure 7A shows a binomial distribution of coding changes ( ⁇ ) in the 11 selected CDRl and CDR3 positions Qn) when the coding change rate (s) is set at 6.26 per molecule.
  • This rate was selected to maximize the diversity of a library of 10 independent clones with respect to clones bearing 3-5 coding changes among the 11 selected positions, which comprise ⁇ 31 % of the library.
  • Figure 8 shows the doping codons to be used at each mutagenized position in CDRl and CDR3 of the Al 8 VK domain.
  • the parent amino acid pAA
  • its preferred codon Code
  • Dope selected doping codon
  • the parent amino acid is present at each position in 43% of clones, thus, each position will be altered in 57% of clones in the library.
  • each set of non-parental amino acids includes essentially all conservative changes as well as a broad selection of non-conservative changes.
  • the library will contain all of the listed amino acids at each position in every permutation of n-3 coding changes. Given the high degree of sequence redundancy with respect to protein structure and activity, such a library should contain most of the highest proteolytic activities in the Al 8 human VK structure/activity space.
  • Similar libraries may be constructed for each of the other 5 structural groups of human VK domains, based on A30 (VKI), L20 (VKIII), B3 (VKIV), B2 (VKV), and A26/10 (VKVI), respectively. When these libraries are combined with the Al 8 library, the resulting library may be expected to contain high proteolytic activities for most protein and peptide targets.
  • VH human heavy chain variable region domain
  • scFv single-chain Fv
  • Fab single-chain Fv
  • IgG format full-length IgG format.
  • a companion VH domain can make significant contributions to the association rate, proteolytic rate, and/or turnover rate of the abzyme, as well as to its stability. Since a selected target-cleaving VK domain is likely to show a strong preference for a particular site on the surface of the target, a complementing VH domain would be expected to bind a nearby epitope.
  • Such a VH domain can be obtained by pairing the selected VK domain with a panel of human VH domains recognizing diverse epitopes on the target, as would be obtained, e.g., from a phage display library panned against the target antigen, and selecting for increased target-cleaving activity using the present invention.
  • a phage display library panned against the target antigen
  • an unselected VH repertoire could be used.
  • Such libraries in the preferred Fab format would be expressed from a vector such as that shown in Figure 4.
  • proteolytic antibody selection is at which stage in the process VH selection should be introduced.
  • heavy and light chains are selected together from immense repertoires in which both are diversified.
  • the independent activity of human proteolytic VK domains and V ⁇ -containing light chains permits the proteolytic antibody selection process to be subdivided into two steps in which light chain and heavy chain selections are performed separately from smaller libraries and/or larger segments of sequence/structure space, thereby allowing access to greater combinatorial diversity than would be possible by selecting from combined light chain and heavy chain repertoires. Two general strategies are envisioned.
  • a pool of target-binding antibodies are obtained separately, and the Fd chains from these antibodies are combined with the unselected light chain library, or with a pool enriched for target-cleaving light chains by pre-selection at low stringency, i.e., for activities which are minimally detectable above the background activity of a non- cleaving negative control.
  • the second strategy one or a small number of target-cleaving light chains are selected first, and then combined with an unselected human Fd repertoire for selection of Fabs with enhanced target-cleaving activities.
  • a third strategy may be envisioned which may be considered to be contingent on the failure of the other two. That is, if a suitable target-cleaving activity is not obtained by direct selection from a VK PM library, whether as free VK domains, light chains, or Fabs with target-binding Fd chains, then advantage may be taken of the natural ProPheArg- cleaving activity of the wild-type Al 8 VK domain.
  • One or more sites on the surface of the target can be mutated to this sequence, and tested for soluble expression in E. coli as the TFS or IIS proteolytic activity sensor, and for BLAC activation in the presence of the wild- type Al 8 light chain. It is expected that one or more sites should be sufficiently exposed to confer selectable Al 8 substrate activity.
  • the target mutant(s) with the highest Al 8 substrate activity is(are) then reverted to the wild-type target sequence one residue at a time and challenged with an Al 8 PM library.
  • the set of all three single-site revertants for each mutant is challenged with the Al 8 PM library, and selected at a low-to- moderate stringency, e.g. , antibiotic concentrations on which a non-cleaving negative control light chain confers a plating efficiency of ⁇ 10 "4 . It may be expected that many members of the PM library should be selectable under these conditions.
  • the stringency can then be raised to identify the most active Al 8 variants and their cognate single-site target revertants.
  • the best selected Al 8 variants can then be used as the parental sequence for a new PM library, which is made with the same parameters, i.e., coding change frequency and doping codons, as the original.
  • This library is then used to challenge the cognate single-site revertants of the target with each of the remaining two sites reverted. Again, multiple target-cleavers should be selected from this library, and the most active are identified with their cognate two-site target revertants by their selectability at the highest stringency. This process is repeated one final time with the best second-round Al 8 variant(s) serving as parental sequences for a new PM library, which is then used to challenge the wild-type target sequence.
  • this step-wise process might succeed where the other strategies have failed if, as expected, the failure is due to the abundance of selectable sequences in the original PM library being below the false negative threshold of the system, i.e., the abundance at which recovery is below 50%.
  • suitable target- cleaving light chains Once one or more suitable target- cleaving light chains have been obtained, they can be combined with human Fd chains as described above for selection of antibody Fab fragments with enhanced target-cleaving activities.
  • the present invention can also be used to engineer desired target specificities into human proteases.
  • proteases As mentioned above, -2.5% of the coding sequences in the human genome encode proteases, and many of these may be amenable to engineering for altered substrate specificity, particularly those which are present in the circulation such as proteases involved in the complement or coagulation cascades, tissue remodeling, or the regulation of vascular tone.
  • PM can be applied to positions surrounding the active site of the protease, and the resulting protease PM library is co-expressed, e.g., in the E. coli periplasm with the desired target protein or peptide inserted between BLIP and ⁇ -lactamaseE104K, in the proteolytic activity reporter system of the invention. The desired activity is then selected by activation of the reporter, e.g., its ability to confer antibiotic resistance on the cells. Definitions
  • Antibody refers to a polypeptide comprising at least a heavy chain variable region and a light chain variable region that together specifically bind and recognize an antigen, the variable regions being specified by immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chain variable regions respectively.
  • Antibodies exist, e.g., as intact immunoglobulins, as a number of well- characterized fragments produced by digestion with various peptidases, or as well- characterized fragments produced by recombinant gene expression.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CHl (Fd fragment) by a disulfide bond.
  • the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially Fab with part of the hinge region (see Fundamental
  • antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
  • the term antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).
  • Binding refers to the non-covalent adherence of molecules to one another, for example, enzymes to substrates, antibodies to antigens, DNA strands to their complementary strands. Binding occurs because the shape and chemical natures of parts of the molecules surfaces are complementary.
  • Binding affinity is generally expressed in terms of equilibrium association or dissociation constants (K 3 or K d , respectively), which are in turn reciprocal ratios of dissociation and association rate constants (k ⁇ j and k a respectively).
  • equivalent affinities may comprise different rate constants, so long as the ratio of the rate constants remains the same.
  • a “complex” as used herein refers to an assemblage of components that are linked, either covalently or non-covalently, e.g., via a peptide bond, or via a binding interaction. As appreciated by one of skill in the art, components that are linked by a binding interaction will typically be in an equilibrium, depending on the affinity and concentration of the components.
  • Domain refers to a unit of a protein or protein complex, comprising a polypeptide subsequence, a complete polypeptide sequence, or a plurality of polypeptide sequences where that unit has a defined function.
  • the function is understood to be broadly defined and can be catalytic activity, structural activity, binding activity, or can have a stabilizing effect on the structure of the protein.
  • Domain also refers to a structural unit of a protein or protein complex, comprising one or more polypeptide sequences where that unit has a defined structure which is recognizable within the larger structure of the native protein.
  • the domain structure is understood to be semi-autonomous in that it may be capable of forming autonomously and remaining stable outside the context of the native protein.
  • Domain as so defined may also be called a "subdomain”.
  • expression vector includes vectors which are capable of expressing nucleic acid sequences contained therein, i.e., any nucleic acid sequence which is capable of effecting expression of a specified nucleic acid code disposed therein (the coding sequences are operably linked to other sequences capable of effecting their expression). Some expression vectors are replicable in the host organism either as episomes or as an integral part of the chromosomal DNA.
  • a useful, but not a necessary, element of an effective expression vector is a marker encoding sequence—i.e. a sequence encoding a protein which results in a phenotypic property (e.g. tetracycline resistance) of the cells containing the protein which permits those cells to be readily identified.
  • Heterologous when used with reference to portions of a protein, indicates that the protein comprises two or more domains that are not found in the same relationship to each other in nature.
  • a protein e.g., a fusion protein or a conjugate protein, contains two or more domains from unrelated proteins arranged to make a new functional protein.
  • Heterologous may also refer to a natural protein when it is found or expressed in an unnatural location such as when a mammalian protein is expressed in a bacterial cell.
  • heterologous is used with reference to portions of a nucleic acid, e.g., an expression vector, the term indicates that the nucleic acid comprises regions that are not found in the same relationship to each other in nature.
  • An "inhibitor” refers to a molecule that can inhibit the reporter when both are in the reporter complex.
  • a "low-affinity inhibitor” is a relative term referring to an embodiment of the inhibitor where the inhibitor has a K ⁇ i (equilibrium dissociation constant) for the reporter which is at least ten- fold higher than the working concentration of the inhibitor, such that the inhibitor cannot bind to the reporter to an appreciable extent without a heterologous mechanism for bringing the two together.
  • interaction refers generally to attractive physical interactions, i.e., the binding of two or more molecules into a supra-molecular complex which is stable in the sense that each component has an affinity for at least one other member of the complex corresponding to & Kj>l mM.
  • Binding or "interacting” as used herein refers to noncovalent associations, e.g., hydrogen bonding, ionic bonding, electrostatic bonding, hydrophobic interaction, Van der Waals associations, and the like.
  • library of expressed sequences refers to any population of nucleotide sequences which are derived from messenger RNA, and which are therefore understood to encode polypeptide sequences, or fragments thereof, that are produced naturally in cells.
  • a “linker” or “spacer” refers to a molecule or group of molecules that covalently connects two molecules, such as a binding pair member and a reporter or an inhibitor, and serves to place the two molecules in a preferred configuration, e.g., so that a reporter can interact with an activator or inhibitor with minimal steric hindrance from a binding pair member, and a binding pair member can bind to a binding partner with minimal steric hindrance from the reporter or inhibitor.
  • a “flexible linker” refers to a peptide linker of any length in which the amino acid composition minimizes the formation of rigid structure by interaction of amino acid side chains with each other or with the polypeptide backbone. Typically a “flexible linker” is rich in glycine. An example of such a linker has the composition (GIy 4 SCr) x , where "x" may typically vary from 1 to 10.
  • Link or “join” or “fuse” refers to any method of functionally connecting peptides, typically covalently, including, without limitation, recombinant fusion of the coding sequences, and covalent bonding (e.g., disulfide bonding).
  • an inhibitor molecule is typically linked or joined or fused, often using recombinant techniques, to a target polypeptide and reporter.
  • "Linked” may also refer to a non-covalent physical association, particularly one which is constitutive under operating conditions.
  • each component is linked to at least one other component, either covalently, e.g., via peptide linkage, or non-covalently, via high-affinity binding interaction.
  • operably linked refers to a linkage of polynucleotide or polypeptide elements in a functional relationship.
  • a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • polypeptides are said to be operably linked when they are placed in a functional relationship, e.g., when a reporter and inhibitor are placed in sufficient proximity to allow inhibition of the reporter by the inhibitor.
  • Recombinant nucleic acid refers to a nucleic acid in a form not normally found in nature. That is, a recombinant nucleic acid is flanked by a nucleotide sequence not naturally flanking the nucleic acid or has a sequence not normally found in nature. Recombinant nucleic acids can be originally formed in vitro by the manipulation of nucleic acid by restriction endonucleases, or alternatively using such techniques as polymerase chain reaction.
  • nucleic acid once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non- recombinantly, are still considered recombinant for the purposes of the invention.
  • Recombinant polypeptide refers to a polypeptide expressed from a recombinant nucleic acid, or a polypeptide that is chemically synthesized in vitro
  • reporter refers to any protein that produces a detectable signal, including, but not limited to detectable signals such as fluorescence, enzymatic activity, a selectable phenotype (e.g., antibiotic resistance), a screenable phenotype, or that produces an activity that results in a phenotypic change or provides a functional product that is detectable.
  • reporter also refers to a fragment or subsequence of a reporter, or a subunit of a reporter, that retains reporter activity, i.e. produces a detectable signal, or that confers a selectable phenotype.
  • reporter complex refers to a complex comprised of the reporter, the inhibitor, and a target polypeptide of interest. At least one member of the reporter complex may be in the same polypeptide chain as at least one other member. In a preferred embodiment, all three members of the reporter complex are in the same polypeptide chain. In another embodiment, the inhibitor and the target are in the same polypeptide chain.
  • single-chain antibody refers to a polypeptide comprising a Vn domain and a Vi_domain in polypeptide linkage, generally linked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser] x ), and which may comprise additional amino acid sequences at the amino- and/or carboxyl -termini.
  • a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.
  • a scFv is a single-chain antibody.
  • Single-chain antibodies are generally proteins consisting of one or more polypeptide segments of at least 10 contiguous amino acids substantially encoded by genes of the immunoglobulin superfamily (e.g., see The Immunoglobulin Gene Superfamily, A. F. Williams and A. N. Barclay, in Immunoglobulin Genes, T. Honjo, F. W. Alt, and T. H. Rabbitts, eds., (1989) Academic Press: San Diego, Calif, pp.361-387, which is incorporated herein by reference), most frequently encoded by a rodent, non-human primate, avian, porcine, bovine, ovine, goat, or human heavy chain or light chain gene sequence.
  • a functional single-chain antibody generally contains a sufficient portion of an immunoglobulin superfamily gene product so as to retain the property of binding to a specific target molecule, typically a receptor or antigen (epitope [0086]
  • target molecule or “target polypeptide” is used herein to refer to a polypeptide of interest, for which a proteolytic activity is being sought, or is to be detected.
  • a "scaffolded peptide” refers to a peptide, typically of up to about 20 amino acids in length, that is inserted into a natural protein at a location known to accept such insertions without interfering with the folding or native configuration of the protein (A Skerra, J MoI Recognit 13: 167-87, 2000).
  • the location is on the surface of the protein.
  • the peptide is not a known natural sequence, and therefore is not expected to fold into a stable structure on its own, but generally assumes a random coil structure in solution.
  • the peptide when inserted into the scaffold protein the peptide is expected to acquire some degree of stable structure by packing against the surface of the protein.
  • Such structure generally improves the ability of the peptide to bind with high affinity to other molecules, such as other proteins.
  • Many proteins may serve as scaffolds for random peptide libraries.
  • surface loops between elements of secondary structure such as .alpha. -helixes or strands of a .beta.-sheet may accept such insertions without significant perturbation of folding or structure.
  • proteins that have been used as scaffolds include, but are not limited to, thioredoxin (or other thioredoxin-like proteins), nucleases (e.g., RNase A), proteases (e.g., trypsin), protease inhibitors (e.g., bovine pancreatic trypsin inhibitor), antibodies or structurally-rigid fragments thereof, and other domains of the immunoglobulin superfamily.
  • nucleases e.g., RNase A
  • proteases e.g., trypsin
  • protease inhibitors e.g., bovine pancreatic trypsin inhibitor
  • Example 1 Detection of proteolytic activity against a model target using the Proteolytic Activity Sensor of the invention.
  • the Al 8 human VK light chain was co-expressed in E. coli with a fusion of BLAC(E 104K) and BLIP in which two copies of the Al 8 substrate tripeptide PFR were inserted into the middle of the linker between BLAC and BLIP: The sensor and the Al 8 light chain, or a negative control kappa light chain containing a non-proteolytic VK domain linked to the same kappa constant region, were expressed from separate compatible vectors, as described above and illustrated in Figure 4.
  • the doubly-transformed cells were then plated on solid rich medium (2xYT; see Sambrook and Russell, 2001), containing 400 ⁇ M IPTG, increasing amounts of ampicillin from 0 ⁇ g/ml to 400 ⁇ g/ml, and arabinose at 0%, 0.002% and 0.2%. After overnight growth at 33 0 C, plating efficiencies (PE) for each set of conditions were determined as the ratio of colonies to doubly-transformed cells plated. The results are shown in Figure 9.
  • Al 8 variants in a PM library with high enough activity should be enriched against wild-type Al 8 and unimproved variants up to 1000-fold.
  • Induction of A18 with 0.2% ara does not activate more than a small fraction of the ⁇ - lactamase present because reactivation by co-expression of the BLAC(E 104K) - BLIP fusion with an excess of a decoy, BLAC(S70A), which is inactive but which binds BLIP with 1000-fold higher affinity than BLAC(E 104K) does, leads to quantitative plating on >800 ⁇ g/ml ampicillin (not shown).
  • proteolytic activity sensor should be capable of sensitive discrimination of proteolytic activities over a wide dynamic range from wild-type Al 8 at the low end to >1000-fold higher activities at the high end.
  • this example demonstrates the ability of the TFS proteolytic activity sensor to identify target-cleaving activities against large backgrounds of non-target-cleavers.
  • Example 2 Construction of a human proteolytic light chain library based on the Al 8 VKII germline gene with at least IQ 9 independent clones.
  • the PM library for the human germline Al 8 VK domain was constructed from two degenerate oligonucleotide primers, which were used to amplify the sequence from ⁇ 20 bp upstream from the C -terminus of Framework 1 (FRl) through the end of FR4. This product was then amplified a second time by overlap extension PCR with a fragment containing the coding sequence for all of FRl, so that the two fragments overlap in the C-terminal region of FRl.
  • the final product contains unique restriction sites for ligation into the expression vector shown in Figure 4C, in frame with the N-terminal signal peptide and C-terminal kappa constant region (CK).
  • the expression vector is a modified pBAD vector (Guzman et al., 1995) based on pBR322 with the tunable arabinose operon promoter (P BAD ) for precise control of light chain expression, and kanamycin resistance for plasmid maintenance.
  • the pelB signal peptide is appended to the N-terminus of the VK for efficient translocation to the periplasm, and a His 6 tag is present at the C-terminus of CK for expression analysis and purification (Janknecht et al., 1991). Standard procedures are used for all cloning and analyses of structure and expression (Sambrook and Russell, 2001).
  • the degenerate oligonucleotide primers for PM library construction are designed with the aid of a software tool (Balint and Larrick, 1993).
  • the operator specifies the number of sites to be diversified, the coding change frequency, and the desired library size.
  • the coding change frequency is then adjusted to maximize diversity, and the program computes the binomial distribution of « coding changes among the diversified sites, and the number of permutations and clones per permutation in the library for each value of n.
  • the sets of amino acids to be sampled at each position are selected, and the program computes the required doping codon for each position.
  • the program then computes the precise composition of the nucleoside phosphoramidite mixtures required at each position of the coding sequence during primer synthesis to achieve the prescribed frequency and identity of each coding change at each position.
  • FIG. 6-8 The properties of the PM library for the diversification of CDRl and CDR3 of the proteolytic human VK domain Al 8 are illustrated in Figures 6-8.
  • the sequence of Al 8 (minus the 12 FR4 residues) is shown at the top of Figure 6, with catalytic triad in magenta, and the 11 positions selected for PM in blue-green.
  • Figure 6A (side view) and 6B (top view) show the 3-dimensional locations of the catalytic triad (Dl, S27a, and H93) and the surrounding PM sites, respectively.
  • the PM sites were selected for their potential impact on substrate recognition and on the rate of acyl adduct hydrolysis. Notice the density of sites around the S27a hydroxyl.
  • the acyl adduct hydrolysis rate can be accelerated by several mechanisms, including (1) electrophilic activation of the ester linkage toward hydrolysis, (2) nucleophilic activation of water for attack at the ester carboxyl, (3) destabilization of the acyl adduct by repulsive contact, and (4) increased exposure of the ester linkage to the aqueous environment. Strongly conserved positions in this region were avoided since they may play essential structural roles.
  • nt 1- and 2-nucleotide
  • Figure 8 shows the doping codons to be used at each mutagenized position in CDRl and CDR3 of the Al 8 VK domain.
  • the parent amino acid pAA
  • its preferred codon Code
  • Dope selected doping codon
  • the library is validated by sequencing at least 50 clones through the VK domain to determine the frequency of intact reading frames, and to confirm the prescribed coding change frequencies. At least 80% of reading frames should be intact and the coding change frequencies should be between 50 and 65% with the prescribed amino acid distribution. If necessary, adjustments can be made in the cloning strategy or oligonucleotide synthesis to achieve the desired library size and properties.
  • At least 20 clones of the library should be examined by immuno-blotting after PAGE using enzyme-conjugated anti-His 6 tag antibody to assess the soluble expressibility of the full-length light chains of the library.
  • soluble expression is not expected to vary greatly from that of the light chain bearing the germline Al 8 VK, since the few mutations per molecule will be in the most variable region of the antibody surface, and not in the structurally important interior.
  • Example 3 Construction of the TFS proteolytic activity sensor with the Botulinum neurotoxin (BoNT) serotype A light chain (LC) as the Target,
  • BoNT Botulinum neurotoxin
  • BoNTs have been classified by the Centers for Disease Control and Prevention as one of the six highest-risk agents for bioterrorism (the "category A agents").
  • categories A agents the highest-risk agents for bioterrorism
  • MAb monoclonal antibody
  • Oligoclonal MAbs have shown greater promise (Nowakowski et al., 2002), but the large doses likely to be required for efficacy would make the costs per dose of producing such cocktails by mammalian cell fermentation prohibitive. Furthermore, the development of MAb therapy for botulism is complicated by the fact that there are seven BoNT serotypes (A to G) (Hatheway, 1995) that show little, if any, antibody cross-reactivity. [0100] In a preferred application, the present invention is used to select and optimize human antibody Fab fragments with high proteolytic activity against the BoNT serotype A light chain (LC).
  • LC light chain
  • the LC is the catalytic subunit of the toxin, proteolytically inactivating intra-cellular proteins which mediate acetylcholine exocytosis (Lalli et al., 2003; Montecucco and Schiavo, 1995).
  • Fabs are preferred over the light chain alone because, though the active site is expected to reside in CDRs of the light chain, its activity, specificity, and/or stability may be substantially enhanced by providing an appropriate heavy chain Fd fragment, which can make significant contributions to the association rate, proteolytic rate, and/or turnover rate of the abzyme, as well as to its stability.
  • a truncated form of the BoNT serotype A light chain (less 8 N-terminal and 32 C- terminal residues) is used as the target because it is more stable and expresses better in E. coli, while remaining catalytically active (Kurazono et al., 1992).
  • a synthetic DNA molecule corresponding to the optimized coding sequence for the 407-residue truncated LC is obtained commercially ⁇ e.g., Blue Heron Biotechnologies, Inc, Seattle, WA), and used as template for PCR amplification of the LC with primers which include restriction sites for ligation into the linker between BLIP and BLAC(E 104K) in the correct reading frame.
  • the final structure of the 95 kDa sensor fusion protein is BLIP-(G 4 S) 3 -LC-(G 4 S) 3 - BLAC(E104K) (see Figure 3).
  • this gene is preferably expressed from a lactose operon promoter in a pBR322-derived vector, which will provide a signal peptide at the N-terminus of the sensor for efficient translocation into the periplasm.
  • the ligation product is then transformed into E. coli TOPlOF' cells and plated onto chloramphenicol.
  • the sensor gene is recovered from several colonies by colony PCR and sequenced to confirm the accuracy and integrity of the expression cassette.
  • Cells with the correct construct are then plated onto increasing concentrations of ampicillin in the presence of ImM IPTG to induce maximum expression to determine the background BLAC(El 04K) activity of the auto-inhibited complex.
  • western immuno-blot analysis is performed on cells grown in suspension culture to assess the expression level and integrity of the sensor fusion protein.
  • BLAC(E104K) It is useful to test the reactivatability of BLAC(E104K) in the sensor construct. This is done by co-expressing the sensor with a S70A mutant of BLAC. BLAC(S70A) is catalytically inactive, but binds BLIP with wild-type affinity. Thus, it has 1000-fold higher affinity for BLIP than does BLAC(E 104K), and when expressed at moderate levels ⁇ e.g., from pBAD promoter in pl5A vector, fully induced by e.g., 0.2% arabinose) in the same cells as the sensor, it should completely displace BLIP from BLAC(E 104K) (Balint and Her, 2002b).
  • the difference in plating efficiencies (PE) on ampicillin with and without arabinose, i.e., with and without BLAC(S70A), will approximate the dynamic range of the selection system.
  • the PE with BLAC(S70A) expressed should still be 100% at ampicillin levels where the PE in its absence is ⁇ 10 "6 , i.e., less than 1 colony per million cells plated.
  • VK domains For rapid BoNT-neutralizing activity in the circulation, it would be desirable to have LC-cleaving VK domains, as these would have enhanced short-term pharmacokinetics over the four-fold larger Fab fragments. However, it may be possible to obtain Fab fragments with sufficiently higher activity and/or stability to offset the pharmacokinetic advantage of the free VK domains. Thus, VK domains are selected, optimized, and tested first, and if necessary, the best VK domains are then be combined with an unselected human Fd repertoire for selection of Fabs with enhanced BoNT LC-cleaving activities.
  • At least lO ⁇ g of purified, desalted VK PM library expression vector plasmid DNA representing 10 9 -10 10 independent clones is mixed with a molar equivalent of purified, desalted BoNT LC proteolysis sensor expression vector plasmid DNA, and transfected into at least 10 10 -10 1 ! E. coli TOPlOF' cells by high-voltage electroporation (Dower et al., 1988). This may require many electroporations over several days. After a one-hour recovery, an initial enrichment of cells expressing target-cleaving activities is accomplished by growing the library in suspension cultures.
  • the doubly-transformed cells are inoculated at 10 8 cells per ml into 2xYT broth (Sambrook and Russell, 2001) containing 0.4 mM IPTG and 0.1% arabinose and 100 ⁇ g/ml ampicillin. After overnight incubation in an O 2 -rich atmosphere with just enough agitation to keep the cells in suspension, cells not expressing sufficient target-cleaving activity will die, and cells expressing sufficient activity will activate enough BLAC to allow exponential growth. The surviving cells are then washed and spread at a density of ⁇ 10 6 cells per cm 2 onto agar plates containing 2xYT medium, 0.4 mM EPTG, and the pre-determined ampicillin and arabinose concentrations on which the sensor alone has a PE of ⁇ 10 "4 . An aliquot of the cells is also plated onto kanamycin (kan) and chloramphenicol (cam) alone to determine the number of doubly transformed cells.
  • kan kanamycin
  • cam chloramphenicol
  • Example 5 Evaluation and optimization of BoNT-cleaving VK selected in Example 3 with respect to enzyme kinetics, activity against the serotype A holotoxin, and activity against the LCs of other BoNT serotypes [0106]
  • Each selected BoNT LC-cleaving VK is first tested for its ability to inactivate the proteolytic activity of the LC.
  • each VK is purified from osmotic shock lysates of overnight cultures by IMAC.
  • BoNT serotype A purified LC is obtained commercially (List Biological Laboratories, Inc, Campbell, CA; or, Metabiologics, Inc, Madison WI).
  • a fluorogenic substrate for the serotype A LC is also obtained commercially (SNAPtideTM, List Biological Labs, Campbell, CA).
  • BoNT serotype A LC cleaves SNAP-25, a protein required for acetylcholine exocytosis, at a specific site (Schmidt et al., 1998).
  • the SNAPtide LC substrate is based on the peptide sequence around this site with an N-terminal fluorophore quenched by a C-terminal chromophore. Cleavage of the peptide activates the fluorophore.
  • the serotype A LC-cleaving VK domains can also be tested for their ability to cleave the LCs of other serotypes.
  • the light chains of serotypes B, C, D, and E are available commercially (List Biological Labs, Inc; Metabiologicals, Inc). However, fluorescent substrates are not. In some cases antibodies are available, so activity could be monitored by immunoassay after PAGE, and compared in the same way to VK activity against the serotype A LC.
  • BLAC activity sensors could be produced by inserting a fragment of the substrate containing the cleavage site into the linker between BLIP and BLACE(104K).
  • This sensor is then used to measure residual LC activity after treatment with VK domains.
  • Such sensors may also be useful if VK cross-reactivity is poor because high-activity VK could be developed for each serotype, and the assays could be used to rapidly identify the serotype of a deployed toxin, so that the correct VK could be administered to victims.
  • VK cross-reactivity is poor because high-activity VK could be developed for each serotype, and the assays could be used to rapidly identify the serotype of a deployed toxin, so that the correct VK could be administered to victims.
  • the LC must go through to reach its substrate in vivo, including acidification and membrane translocation, it is unlikely that with a severed backbone it can remain active long enough to perform its function. Nevertheless, it is important to confirm the activity of the LC-cleaving VK on both the serotype A holotoxin and the holotoxin complex, i.e., the heavy and light chains with the HA component.
  • These components can be obtained commercially (List Biological Labs and Metabiologics), and the assays described above can be used to assess the activity of the LC-cleaving VK domains against them. However, it is also possible to have these tests performed by certified contract laboratories, such as Metabiologics, Inc of Madison WI.
  • the tests are expected to confirm one or more human VK domains with therapeutically promising activity for proteolytic neutralization of BoNT serotype A and possibly other serotypes as well, which will then be ready for pre-clinical evaluation of efficacy in animal models.
  • Example 6 Construction of an HS system and demonstration of its use in detection of a target-specific proteolytic activity.
  • the Al 8 human VK light chain was co- expressed in E. coli with wild-type BLAC and BLIP, in which a 30-residue peptide containing the preferred Al 8 substrate sequence PFR was inserted between Ala77 and Pro78 in the center of the inter-subdomain loop of BLIP.
  • the sensor and the Al 8 light chain, or a negative control kappa light chain containing a non-proteolytic VK domain linked to the same kappa constant region were expressed from separate compatible vectors, as described above and illustrated in Figure 4B and C, respectively.
  • the doubly-transformed cells were then plated on solid rich medium (2xYT; see Sambrook and Russell, 2001), containing 400 ⁇ M IPTG, increasing amounts of ampicillin from 0 ⁇ g/ml to 400 ⁇ g/ml, and arabinose at 0%, 0.002% and 0.2%. After overnight growth at 33 0 C, plating efficiencies (PE) for each set of conditions were determined as the ratio of colonies to doubly-transformed cells plated. The results are shown in Figure 10.
  • Al 8 variants in a PM library with high enough activity should be enriched against wild-type Al 8 and unimproved variants up to 10,000-fold.
  • Induction of Al 8 with 0.2% ara does not activate more than a small fraction of the ⁇ -lactamase present because cells expressing
  • this example demonstrates the ability of the IIS system to identify target- cleaving activities against large backgrounds of non-target-cleavers.
  • Hifumi E., Mitsuda, Y., Ohara, K. and Uda, T. 2002. Targeted destruction of the HIV-I coat protein gp41 by a catalytic antibody light chain. J. Immunol. Methods, 269, 283- 298. [0138] Hifumi, E., Okamoto, Y. and Uda, T. 1999 Super catalytic antibody [I]:

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Abstract

Cette invention concerne des biocapteurs cellulaires ayant une activité protéolytique et des procédés les utilisant pour remanier les activités protéolytiques souhaitées dans des protéines telles que les anticorps. Dans un mode de réalisation de l'invention, le biocapteur est constitué d'un polypeptide cible lié de telle manière à un rapporteur à inhibition automatique que le clivage de la cible conduit à l'activation du rapporteur. Le rapporteur préféré confère un phénotype sélectionnable sur les cellules exprimant à la fois le biocapteur et un membre quelconque d'une population de protéases candidates qui clive de manière efficace et spécifiquement la cible. Le phénotype sélectionnable permet aux candidates présentant les activités de clivage de cible souhaitées d'être récupérées avec efficacité à partir d'importantes populations de candidats.
PCT/US2008/087342 2007-12-18 2008-12-18 Systèmes et procédés de génie enzymatique WO2009079618A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140178895A1 (en) * 2011-03-23 2014-06-26 Saurabh Rajendra Nirantar Recombinant protein biosensors and a method for detecting the presence of an analyte molecule
WO2016065415A1 (fr) * 2014-10-27 2016-05-06 The University Of Queensland Biocapteur bimoléculaire auto-inhibé
EP4279607A1 (fr) * 2022-05-20 2023-11-22 Siemens Healthcare Diagnostics Products GmbH Test d'activité de l'adamts-13 renforcée par des enzymes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5258289A (en) * 1990-09-05 1993-11-02 Davis Claude G Method for the selecting of genes encoding catalytic antibodies
US20030157579A1 (en) * 2002-02-14 2003-08-21 Kalobios, Inc. Molecular sensors activated by disinhibition

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5258289A (en) * 1990-09-05 1993-11-02 Davis Claude G Method for the selecting of genes encoding catalytic antibodies
US20030157579A1 (en) * 2002-02-14 2003-08-21 Kalobios, Inc. Molecular sensors activated by disinhibition

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140178895A1 (en) * 2011-03-23 2014-06-26 Saurabh Rajendra Nirantar Recombinant protein biosensors and a method for detecting the presence of an analyte molecule
WO2016065415A1 (fr) * 2014-10-27 2016-05-06 The University Of Queensland Biocapteur bimoléculaire auto-inhibé
EP4279607A1 (fr) * 2022-05-20 2023-11-22 Siemens Healthcare Diagnostics Products GmbH Test d'activité de l'adamts-13 renforcée par des enzymes

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