WO1996026733A1 - Toxines controlees par codominance - Google Patents

Toxines controlees par codominance Download PDF

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Publication number
WO1996026733A1
WO1996026733A1 PCT/US1996/002271 US9602271W WO9626733A1 WO 1996026733 A1 WO1996026733 A1 WO 1996026733A1 US 9602271 W US9602271 W US 9602271W WO 9626733 A1 WO9626733 A1 WO 9626733A1
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protein
domain
intracellular
toxin
binding domain
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PCT/US1996/002271
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WO1996026733A9 (fr
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Alexander Varshavsky
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Alexander Varshavsky
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Priority to AU51329/96A priority Critical patent/AU5132996A/en
Priority to EP96907881A priority patent/EP0812204A4/fr
Priority to JP8526307A priority patent/JPH11502407A/ja
Publication of WO1996026733A1 publication Critical patent/WO1996026733A1/fr
Publication of WO1996026733A9 publication Critical patent/WO1996026733A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • Abnormal cells differ from their normal progenitors and other cells of the same organism in a variety of ways, including protein composition.
  • virus-infected cells contain virus-specific proteins; the levels of certain cellular proteins are also altered as a result of viral infection.
  • Cancer cells which can grow at sites of their initial emergence and at distant sites that they are capable of colonizing, differ from their normal progenitors in the patterns of gene expression.
  • Some of the tumor-specific proteins are altered versions of normal proteins, in that they are encoded by genes whose mutations were among the causes of a malignant phenotype.
  • a viral genome encodes relatively few proteins. Since most of these proteins have functional counterparts in cells that the virus infects, effective antiviral drugs remain, by and large, a goal to be reached. With malignant tumors that cannot be eliminated by surgery alone, the problem of finding a drug up to the task is even more complicated, because compositional differences between a tumor cell and its normal progenitor can be subtle and quantitative rather that qualitative. In addition, cells of a tumor are often heterogeneous genetically and in protein composition. These difficulties are a major reason for the failure of present-day therapies to cure most cancers.
  • the problem is not necessarily the insufficient specificity of a drug — some of the cytotoxic compounds used in cancer therapy, for example, methotrexate and vinblastine (which bind, respectively, to dihydrofolate reductase and tubulin), are highly specific for their ligands.
  • methotrexate and vinblastine which bind, respectively, to dihydrofolate reductase and tubulin
  • a single drug target may not define the cell type to be eliminated unambiguously enough.
  • Another approach to cancer therapy involves the linking of a toxin to an antibody or another ligand (for example, a growth factor) that binds to a target on the surface of tumor cells.
  • a toxin for example, an antibody or another ligand that binds to a target on the surface of tumor cells.
  • the tumor selectivity of these drugs called immunotoxins or chimeric toxins, is often higher than that of small cytotoxic drugs such as methotrexate.
  • a surface marker that an immunotoxin recognizes may be present not only on target cells.
  • this marker is often not essential for tumorigenicity, there may be cells in a tumor that lack the marker but are still malignant. These are some of the limitations of present-day immunotoxins.
  • Yet another approach is to increase or redirect the power of the immune system to identify and selectively destroy tumor cells. Immunotherapy of cancer has a long and checkered history.
  • the subject invention is based on the discovery of a new and generally applicable set of methods and reagents for eliminating (or modifying) dangerous cells.
  • the invention relates, in one aspect, to a new class of therapeutic reagents referred to as codominance-mediated toxins.
  • These reagents include (1) an effector domain; (2) a first codominant signaling moiety located proximately to a binding domain for a first protein; and (3) a second codominant signaling moiety located proximately to a binding domain for a second protein.
  • the invention relates to a method for selectively killing or precluding division of a cell known to contain both a first protein and a second protein.
  • a DNA construct is provided which encodes 1) an effector domain; 2) a first codominant degron located proximately to a binding domain for a first protein; and 3) a second codominant degron located proximately to a binding domain for a second protein.
  • the DNA construct is introduced into the cell under conditions appropriate for expression of the intracellular degron- dependent, ligand-regulated toxin.
  • Fig. 1 is a diagram showing an tracellular degron-dependent, /igand- regulated toxzn (termed indelin).
  • An indelin designed to kill [Pl + P2 + ] cells that express proteins PI and P2, but to spare the other cell types — [Pl + P2 ], [PI " P2 + ] and [PI " P2 ' ] — contains a cytotoxic effector domain and two degradation signals (degrons) dl and d2, placed within or near two domains PI* and P2* that bind, respectively, to PI and P2.
  • Fig. 2 is a diagram showing an intracellular translocation signal-dependent, igand regulated tox (termed intralin).
  • An intralin designed to kill [Pl + P2 + ] cells but to spare the other cell types — [Pl + P2 ], [PI " P2 + ] and [PI " P2 ] — is a fusion containing an effector domain that is toxic in the cytosol but not in the nucleus and two nuclear localization signals (NLSs), placed within or near two domains PI* and P2* that bind, respectively, to PI and P2.
  • NLSs nuclear localization signals
  • FIG. 3 is a diagram showing a hybrid c ⁇ dominance-mediated toxin (termed comtoxi ).
  • a hybrid comtoxin contains: an effector domain that is active in the nucleus but not in the cytosol; a degron placed within or near the domain PI*; and a nuclear localization signal (NLS) placed within or near the domain P2*.
  • PI* and P2* bind, respectively, to intracellular proteins PI and P2.
  • this comtoxin would kill exclusively [Pl + P2 ] cells, which contain PI but lack P2.
  • PI must be located at least in the nucleus, whereas P2 must be a cytosolic protein.
  • Another constraint is that the degron dl must be active at least in the nucleus.
  • the actually indicated state of comtoxin in [Pl + Pl + ] cells requires that PI is present in both the cytosol and the nucleus, and that the degron dl is active in both of these compartments.
  • Fig. 4 is a diagram showing a split toxin. In this design, two subdomains of a toxic effector domain are separated by an insert whose sequence contains a binding site for an intracellular protein PI. Unlike the PI* domains of other comtoxins (Figs. 1-3), the Pl-binding site of a split comtoxin should be a relatively short (peptide-size) region that remains conformationally flexible unless it is bound by PI.
  • the affinity between subdomains of a toxic domain should be low enough to make their interaction substantially reversible.
  • a flexible insert between subdomains of a toxin can also contain two binding sites for intracellular proteins PI and P2, arranged so that the binding of either PI or P2 would be sufficient to impair the reconstitution of active toxin. The resulting comtoxin would kill exclusively cells that lack both PI and P2.
  • Other split-toxin designs are mentioned elsewhere.
  • the present invention is based on the identification of a new and generally applicable strategy for eliminating, or modifying, dangerous cells in a multicellular organism.
  • the main idea of this strategy is grounded in the property of codominance which is exhibited by a variety of signals that reside in biopolymers such as, for example, proteins and nucleic acids.
  • protein signals and their use in connection with the invention will be used as examples.
  • Protein signals include, but are not limited to, degradation signals (degrons) and various translocation signals, including nuclear localization signals (NLSs), which confer on a protein the ability to be transported from the cytosol into the nucleus of a cell.
  • Two signals present in a protein are termed codominant when each of these signals can exert its effect on the protein independently from, and without a significant interference by, the other signal in the protein.
  • the main idea of this invention is that the rational and novel combination of codominant signaling moieties, protein-interacting domains and an effector domain can result in the creation of a new class of drugs having previously unattainable selectivity.
  • These new pharmaceutical reagents are referred to herein as comtoxins (codominance- ediated toxins), and specific examples are shown in Figs. 1-4.
  • Figure 1 shows a comtoxin bearing degradation signals.
  • Comtoxins of this class will be referred to herein as indelins (intracellular rfegron-dependent, /igand- regulated toxins).
  • the left-hand panel of Fig. 1, labeled “Cell Type”, indicates either the absence (PI " or P2 " ), or the presence (Pl + or P2 + ) of the first protein or polypeptide (PI) or the second protein or polypeptide (P2) in the cell in question.
  • the right-hand panel shows each of the three required elements of a comtoxin in diagrammatic form.
  • a fusion protein of the type shown in the right-hand panel is delivered into cells in particular by introducing an appropriate expression vector encoding the fusion protein into the cells.
  • the toxic domain is a protein or polypeptide that is toxic at least in the cytosol of the cell.
  • the N- terminal domain of the comtoxin of Fig. 1 comprises a first degron
  • (dl) which is located proximately to a binding domain (PI * ) for a first intracellular protein.
  • the domain which is flanked by the N-terminal domain and the toxic domain comprises a second degron (d2), located proximately to a binding domain (P2 * ) for a second intracellular protein.
  • the first and the second protein are indicated in the right-hand panel as PI and P2, respectively.
  • the fusion protein is either short-lived or long-lived. If the fusion protein is long-lived, the toxic domain-containing comtoxin will persist in the cytoplasm for a period of time sufficient to kill the cell. If the fusion protein is short-lived, the toxic domain will be degraded by the highly processive ubiquitin-dependent proteolytic pathways that recognize and target either one or both of the comtoxin 's degrons, dl and d2. Whether the fusion is long-lived or short-lived is dependent upon the presence or absence of PI or P2 in the cytosol of the cell.
  • the fusion protein is short ⁇ lived due to the presence of the unobstructed (unmasked) degrons dl and d2. If either PI or P2 is present, but not both, the fusion protein is still short-lived due to the fact that one of the two codominant degrons remains unobstructed
  • the comtoxin of Fig. 1 is designed to kill cells that express both PI and P2, but spare cell that lack PI and/or P2.
  • FIG. 2 Another example of comtoxin design is shown diagramatically in Fig. 2.
  • the left-hand panel of Fig. 2, labeled "Cell Type" indicates either the absence (PI or P2 ) or the presence (Pl + or P2 + ) of the first protein (PI) or the second protein (P2) in the cell in question.
  • Panel A of Fig. 2 relates to a comtoxin molecule bearing a cytosol-specific toxic domain
  • Panel B relates to a comtoxin molecule bearing a nucleus-specific toxic domain.
  • the intracellular signaling domain is a nuclear localization signal (NLS).
  • Molecules of comtoxin bearing at least one NLS which is not sterically masked will be transported from the cytosol to the nucleus.
  • the comtoxin shown in Fig. 2, Panel A will be transported to the nucleus if neither PI nor P2, or if either PI or P2 (but not both) are present in the cytosol. Since the toxic domain of this comtoxin is cytosol-specific (see below), the toxic effect will result if, and only if, the comtoxin is retained in the cytosol, and this will occur only if both codominant NLSs are sterically masked (i.e. , only when both PI and P2 are present in the cell's cytosol).
  • Comtoxins of the type shown in Fig. 2 are referred to herein as intralins (mtracellular tr ⁇ nslocation signal-dependent, ⁇ gand-regulated toxins).
  • Panel B of Fig. 2 shows the inverse situation where the effector domain of an intralin is toxic in the nucleus but not in the cytosol (see below).
  • This type of intralin would kill cells that lack PI or P2, or both of them — the selectivity opposite to that of the intralin considered above (Fig. 2 A versus Fig. 2B).
  • a comtoxin bearing NLSs would stay in the cell's cytosol if, and only if, both PI and P2 are present in the cytosol, masking both NLSs of the intralin (Fig. 2B).
  • an indelin a comtoxin bearing degrons
  • conditional toxins of the present invention which bear a single P*-type domain represent a new drug design as well. While their selectivity is limited to one protein target, this target is intracellular, in contrast to the cell surface-expressed targets of present-day immunotoxins (also called chimeric toxins).
  • present-day immunotoxins also called chimeric toxins.
  • the single-P*-type-domain conditional toxins of the present invention are "natural" intermediates in the construction of multiple- P*-type-domain comtoxins, and should also be useful as drugs under conditions in which the targeting of a single, predetermined intracellular protein is sufficient for the goals of a given therapy or cytotoxic cell selection in a mammalian cell culture in vitro.
  • a hybrid comtoxin Another type of comtoxin, referred to herein as a hybrid comtoxin, is shown diagrammatically in Fig. 3.
  • This type of comtoxin bears both degron and NLS signaling moieties.
  • the simplest comtoxin of this class, shown in Fig. 3 contains an effector domain that is toxic in the nucleus but not in the cytosol; a degron placed within or near domain PI * ; and an NLS placed within or near domain P2 * .
  • the PI' and P2 * domains should be able to bind, respectively, to intracellular proteins PI and P2.
  • a pure indelin or intralin
  • this "hybrid" comtoxin would kill exclusively cells that contain nuclear protein PI but lack cytosolic protein P2, for only in such cells would the comtoxin be both nuclear (because its NLS is not masked, owing to the absence of P2) and long-lived (because its degron is masked by the PI -PI* complex).
  • FIG. 3 illustrates a class of conditional toxins whose mode of selectivity addresses, in particular, this problem.
  • a single-domain protein whose subdomains are separated (at a surface loop) by a conformationally flexible insert can adopt a (nearly) normal conformation, in which the insert is extruded to the outside of the folded domain.
  • ubiquitin a 76-residue protein
  • Fig. 4 The idea of a "split" toxin (Fig. 4) stems from these and analogous data, and also from the concept that the extent of conformational flexibility of an insert between two subdomains of a protein can influence both kinetic and equilibrium aspects of the protein's folding.
  • a conformationally rigid insert would be expected to perturb or preclude the interaction between the protein's subdomains.
  • two subdomains of a toxic domain are separated by a sequence that contains a binding site (PI * ) for an intracellular protein PI.
  • the PI * site of a split comtoxin should be a relatively short "peptide-size" ( ⁇ 10 to -40 residues) region that remains conformationally flexible unless it is bound by PI.
  • the construct of Fig. 4 would be toxic in cells that lack PI but relatively nontoxic in Pl-containing cells.
  • a flexible insert between subdomains of a toxin can also contain two binding sites for intracellular proteins PI and P2, arranged so that the binding of either PI or P2 would be sufficient to impair the reconstitution of active toxin.
  • the resulting comtoxin would kill exclusively cell that lack both PI and P2.
  • a split toxin containing a single PI -binding site represents a novel design and useful therapeutic agent, it is not yet comtoxin in the strict definition, because no codominance and multiple protein binding sites are involved here as yet.
  • the simplest "true" split comtoxin would contain at least two protein- binding sites for proteins PI and P2.
  • the construct shown in Fig. 4 will be referred to as a "comtoxin" as well.
  • the affinity between subdomains of a toxic domain should be low enough to make their interaction substantially reversible. This would preclude irreversible activation of the toxic domain before its encounter with PI (Fig. 4).
  • the affinity between subdomains of the toxic domain can be adjusted, if necessary, through mutational alterations analogous to those that have been used to adjust the affinity between the subdomains of ubiquitin (Johnsson and Varshavsky, Proc. Natl. Acad. Sci. USA 91: 10340 (1994)).
  • a split-toxin design utilizes barnase, a ribonuclease secreted by the bacterium Bacillus amyloliguefaciens.
  • Bamase is a 110-residue protein lacking disulfide bonds. It has been studied extensively as a model for protein structure and folding. In particular, it has been shown that two fragments of barnase (residues 1-36 and residues 37-110) can reassociate upon mixing to form the active enzyme with a nearly normal structure and thermal stability (Sanco and Fersht, J. Mol. Biol. 224: 741 (1992)).
  • SH2 domains are present in a variety of regulatory proteins, including the cytosol- exposed regions of transmembrane receptors.
  • a common feature of SH2 domains is their ability to form tight complexes with sequences containing a phosphotyrosine residue, with each SH2 domain recognizing a specific phosphotyrosine-containing sequence motif.
  • a split toxin that comprises a split barnase with a relatively short (10-20 residues) linker sequence that connects the two barnase subdomains and binds to a specific SH2-containing intracellular protein
  • the activity of this toxin is modulated by the binding of an SH2-containing intracellular protein to the peptide linker, whose conformation (flexible in the absence of a bound SH2 domain, relatively rigid in its presence) would determine the efficiency of reconstitution of active barnase and hence the overall activity of this barnase- based split toxin.
  • a more complex split comtoxin can be designed, for example, to distinguish [Pl + P2 ] cells from other cells types even if both PI and P2 are nuclear proteins.
  • this comtoxin would also bear a nonconditional NLS, a P2 * domain — the ligand of nuclear protein P2, and an adjacent degron that can be masked by a complex between P2 and P2 * .
  • This split comtoxin would kill exclusively cells that contain nuclear protein P2 but lack nuclear protein PI, for only in such cells would the comtoxin be long-lived (because a degron is masked by the P2-P2 * complex) and bear an active toxic domain (because the PI '-containing insert between subdomains of the toxic domain is not bound by PI and therefore remains flexible). Cytosol- specific versions of these designs are possible as well. Since other polymers, for example, RNA, can also fold into ligand-binding domains and bear signals such as degrons, nucleic acid-based comtoxins should also be feasible. Having discussed specific examples of comtoxins, it is important to more fully discuss their elements.
  • comtoxins are amino acid copolymers (e.g., fusion proteins) which include at least three elements: (1) an effector domain; (2) a first codominant intracellular signaling moiety located proximately to a binding domain for a first intracellular protein; and (3) a second 5 codominant intracellular signaling moiety located proximately to a binding domain for a second intracellular protein.
  • first and second intracellular protein be different.
  • intracellular protein should be understood to encompass intracellular peptides and polypeptides as well.
  • Comtoxins can be produced by any of the known methods for producing an amino acid copolymer having a predetermined sequence identity.
  • the preferred method for constructing and producing a comtoxin employs recombinant DNA techniques. Through the use of such conventional techniques, DNA encoding the required comtoxin elements is isolated from a biological source or sources and modified as necessary using techniques such as site-directed mutagenesis.
  • the minimum number of nucleotides required to encode an amino acid segment (e.g., a peptide, polypeptide or protein) which confers the required function is employed. Since each of the required elements of the comtoxin molecule is easily assayable, it is a matter of routine experimentation to determine the minimum length of a DNA fragment which will encode a functional comtoxin element. The resulting DNA fragments are linked together, using standard recombinant DNA techniques, yielding an open reading frame which translates into a fusion protein comprising all of the required comtoxin elements.
  • an amino acid segment e.g., a peptide, polypeptide or protein
  • interdomain linkers sequences that form a hydrophilic and flexible segment of the polypeptide chain relatively resistant to endoproteases present in the bloodstream, intercellular spaces and inside the cells.
  • linker sequence connects a light-chain antigen-binding domain to an analogous heavy-chain domain.
  • DNA expression vector which includes a transcriptional promoter and other sequences required for expression.
  • the choice of expression vectors from among the many available options is largely dependent upon the cell type in which expression is desired. As discussed more fully below, eukaryotic expression vectors are preferred for many applications.
  • a comtoxin can also be produced by expressing it, through the intermediacy of prokaryotic expression vectors, in bacteria (e.g., E. coif), purifying the resulting overexpressed protein, and contacting the purified protein with target cells directly.
  • bacteria e.g., E. coif
  • purifying the resulting overexpressed protein and contacting the purified protein with target cells directly.
  • a comtoxin should also bear an additional domain that enables its translocation into the cell's cytosol.
  • cytoplasm denotes the interior of a cell outside of its nucleus
  • cytosol is the cytoplasmic milieu outside of other membrane-enclosed compartments that reside in the cytoplasm.
  • the fundamental, qualitative differences between the current chimeric toxins and comtoxins of the present invention are: (1) the current chimeric toxins are able to recognize surface markers but not intracellular ones; and (2) the current chimeric toxins are in principle incapable of multiple-target, combinatorial selectivity that is characteristic of comtoxins.
  • an effector domain is a protein or polypeptide which is able to exert a specific effect (e.g., to cause the death of a cell, or its terminal differentiation) when the effector is delivered to a predetermined intracellular location.
  • the effector domain of a comtoxin can be derived from a protein or polypeptide which acts as a toxin when delivered to a cell.
  • Many such toxins are known in the art, including, for example, the A-chain of ricin (and analogous plant toxins), the toxic domain of the Pseudomonas exotoxin and the toxic domain of diphtheria toxin.
  • proteins or polypeptides include, for example, the diphtheria toxin and the Pseudomonas exotoxin A, both of which inhibit protein synthesis by ADP-ribosylating (and thereby inactivating) elongation factor 2 (EF2).
  • EF2 elongation factor 2
  • JE-coRI has been shown to cleave nuclear DNA in vivo (in the yeast Saccaromyces cerevisiae), killing the cells (Barnes and Rine, Proc. Natl. Acad. Sci. USA 82:
  • Examples of toxic domains whose substrates are present in both the cytosol and the nucleus include, in addition to the A-chain of ricin, ribonucleases such as RNAase A and barnase, which have been used to produce conventional chimeric toxins (Rybak et al. , J. Biol. Chem. 266: 21202 (1991); Prior et al , Cell 64: 1017 (1991)). Since the goal of therapy is to render dangerous cell harmless, the set of useful effectors is not confined to cytotoxic proteins. This set includes, for instance, transcription factors whose presence in a cell results in growth arrest and terminal differentiation (Weintraub, Cell 75: 1241 (1993)).
  • a muscle-specific transcription factor such as MyoD or myogenin in nonmuscle cells causes them to differentiate into muscle-like cells. Under normal conditions, these proteins are expressed only in the muscle and only at appropriate times.
  • a comtoxin whose effector domain is based on MyoD, myogenin or an analogous transcription factor should be able to cause a terminal differentiation of the targeted cells but not of other cells that receive the drug.
  • codominant intracellular signals With regard to codominant intracellular signals, a variety of such signals have been described in the literature. For example, a short in vivo half-life can be conferred on a protein by one or more of distinct degradation signals, or degrons (Varshavsky, Cell 64: 13 (1992); Hershko & Ciechanover, Ann. Rev. Biochem. 61: 761 (1992)).
  • the best understood intracellular degradation signal is called the N-degron (Varshavsky, Cell 64: 13 (1991)). This signal comprises a destabilizing N-terminal residue and an internal lysine (or lysines) of a protein substrate.
  • N-end rule a relation between the in vivo half-life of a protein and the identity of its N-terminal residue.
  • the lysine residue of an N-end rule substrate is the site of formation of a multiubiquitin chain, which is required for the substrate's degradation.
  • Ubiquitin is a protein whose covalent conjugation to other proteins plays a role in a number of processes, primarily through routes that involve protein degradation.
  • the recognition of an N-end rule substrate is mediated by a targeting complex whose components include a ubiquitin-conjugating enzyme (one of several such enzymes in a cell) and a protein called N-recognin or E3.
  • a targeted, multiubiquitinated substrate is processively degraded by the 26S proteasome — a multicatalytic, multisubunit protease.
  • the amino acid sequences that function as degradation signals and are transplantable to other proteins include, for example, the "destruction boxes" of short-lived proteins called cyclins (Glotzer et al , Nature 349: 132 (1991); Ciechanover, Cell 79: 13, 1994)) and two specific regions of Mat ⁇ 2, the short-lived transcriptional regulator S. cerevisiae (Hochstrasser and Varshavsky, Cell 61: 691 (1990)).
  • NLSs are short sequences (10-20 residues) rich in lysine and arginine; their steric accessibility in a target protein appears to be sufficient for their activity as nuclear translocation signals.
  • Many NLS-bearing proteins enter the nucleus shortly after their synthesis in the cytosol, but the transport of some proteins is not constitutive: their NLSs have to be "activated", often by unmasking sterically shielded NLSs.
  • comtoxins are the mammalian protein pi 10, an NLS -containing precursor of the p50 subunit of the transcription factor NF-KB.
  • pi 10 has been shown to remain in the cytosol because a region in the C-terminal half of pi 10 sterically masks an NLS located in the N- terminal half (p50 region) of the same protein.
  • the protein p50 the product of proteolytic processing of pi 10 — bears an active (unobstructed) NLS and is transported into the nucleus.
  • Other instances of subtle and precise regulation of nuclear uptake through the masking of NLSs in the NF- ⁇ B family of transcription factors have also been described (Liou and Baltimore, Curr. Op.
  • Comtoxin function is based on the fact that either the metabolic stability (in vivo half-life) of a comtoxin or its intracellular location (for example, the cytosol versus the nucleus) depend on the occupancy of the comtoxin 's binding domains (referred to as P*-type domains in this discussion) (see Figs.
  • a signaling moiety in a protein is a distinct area of the protein's surface that is recognized by a large (protein-size) targeting complex specific for a signaling moiety. Given the physical bulkiness of targeting complexes, and relatively large areas occupied by signaling moieties on the surfaces of proteins, even a partial obstruction (steric masking) of a signaling moiety in a protein by another protein globule bound nearby would be sufficient to prevent a productive binding of the signaling moiety by a respective targeting complex.
  • a P*-type domain of a comtoxin adjacent to a chosen signaling moiety such as, for example, a degron or an NLS
  • a chosen signaling moiety such as, for example, a degron or an NLS
  • the P * -type domains of a comtoxin are selected to bind to specific, predetermined, intracellular ligands (e.g., specific intracellular proteins PI, P2, etc.).
  • specific intracellular ligands e.g., specific intracellular proteins PI, P2, etc.
  • the set of intracellular ligands of a comtoxin should be chosen by carrying out a detailed survey of proteins produced by a targeted cell population.
  • the protein composition of specific cell types, in particular of normal and tumor-derived human cells, is being determined (for example, Hall et al. , Proc. Natl Acad. Sci. USA 90: 1927
  • a tumor cell lacking the wild-type version of a tumor suppressor protein (expressed in the normal progenitors of this cell) and overproducing another protein, perhaps as a result of a gene amplification that contributed to the cell's malignant phenotype.
  • many (but not all) human breast carcinomas lack the tumor suppressor protein p53 or contain its functionally inactive variants. Further, many of these tumors overproduce the protein c-Myc (in addition to several other proteins).
  • a comtoxin that kills exclusively cells which contain a PI protein but lack a P2 protein has the requisite selectivity for such a setting.
  • the PI * domain of this comtoxin would bind to c-Myc, while the comtoxin 's P2 * domain would bind to wild-type p53 (but not to its mutant variant in a given carcinoma). Even nonselective intracellular delivery of this comtoxin would kill Myc-overexpressing, p53-lacking carcinoma cells but spare most, if not all, other cells of the organism, because normal cells contain p53 — the few, if any, normal cell types that lack p53 are unlikely to overproduce c-Myc.
  • this comtoxin can be increased further, if necessary, by adding to it a degron- or NLS-containing P3 * domain that binds to a third intracellular protein, P3, chosen to sharpen the description of target cells.
  • cancer cells were found to differ from their normal progenitors by a combination of at least the following traits: the absence (or decreased production) of certain proteins expressed in the progenitor cells; the presence of mutant variants of normally expressed proteins; and an overproduction of either wild-type or mutant proteins that were not expressed (or expressed weakly) in the progenitor cells.
  • P * -type domains In selecting protein binding sites, referred to as P * -type domains, it should be recognized that a homodimerization of the P * -type domains in a single comtoxin molecule is undesirable, because it would interfere with the function of the comtoxin. Thus, a P * -type domain should be able to form a heterodimer with its intracellular partner (a PI '-type protein) while not forming a high affinity homodimer.
  • Examples of alternative P * -type domains include: (1) natural protein ligands of a Pl-type protein; (2) peptide-size fragments of the natural ligand that retain affinity for a Pl-type protein; (3) single-chain antibodies, or fragments of same, which are specific for a Pl-type protein; and (4) nonpepude, low M r ligands of a Pl-type protein. This latter possibility is confined to directly delivered comtoxins (as distinguished from comtoxins delivered through the intermediacy of expression vectors).
  • This class of P * -type domains includes single-chain antibodies to the junctional regions of tumor-specific fusion proteins produced by tumorigenic chromosome translocations. These mutant proteins form early in the evolution of a tumor cell lineage, are likely to be required for the malignant phenotype, and therefore are an especially pertinent class of Pl-type proteins.
  • the construction of a single-chain antibody against a specific protein or a specific region of a protein includes, first, the production of a monoclonal antibody with a requisite specificity, and, second, the assembly of a DNA fragment containing an open reading frame that encodes a single-chain counterpart of this monoclonal antibody.
  • the latter step is carried out using Polymerase Chain Reaction (PCR) to amplify and fuse in-frame the relevant non-contiguous regions of the two genes that encode the protein-binding domains of the antibody's heavy chain and the light chain in the hybridoma cells that produce the antibody.
  • PCR Polymerase Chain Reaction
  • a comtoxin For delivery via the intravascular route (Gilman et al, Eds., The Pharmacological Basis of Therapeutics (Pergamon Press, New York, 1990), a comtoxin should possess not only a toxic domain but also a domain that mediates the translocation of a protein fusion from the cell surface to the cytosol.
  • This aspect of comtoxins is confined to direct-delivery (as distinguished from expression-based) strategies, and is similar to the analogous aspects of current immunotoxins (Vitetta et al , 1mm. Today 14: 252 (1993); Pastan et al. , Annu. Review Biochem. 61: (1992); and Olsnes et al , Som. Cell Biol. 2: 1 (1991)).
  • the initial step of a comtoxin 's delivery as a protein can be made partially cell type-selective by fusing the comtoxin to a domain such as, for example, an antibody that binds to a surface marker on target cells.
  • a domain such as, for example, an antibody that binds to a surface marker on target cells.
  • the said surface marker can be present on more that just target cells without significantly increasing the comtoxin's nonspecific toxicity.
  • the conditional toxicity of a comtoxin is decided by the environment it encounters after entering the cell; therefore, a comtoxin would not affect cells whose intracellular protein "signatures" differ from the targeted one.
  • One advantage of the direct-delivery strategy is that it bypasses potential problems associated with the vector-mediated delivery of an indelin.
  • One potential drawback of the direct delivery is a larger size of a multidomain comtoxin (due to the presence of additional domains), in comparison to an otherwise identical indelin that is delivered through the intermediacy of an expression vector. Since the testing of comtoxins using either of these delivery strategies is technically straightforward, involves exclusively the existing technologies, and can be assessed directly and objectively, a prudent experimental approach would be to use both strategies in evaluating a given comtoxin, and to compare the results.
  • Comtoxins can also be delivered into cells through the intermediacy of expression vectors. Both of these approaches are a part of ongoing efforts to improve bioavailability of protein drugs used in medical interventions, from cytotoxic treatments to gene therapies.
  • the problem of insufficient selectivity is common to all of the current cytotoxic strategies: once the effector reaches its intended intracellular compartment, the cell is likely to be killed irrespective of whether it was a target or an innocent bystander.
  • one difficulty with the current immunotoxins is their nonspecific toxicity — largely, but not only, to the liver.
  • comtoxins of the present invention By contrast, even nonselective delivery of the comtoxins of the present invention would not affect most nontarget cells. This feature of comtoxins will yield a much higher therapeutic index (i.e., a much higher tolerated intensity and duration of treatments).
  • the initial step of a comtoxin 's delivery can be made selective by fusing the comtoxin to a domain that binds to a surface marker on target cells.
  • this marker can be present on more than just target cells without significantly increasing the comtoxin 's nonspecific toxicity.
  • Comtoxins designed for delivery by an expression vector would lack the "compartment-crossing" domain required for their directly delivered counterparts.
  • the vector can be either a retroviral vector, adenoviral vector, another viral vector, or simply naked DNA within a gene delivery system, for example, a liposome-based delivery system. Both viral vectors and liposome-based gene delivery systems have been successfully used in approaches to gene expression in whole animals.
  • a retroviral vector for gene therapy and other applications have been successfully used in approaches to gene expression in whole animals.
  • liposome-based gene delivery systems have been successfully used in approaches to gene expression in whole animals.
  • Several types of vectors for gene therapy and other applications are already available (reviewed by Yee et al, Proc. Natl. Acad. Sci. USA 91: 9564 (1994); see also Mulligan, Science 260: 926 (1993) and Anderson, Science 256: 808 (1992)). These vectors can be used for the delivery of comtoxins in cell cultures, in whole animals, and, with appropriate preliminary testing, in human patients as well.
  • Comtoxins can also be used in a variety of in vitro applications.
  • comtoxins in a bone marrow culture explant of a leukemia patient, can be used to selectively eliminate leukemic cells from a sample of the patient's bone marrow (for subsequent reinfusion) without perturbing the marrow's normal cells.
  • a high-dose chemotherapy with bone marrow stem cell rescue is, at present, a standard, and sometimes curative, treatment for a variety of cancers, including leukemias, lymphomas, and, more recently, glioblastomas and other metastatic solid tumors.
  • a common step in all of the current stem cell-rescue protocols involves withdrawing a portion of the patient's bone marrow prior to the marrow- ablating chemotherapy.
  • the resulting short-term in vitro culture of the marrow cells is then treated to eliminate, if possible, any malignant cells present in it before the reinfusion of the purged bone marrow back to the patient whose resident bone marrow has been ablated by a high-dose chemotherapy.
  • chimeric toxins Vitetta et al , 1mm. Today 14: 252 (1993)
  • chimeric toxins comprise a cytotoxic effector domain and a domain (typically antibody-based) that binds to a structure on the surface of target cells.
  • This design of chimeric toxins is often, but not always, sufficient for eliminating most leukemic cells (the bulk of which bears lineage-specific surface markers) from an explant culture of a bone marrow.
  • many types of tumor cells including nonhematologic cancers, are much more heterogeneous, surface marker-wise, than certain types of leukemias.
  • the comtoxins of the present invention by virtue of their higher, multitarget selectivity and sensitivity to intracellular markers (as distinguished from cell surface ones) should provide an alternative and more selective means for in vitro bone marrow purging of cancer cells.
  • Example 1 Construction and testing of an indelin
  • the testing of a [PI + P2 + ]-specif ⁇ c indelin involves determining whether this reagent, once it is introduced (directly or through the intermediacy of an expression vector) in the targeted [Pl + P2 + ] versus nontargeted ([Pl + P2 ], [PI " P2 + ], or [PI ' P2 ]) cells, will kill exclusively (or nearly exclusively) [Pl + P2 + ] cells.
  • the indelin to be designed in this Example is a ultidomain fusion whose C-terminal domain is the A-chain of ricin, linked to the nearest "upstream” domain (P2*) by a short (5 to 20 residues) linker sequence.
  • the domain P2* is linked, in turn, to the domain PI*.
  • a simple-sequence, relatively hydrophilic linker GGGSGGGSGGGSGGGS (in single-letter amino acid abbreviations) will be used initially to join these domains.
  • a first and a second degron will be positioned within or near domains PI* and P2* such that the binding of a protein to PI* inhibits the activity of the first degron and the binding of a protein to P2* inhibits the activity of the second degron.
  • the nucleic acids which encode the various elements of the indelin molecule will be isolated from naturally occurring sources and are assembled by conventional techniques within a suitable expression vector such as, for example, the pSG5 vector sold by Stratagene Inc. This vector is a 4.1 kb E. coli- mammalian cells "shuttle" plasmid containing the SV40 early promoter and other relevant features for expression of inserted genes of interest in mammalian cells.
  • the toxic domain employed will be the ricin A-chain (other cytotoxic effectors, for example, the toxic domain of the Pseudomonas exotoxin or the toxic domain of the diphtheria toxin, can also be used to construct an indelin).
  • the deduced amino acid sequence of the A-chain of ricin is provided, for example, in Funatsu et al (Biochimie 73: 1157 (1991)). In this experiment, no effort will be made to reduce the size of the toxic domain by deletion or other methods.
  • a cDNA-containing fragment encoding the complete ricin A-chain will be isolated from, for example, the plasmid pRAP229 (Ready et al, Proteins 10: 270 1991)), using conventional techniques.
  • the degradation signals (degrons), to be positioned within or immediately adjacent to the P*-type domains, can be chosen from among several presently known degrons.
  • the degradation signals employed in initial studies will be the N-degron and the degron defined by the cyclin "destruction box".
  • the N-degron has been dissected biochemically and genetically, and is understood in considerable detail (Varshavsky, Cell 69: 725 (1992)).
  • Varshavsky Cell 69: 725 (1992)
  • the portable N-degron employed in the initial studies will be the one described by Bachmair and Varshavsky (Cell 56: 1019 (1989)).
  • This N-degron comprises a destabilizing N-terminal residue, such as arginine (Arg), followed by the 45-residue sequence derived from E. coli Lac repressor.
  • Arg arginine
  • 45-residue sequence derived from E. coli Lac repressor.
  • the corresponding procedures, sequences, and the plasmids, together with their restriction maps, are described in detail by Bachmair and Varshavsky (Cell 56: 1019 (1989)).
  • the sequences involved are encoded by a specific fragment of the plasmid pAG132 (Glotzer et al , Nature 349: 132 (1991)).
  • P*-type domains that bind to at least the following intracellular proteins will be used: 1) the E6 protein of an oncogenic human papillomavirus (HPV) such as HPV16; 2) the E7 protein of the same oncogenic HPV.
  • HPV human papillomavirus
  • the E6 and E7 proteins of the oncogenic human papillomaviruses are among the preferred initial intracellular targets for the design and testing of comtoxins of the present invention.
  • HPVs human papillomavirus
  • HPV- 16 and HPV- 18 are associated with squamous intraepithelial neoplasias which are potentially precancerous.
  • more than 90% of cervical cancers can be shown to contain DNA of one of the high-risk HPV types (Scheffner et al. , Curr. Topics Microbiol Immunol 186: 83 (1995)).
  • HPV proteins E6 and E7 are consistently expressed in cervical carcinomas, strongly suggesting that the tumorigenic effect of HPV is mediated specifically by these proteins.
  • E7 protein acts by forming a specific complex with a nuclear protein called retinoblastoma protein (Rb) — a regulatory component of the networks that prevent an uncontrolled cell proliferation.
  • Rb protein thus acts as a tumor suppressor.
  • the formation of a complex between the E7 protein of HPV and Rb protein in an HPV-infected cell interferes with the tumor suppressor function of Rb thereby contributing to the malignant transformation of the corresponding cell lineage.
  • E6 specifically binds to another cellular tumor suppressor protein, called p53.
  • the formation of E6-p53 complex not only perturbs the growth-suppressing function of p53 but also results in the accelerated degradation of the E6-bound p53, decreasing p53 concentration and thereby, in effect, inhibiting the function of p53 in more ways than one (Huibregtse et al, EMBO J. 10: 4129 (1991)). No curative treatment of HPV-derived metastatic carcinomas is available at the present time.
  • P*-type domains For describing the construction of an E6/E7-specific indelin, general requirements for PI* and P2* domains of an indelin (these domains are denoted collectively as P*-type domains) are briefly considered. These requirements are as follows: (i) a P*-type domain should bind to an intracellular (cytosolic or nuclear) protein PI or P2 (these proteins, chosen by the designer, comprise the target cell's protein "signature"); (ii) a P*-type domain should not form a high-affinity homodimer; (iii) a P*-type domain should contain, either as a part of the domain or in its immediate vicinity, a sequence (degradation signal) that confers a short half-life on the indelin in the cytosol and/or in the nucleus.
  • a P*-type domain should bind to an intracellular (cytosolic or nuclear) protein PI or P2 (these proteins, chosen by the designer, comprise the target cell'
  • a suitable P*-type domain can be either a natural protein ligand of the proteins PI or P2, or, for example, a single-chain antibody that binds to a PI or P2.
  • the use of single-chain antibodies as P*-type domains eliminates the problem of homodimerizaton that a natural ligand might be prone to; it also makes possible a standardization of the design of a P*-type domain, since single-chain antibodies against different protein domains have a common "core" antibody structure.
  • the use of a single-chain antibody as a P*-type domain makes the vector-mediated delivery of an indelin technically straightforward, because no assembly of a conventional (multi-chain) antibody molecule is required with single-chain antibodies.
  • E6/E7-indelin The actual construction of an E6/E7-indelin will proceed via two routes that produce, separately at first, an E6-indelin and an E7-indelin.
  • E6-indelin and E7-indelin These single- P*-type-domain indelins can be tested and, if necessary, fine-tuned in human cell cultures that either lack or express E6 and/or E7 proteins of HPV 16 (see below).
  • E6/E7-indelin a more complex, codominance-based comtoxin (E6/E7-indelin) will be constructed from its single-P*-type-domain precursors (E6-indelin and E7-indelin).
  • an E7-indelin (or, alternatively, an E6-indelin) may actually prove sufficient for selective elimination of these carcinoma cells, in a cell culture setting and, later, in a human patient. Why then would one construct and use the more complex, codominance mediated, comtoxin-type E6/E7-indelin? The answer, discussed in the following paragraphs, is two-fold.
  • E6/E7-indelin kill exclusively cells that contain both E6 and E7, and to spare cells that lack even one of these proteins makes the comtoxin-type E6/E7-indelin much less perturbable (than a single-P*-type-domain indelin such as E6-indelin or E7-indelin) by the in vivo "noise" of relatively weak and nonspecific protein-protein interactions.
  • E6-indelin For example, if a single-chain antibody that forms a P*-type domain of E6-indelin and binds, with high affinity, to E6 protein (see below), accidentally binds (crossreacts), even with a relatively low affinity, to a normal cellular protein in a normal cell that lacks E6 and E7 proteins of HPV, this crossreaction may adversely affect the desired selectivity of E6-indelin for the E6/E7-containing carcinoma cells. The same would be true of E7-indelin.
  • the comtoxin-type E6/E7-indelin would be remarkably more resistant to this selectivity-reducing perturbation because, for it to occur, both the E6-binding and the E7-binding P*-type domains of this E6/E7-indelin must also bind to normal cellular proteins — a much less probable event in any specific cell lineage.
  • a comtoxin-type E6/E7-indelin is not only more selective than its single-P*-type-domain counterparts, but its selectivity is in addition more robust against perturbations of the kind described above.
  • E6 or E7 proteins of HPV 16 are available, but a monoclonal antibody has been prepared, thus far, only against E7 protein (Scheffner et al , EMBO J. 11: 2425 (1992)).
  • the genes encoding E6 and E7 proteins of HPV 16 (and several other strains of human papillomavirus) have been cloned by several groups; the corresponding proteins have been overexpressed in E. coli, purified to homogeneity and used as antigens to raise antibodies (Scheffner et al. , Curr. Topics Microbiol. Immunol 186: 83 (1994), and references therein).
  • a DNA fragment that encodes a single-chain counterpart of the anti-E7 monoclonal antibody 100201 will be produced using Polymerase Chain Reaction (PCR) and purified genomic DNA of the 100201 hybridoma cells to amplify and fuse in-frame the relevant noncontiguous regions of the two genes that encode the E7-binding domains of the 100201 antibody's heavy chain and the light chain.
  • PCR Polymerase Chain Reaction
  • the PCR-mediated construction of a DNA-based open reading frame (ORF) encoding an antibody specific for a hybridoma of interest is a routine procedure for those skilled in the art.
  • the resulting single-chain 100201 antibody-encoding ORF will be linked in-frame, using standard techniques for manipulating recombinant DNA, to a reading frame encoding the A chain of ricin (see above), using the flexible, relatively protease-resistant and hydrophilic linker GGGSGGGSGGGSGGGS — one of the many suitable linkers described in the prior art, in particular in the art of producing single-chain antibodies (Whitlow and Filpula, Methods 2: 97 (1991)).
  • the resulting larger ORF will encode a fusion of the anti-E7 single-chain antibody (a P*-type domain of the indelin being designed) to the toxic domain (A-chain) of ricin, with the latter domain being the C-terminal domain of the fusion.
  • the N-degron of Bachmair and Varshavsky comprises a destabilizing N-terminal residue, such as arginine (Arg), followed by the 45-residue sequence derived from E. coli Lac repressor. Both the nucleic acid sequence and the corresponding amino acid sequence of this N-degron have been described by Bachmair and Varshavsky (Cell 56: 1019 (1989)).
  • an N-degron contains a destabilizing N-terminal residue, it should be placed, by definition, at the N-terminus of an indelin fusion construct.
  • ubiquitin fusion technique developed earlier by the author of the present invention (Varshavsky, Cell 69: 725 (1992); US patents issued to Varshavsky et al , including U.S. Patent Numbers 5, 132,213; 5,212,058; 5,122,463; 5,093,242 and 5, 196,321).
  • Ubiquitin is a 76-residue protein essentially unchanged from yeast to humans. Many previously sequenced recombinant DNA plasmids encoding ubiquitin are available (for example, the plasmid pUB23 by Bachmair et al , Science 234: 179 (1986)).
  • the fully assembled DNA-based ORF of the ⁇ 7-specific indelin will encode the following protein regions or domains, beginning from the N-terminus:
  • an internal degradation signal such as, for example, the cyclin destruction box (Glotzer et al , Nature 349: 132 (1991)), will be positioned adjacent to the E6-binding single-chain antibody domain.
  • the rest of the construction protocol will be essentially identical to the one described above for the construction of the E7-indelin.
  • the fully assembled DNA-based ORF encoding the E6-specific indelin will encode the following protein regions or domains, beginning from the N-terminus:
  • the DNA ORF encoding the E7-indelin will be positioned downstream of a moderately active promoter such as the metalothionein or Moloney leukemia virus promoter within a mammalian expression vector such as, for example, the pE7Mo plasmid (Barbosa et al, EMBO J. 9: 153 (1990)).
  • a moderately active promoter such as the metalothionein or Moloney leukemia virus promoter
  • a mammalian expression vector such as, for example, the pE7Mo plasmid (Barbosa et al, EMBO J. 9: 153 (1990)).
  • E7-indelin-expressing plasmid will be introduced, under identical conditions (see below), into two otherwise identical test cell cultures that differ by the presence of E7 protein.
  • Such pairs of E7-lacking and E7-expressing human cultures have been described, in particular, by Barbosa et al. (EMBO J. 9: 153 (1990)); they can also be produced de novo from any HPV-lacking human cell line by stably transfecting it with the cloned E7 HPV gene, as described previously for E7 and E6 HPV proteins (for example, Kessis et al. , Proc. Natl Acad. Sci. USA 90: 3988 (1993)).
  • the region of the DNA ORF encoding the cytotoxic (ricin) domain of the indelin will be (temporarily) replaced, using standard recombinant DNA techniques, with a short DNA region encoding one of several epitopes for commercially available monoclonal antibodies, for example the 9-residue epitope, termed "ha", derived from hemagglutinin of the influenza virus and the corresponding anti-ha monoclonal antibody available from Boehringer Inc.
  • This modification will make it possible to vary the distance between the N-degron and the E7-binding single- chain antibody domain of the E7-indelin without having to deal with the intrinsic toxicity of the indelin's ricin domain.
  • the adjustment protocols, for this or any other indelin will vary the distance between a degron and an E7-binding domain of the indelin.
  • the resulting constructs, in which the indelin's toxic C-terminal domain such as ricin had been replaced by an epitope tag such as ha (see above), will be expressed in either E7- containing or E7-lacking human cell cultures, using cell lines and expression vectors described above.
  • Pulse-chase analysis of the metabolic stabilities of these constructs will then be carried out to determine directly their in vivo half-lives.
  • a pulse-chase assay a set of protein molecules labeled with a radioactive tracer (for example, a radioactive amino acid) for a relatively short period of time (a "pulse") is followed by a "chase", which is accomplished through the immunoprecipitation and electrophoretic analysis (and quantitation) of a protein of interest at different times after the termination of pulse.
  • Pulse-chase analysis is a routine assay; its protocol and logic are well known to those skilled in the art (for example, Bachmair et al , Science 234: 179 (1986)).
  • the aim of the adjustment of an E7-indelin is to produce a construct that is short-lived (as much as possible) in vivo in the absence of E7 protein, and is long- lived (as much as possible) in the presence of E7 protein.
  • the final, satisfactory construct is fused to the cytotoxic domain (see above), restoring the indelin's organization.
  • the resulting E7-indelin is then retested for conditional toxicity in E7-containing and E7-lacking cell cultures as described above. It should be emphasized that even the very first, initial E7-indelin is likely to possess the required qualities.
  • the adjustment procedure would be utilized only if the metabolic properties of an initial E7-indelin are less than satisfactory according to the criteria stated above.
  • E6/E7-indelin a monoclonal antibody is available for E7 but not E6 protein (see above). Therefore one preliminary step in the construction of E6/E7-indelin will be to produce a panel of monoclonal antibodies to E6 protein (which had been purified from E6-overproducing E. coli), to choose an appropriate (highest-affinity) antibody among those produced, and to use the corresponding hybridoma cells as described above to construct an ORF encoding the corresponding single-chain antibody to E6 protein. Both steps of this protocol (the production of hybridomas secreting a monoclonal antibody of requisite specificity and the PCT-mediated construction of an ORF encoding a single-chain counterpart of this antibody) are standard procedures for those skilled in the art.
  • Intralins are comtoxins that bear translocation signals, in particular nuclear localization signals (NLSs) ( Figure 2).
  • NLSs nuclear localization signals
  • nsP2/4-intralin The P*-type domains of this intralin, termed "nsP2/4-intralin”, will be designed to bind the following "early" proteins of the Sindbis virus: a. the nsP2 protein (a site-specific protease). b. The nsP4 protein (RNA polymerase).
  • cytoplasmic (cytosolic) proteins of the Sindbis virus a plus- stranded RNA virus that infects humans and several other metazoan hosts — are among the preferred (initial) intracellular targets for the design and testing of comtoxins of the present invention.
  • the Sindbis virus is a member of the family of alphaviruses. This family has 26 presently recognized members (reviewed by Strauss and Strauss, Microbiol Rev. 58: 491 (1994)). Many strains of the alphavirus family are a serious threat to human health. For example, eastern and western equine encephalitis viruses cause fatal encephalitis in both North America and South America.
  • the Ross River virus and related alphaviruses cause epidemic polyarthritis in humans; the crippling symptoms of this disease can persist for years.
  • the Sindbis virus is nearly (though not entirely) avirulent for humans, and at the same time is closely related to virulent members of the alphavirus family such as the viruses mentioned above.
  • the Sindbis virus and other alphaviruses replicate exclusively in the cytoplasm (more specifically, in the cytosol) of infected cells. Given the logic of intralins in Figure 2A, the cytosolic localization of the Sindbis virus' life cycle is one reason for choosing this virus as the first targets for intralins.
  • the Sindbis genomic RNA is 11,703 nucleotides (nt) in length. It comprises two major regions: a nonstructural domain encoding the nonstructural proteins, including RNA polymerase, and a structural domain encoding the three structural proteins of the Sindbis virion.
  • the nonstructural (“early") proteins nsPl-nsP4 are translated as one or two polyproteins from the genomic RNA itself.
  • nsP2 which is a site-specific protease that processes (cleaves) the Sindbis polyproteins (including the polyprotein of which nsP2 is initially a part) to produce individual proteins of the Sindbis virus
  • nsP4 which is the viral RNA polymerase that replicates the Sindbis genomic RNA (reviewed by Strauss and Strauss, Microbiol Rev. 58: 491 (1994)).
  • nsP2 and nsP4 are located in the cytosol, are distinct from normal cellular proteins, are produced early in the infection cycle, and therefore are good targets of an intralin whose function would be to selectively kill virus-infected cells before the formation of significant amounts of mature Sindbis virions in these cells.
  • Sindbis nsP2 protein is a protease, its highly restricted substrate specificity render it nontoxic to mammalian cells and incapable of cleaving most antibodies, which will be used below as domains that bind nsP2 and nsP4).
  • the assembled DNA ORF of the nsP2/4-intralin will encode the following protein regions or domains, beginning from the N-terminus: a) a single-chain antibody to the nsP2 protein of the Sindbis virus; b) the nuclear localization signal (NLS) Pro-Lys-Lys-Lys-Arg-Lys-Val, positioned in proximity to the domain in item a; c) a single-chain antibody to the nsP4 protein of the Sindbis virus; d) the NLS sequence Pro-Lys-Lys-Lys-Arg-Lys-Val (same as in item b above), positioned in proximity to the domain in item c; and e) the toxic domain (A-chain) of diphtheria toxin.
  • the NLS to be used in items b and d above is the one present in the large T antigen of the SV40 virus. This strong and at the same time short, portable NLS has been extensively characterized (Dingwall and Laskey, Trends Biochem. Sci. 16: 478 (1991));
  • the adjustment strategies for the nsP2/4-intralin (if they prove necessary) will be implemented similarly to the adjustment strategies described above for the E6/E7-indelin. Specifically, the position of an NLS will be varied relative to the position of the nearby nsP2- or nsP4-binding domain of the intralin;
  • Figure 2A a cytotoxic domain of an intralin of this type should be toxic in the nucleus but not in the cytosol. Indeed, unlike the A- chain of ricin, which is toxic in both the nucleus and the cytosol, the A-chain of diphtheria toxin acts by ADP-rybosylating (and thereby inactivating) the elongation factor 2 (EF2).
  • EF2 elongation factor 2
  • EF2 Since the bulk of EF2 is cytosolic, the translocation of a intralin containing the diphtheria-type toxic domain from the cytosol to the nucleus would physically separate a toxin from its substrate; 5) the testing of efficacy of either intermediate designs (nsP2-intralin and nsP4-intralin) or the final construct (nsP2/4-intralin) will be carried out similarly to the testing of the E6/E7-indelin and its precursors, except that Sindbis virus-infected and uninfected human cells will be used as test targets.
  • the cells and viral strain to be used will be the SW-13 line of human cells and the Toto-1000 strain of the Sindbis virus, as described by Li and Rice (J. Virology 63: 1326 (1989)).

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Abstract

L'invention concerne une nouvelle classe d'agents thérapeutiques constitués par des toxines contrôlées par codominance. Ces réactifs contiennent (1) un domaine effecteur; (2) une première portion de signalisation codominante située près d'un domaine de fixation pour une première protéine; et (3) une seconde portion de signalisation codominante se trouvant près d'un domaine de fixation pour une seconde protéine. La possibilité de masquer et supprimer le masquage de la portion de signalisation par la fixation de réactifs intracellulaires et la codominance des portions de signalisation sont mises à profit pour réaliser une nouvelle classe de réactifs ayant une sélectivité qu'il n'était pas possible d'atteindre précédemment.
PCT/US1996/002271 1995-03-01 1996-02-28 Toxines controlees par codominance WO1996026733A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU51329/96A AU5132996A (en) 1995-03-01 1996-02-28 Codominance-mediated toxins
EP96907881A EP0812204A4 (fr) 1995-03-01 1996-02-28 Toxines controlees par codominance
JP8526307A JPH11502407A (ja) 1995-03-01 1996-02-28 共優性で媒介される毒素

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WO2000050089A2 (fr) * 1999-02-26 2000-08-31 Mindset Biopharmaceuticals (Usa) Ltd. Regulation de la stabilite de proteines de recombinaison, anticorps et produits convenant pour cette regulation
WO2012091590A1 (fr) * 2010-12-31 2012-07-05 Bioinfobank Sp. Z O.O. Cytotoxine recombinante et son procédé de production

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US5132213A (en) * 1986-10-02 1992-07-21 Massachusetts Institute Of Technology Method for producing proteins and polypeptides using ubiquitin fusions
US5122463A (en) * 1990-05-17 1992-06-16 Massachusetts Institute Of Technology Methods for trans-destabilization of specific proteins in vivo and dna molecules useful therefor
US5677274A (en) * 1993-02-12 1997-10-14 The Government Of The United States As Represented By The Secretary Of The Department Of Health And Human Services Anthrax toxin fusion proteins and related methods

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CELL, 29 May 1992, Vol. 69, VARSHAVSKY A., "The N-end Rule", pages 725-735. *
PROC. NATL. ACAD. SCI. U.S.A., 1995, Vol. 92, VARSHAVSKY A., "Codominance and Toxins: A Path to Drugs of Nearly Unlimited Selectivity", pages 3665-3667. *
See also references of EP0812204A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050089A2 (fr) * 1999-02-26 2000-08-31 Mindset Biopharmaceuticals (Usa) Ltd. Regulation de la stabilite de proteines de recombinaison, anticorps et produits convenant pour cette regulation
WO2000050089A3 (fr) * 1999-02-26 2001-03-29 Mindset Biopharmaceuticals Usa Regulation de la stabilite de proteines de recombinaison, anticorps et produits convenant pour cette regulation
WO2012091590A1 (fr) * 2010-12-31 2012-07-05 Bioinfobank Sp. Z O.O. Cytotoxine recombinante et son procédé de production
US9315552B2 (en) 2010-12-31 2016-04-19 Bioinfobank Sp. Z O. O. Recombinant cytotoxin as well as a method of producing it

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EP0812204A4 (fr) 2000-05-03
EP0812204A1 (fr) 1997-12-17
AU5132996A (en) 1996-09-18
JPH11502407A (ja) 1999-03-02

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