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WO1998003538A2 - Toxines a regulation par signaux et mediation par clivage - Google Patents

Toxines a regulation par signaux et mediation par clivage Download PDF

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Publication number
WO1998003538A2
WO1998003538A2 PCT/US1997/010941 US9710941W WO9803538A2 WO 1998003538 A2 WO1998003538 A2 WO 1998003538A2 US 9710941 W US9710941 W US 9710941W WO 9803538 A2 WO9803538 A2 WO 9803538A2
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domain
sitoxin
signal
cell
protease
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WO1998003538A3 (fr
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Alexander Varshavsky
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California Institute of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2319/50—Fusion polypeptide containing protease site
    • 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

  • Virus-infected cells differ from their uninfected counterparts in particular by the presence of virus- specific proteins. The levels of certain cellular proteins are also altered as a result of viral infection. Most of the relatively few proteins that are encoded by a viral genome have functional counterparts in cells that a virus infects. In part for this reason, effective antiviral drugs remain, by and large, a goal to be reached.
  • a drug that does not necessarily kill an infected cell but acts by directly suppressing the formation of infectious virions through the inhibition of a step in the viral reproduction cycle is administered.
  • An example of the strategy outlined in item 3a is the treatment of herpesvirus-infected cells with ganciclovir (a purine nucleoside analog) .
  • Thymidine kinase (TK) of a herpesvirus phosphorylates ganciclovir more efficiently than the cellular TK.
  • ganciclovir monophosphate is then converted by cellular kinases into ganciclovir triphosphate, which inhibits viral DNA replication and also (less strongly) cellular DNA replication; the latter effect results in death of a herpesvirus-infected cell at a pre-virion stage of the viral reproduction cycle.
  • a herpesvirus such as, for example, herpes simplex virus or cytomegalovirus, are more sensitive to killing by ganciclovir than their uninfected counterparts.
  • ganciclovir or its analogs are among the few relatively efficacious antiviral therapies available at the present time. Unfortunately, these therapies are limited to herpesviruses and closely related viruses. Moreover, since the cellular TK enzymes also phosphorylate ganciclovir (albeit less efficiently than herpesviral TK) , a systemic treatment with ganciclovir or analogous drugs is accompanied by a multitude of side effects that confine this class of treatments largely (though not exclusively) to herpesviral infections of skin and mucosal surfaces.
  • An example of the strategy outlined in item 3b is the treatment of cells infected by a retrovirus such as, for example, the human immunodeficiency virus (HIV) , with azidothy idine (AZT) .
  • a retrovirus such as, for example, the human immunodeficiency virus (HIV)
  • HIV human immunodeficiency virus
  • AZT azidothy idine
  • This analog of thymidine is phosphorylated in vivo by cellular enzymes " , and in the form of a triphosphate preferentially inhibits viral reverse transcriptase.
  • AZT that becomes incorporated into a growing DNA chain terminates further chain growth, thereby enhancing the overall inhibitory effect of AZT on the viral reproduction cycle.
  • AZT also interferes with normal cellular functions, including cellular DNA replication, which limits its utility as an antiviral drug, and in most cases (including AIDS) renders AZT incapable of effecting a cure at doses that do not cause unacceptable side effects.
  • PR HIV processing protease
  • the present invention relates to a novel class of molecules referred to herein as sitoxins (signal-related, cleavage-mediated toxins) , and to nucleic acids encoding the molecules.
  • a sitoxin combines several functional domains to produce a therapeuticaliy effective molecule. More specifically, a sitoxin is comprised of an effector domain; a domain bearing an intracellular signalling moiety; and a domain located between the effector domain and the domain bearing the intracellular signalling moiety which specifies a cleavage site for a predetermined protease.
  • the invention in another aspect, relates to methods for selectively killing a target cell which is known to contain a predetermined protease.
  • Such methods involve the introduction of a sitoxin into the target cell.
  • the sitoxin can be introduced directly, or through the intermediacy of an expression vector.
  • cleavage by the predetermined protease activates the effector domain of the sitoxin.
  • a degron-based sitoxin contains a toxic effector domain, an N-terminal domain bearing a degradation signal D (any degradation signal that results in the processive intracellular degradation of sitoxin) , and the "target" domain Tl containing a site that can be recognized and cleaved by a viral processing protease Pl.
  • B The same design as in A but with the N-degron as the degradation signal.
  • Figure 2. The nuclear localization signal (NLS) -based sitoxin is analogous to the construct in Fig. 1A but contains an NLS instead of degron in the N-terminal part of the fusion.
  • NLS nuclear localization signal
  • the present invention is based on the discovery of a new and generally applicable strategy for eliminating, or modifying, cells of a multicellular organism that are known to contain a predetermined protease (e.g., virus-infected cells) in the absence of a significant damage to unintended targets that do not contain the predetermined protease (e.g., cells not infected by the aforementioned virus) .
  • a predetermined protease e.g., virus-infected cells
  • the main idea of this strategy is illustrated, for example, in Fig. 1, which shows a tripartite fusion protein comprising: 1) an intracellular signalling domain (a degradation signal (degron) is illustrated) ; 2) a protease cleavage site; and 3) an effector domain (a toxic domain is illustrated) .
  • a degron in particular the N-degron, is an intracellular signalling domain which targets a protein for rapid destruction.
  • the tripartite fusion protein of Fig. 1 is rapidly degraded, and is therefore nontoxic to a cell, provided that a degron remains linked to the toxic domain.
  • the tripartite fusion protein is introduced into a cell which contains a protease that specifically recognizes and cleaves the fusion protein at the protease cleavage site, the degradation signal and the effector domain become unlinked. As a result, the cleavage by the protease greatly extends the intracellular half-life of the effector domain.
  • Conditionally toxic protein reagents of this new class will be referred to as sitoxins (signal-regulated, cleavage-mediated toxins) .
  • Sitoxins 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 sitoxin employs recombinant DNA techniques.
  • DNA encoding the required sitoxin elements is isolated from a biological source or sources and modified as necessary using standard techniques such as site-directed mutagenesis.
  • the minimum number of nucleotides required to encode an amino acid sequence e.g., a peptide, polypeptide or protein (these terms will be used interchangeably in the present application) ) that confers the required function is employed.
  • the open reading frame is inserted into a DNA expression vector which includes a transcriptional promoter and other sequences required for expression.
  • a 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 sitoxin can also be produced by expressing it, through the intermediacy of prokaryotic expression vectors, in bacteria (e.g., E . coli ) , purifying the resulting overexpressed protein, and contacting the purified protein with target cells directly.
  • bacteria e.g., E . coli
  • a sitoxin 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 many membrane-enclosed compartments (including the nucleus) that reside in the cytoplasm.
  • translocation domains present in a variety of natural toxins such as, for example, whole ricin, whole Pseudomonas exotoxin A, and whole diphtheria toxin, is widely reported in the prior art. Typically, such reports relate to conventional, present-day chimeric toxins that recognize cell surface markers (Vitetta et al . , Imm . Today 14 : 252 (1993); Pastan et al . , Annu . Rev . Biochem . 61 : 331 (1992)).
  • the fundamental, qualitative difference between the current chimeric toxins and sitoxins of the present invention is the sensitivity of sitoxins to intracellular (as distinguished from cell surface) molecular targets, in contrast to the present- day chimeric toxins whose limitations stem in part from the confinement of their selectivity to the surface of a cell — from their inability, upon entering the cell, to "adjust" their toxicity in response to the intracellular protein composition.
  • An effector domain is preferably a protein or polypeptide that is able to exert a specific effect (e.g., to cause the death of a cell, or to disrupt a viral replication cycle) when the effector is delivered to a predetermined intracellular location.
  • the effector domain of a sitoxin 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) . Since the bulk of EF2 is cytosolic, the translocation of sitoxin containing a Pseudomonas-type toxic domain from the cytosol to the nucleus would physically separate a toxin from its substrate.
  • EF2 elongation factor 2
  • An effector domain can be derived from any protein or polypeptide the introduction of which into a predetermined cellular location would cause cell death.
  • a deoxyribonuclease would act as a toxic effector domain if introduced into the nucleus (but not into the cytosol) of a target cell.
  • -ScoRI 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 : 1353 (1985)).
  • 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 (cell surface-recognizing) chimeric toxins (Rybak et al . , J. Biol . Chem . 266 : 21202 (1991); Prior et al . , Cell 64 : 1017 (1991)). Since a major aspect of the present invention relates to antiviral therapy, the goal of which is to eliminate an ongoing viral infection, the set of useful effector domains is not confined to cytotoxic proteins.
  • an effector domain of a degron-based sitoxin can be an enzyme whose activity perturbs the viral reproduction cycle while not perturbing the viability of a cell to a similar extent.
  • a degron-based sitoxin of this type would be (by definition) short-lived and therefore nontoxic to the cell, whereas in a virus-infected cell the effector domain of a sitoxin would be separated from the sitoxin's degron ( Figure 1A) , rendering the effector domain long-lived and toxic to a virus; whether the same effector domain would also be toxic to the infected cell would be determined by the intrinsic selectivity of the effector domain.
  • Cytotoxic effector domains of a sitoxin are emphasized in the present invention because they provide a sufficient solution to the problem of stopping the spread of an ongoing viral infection. Indeed, since the processing proteases of most viruses are produced early in the viral reproduction cycle, an antiviral sitoxin bearing a conditional cytotoxic domain ( Figures 1A and 2) would be activated by a viral processing protease early in the infection cycle. As a result, an infected cell would be killed before the bulk of mature (infectious) virions could form.
  • sitoxins may also employ small molecular weight compounds as cytotoxic effectors. An example of such a compound, methotrexate, in the context of a sitoxin, is discussed below.
  • intracellular signal component With regard to the intracellular signal component, 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, ultiubiquitinated substrate is processively degraded by the 26S proteasome — a multicatalytic, multisubunit protease.
  • Aspects of the N- end rule, ubiquitin fusions and related technologies are the subject of a number of U.S. Patents issued to Varshavsky et al . , including U.S. Patent Nos. 5,132,213; 5,212,058; 5,122,463; 5,093,242 and 5,196,321, the disclosures of which are incorporated herein by reference.
  • the in vivo half-life of a protein bearing a strongly destabilizing N-terminal residue such as arginine can be as short as l minute, whereas an identically expressed and otherwise identical protein bearing a stabilizing N-terminal residue such as valine has a half-life of more than 20 hours, resulting in a greater that 1, 000-fold difference between the steady- state concentrations of these proteins in a cell.
  • Ubiqui in-dependent proteolytic systems (including the N- end rule pathway) share many components of the 26S proteasome. Differences among these systems encompass their distinct targeting complexes, whose recognins bind to degradation signals other than N-degrons.
  • amino acid sequences that function as degradation signals and are transplantable to other proteins including, 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 of S . cerevi ⁇ iae (Hochstrasser and Varshavsky, Cell 61 : 697 (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.
  • An NLS-based sitoxin is a fusion that includes (1) a toxic domain that exerts its effect exclusively or preferentially in the cytosol (see below) ; (2) an NLS- bearing domain; and (3) a recognition/cleavage site for a specific viral processing protease inserted between the toxic domain and the NLS-bearing domain of the fusion ( Figure 2) .
  • the mechanism of an NLS-based sitoxin is as follows. The introduction of an NLS-based sitoxin into an uninfected cell would result in a rapid, NLS-mediated translocation of the NLS-sitoxin into the nucleus, where the sitoxin' s toxic domain, being cytosol-specific, would be unable to exert its toxic effect.
  • an NLS- based sitoxin can readily be "inverted” by employing a toxic domain that is active in the nucleus but not in the cytosol. Such a sitoxin would kill cells that lack a protease that cleaves a sitoxin between its NLS and toxic domain, but would spare cells that contain such a protease.
  • the region (domain) of a sitoxin between its effector domain and the domain bearing the intracellular signaling moiety specifies the cleavage site for a predetermined protease. It is this protease-specific cleavage site which enables the constructs (sitoxins) of the present invention to become activated in a cell bearing the predetermined protease and to remain relatively inactive in a cell lacking such a protease.
  • a particularly well-defined group of proteases that are relevant to the present invention are virus-encoded processing proteases, whose main (and often the only) normal function is to convert a viral polyprotein — the initial translation product — into mature viral proteins, in particular the enzymes for viral replication, as well as structural proteins of the virions.
  • processing proteases of different viruses form a family that is diverse both structurally and mechanistically, most of these enzymes share at least the following properties:
  • proteolytic activity which does not require the presence of ATP or other high-energy cofactors ;
  • a viral protease is often encoded as a part of a polyprotein precursor, from which the protease excises itself through the same pathway of sequence- specific cleavages that yields other mature protein products of the polyprotein;
  • a viral protease typically makes just a single, sequence- specific cut, the exact location of which is dictated by short (e.g. , approximately 10 residues) amino acid sequences on both sides of (and immediately adjacent to) the cleavage site; and (5) the relative efficiency of the protease-mediated cleavage at a given site is determined by the specific amino acid sequences flanking the site, and also by the extent of steric shielding of the site within a polyprotein — as a result, different mature proteins can be excised by the protease from the polyprotein precursor at different, physiologically relevant intrinsic rates.
  • the crucial property of a viral processing protease is its ability to recognize and cleave within an amino acid sequence motif that is sufficiently short (e.g., approximately 20 residues) to be readily portable (transplantable) to artificial (engineered) protease substrates and, at the same time, sufficiently long to be a unique site, in the sense that such a site would be left uncleaved by other intracellular (cytosolic) proteases in an uninfected cell that lacks the viral protease.
  • the crucial idea of the present invention is to combine the use of degrons or translocation signals for changing the state of a cytotoxic protein in a cell with the possibility of making this change of state conditional on the presence of a virus-specific processing protease.
  • the latter is achieved through the utilization of the proteases's ability to cleave a protein bearing an appropriate, protease-specific cleavage site.
  • the most important factor considered in selecting an appropriate viral protease recognition sequence is the identity of virus of interest. Once the identity of the virus of interest is determined, one of skill in the art can consult the literature to determine an appropriate protease recognition sequence to be included as a component of the sitoxin. Two recent review articles containing such information, as well as citations to additional sources of such information, are Kr&usslich and Wimmer (Annu . Rev . Biochem . 57 : 701 (1988)) and Dougherty and Semler (MicroJiol. Rev . 57 : 781 (1993)).
  • the specific protease recognition sequence which can be incorporated in the sitoxin molecule is not limited, however, to those specifically discussed in these references or the citations contained therein.
  • the typical in vivo application of a sitoxin is in a therapeutic regimen designed to eliminate cells known to contain a predetermined protease.
  • a sitoxin whose protease cleavage site is recognized by a protease encoded by the virus has been designed as described above. (A large fraction of pathogenic human and animal viruses utilize polyproteins and a virus-specific protease as an essential part of their life cycle.)
  • the mechanistic routes of comtoxin delivery can be either intravascular (for both protein-based and vector-based forms of sitoxin) or any other route (for example, a skin or mucosal surface application) that would serve to deliver a sitoxin to the bulk of virus-infected cells.
  • intravascular for both protein-based and vector-based forms of sitoxin
  • any other route for example, a skin or mucosal surface application
  • sitoxins are confined to direct-delivery (as distinguished from expression-based) strategies, and is similar to the analogous aspects of current chimeric toxins discussed above (Vitetta et al . , Imm . Today 14 : 252 (1993); Pastan et al . , Annu . Review Biochem . 61 : (1992); and Olsnes et al . , Som . Cell Biol . 2 : 7 (1991)).
  • the initial step of a sitoxin' s delivery as a protein can be made partially cell type-selective by fusing the sitoxin 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 surface marker can be present on more that just target cells without significantly increasing the sitoxin' s nonspecific toxicity.
  • the conditional toxicity of a sitoxin is decided by the environment it encounters after entering the cell; therefore, a sitoxin against a specific virus would not affect uninfected cells, which lack the virus-specific intracellular protease characteristic of cells infected by the virus.
  • the advantage of the direct-delivery strategy is that it bypasses potential problems associated with the vector-mediated delivery of a sitoxin.
  • One potential drawback of the direct delivery is a larger size of a multidomain sitoxin (due to the presence of additional domains) , in comparison to an otherwise identical sitoxin that is delivered through the intermediacy of an expression vector. Since the testing of sitoxins 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 sitoxin, and to compare the results.
  • This toxicity which imposes a limit on both duration and intensity of treatments, stems in part from the clearance of an intravenously administered immunotoxin by cells of the reticuloendothelial system that are killed as a result of the toxin's entry into these normal cells (Vitetta et al . , I m . Today 14 : 252 (1993) ) .
  • sitoxins of the present invention would not affect most nontarget cells.
  • This feature of sitoxins will yield a much higher therapeutic index (i.e., a much higher tolerated intensity and duration of treatments) .
  • Sitoxins 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.
  • 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)).
  • vectors can be used for the delivery of sitoxins in cell cultures, in whole animals, and, with appropriate preliminary testing, in human patients as well.
  • Recent advances in the design of viral and plasmid-based vectors see e.g., Nabel et al . , Proc . Natl . Acad . Sci . USA 90 : 11307 (1993) and Mulligan, Science 260 : 926 (1993)) resulted in tailor-made, nonreplicating vectors that can transfect both growing and quiescent cells, and are either specific for cells that bear a predetermined surface marker or almost nonselective.
  • Such vectors in use for gene therapy and other applications (for example.
  • Sitoxins can also be used in a variety of in vitro (cell culture-level) applications. A common feature of these applications is based on the ability of sitoxins to selectively eliminate virus-infected cells from a cell population that contains both infected and uninfected cells. For example, in a commercial production setting, sitoxins can be employed to purge a valuable cell culture from cells that have been infected with a specific virus.
  • sitoxins that is likely to prove especially valuable is to use them for selective elimination of virus-infected cells from, for example, the patient's bone marrow — for subsequent reinfusion of the virus-free marrow into the same or another (immunologically compatible) patient whose own bone marrow was deliberately destroyed by a marrow-ablating chemotherapy.
  • sitoxins their ability to kill cells infected bearing a specific virus while sparing uninfected cells — makes possible such prophylactic purgings of cell populations that one would like to be definitely free of a virus prior to a "downstream" utilization of the cells, especially if this utilization involves the infusion of cells into a patient.
  • One remarkable advantage of sitoxins is that the probability of emergence of a sitoxin-resistant mutant virus is expected to be negligible. Indeed, since the cleavage site of an antiviral sitoxin is functionally indistinguishable from the natural cleavage sites in a viral polyprotein (Figs.
  • the sitoxin concepts disclosed herein are compatible with a low molecular mass design as well.
  • the effector domain of a sitoxin construct can be a low molecular mass cytotoxic compound such as, for example, methotrexate (MTX).
  • MTX methotrexate
  • This compound would be linked to a short polypeptide (less than -15 residues in length) that contains the recognition/cleavage site for a viral processing protease, such as, for example, the HIV protease.
  • the cleavage site-containing peptide would be linked to MTX at a position that renders MTX inactive for the binding to its target — the cellular enzyme dihydrofolate reductase (DHFR) .
  • DHFR dihydrofolate reductase
  • the above construct will be cleaved, resulting in MTX bearing a shorter peptide (or peptide-like) extension that corresponds to the C-terminal part of the initial recognition/cleavage site.
  • the additional requirements for this design is that the remainin ⁇ peptide (or peptide-like) extension attached to MTX after the above cleavage event must be readily degradable by one of the ubiquitous exopeptidases normally present in the cell's cytosol.
  • This degradation would release free (and toxic) MTX in cells containing the viral protease but not in cells lacking the protease, because the initial MTX-linked peptide (or peptide-like) extension is constructed to be insensitive to cytosolic exopeptidases.
  • the initial testing of a sitoxin specific for a predetermined virus would ask whether this sitoxin would kill cells expressing the viral processing protease while sparing otherwise identical cells that lack the protease.
  • the yeast-based cell system with its powerful "reverse-genetics" techniques, will be used. Once a working sitoxin design has been established at the level of yeast cell culture. the analogous tests will be carried out with human cells, as described below.
  • the sitoxin to be designed in this example is a multidomain protein fusion whose c-terminal domain is the A-chain of ricin, linked to the nearest upstream domain Tl ( Figure IB) by a short (5 to 10 residues) linker sequence.
  • the domain Tl contains a cleavage site for a processing protease of a specific virus, as described below.
  • Simple-sequence, relatively hydrophilic linkers see, e.g., Johnson and Bird, Meth . Enzymol . 203 : 88
  • the nucleic acids which encode the various elements of the sitoxin molecule will be isolated from naturally occurring sources (or engineered and publicly available sources) and assembled using standard recombinant DNA techniques and either yeast or mammalian expression vectors described below.
  • the toxic domain to be 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 a sitoxin) .
  • the deduced amino acid sequence of the A-chain of ricin is provided, for example, in Funatsu et al . , Biochimie 73 : 1157 (1991).
  • the degradation signals (degrons) to be positioned upstream of both the toxic domain and the Tl (cleavage site-containing) domain can be chosen from among several presently known degrons.
  • the degradation signal to be employed in the first construct will be the N-degron (see above) , which has been dissected biochemically and genetically, and is understood in considerable detail (Varshavsky, Cell 69 : 725 (1992)).
  • N-degron see above
  • Several portable variants of the N-degron have been described and analyzed (Varshavsky, Cell 69 : 725 (1992)).
  • the actual portable N-degron employed in the construction below 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 plasmids, together with their restriction maps, are described in detail by Bachmair and Varshavsky, Cell 56 : 1019 (1989) .
  • the construction of the Tl (cleavage site-containing) domain between the sitoxin's C-terminal toxic domain and the N-degron is described below.
  • HIV human immunodeficiency virus
  • PR HIV protease
  • the A-chain of ricin as a toxic domain of the HIV- specific sitoxin is described above. Also described above are the design and sources of the N-degron that will be used to confer conditional instability of the HIV-specific sitoxin. These two elements are generic elements of any N-degron-, ricin-based sitoxin.
  • the HIV specificity of this design will be conferred by its Tl region ( Figure IB) , which will contain a cleavage site for the HIV protease.
  • the Tl region can be as short as 20 residues, with the cleavage site in the middle of the region, the region itself being joined to the N-degron upstream of Tl and to the A-chain of ricin downstream of Tl.
  • the actual Tl region to be used can be chosen from several natural HIV protease cleavage sites in the HIV polyproteins (Dougherty and Semler, Microhiol . Rev . 57 : 781 (1993)). Many of these sites are cleaved at similar rates by the HIV protease.
  • Initial experiments will employ site No. 6, the one between HIV protease and reverse transcriptase in the HIV precursor polyprotein (Debonck, AIDR Res . Human Retroviruses 8 : 153 (1992)), but other sites of comparable efficiency can also be used to design the Tl region of HIV-specific sitoxin.
  • the amino acid sequence abutting the HIV cleavage site No.
  • the fully assembled DNA open reading frame of the HIV-specific sitoxin will encode the following protein regions or domains, beginning from the N-terminus:
  • the above open reading frame can be cloned into any of a number of readily available yeast expression vectors that allow regulated expression of the HIV-specific sitoxin.
  • the pUB23 plasmid will be used (Bachmair et al . , Science 234 : 179 (1986)), which contains a galactose-inducible promoter and allows a tight regulation of the expression of HIV-specific sitoxin (no expression in the presence of glucose in the medium, and active expression upon the substitution of glucose with galactose) .
  • Two otherwise identical yeast strains will be used, one of which (the "control" strain) is one of the many "wild-type" S .
  • the cerevisiae strains for example, S228c
  • the other is an otherwise identical strain that constitutively expresses the HIV protease.
  • This latter strain can be constructed using standard methods, well known to those skilled in the art. Specifically, the open reading frame encoding the HIV protease can be subcloned from the plasmid PR04 (Strickler et al . , Proteins 6 : 139 (1989)). This open reading is placed downstream of a moderately active yeast promoter such as the CUP1 promoter of the expression vector pRS314 (Sikorski and Hieter, Genetics 122 : 19 (1989) ) . This construct, upon its introduction into the control yeast strain, will yield the test strain.
  • the plasmid encoding the HIV-specific sitoxin described above will then be introduced (using standard transformation techniques (Rothstein, R. , Meth . Enzymol . 194 : 281 (1991)) into both the control and test yeast strains in the presence of glucose in the medium. Under these conditions the galactose-inducible promoter of the plasmid is repressed, resulting in negligible levels of HIV-specific sitoxin in the cells. At this point, both the control and test strains are transferred to a galactose-containing medium to induce the expression of the HIV-specific sitoxin.
  • the ubiquitin moiety at the N-terminus of the nascent HIV-specific sitoxin is rapidly (nearly cotranslationally) cleaved off by ubiquitin-specific proteases, which are present in all eukaryotic cells (Varshavsky, Cell 69 : 725 (1992)), yielding the mature sitoxin ( Figure IB) that bears a destabilizing residue such as, for example, Arg at the newly formed N-terminus.
  • the A-chains of ricin and diphtheria toxin are toxic to both S. cerevisiae and mammalian cells, enabling a simpler yeast setting to be employed first.
  • the control ("wild-type") S. cerevisiae strain will be compared with an otherwise identical strain that has been transformed with the above-described plasmid expressing HIV protease.
  • both strains will be also transformed with another of the above-described plasmids encoding the N-degron-mediated sitoxin construct, both strains being grown on glucose-containing media, where the galactose-inducible, glucose-repressible promoter of the sitoxin-encoding plasmid is inactive.
  • both strains will be transferred to a galactose- containing medium, which induces the promoter and allows the expression of the sitoxin construct.
  • the sitoxin will be short-lived, owing to the presence of the N-degron, resulting in a low or negligible overall toxicity.
  • the otherwise identical strain expressing HIV protease at least a fraction of the newly formed sitoxin will be cleaved by HIV protease, resulting in separation of the sitoxin's toxic domain and the N-degron ( Figure 1).
  • the resulting "free" toxic domain will be a relatively long- lived protein (in comparison to the same domain bearing the N-degron) , accumulating in a cell to a higher steady- state level and as a result causing a much greater overall toxicity.
  • this difference will be manifested in the death of cells that express both the sitoxin and HIV protease versus survival of otherwise identical cells that express the sitoxin alone.
  • the fate of cell populations in these experiments can be followed using several well-known and technically straightforward assays, for example, by determining growth curves of the corresponding cultures after their transfer to a galactose-containing medium (which induces sitoxin's expression) , or, independently, by comparing the numbers of colonies formed by the above two strains under the same conditions.
  • the in vivo half-life of sitoxin can be followed explicitly in such settings, if necessary, through the use of a standard pulse-chase assay.
  • the sitoxin design has been tested in the S . cerevisiae setting, it will be used for a similar test in a human cell culture. Since an overexpression of HIV protease has been shown to be toxic to mammalian cells (Vaishnav and Wong-Staal, Annu. Rev . Biochem . 60 : 577 (1991)), a stable human cell line will be constructed in which HIV protease is expressed from a tetracycline- repressible promoter, using the system developed by Gossen and Bujard (Proc . Natl . Acad . Sci .
  • HeLa cells derivatives expressing the tetracycline-repressible transactivator protein (Gossen and Bujard (Proc . Natl . Acad . Sci . USA 89 : 5547 (1992)), will be employed in these experiments.
  • Bujard's system which is, by now, a standard method in the field of mammalian gene expression, makes possible a titration of the in vivo concentration of HIV protease to an optimal level for the tests with protease-expressing and protease-lacking cell lines — the tests closely analogous to those described above for S . cerevisiae .
  • the sitoxin tests in human cells will determine the extent of killing by sitoxin of cells that lack HIV protease versus otherwise identical cells that express HIV protease.
  • the conceptual and technical similarities between the yeast- and human cell-based tests of sitoxin extend to the adjustment protocols as well (if such adjustment procedures prove necessary at all) .
  • the half-lives of sitoxin constructs can be measured in human cells using a standard pulse-chase assay, and modifications of the open reading frames encoding the relevant constructs can also be carried out as described above.

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Abstract

L'invention concerne une nouvelle classe de molécules dénommées sitoxines (toxines à régulation par signaux et médiation par clivage). Une sitoxine associe plusieurs domaines fonctionnels de façon à produire une molécule active sur le plan thérapeutique. Plus spécifiquement, une sitoxine est constituée d'un domaine effecteur; d'un domaine portant un fragment de signalisation intracellulaire; et d'un domaine situé entre le domaine effecteur et le domaine portant le fragment de signalisation intracellulaire, qui spécifie un site de clivage pour une protéase prédéterminée. L'invention concerne également des méthodes permettant de tuer sélectivement une cellule cible dont on sait qu'elle contient une protéase prédéterminée. Ces méthodes font appel à l'introduction d'une sitoxine dans la cellule cible. La sitoxine peut être introduite soit directement, soit par l'intermédiaire d'un vecteur d'expression. Après introduction de la sitoxine dans la cellule cible, le clivage par la protéase prédéterminée sépare le domaine effecteur de la sitoxine du fragment de signalisation intracellulaire. Le domaine effecteur, une fois séparé, a une vie plus longue (il est donc plus toxique), ou bien il passe d'un compartiment cellulaire où il n'est pas toxique à un compartiment cellulaire où il est capable d'exercer son effet.
PCT/US1997/010941 1995-10-27 1997-10-24 Toxines a regulation par signaux et mediation par clivage Ceased WO1998003538A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023593A3 (fr) * 1998-10-16 2000-07-27 Fraunhofer Ges Forschung Pathogenicide moleculaire induisant une resistance a la maladie chez des vegetaux
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
WO2012139112A1 (fr) * 2011-04-08 2012-10-11 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Fusions d'ubiquitine pour améliorer l'efficacité de toxines ciblées à action cytosolique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCONJUGATE CHEM., 1993, Vol. 4, COOK J.P. et al., "Biologically Active Interleukin 2-Ricin A Chain Fusion Proteins May Require Intracellular Proteolytic Cleavage to Exhibit a Cytotoxic Effect", pages 440-447. *
EUR. J. BIOCHEM., 1991, Vol. 199, LOUIS J.M. et al., "Autoprocessing of the HIV-1 Protease Using Purified Wild-Type and Mutated Fusion Proteins Expressed at High Levels in Escherichia Coli", pages 361-369. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023593A3 (fr) * 1998-10-16 2000-07-27 Fraunhofer Ges Forschung Pathogenicide moleculaire induisant une resistance a la maladie chez des vegetaux
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
WO2012139112A1 (fr) * 2011-04-08 2012-10-11 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Fusions d'ubiquitine pour améliorer l'efficacité de toxines ciblées à action cytosolique

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