JPH11502407A - Toxins mediated by co-dominance - Google Patents

Abstract

A novel class of therapeutic reagents, called co-dominantly mediated toxins, is disclosed. These reagents comprise (1) an effector domain, (2) a first codominant signal portion located proximate to the binding domain of the first protein, and (3) a proximate binding domain of the second protein. And a second co-dominant signal portion located at The ability to block and unblock signal moieties due to intracellular binding of reagents and the co-dominance of signal moieties is exploited to design new classes of reagents with selectivity previously unattainable.

Description

DETAILED DESCRIPTION OF THE INVENTION                          Toxins mediated by co-dominance                                Background of the Invention   Aberrant cells are their normal ancestors in a variety of ways, including their protein composition. And different from other cells of the same organism. For example, a cell infected with a virus is a virus Contains specific proteins and the level of certain cellular proteins is also viral infection Can be changed as a result of Where they first appeared and where they formed colonies Cancer cells that can grow at distant sites where possible Different from their normal ancestors. Some of the tumor-specific proteins are normal Are altered variants of the protein, where they have a mutation of the malignant phenotype. It is encoded by a gene that was one of the causes.   The viral genome encodes relatively few proteins. These proteins Most of these cells have functional counterparts in cells infected by the virus, so that effective anti-virus Irs drugs generally remain the ultimate goal to be reached. Surgery alone The problem of finding drugs by engaging in tasks in malignant tumors that cannot be removed Even sloppy. Because the difference in composition between tumor cells and their normal ancestors is not Because it can be difficult and quantitative rather than qualitative. In addition, tumor cells Often heterogeneous in genetics and protein composition. These difficulties are great It is a major reason for the failure of today's treatment to cure some cancers. The problem is not necessarily drug Not of insufficient specificity. Some of the cytotoxic compounds used in cancer therapy, for example For example, methotrexate and vinblastine (dihydrofolate reductase and And tubulin) bind to their ligands Highly specific. The problem is that only one drug target is That is, sometimes it is not clear enough.   Another approach to cancer therapy is to use antibodies or antibodies that bind to targets on the surface of tumor cells Requires binding of the toxin to another ligand (eg, a growth factor). Immunotoxicity The tumor selectivity of these drugs, called Often larger than that of small cytotoxic drugs such as sate. Unfortunately, Surface markers recognized by immunotoxins are not only present on target cells Absent. In addition, since this marker is often not essential for tumorigenicity, Cells that lack but are still malignant may be present in the tumor. These are today ’s Some of the limitations of munotoxins.   Yet another approach is the ability of the immune system to identify and selectively destroy tumor cells. To reinforce or redirect. Cancer immunotherapy is long and authentic Has a history of history. Advances in antigen presentation and understanding of lymphocyte-mediated cell killing The recent revival of this strategy, brought about by the step, is a rational and curative therapy Retain the prospect of. Since this end goal remains to be reached, the immune system Rational manipulation may prove to be sufficient for the complete and reliable cure of most cancers It is not at all certain. Summary of the Invention   The subject invention is a novel and novel method for removing (or modifying) dangerous cells. Based on the discovery of a series of generally applicable methods and reagents. The present invention provides one station In terms of a new class of so-called co-dominantly mediated toxins, Medical therapeutic reagents. These reagents include (1) the effector domain, (2 A) a first co-dominant signal located in close proximity to the binding domain of the first protein; Part, and (3) a second protein located in close proximity to the binding domain of the second protein. A co-dominant signal portion. Blocks the signal area by binding intracellular reagents The ability to mask and unmask and the co-dominance of the signal portion can be achieved previously. It is used to design new classes of reagents with unprecedented selectivity.   In another aspect, the invention provides both a first protein and a second protein. A method for selectively quenching or eliminating cell division known to contain Related. In this method, 1) the effector domain, 2) the binding of the first protein A first co-dominant degron located in close proximity to the integration domain, and 3) a second tan Encodes a second co-dominant degron located in close proximity to the binding domain of protein A DNA construct is provided. The DNA construct is intracellular degron dependent and ligated. The cells are introduced into cells under conditions appropriate for expression of the toxin that is regulated by the enzyme. BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows a ligand-regulated toxin dependent on intracellular degron (intracellula rdegron-dependent,ligand-regulated toxinNamed indelin from FIG. Expresses proteins P1 and P2 [P1⁺P2⁺ ] But kills the cells but other cell types, ie [P1⁺P2^-], [P1^-P2⁺] And [P1^-P2^-Inderin is designed to save Effector domain and two domains that bind to P1 and P2, respectively. Inn P1^*And P2^*Two degradation signals placed in or near (degro N) Contains d1 and d2. [P1⁺P2⁺], Cells other than at least one Phosphorus degron, ie, d1 and / or d2, is active (uninterrupted) Results in a short-lived, and therefore relatively non-toxic, indelin. Contrast [P1⁺P2⁺In cells, both d1 and d2 are P1 and P2 (each Inderin P1^*And P2^*Appears to be shielded by Resulting in a long-lived and therefore toxic indelin. This design is , P1, P2 and indelin are in the same compartment, ie, the toxicity of indelin therein What the domain exerts its effect on (the cytosol, the nucleus, and / or (Which may be another compartment). In addition, degron d1 and d2 must be active in the relevant compartment. They may or may not be the same Well, this design is consistent with either choice.   FIG. 2 shows that intracellular translocation signal-dependent and ligand-regulated Toxin (intracellular translocation signal-dependent, ligand regulated t oxin Is designated as intralin. (A) [ P1⁺P2⁺] But kills the cells but other cell types, ie [P1⁺P2^-], [P 1^-P2⁺] And [P1^-P2^-Is designed to save] Effector domains that are toxic in the cytosol but not in the nucleus; and Two domains P1 binding to P1 and P2 respectively^*And P2^*Inside Is a fusion containing two nuclear localization signals (NLS) placed in close proximity. [P1⁺P2⁺In at least one other cell, at least one of the NLS of indelin is active (interfered). Not), resulting in nuclear and thus non-toxic intralin. Contrast [P1⁺P2⁺In cells, both NLSs are P1 and And P2 (intraline P1 respectively)^*And P2^*To the domain) It appears to be more screened and also contains cytosolic and thus toxic intralin. Sprinkle. [P1⁺P2⁺In cells, P1 and P2 are at least located in the cytosol. Must be placed. (B) Same as A but with intraline The effector domain is toxic in the nucleus but not in the cytosol. this Intraline is one of two cytosolic proteins, P1 and P2, Both lack one [P1⁺P2^-], [P1^-P2⁺] And [P1^-P2^-] Kill cells Appears to be, but contains both P1 and P2 [P1⁺P2⁺]cell Will be saved.   FIG. 3 shows toxins mediated by hybrid dominance (codominance-mediatedt oxin (Named comtoxin). Hybrid Docomotoxin is an effector that is active in the nucleus but not in the cytosol -Domain, domain P1^*Degrons placed in or near P2^*Contains a nuclear localization signal (NLS) located within or near. P 1^*And P2^*Binds to intracellular proteins P1 and P2, respectively. Illustration This comtoxin is exclusively [P1⁺P2^-] When you kill cells Be looked at. This is P1containsBut P2Lack. In these cells, P1 Must be located at least in the nucleus, while P2 is a cytosolic Must be quality. Another constraint is that degron d1 must be at least nuclear active. It must be. [P1⁺P2⁺] Actually pointed out in cells Mutoxin status indicates that P1 is present in both cytosol and nucleus, and In addition, degron d1 is the opposite of these compartments. Need to be more active. If P1 is mainly in the nucleus, or If d1 is active only in the nucleus, the metabolic properties of this comtoxin are indicated And its selectivity ([P1⁺P2^-] Kill cells ) Appears to remain the same.   FIG. 4 is an illustration showing fission toxin. With this design, toxic effector The two main subdomains have sequences whose binding sites for intracellular protein P1 Separated by the containing insert. P1 of other comtoxins^*Unlike domain (FIGS. 1-3), the P1 binding site of fission comtoxin is such that it is bound by P1. Relatively short (peptide large) that remains conformationally mobile unless otherwise Area). In order for this design to work, The affinity between the subdomains of the proteins is sufficient to render their interactions essentially reversible. Should be a minute smaller. A flexible insert between the subdomains of the toxin Binding of either protein P1 or P2 prevents reconstitution of the active toxin Also contains the two binding sites of P1 and P2 arranged so that they will be sufficient for obtain. The resulting comtoxin lacks both P1 and P2 exclusively It is expected to kill cells. Other fission toxin designs are listed elsewhere. Detailed description of the invention   The present invention provides a new method for removing or modifying dangerous cells in multicellular organisms. It is based on the identification of regular and generally applicable strategies. The main idea of this strategy is, for example, Represented by various signals present in biopolymers such as proteins and nucleic acids Based on the characteristics of co-dominance. For simplicity of discussion, the protein system Gunnar and their use as examples Will be used. One of skill in the art will appreciate that the principles described herein may be applied to, for example, nucleic acids. Related extensions, and such extensions are within the scope of the present invention. Will recognize. The protein signal is a degradation signal (degron) and Various translocation signals including nuclear localization signal (NLS) Including but not limited to. NLS is derived from the cytosol of cells Gives proteins the ability to be transported into.   The two signals present in the protein are Independent of other signals of the protein and without significant interference thereby, It is termed codominant if it can exert its effect on protein. More details below The main idea of this invention is to describe the codominant signal part, the protein, as described in A rational and novel combination of the protein interaction domain and the effector domain Can result in the creation of a new class of drugs with selectivity not previously achieved That's what it means. These novel pharmaceutical reagents are referred to herein as comtoxins Dominantly mediated toxins) and a specific example is shown in FIGS. 1-4.   FIG. 1 shows a commutoxin with a degradation signal. Comtoxin of this class Stands for indelin (ligand-regulated toxin dependent on intracellular degron) Would be called. The left column of FIG. 1 labeled “Cell Type” The first protein or polypeptide (P1) or the second protein in the cell in question Absence of protein or polypeptide (P2) (P1^-Or P2^-)Also Exists (P1⁺Or P2⁺) To point out. The column on the right is comtox Each of the three required elements of the component is shown in schematic form. Shown in the right column Thailand Fusion proteins, among others, are suitable expression proteins that encode the fusion protein. It is delivered intracellularly by introducing the vector into the cell. Intracellular expression The introduction of the vector is followed by the transcription and translation of the encoded fusion protein. This In examples, the toxic domain is a protein that is at least toxic in the cytosol of the cell. Quality or polypeptide.   The N-terminal domain of comtoxin in FIG. 1 is the binding domain of the first intracellular protein. Main (P1^*) Is disposed adjacent to the first degron (d1). N-terminal The domain flanked by the domains and the toxic domain is the second intracellular protein Protein binding domain (P2^*) And a second degron (d2) Including. The first and second proteins are designated P1 and P2 respectively in the right hand column. Is pointed out.   For the comtoxin of FIG. 1, two alternative metabolic Destiny is possible. That is, whether the fusion protein has a short or long life It is misplaced. If the fusion protein has a long lifespan, The toxin will remain in the cytoplasm for a time sufficient to kill the cell. Fusion If the life span of the protein is short, the toxic domain is the degron of commutoxin, highly recognizing and targeting either or both d1 and d2 A ubiquitin-dependent proteolytic pathway that processes Will be understood. The long or short life span of the fusion depends on the P1 in the cell cytosol. Or it depends on the presence or absence of P2. Neither P1 nor P2 is present in the cell In this case, the fusion protein is unhindered (removed of shielding) degron d1 and The life is short due to the presence of d2. Either P1 or P2 exists but If not, there are two fusion proteins One of the co-dominant degrons in the mosquito remains unobstructed (shielding removed) The fact that it would retain its ability to signal the degradation of comtoxin Therefore, the life is still short. However, if both P1 and P2 are present, The other degron (d1 and d2) will be sterically shielded and The lifespan of the composite protein will be long. Thus, the comtoxin of FIG. Kill cells expressing both and P2, but not P1 and / or P2. Designed to save missing cells.   Another example of a comtoxin design is shown schematically in FIG. "Cell type The left column of FIG. 2 labeled "" is the first protein in the cells in question. (P1) or the absence of the second protein (P2) (P1^-Or P2^-)Also Exists (P1⁺Or P2⁺) To point out. Panel A in FIG. Panel B, on the other hand, relates to comtoxin molecules with toxin-specific toxic domains. Comtoxin molecules with nuclear-specific toxicity domains. All drawn in Figure 2 In all comtoxin molecules, the intracellular signal domain has a nuclear localization signal (NLS). ). Comtoxin molecules with at least one NLS that are not sterically hindered are It will be transported from the itsol to the nucleus.   Thus, the comtoxin shown in panel A of FIG. 2 also has P1 in the cytosol. If P2 is also absent, or either P1 or P2 (but not both) Will be transported to the nucleus if present. The toxicity of this comtoxin Because ins are cytosol-specific (see below), their toxic effects Will occur if, and only if, the And this is both Only when the NLS is spatially shielded (ie, both P1 and P2 Will occur only if present in the cytosol of the cell). The tag shown in FIG. Ip comtoxin is referred to herein as intralin (intracellular translocation). Signal-dependent, ligand-regulated toxin).   Panel B of FIG. 2 shows that the effector domain of intralin is nuclear and toxic However, the opposite is true in the cytosol (see below). Intralin of this type is used in cells lacking P1 or P2 or both. It is expected to kill. That is, the selectivity opposite to that of intralin discussed above. (FIG. 2A vs. FIG. 2B). In fact, NLS-bearing commutoxins are P1 and And if and only if both are present in the cytosol, the cell Will remain in the cytosol and will block both NLSs of intratraline (No. 2B). Thus, indelin (comtoxin with degron) (Figure 1) While killing cells containing exclusively P1 and P2, the toxicity domain Intralin, which is nucleus-specific (FIG. 2B), rescues these cells exclusively. In advance The ability to target cells that lack a defined set of proteins is important. Why Thus, cancer cells are dependent on certain regulatory proteins ("tumors") that are present in their normal ancestors. Ulcer suppressor ") (or contains a functionally inactive variant thereof) It is.   Only one P^*Contrasts of the constructs of FIGS. 1 and 2 with a type domain It should be noted that it can function as delin or intralin. However While these constructs are not yet commutoxins (The dynamics of codominance is more than one P^*Requires a domain). At the same time, these Ligand-controlled toxicity of the new construct appears to be similar to that of commutoxin Can be Thus, it is a natural intermediate in the design of comtoxins. In addition to just one P^*Indelin or int with type domain Larin can also be used as a drug specific to only one intracellular target.   Only one P^*Conditional toxins of the invention having a type domain It should be emphasized that new drug designs are also representative. Them While selectivity is limited to one protein target, this target is Targets and controls expressed on the cell surface of toxins (also called chimeric toxins) And is intracellular. Only one P of the present invention^*Conditional poison of type domain The element is poly-P^*A "natural" intermediate in the construction of type domain comtoxins And targeting only one predetermined intracellular protein, Selection of cytotoxic cells in the resulting therapeutic or in vitro mammalian cell culture system It should also be useful as a drug, under conditions that are sufficient for the end goal of choice.   Another type of comtoxin, referred to herein as hybrid comtoxin, is This is shown schematically in FIG. Comtoxins of this type are degron and NLS It has both signal parts. The simplest comb ibis of this class shown in FIG. Syn is an effector domain that is toxic in the nucleus but not in the cytosol. Domain P1^*Degron placed in or near, and domain P 2^*Contains NLS placed within or near. As before, P1^*and P2^*The domain is It should be possible to bind to the intracellular proteins P1 and P2, respectively. is there. However, "pure" indelin or intralin (first and In contrast to FIG. 2), this “hybrid” comtoxin is exclusively composed of nuclear proteins. Kills cells containing cytoplasmic P1, but lacking cytosolic protein P2 It is. Only in such cells is commutoxin nuclear (because its N LS is not shielded by the absence of P2 and has a long lifetime (because its degro Is P1-P1^*(Shielded by the complex).   Comtoxin with degron and NLS (FIG. 3) is the dual of P1 and P2. [P1⁺P2^-Identify cells from other cell types It seems to fail. Figure 4 shows that the form of selectivity addresses this problem, among others. FIG. 4 illustrates a conditional toxin in a class of mating. The subdomain is conformation Of only one domain, separated (by surface loops) by a chemically movable insert The protein may adopt a (nearly) normal conformation. This conformation In the alternative, the insert protrudes outside the folded domain. An example For example, in vivo folding of the 76-residue protein ubiquitin is By inserting an unrelated 80 residue sequence at the site between the two subdomains of chitin It was shown that they were not actually confused (Johnsson and Warsawski (V arshavsky), Proc. Natl. Acad. Sci. USA 91: 10340 (1994)). Of the "split" toxin The idea (FIG. 4) is based on these and similar data, and also on the protein Of the conformational mobility of the insert between the two subdomains of Can affect both dynamics and equilibrium aspects of quality folding It also comes from concepts. In particular, the conformationally fixed insert is a protein subdomain It is expected to disrupt or eliminate the interaction between them.   According to the diagram in FIG. 4, the two subdomains of the toxic domain contain intracellular proteins. Binding site of protein P1 (P1^*)). Other Comt P1 of Xin^*Unlike the domain (Fig. 1-3), split-comtoxin (Fig. 4) P1^*The site is conformationally accessible, unless it is bound by P1. A relatively short “peptide size” (about 10 to about 40 residues) that remains mobile Area. The construct of FIG. 4 is toxic in cells lacking P1, but Cells containing P1 appear to be relatively non-toxic. Toxin subdomain The mobile insert between the two is either binding of the intracellular protein P1 or P2. P1 and P1 arranged to appear to be sufficient to impair the reconstitution of the active toxin And two binding sites for P2. The resulting comtoxin is It seems to kill cells that lack exclusively both P1 and P2. (Only one Schizophrenia containing a P1 binding site represents a novel design and useful therapeutic agent On the other hand, note that it is not yet a commutoxin by strict definition. Because co-dominance and multiprotein binding sites are so far needed here Because it is not. The simplest "true" fission comtoxin is protein P1 and And appears to contain at least two protein binding sites, P2 and P2. Easy terminology To achieve this, the construct shown in FIG. 4 is also referred to as "comtoxin". Would. )   In order for split-comtoxin to work, a subdomain of the toxicity domain Affinities between should be small enough to make their interactions essentially reversible It is. This eliminates its irreversible activation before encountering P1 of the toxicity domain (Fig. 4). The affinity between the subdomains of the toxicity domain In some cases, it has been used to regulate the affinity between ubiquitin subdomains. (Johnsson and Walsh Varshavsky, Proc. Natl. Acad. Sci. USA 91: 10340 (1994)).   One particular example of a fission toxin design is barnase, a bacterial bacillus.   Ribobo secreted by amyloliquefaciens (Bacillus amyloliquefaciens) Use nucleases. Barnase is a 110-residue tandem that lacks a disulfide bond. It is a park. It is a model for protein structure and folding Extensively studied. In particular, two fragments of barnase (residues 1-36 and And residues 37-110) reassociate upon mixing and have almost normal structure and thermal stability. Have been shown to be able to form an active enzyme (Sanco and Verst ( Fersht), J. et al. Mol. Biol. 224: 741 (1992)). These findings, as well as Barner Knowledge of the three-dimensional structure of zeolites and similar studies of split ubiquitin cited above (Di Johnsson and Varshavsky, Proc. Natl. Acad. Sci. U SA 91: 10340 (1994)) shows a mobility inserted between residues 36 and 37 of barnase. Peptide linker is involved in the assembly and coalescence of the two subdomains of barnase. Finger that appears to protrude outside the folded Pluck.   P1 binding site of fission toxin that regulates reconstitution of active barnase (Figure 4) From various candidate sequences Can be chosen. For example, tyrosine residues phosphorylated in a predetermined sequence context Is a synthetic peptide of 10-15 residues in length, which is a so-called SH of a specific protein. It has been shown that it can form a tight complex with the two domains (Felder) Et al., Mol. Cell. Biol. 13: 1449 (1993)). SH2 domain is a transmembrane receptor Are present in a variety of regulatory proteins, including regions exposed to the cytosol. S A common feature of the H2 domain is a tight complex with sequences containing phosphotyrosine residues. Each SH2 domain is capable of forming phosphotyrosine. Recognize specific sequence motifs contained. Thus, the two sub-domains of barnase Relatively linking the main and binding to specific intracellular proteins containing SH2 The present invention relates to fission toxins containing fission barnase with short (10-20 residues) linker sequence. May function as a conditional fission toxin. The activity of this toxin is It is regulated by the binding of the contained intracellular protein to the peptide linker, Conformation (mobile in the absence of the bound SH2 domain; Relatively fixed in the presence), but the efficiency of reconstitution of active barnase and hence Will determine the overall activity of this fission toxin based on barnase.   More complex fission comtoxins, for example, have both P1 and P2 nuclear proteins. Even if it is quality [P1⁺P2^-] Design to distinguish cells from other cell types Can be In addition to the P1 binding, effector domain of the fission toxin (FIG. 4) , This comtoxin is an unconditional NLS, P2^*Domain or nuclear tan Ligands for protein P2 and P2 and P2^*Can be shielded by the complex between More adjacent degrons are also found. This split commutoxin is exclusively , Nuclear protein P 2 but is expected to kill cells lacking nucleoprotein P1. That Indicates that only in such cells, the longevity of commutoxin is long (because degron is -P2^*Has a toxicity domain that is shielded by the complex and is active (because of toxicity P1 between subdomains of a domain^*The insert containing is bound by P1 And therefore remain mobile). These designs Cytosolic-specific variants of the protein are also possible. Other polymers, such as RNA It can also fold into the ligand binding domain and, like degrons Comtoxins based on nucleic acids should also be viable due to possible signal It is.   Having discussed specific examples of comtoxin, we will discuss those elements more completely This is very important. In a preferred embodiment, the comtoxin has at least three components. (1) Effector domain, (2) First intracellular protein binding domain A first co-dominant intracellular signal portion located in close proximity to Second co-dominant intracellular signaler located in close proximity to the intracellular protein binding domain An amino acid copolymer (eg, a fusion protein) that includes an amino acid moiety. first There is no strict requirement that the first and second intracellular proteins be different. In addition, as used in this context, the term "intracellular protein" It should be understood that the peptides and polypeptides within are also encompassed. Before In vivo half-life and / or intracellular of commutoxin as discussed in Determining the selectivity of commutoxin by affecting its localization is Or to their binding site located in close proximity to the second co-dominant intracellular signaling moiety Of the first or second protein It is a union.   Comtoxin is an amino acid copolymer having a predetermined sequence identity. It can be produced by any of the known methods of producing. However, Comtoxy The preferred method of constructing and producing proteins employs recombinant DNA technology. so DN encoding the required comtoxin element by the use of conventional techniques A is isolated from a biological source (s) and site-directed mutagenesis Modified as needed using techniques such as firing.   Preferably, amino acid segments that provide the required function (eg, peptides, The minimum number of nuclei required to encode Leotide is employed. Each of the required elements of the comtoxin molecule is easy To encode a functional comtoxin element Determining the minimum length of an NA fragment is a matter of routine experimentation. As a result The resulting DNA fragments are ligated together using standard recombinant DNA techniques. Reads translated into a fusion protein containing all required comtoxin elements Create a frame. Over the past decade, many amino acid sequences have Ker, an endoprotein present in the bloodstream, intercellular voids and inside cells A sequence that forms a hydrophilic and mobile segment of the polypeptide chain that is relatively resistant to It is shown that it can be used. Especially wide range of suitable linkers The collection emerges from the design of a single-chain antibody, in which the linker sequence is the antigen of the light chain. The binding domain is linked to a similar heavy chain domain.   The open reading frame prepared as described above contains a transcription promoter and And inserted into a DNA expression vector containing other sequences required for expression . Selection of an expression vector from among many available choices is primarily It depends on the cell type for which expression is desired. True, as discussed more fully below Nuclear expression vectors are preferred for many applications.   Alternatively, comtoxin is prokaryotic in bacteria (eg, E. coli). Expression of it via the expression vector and the resulting overexpressed protein Purifying the protein and bringing the purified protein into direct contact with target cells Can also be produced. Comt when produced for use in this manner Xin is an addition that allows its translocation into the cytosol of cells Should also have a generic domain. (For purposes of clarity, the term "cytoplasm" The term refers to the interior of the cell outside its nucleus, while "cytosol" It is mentioned that there is a cytoplasmic environment outside of other compartments surrounded by membranes. For example, complete lysine, complete Pseudomonas exotoxin A and These transcripts are present in a variety of natural toxins, such as The use of "slocation" domains has been widely reported in the prior art. Typically , Such reports refer to conventional today's chimeric toxins (Vitetta ) Et al., Imm. Today 14: 252 (1993), Pastan et al., Annu. Rev. Biochem. 6 1: 331 (1992)). The basics between the current chimeric toxin and the comtoxin of the present invention The major qualitative differences are (1) that the current chimeric toxin recognizes a surface marker. Possible but not intracellular, and (2) the current chimeric ibis Syn is, in principle, a multi-target combination that is characteristic of comtoxin. It does not allow selectivity.   An effector domain places its effector at a predetermined location in the cell. When delivered, they cause certain effects, such as cell death or its terminal differentiation. Is a protein or polypeptide that can exert . Thus, the effector domain of comtoxin, when delivered to cells, It can be derived from a protein or polypeptide that acts as a toxin. For example, Rishi A chain (and similar plant toxins), exotoxin of Pseudomonas And many others including the toxicity domain of diphtheria toxin Toxins are known in the art. As previously discussed, certain toxic proteins Intracellular compartment specificity of a protein or a polypeptide, the substrate is located in the cytosol Comtoxin to alter selectivity of but not nucleared comtoxin It can be used together with the design. Such proteins or polypeptides Examples include diphtheria toxin and Pseudomonas exotoxin A Both of which extend ribosylation (and thereby inactivate) ADP. Inhibits protein synthesis by long factor 2 (EF2). Most of EF2 is a site Contains toxic domain of Pseudomonas type because it is sol Comtoxin translocation from the cytosol to the nucleus It appears to be physically separated from the substrate.   The effector domain states that its introduction into a predetermined cell location May be from any protein or polypeptide that is thought to cause . For example, deoxyribonuclease is located in the nucleus of target cells (but in the cytosol). Not) if introduced into a toxic effect It appears to act as a ter domain. In fact, EcoRI is in vivo (yeast yeast Cuts the nuclear DNA in Saccaromyces cerevisiae) Cells have been shown to kill (Barnes and Rine, Proc. Natl. Acad. Sci. USA 82: 1353 (1985)). The substrate is present in both the cytosol and the nucleus. Examples of toxic domains that are present include, in addition to the ricin A chain, RNase A and Balner. Ribonucleases such as zeolites. These produce the traditional chimeric toxins (Rybak et al., J. Biol. Chem. 266: 21202 (199 1), Prior et al., Cell 64: 1017 (1991)).   Because the goal of treatment is to render harmless cells harmless, a series of useful Vectors are not limited to cytotoxic proteins. This series includes, for example, cells Include transcription factors whose presence in E. coli leads to growth arrest and terminal differentiation (Vai Weintraub, Cell 75: 1241 (1993)). In particular, My muscle cells Expression of muscle-specific transcription factors, such as oD or myogenin, is Causes the cells to differentiate. Under normal conditions, these proteins It is expressed only in meat and only when appropriate. Its effector domain is Myo Comtoxins based on D, myogenin or similar transcription factors are Should be able to cause terminal differentiation, but Other cells are not possible.   Regarding co-dominant intracellular signals, a variety of such signals have been described in the literature. ing. For example, a short in vivo half-life may result in a distinct degradation signal, The protein may be provided to the protein by one or more of the degrons (Warsaw Varshavsky, Cell 64: 13 (1992), F Hershko and Ciechanover, Ann. Rev. Biochem. 61:  761 (1992)). The best understood intracellular degradation signal is called N-degron. (Warshavsky, Cell 64: 13 (1991)). This signal is The destabilizing N-terminal residue of the protein substrate and an internal lysine (one or more) )including. The propensity of N-degrons with different destabilizing residues is called the N-terminal rule. Ie, the relationship between the in vivo half-life of a protein and the identity of its N-terminal residue. It is a ream. The lysine residue of the N-terminal substrate is a site for forming a multi-ubiquitin chain, It is required for substrate degradation.   Ubiquitin is primarily associated with proteolysis because of its covalent binding to other proteins It is a protein that plays a role in many processes through the pathway. Recognition of N-terminal substrate Are enzymes whose components are ubiquitin-binding enzymes (one of several such enzymes in cells) And N-recognin or proteins called E3 Mediated by a complex that defines Targeted, multi-ubiquitinated groups Quality is due to the 26S proteasome, a multicatalytic, multisubunit protease. Decomposed like processing. N-terminal rule, ubiquitin fusions and related Aspects of the technology are described in U.S. Pat.Nos. 5,132,213, 5,212,058, 5,122,463, Varshavsk, including 5,093,242 and 5,196,321. y) et al. are the subject of a number of U.S. patents assigned to U.S. Pat. Incorporated in   In vivo of proteins with strongly destabilizing N-terminal residues such as arginine Half-life can be as short as 1 minute, while the stabilized N-end like valine Identically expressed with terminal residues and otherwise identical The proteins have a half-life of more than 20 hours, and the steady state of these proteins in cells It results in a difference of more than 1,000 times between the concentrations of the states. Ubiquitin-dependent tan Protein degradation systems (including the N-terminal rule pathway) share many components of the 26S proteasome I do. The differences between these systems include their distinct complexes that target, and Recognin binds to degradation signals other than N-degron. In addition to N-degron Amino acid sequence that functions as a degradation signal and is transplantable to other proteins Is, for example, a “break box” of a short-lived protein called cyclin ( Glotzer et al., Nature 349: 132 (1991), Ciechanov er), Cell 79:13 (1994)) and S.M. Short-lived S. cerevisiae Two specific regions of the transcriptional regulator Matα2 (Hochstras ser) and Warshavsky, Cell 61: 697 (1990)).   The property of codominance is not limited to degradation signals. Degron discussed above A sig that gives proteins the ability to enter a compartment surrounded by a membrane for similar reasons Null (Schatz, Protein Sci. 2: 141 (1993), Osborne and Silver, Annu. Rev. Biochem. 62: 219 (1993), and Dingwoo Dingwall and Lasky, Trends Biochem. Sci. 16: 478 (1991)) , Independent of each other if they are in spatially distinct regions of the same protein Will work. Discussions herein are limited to nuclear localization signals (NLS) But, but also other autonomous signals, especially other translocation signals It is also related to comtoxins.   Proteins smaller than about 60 kDa are nucleated by diffusing through nuclear pores. Pore-mediated transport of larger proteins, Requires the presence of at least one NLS accessible to the components of the slocation system. N LS is a short sequence (10-20 residues) rich in lysine and arginine, Their steric accessibility in proteins can be attributed to nuclear translocation signals Seems to be sufficient for their activity as. Many proteins with NLS Enters the nucleus shortly after their synthesis in the cytosol, but some protein Transport is not structural. These NLS are often sterically shielded NLS Must be "activated" by removing the shielding. Comtoxin concept An example related to is the mammalian protein p110, the transcription factor NF-κB Is a precursor containing the NLS of the p50 subunit. p110 is cytosol It is shown to survive. Because the C-terminal half region of p110 is the same Three-dimensionally shields NLS located in the N-terminal half of the protein (p50 region) Because. In contrast, it is the product of proteolytic processing of p110 The protein p50 has an active (unhindered) NLS and is transported into the nucleus Is done. Nuclear uptake by shielding NLS in the NF-κB family of transcription factors Other examples of subtle and precise adjustments of the object are also described (Liou and Balti) More (Baltimore), Curr. Op. Cell Biol. 5: 477 (1993), Henkel et al., Cell 68: 1121 (1992), and Beg and Baldwin, Genes Dev. 7: 2064 (1993)).   The concept of the present invention is to produce a new class of reagents named comtoxins. Based on the co-dominant properties of biopolymer signals. Comtoxin Function of metabolic stability of comtoxin (half in vivo) Target) or its location within the cell (eg, cytosol versus nucleus) Comtoxin binding domain by their protein ligands in cells (this paper By the way, P^*Depends on occupancy (referred to as type domain) (see Figures 1-3) Based on the fact that. One important feature of comtoxin, this dependence, The signal portion of the protein is a large (tan) that targets specifically to the signal portion. Distinct regions of the protein surface recognized by the complex (of the size of the protein) Made possible by the fact that The physical bulk of the targeting complex, and Given the relatively large area occupied by the signal portion on the protein surface Part of the signal part of the protein by another protein sphere bound nearby Signal due to the respective complex targeting even the primary disturbance (steric occlusion) It appears to be sufficient to prevent productive joining of the parts.   Thus, the P of comtoxin^*Type domain eg degron or NL Positions adjacent to selected signal moieties, such as S, are intracellular protein ligands Comtoxin P^*When binding to the type domain, three-dimensional shielding of the signal part Enables shielding (FIG. 1-3). The term "adjacent" or "adjacent" It is not possible to specify clearly and in advance the range of distances covered by On the other hand, the above considerations suggest that the screenable signal portion and P^*Suitable for type domain Relative to each other for a particular comtoxin design Point out that there will be many. Therefore, the protein binding domain Synthesizing fusion proteins located at varying distances from the main is routine Will be a problem for the experiment. Intracellular signal domain functions Given the ease with which they can be assayed, many such fusion proteins can be To determine the extent to which protein binding interferes with signal domain function Can be assayed. This impairment of the intracellular signal domain by steric shielding Inactivation results in alteration of the intracellular fate of the commutoxin molecule.   Comtoxin P^*Type domains are specific, predetermined intracellular triggers. (Eg, specific intracellular proteins P1, P2, etc.) It is. Ideally, a series of intracellular ligands of comtoxin, ie, eliminated One group that defines the cell type to be used is the amount of protein produced by the targeted cell population. It should be selected by conducting a detailed survey of the park quality. Specific cell ties Protein composition, especially of normal and tumor-derived human cells. (Eg, Hall et al., Proc. Natl. Acad. Sci. USA 90: 1927 (1993)). However, the information gathered so far is incomplete for most cell types. You. Therefore, useful strategies are overproduced or present in target cells. From a series of intracellular proteins already known to either The choice is to choose a ligand. Most (but not all) of the latter protein Non-target cells.   Tumor suppression possibly as a result of gene amplification contributing to the malignant phenotype of cells Lacks and differentiates wild-type variants of the protein (expressed in the normal ancestor of this cell) Consider a tumor cell that overproduces this protein. For example, many (but not all) No) Human breast cancer lacks or functionally suppresses the tumor suppressor protein p53. Contains inactive mutants. Furthermore, many of these tumors contain the protein c-My c (some other tampa Overproduction). Contains P1 protein but not P2 protein Comtoxins, which exclusively kill cells that lack quality, have essential selectivity for such settings. I do. P1 of this comtoxin^*While the domain appears to bind to c-Myc , P2 of this comtoxin^*The domain appears to bind to wild-type p53. However, it does not bind to that mutant variant in certain cancers). This non-comtoxin Even selective intracellular delivery kills cancer cells lacking p53 that overexpress Myc However, if not all of the organism is rescued, Be looked at. Because normal cells contain p53, some, if any, p5 Because normal cell types lacking 3 do not appear to overproduce c-Myc. You. In addition, the selectivity of this comtoxin, if necessary, will sharpen the description of the target cells. Degron, which binds to a third intracellular protein, P3, which is selected to be Or P3 containing NLS^*By adding a domain to it, Can be increased.   This example illustrates a strategy that can also be used with other anti-tumor comtoxins. Details In the cases that have been analyzed in detail, cancer cells have at least the following characteristics: Absence (or reduced production) of a protein expressed in the preceding cell, normal Of mutants of proteins expressed in ancestral cells and expressed in ancestral cells Wild-type or mutated protein not expressed (or weakly expressed) Different from their normal ancestors by the combination of any overproduction of Was found. Thus, infected by a specific virus against a specific cancer Comtoxin design to cells and also to other unwanted cells It is possible to select the optimal group of intracellular ligands for in Should be. Examples of the latter include cells forming atherosclerotic plaques and intravascular neovasculature. Skin cells, their selective resection appears to block blood supply to metastatic tumors It is.   P^*Selecting a protein binding site, called the type domain, requires only one P in mutoxin molecule^*Unwanted homodimerization of type domains Should be recognized. Because it appears to interfere with the function of the commutoxin That's because. As you can see, P^*Type domains form high-affinity homodimers While not forming its intracellular partner (P1^*Type protein) and heteroda It should be possible to form an immer. Alternative P^*An example of a type domain is (1) a natural protein ligand of a P1 type protein, (2) a P1 type A peptide-sized fragment of the natural ligand that retains affinity for the protein, (3) a single-chain antibody or a fragment thereof specific to a P1-type protein; And (4) non-peptidic low M of P1-type protein_rLigands of You. This latter possibility is delivered directly (delivered via expression vector mediated Comtoxin (as distinguished from comtoxin).   The single-chain antibodies described in item (3) above are described in the prior art (eg, Whitlow and Filpula, Methods 2: 97 (1991), Vietlow Vitetta et al., Imm. Today 14: 252 (1993) and Pastan et al., Annu. R eview Biochem. 61: 331 (1992)). P of this class^*The type domain is Tumor-specific fusion tags produced by oncogenic chromosomal translocation Includes single-chain antibodies to the protein junction region. These mutant proteins Quality is tumor cells Occurs early in the evolution of this lineage, seems to be required for a malignant phenotype, and Is a particularly suitable class of P1 type proteins.   Construction of single-chain antibodies against specific proteins or specific regions of proteins First, the production of monoclonal antibodies with the required specificity, and second, D containing a reading frame encoding a single-chain control of this monoclonal antibody. Association of NA fragments. The latter stage involves the production of antibody-producing hybridoma cells. In the cell, two genes encoding the protein binding domains of the heavy and light chains of the antibody Polymerase that amplifies related non-adjacent regions of the gene and fuses them in the same open reading frame It is performed using a chain reaction (PCR). Both stages of this process (essential singularities Production of hybridomas secreting neutral antibodies and single-chain variants of these antibodies PCR-mediated construction of transgenes) is now standard and routine for those skilled in the art. This is the treatment of the patient. In addition, many potential targets of comtoxin (ie, Proteins P1, P2, etc.) are cancer proteins, tumor suppressors and other Is a regulatory protein for Antibodies (and the corresponding hybridoma cell clones that produce these antibodies) ) Can be selected for the construction of the corresponding single-chain antibody; In many cases, it will be possible to bypass the initial hybridoma production step. ( This latter step involves the development and development of years of technology for producing monoclonal antibodies. And completion (eg Harlow and Lane) (Ed.), Antibodies, A Laboratory Manual (Cold Spring Harbor)   Laboratory (Cold Spring Harbor Labratory), Cold Spring Harbor Bar, New York (1988)). )   Both in vivo and in vitro applications for the comtoxin molecules of the present invention Disclosed. A typical in vivo application is to remove dangerous cells or In a treatment regimen designed to be harmless (non-split). For example, Cancer has been diagnosed in a patient and its selectivity targets tumor cells in this patient Comtoxins that are appropriate to be designed as described above Think about the situation. Two alternative delivery routes for this comtoxin are (1) Comtoxin expression in blood vessels or (2) target cells.   Delivery via intravascular route (Gilman et al., Eds., The Pharmacological Ba sis of Therapeutics (Pergamon Press, New York, 1 990), not only is the toxin domain from the cell surface Also possesses a domain that mediates the translocation of protein fusions to the sol Should. This aspect of comtoxin delivers directly (as distinguished from expression-based Strategy), as well as similar aspects of current immunotoxins. (Vitetta et al., Imm. Today 14: 252 (1993), Pastan) Et al., Annu. Review Biochem. 61: (1992), and Olsnes et al., Som. Ce ll Biol. 2: 7 (1991)). If necessary, send comtoxin as protein. The first step is to use a drug such as an antibody that binds to a surface marker on the target cell. By fusing comtoxin to the main, it becomes partially cell type selective. obtain. Critically, and in contrast to the current situation with chimeric toxins, the aforementioned table Area markers are correct without significantly increasing the nonspecific toxicity of comtoxin. May be present in more than a single target cell. In fact, the conditional toxicity of comtoxin is After it enters the cell Is determined by the environment in which The quality "feature" does not appear to affect cells different from the targeted one.   One advantage of the direct delivery strategy is that it is vector-mediated Circumvent the potential problem associated with the delivery of One of direct delivery A potential drawback is that otherwise identical in vivo delivery is mediated by the expression vector. Compared to delin, multi-domain commutoxin (due to the presence of additional domains ) Larger size. Comtoxy using any of these delivery strategies Testing is technically straightforward, requires exclusively existing technology, and A prudent experimental approach is important because it can be directly and objectively evaluated. Use both strategies in the assessment of mutoxin and compare the results. It seems to be.   Comtoxin can also be delivered into cells by mediation of an expression vector. This Both of these approaches involve medical interventions from cytotoxic treatment to gene therapy. Progress in improving the bioavailability of proteinaceous drugs used Part of the ongoing effort. Insufficient selectivity issues are all current cytotoxic strategies Is common to That is, the effector reaches its intended subcellular compartment Cells seem to be killed regardless of whether they were targeted or non-specific bystanders is there. For example, one difficulty with current immunotoxins is that they mainly affect the liver. But but not only their non-specific toxicity. Duration and intensity of treatment This toxicity, which imposes limits on both, is due, in part, to intravenously administered immunotoxins. Arising from the clearance of the toxin by reticulum cells (Vitetta itetta) et al., Imm. Today 14: 252 (1993)).   In contrast, even the non-selective delivery of the comtoxins of the invention to most non-target cells Not expected to have any effect. This feature of comtoxin makes for a much larger treatment Produces an index (ie, significantly greater tolerable intensity and duration of treatment) . If necessary, the first step in the delivery of comtoxin is to target It can be made selective by fusing it to a domain that binds to the above surface marker. Critically, this marker greatly increases the nonspecific toxicity of comtoxin Can be present in more than the correct target cell without the need.   Comtoxins designed for delivery by expression vectors are It appears that it lacks the "cross-compartment" domain required for indirectly delivered controls. This Vector of retrovirus vector, adenovirus vector, different virus Vector, or simply naked in a gene delivery system such as a liposome-based delivery system May be any of the following DNAs. Virus vector and liposome-based Both gene delivery systems have been successfully used in approaches to gene expression in intact animals. ing. Several types of vectors for gene therapy and other applications are already available (Yee et al., Proc. Natl. Acad. Sci. USA 91: 9564 (1994)). Mulligan, Science 260: 926 (1993) and Anda See also Anderson, Science 256: 808 (1992). These vectors are In cell culture systems, in complete animals, and in human patients with appropriate preliminary testing. It can be used for delivery of comtoxin. Viral and plasmid based vectors Recent advances in the design of tars (eg, Nabel et al., Proc. Natl. Aca d. Sci. USA 90: 11307 (1 993) and Mulligan, Science 260: 926 (1993)) And quiescent cells can be transfected and Either specific or almost non-selective for cells with surface markers , Resulting in a non-replicating vector that is useful. For gene therapy and other applications Such vectors in use (eg, Mulligan, Science 260: 926 (1 993) and Anderson, Science 256: 808 (1992)) It is a powerful means of delivery for delivery.   Comtoxin can also be used for various in vitro applications. For example, white blood Comtoxin disrupts normal bone marrow cells in bone marrow transplantation of diseased patients Selective for leukemic cells from patient bone marrow samples (for subsequent reinfusion) Can be used to remove High-dose chemotherapy with bone marrow stem cell rescue , Currently leukemia, lymphoma, and more recently glioblastoma and other metastatic solids It is a standard and sometimes curative treatment for a variety of cancers, including tumors. Current trunk A common step in all cell rescue protocols is bone marrow ablation chemotherapy Requires that a portion of the patient's bone marrow be recovered prior to use. Resulting short-term The in vitro bone marrow cell culture system is then Prior to reinfusion of the purged bone marrow into a patient who has been removed by dose chemotherapy, It is treated to remove any malignant cells present therein.   The current method of selectively killing malignant cells in bone marrow cell cultures is based on conventional chimeric Requires processing in thin (Vitetta et al., Imm. Today 14: 252 (1993)). ), Which are cytotoxic effector domains and markers A domain (typically based on an antibody) that binds to structures on the surface of the target cell. Ki This design of mela toxin has led to the majority of leukemias from bone marrow explant tissue culture systems. Often removes sexual cells, most of which have lineage-specific surface markers Is sufficient, but not always. Unfortunately, non-hematological cancer Many types of tumor cells include more surface markers than certain types of leukemia Considerably more heterogeneous with respect to Thus, bone marrow in vitro prior to its reinfusion Complete, reliable and selective removal of metastatic tumor cells from a given culture system is generally a solution. It remains an issue to be addressed.   In contrast, the comtoxins of the present invention (as distinguished from those on the cell surface) Due to their higher multi-target selectivity and sensitivity to intracellular markers, Provides an alternative and more selective means of in vitro bone marrow purging of cancer cells It is. Example Example 1: Construction and testing of indelin   The first test of the efficacy and selectivity of indelin is not in whole animals, Hosted in a technically less damaging setting for mammalian (human) cell culture systems There will be. The use of human cell culture systems, e.g., thymus Indelivery in laboratory animals, such as mice, using incomplete "nude" mice Subsequent testing of the efficacy and selectivity of the application. Vector in cell culture system -Mediated delivery of indelin is technically straightforward. Because (human Short-term or long-term transfection of mammalian cells (including cells) Several high-efficiency vectors and protocols are available for You. Briefly, [P1⁺P2⁺The specific indelin test shows that this reagent Is not performed ([P1⁺P2^-], [P1^-P2⁺] Or [P1^-P2^-]) On cells [P1⁺P2⁺] (Directly or by mediation of an expression vector) When introduced, it is exclusively (or almost exclusively) [P1⁺P2⁺] Need to decide whether to kill the vesicles.   Indelin to be designed in this example has a short C-terminal domain. (5-20 residues) the "upstream" domain closest to the linker sequence (P2^*) This is a multidomain fusion that is the A chain of lysine linked. Domain P2^*Is Domain P1 in order^*Is combined with Simple sequence, relatively hydrophilic linker GG GSGGGSGGGSGGGS (in single letter amino acid abbreviations) Will be used first to join the main. First and second degron Is the protein P1^*Binding to the first degron inhibits the activity of Parkinous P2^*Domain P, such that binding to 1^*And P2^*Will be located in or near it.   Nucleic acids encoding the various elements of the indelin molecule are derived from naturally occurring sources. Will be separated and by conventional techniques, e.g. A suitable expression vector such as the pSG5 vector sold by Meeting within the clinic. This vector contains the SV40 early promoter and the mammalian Contains other suitable features for expression of the inserted gene of interest in animal cells , A 4.1 kb "shuttle" plasmid for E. coli and mammalian cells. Re Due to short sequences such as anchor sequences, DNA is custom made by synthetic techniques Will. The toxicity domain employed would be the A chain of ricin (another cytotoxic effector, For example, the toxic domain of the exotoxin of Pseudomonas The toxic domain of ria toxin can also be used to construct indelin). Re The deduced amino acid sequence of the A chain of syn can be found, for example, in Funatsu et al. 3: 1157 (1991)). In this experiment, the effort was This would not be done to reduce the size of the toxicity domain by law. Like In addition, a fragment containing the cDNA encoding the complete ricin A-chain was For example, plasmid pRA229 (Ready et al., Proteins 10:27). 0 (1991)).   P^*Degradation signals to be located within or immediately adjacent to the type domain (data Glon) may be selected from among several currently known degrons. First study The degradation signals employed in are the N-degron and cyclin Degron as defined by N-degron is biochemical and genetic Has been analyzed in great detail and is understood in considerable detail (Warsawski (V arshavsky), Cell 69: 725 (1992)). Some simple variants of N-degron Described and analyzed (Varshavsky, Cell 69: 725 (199 Commented on by 2)). The simple N-degron adopted in the first study is By Bachmair and Varshavsky (Cell 56: 1019 (1989)) Would have been described. This N-degron was subsequently used for E. coli La. Arginine (Arg) -like instability followed by a sequence of 45 residues from the c repressor Includes N-terminal residues that The corresponding treatments, sequences, and plasmids Along with the restriction enzyme cleavage map, In Bachmair and Varshavsky (Cell 56: 1019 (1989)) Described in more detail. Degro defined by cyclin's "destruction box" As for the sequence involved, the sequence involved is encoded by a particular fragment of plasmid pAG132. (Glotzer et al., Nature 349: 132 (1991)).   In the first experiment, at least the following intracellular proteins: 1) HPV16 E6 protein of oncogenic human papillomavirus (HPV) such as Binding to the E7 protein of HPV^*Type domain is used Will be. The E6 and E7 proteins of the oncogenic human papillomavirus are One of the preferred intracellular targets for the design and testing of the comtoxins of the invention One. Anogenital cancer, especially female cervical cancer and certain human papillomaviruses A strong association with HPV (HPV) has been established by a number of detailed studies. Uterus Cervical cancer alone causes more than 500,000 deaths worldwide annually. High risk HP V includes HPV-16 and HPV-18, which are potentially precancerous and Associated with some desquamating intraepithelial neoplasia. Overall, over 90% of cervical cancers May contain DNA of one of the high-risk HPV types. Scheffner et al., Curr. Topics Microbiol. Immunol. 186: 83 (1995)).   Although the HPV genome encodes about six proteins, Only two, HPV proteins E6 and E7 are consistently expressed in cervical cancer And that the tumorigenic effects of HPV are specifically mediated by these proteins. And strongly suggest. Recent studies have shown that the E7 protein is a retinoblastoma protein (Rb ), A nuclear protein called Specific complexes containing regulatory components of networks that prevent uncontrolled cell growth Are shown to act. The Rb protein is therefore a tumor Acts as a suppressor. HPV E7 protein and cells infected with HPV Complex formation between the Rb protein and the corresponding cell line Interfering with the tumor suppressor function of Rb that contributes to transformation.   A second cancer-associated HPV protein, E6, activates another cellular tumor support called p53. Specific binding to the Lesser protein has also been shown. E6-p53 The formation of the complex not only disrupts the function of p53 to suppress proliferation, but also It also results in accelerated degradation of bound p53, reducing the concentration of p53, and Thereby effectively inhibiting the function of p53 in more than one way (Fibreg Huibregtse et al., EMBO J. 10: 4129 (1991)).   No curative treatment for HPV-derived metastatic cancer is currently available. This A common reason for and other non-curable metastatic cancers, which are the majority of cancers, are cell surface Low molecular weight cytotoxic drugs that recognize the above markers and current chimeric toxins That means we can't distinguish between cancer and normal cells. In contrast, the present invention E6 / E7-specific comtoxin (more specifically, indelin) is expressed in target cells Seems to be sensitive to the presence of parts E6 and E7, and therefore It is likely to rescue normal cells that lack these HPV-derived intracellular (nuclear) proteins .   Before describing the construction of E6 / E7-specific indelins, the P1^*You And P2^*Domains (these domains are collectively P^*Show as type domain General requirements) are easily considered. These requirements are as follows. That is, (i) P^*The type domain is Intracellular (cytosolic or nuclear) protein P1 or P2 (by designer These proteins are selected to include the “features” of the target cell protein). (Ii) P^*Type domains form high-affinity homodimers Should not, (iii) P^*A type domain can be either as part of that domain or Either in its immediate vicinity, to cytosolic and / or nuclear indelin It should contain sequences that give a short half-life (degradation signals).   Given these constraints, a suitable P^*The type domain is the protein P1 Or binds to a natural protein ligand of P2 or, for example, P1 or P2 Can be any of the single chain antibodies. P^*Single-chain antibodies as type domains Use eliminates the problem of homodimerization that natural ligands may be prone to, It is also P^*Also enables standardization of type domain design. That Suggest that single-chain antibodies to different protein domains share a common "nuclear" antibody structure. Because it has. Finally, P^*The use of single-chain antibodies as type domains Vector-mediated delivery of indelin is technically unscrupulous. Because conventional (multi-chain) antibody molecule association is not required for single-chain antibodies It is.   The actual construction of E6 / E7-indelin is initially separate, E6-inderin and And E7-indelin. these Only one P^*Indelin of the type domain is E6 of HPV16 and / or Or a human cell culture system that either lacks or expresses the E7 protein Can be tested and, if necessary, Can be adjusted (see below). Then the more complex, co-dominant comtoxin (E6 / E7-Indelin) has only one P^*Type domain precursor (E6 -Indelin and E7-indelin). These "middle Even the "body" is the only P in the design of E6 / E7-Indelin^*Typed Mei It should be emphasized that protein constructs are expected to be useful drugs on their own. It is. This type of conditional toxin regulated by ligand (E6-indeli Or E7-indelin) are sensitive to only one ligand per drug That said, the ligands themselves are intracellular proteins (E6 or E7). Thus, the indelin of these single P * -type domains (E6-Indelin and E7-Indelin) recognize cell surface markers but However, their design does not allow them to be sensitive to specific intracellular markers Have a selectivity that differs from and is not achievable with current chimeric toxins. Would own in. HPV-induced cancers have both E6 and E7 tamper E7-Indelin (or should be replaced) to produce protein E6-Indelin) has been demonstrated in cell culture settings and later in human patients. May be found to be sufficient for the selective removal of cancer cells. So why is it more complex Construction of a crude, co-dominantly mediated comtoxin-type E6 / E7-indelin And then try to use it? The answers discussed in the following sections are two-sided It is.   First, cells containing both E6 and E7 of E6 / E7-indelin were identified. The ability to exclusively kill and rescue cells that lack even one of these proteins is E6 / E7-indelin of the xin type is relatively weak In vivo "noise" of complex and non-specific protein-protein interactions (Only one P such as E6-inderin or E7-inderin^*Typed Considerably smaller (than main indeline) making it confusing. For example, E6-A Ndellin's P^*E6 protein that forms a type domain and has high affinity (see below) Normal cells lacking HPV E6 and E7 proteins By chance, even with relatively low affinity (normally cross-reacts) In this case, this cross-reactivity is due to the E6-indelivery against cancer cells containing E6 / E7. Can adversely affect the desired selectivity of the protein. Same is true with E7-Inderin It seems to be. In contrast, the E6 / E7-indelin of the comtoxin type is selective It appears to be significantly more resistant to this confusion that reduces gender. Because For it to be present, this E6 / E7-indelin's E6 binding and E6 7-linked P^*Type domains also do not bind to normal cellular proteins Must be significantly smaller for any particular cell line. This is a likely event. Thus, comtoxin-type E6 / E7-in Delin is the only P^*Only selective over type domain counterparts Rather, its selectivity is also stronger against the other types of confusion described above.   Second, as a basis for the first test for indulin class commutoxin, H By focusing on PV-induced cancers, one can actually draw on the virus. Not caused but instead the result of a (multiple) mutation of a particular cellular gene Is an area of convenient testing for other relatively prevalent and non-curable cancers. Choose. As discussed above, these cancer cells differ in their protein composition from their normal Often very close to great ancestors And differ mainly from the specific protein concentration. For example, cancer cells may overexpress the c-Myc protein and some other proteins, But some of these proteins are also low in some normal cells of the same organism It appears to exist at the level. In other words, E6 / E7 features of HPV-induced cancer Heterosexuality is less severe about the selectivity of commutoxin than typical cancer does Make a request. Thus, HPV inducibility as a basis for a first test of comtoxin Cancer selection is justified by current incurability and high frequency of these cancers On the other hand, it is more clear between HPV-induced cancer cells and their normal ancestors. It is also suggested by the compositional differences defined in. For these reasons, Comt Of the toxin type of indelin (the only P^*Type domain counterpart Greater selectivity is associated with a curative level in most non-viral cancers. Will be critical in achieving target selectivity. a. Construction of E7-Indelin   At present, HPV16 has various high affinity for E6 or E7 proteins. Polyclonal antibodies are available, but monoclonal antibodies are So far, it has been prepared only for the E7 protein (Scheffner ) Et al., EMBO J .; 11: 2425 (1992)). HPV16 (and human papillomavirus) How many genes encode the E6 and E7 proteins Has been cloned by E. coli and the corresponding protein is E. coli (E. coli, which are overexpressed in E. coli, purified to homogeneity, and (Scheffner et al., Curr. Topics Microbiol. Immun. ol. 186: 83 (1994), and references therein). HPV16 E7 The production of single-chain antibodies against proteins is based on monoclonal antibodies against E7 protein. By Scheffenr et al. (EMBO J. 11: 2425 (1992)), which secretes antibodies. The described mouse hybridoma cell line 100201 will be utilized. Specific things Methods for producing single chain controls of clonal antibodies have been extensively described in the prior art. (Eg Whitlow and Filpula, Methods 2: 97 (19 91), Vitetta et al., Imm. Today 14: 252 (1993), and pasta (Pas tan) et al., Annu. Rev. Biochem. 61: 331 (1992)).   The DNA fragment encoding the single-chain control of anti-E7 monoclonal antibody 100201 was Of the two genes encoding the E7 binding domains of the H and L chains of the 100201 antibody Polymerase chain reaction amplifies related non-adjacent regions and fuses in the same reading frame PCR and using purified genomic DNA from 100201 hybridoma cells Will be produced. Encodes an antibody specific to the desired hybridoma, PCR-mediated construction of a DNA-based open reading frame (ORF) is known to those of skill in the art. This is a routine action. OR encoding the resulting single chain 100201 antibody F is a conventional technique, especially a technique for producing single chain antibodies (Whitl (Whitl) ow) and Filpula, Methods 2: 97 (1991)). One of the linkers is a mobile, relatively protease-resistant and hydrophilic phosphorus. Using the car GGGSGGGSGGGSGGGS, the A-chain of ricin (see above) Encoding open reading frames use standard techniques for manipulating recombinant DNA. And will be combined in the same open reading frame. The resulting larger ORF Is an anti-E7 single chain antibody against the toxic domain of lysine (A chain) (P^* Type domain Is designed), and the toxicity domain of ricin is Will be the C-terminal domain. All of these, as well as mammalian expression vectors Subsequent manipulation of the recombinant DNA prior to placement of the final construct in (see below) Is a versatile E. coli based vector pUC19 (Maniatis ( Maniatis) et al., Molecular Cloning, Cold Spring Harbor Laboratory Lee (Cold Spring Harbor Laboratory), Cold Spring Harbor, D New York (1989)).   A degradation signal (degron) was placed in close proximity to the anti-E7 single chain antibody domain. For example, one can choose from a variety of different convenient degradation signals (see above). This feature For a regular construction, we have Bachmair and Varshawski. avsky) (Cell 56: 1019 (1989)) . However, it must be emphasized that this choice is by no means unique. No. Other variants of N-degron and other degradation signals unrelated to the N-terminal rule pathway Should be relatively suitable for the design of Indelin. As described above In addition, Bachmair and Varshavsky (Cell 56: 1019 (19 89)) N-degron was subsequently purified from the E. coli Lac repressor Contains a destabilizing N-terminal residue, such as arginine (Arg), followed by a sequence of residues . Both the N-degron nucleic acid sequence and the corresponding amino acid sequence By Oach (Bachmair) and Warshavsky (Cell 56: 1019 (1989)) It has been described.   Since N-degron contains a destabilizing N-terminal residue, it is by definition It should be placed at the N-terminus of the Ninderin fusion construct. Protein To produce a destabilizing residue at the quality N-terminus, we Utilizing earlier developed ubiquitin fusion technology (Warshavsk y), Cell 69: 725 (1992), U.S. Patent Nos. 5,132,213, 5,212,058, and 5,12. Warsawski, including 2,463, 5,093,242 and 5,196,321 Varshavsky) et al.). Ubiquitin is essential from yeast to humans This is a 76-residue protein that has not changed to. Encoding ubiquitin, previously Many sequenced recombinant DNA plasmids are available (eg, Plasmid pUB23 by Bachmair et al., Science 234: 179 (1986). ).   The E7-specific indelin, fully-assembled DNA-based ORF, The following protein regions or domains starting from the end: 1) Ubiquitin, 2) Bachmair and Varshavsky (Cell 56: 1019 (19) 89)), N-degron, 3) 100201 single strand against E7 protein of HPV16 human papillomavirus Antibodies, and 4) Lysine toxic domain (A chain) Would code b. Construction of E6-Indelin   Similar procedures construct an ORF of DNA encoding E6 specific indelin Will be used to The only major differences are as follows. First, H Monoclonal antibodies against the E6 protein of PV Because a variety of polyclonal antibodies is not available at this time, E6 / HP V), one step in the construction of E6-indellin E. coli overexpressing proteins (Scheffner et al., Curr. Topics  Microbiol. Immunol. 186: 83 (1994)) and purified by those skilled in the art. Known routine techniques for producing monoclonal antibodies (D. Lane ), Antibodies, Cold Spring Harbor Laboratory Harbor Laboratory), New York (1989)). Would produce a panel of antibodies. Highest affinity anti-E6 cell thus obtained Noclonal antibodies are labeled as described above in the protocol for making E7-indelin. Using standard techniques, single-chain (sFv) antibodies (or rather, (ORF of the DNA to be loaded).   Second, an alternative to N-degron that would be used to construct E7-indelin Instead (see above), for example, the cyclin's destruction box (Glotzer) Et al., Nature 349: 132 (1991)). Will be located adjacent to the chain antibody domain. The rest of the construction protocol is E It will be essentially identical to that described above for the construction of 7-indelin.   OR based on fully assembled DNA encoding E6 specific indelin F is the following protein region or domain starting from the N-terminus: 1) Ubiquitin, 2) Internal degrons identified by the cyclin's destruction box (Glozer tzer) et al., Nature 349: 132 (1991)), 3) a single-chain antibody against the E6 protein of HPV16 human papillomavirus, and 4) Lysine toxic domain (A chain) Would code   The resulting E6- and E7-indelins are well known to those skilled in the art. Separately tested using essentially the same protocol utilizing the Will be. Therefore, for brevity, the description below deals exclusively with E7-inderin. There will be. First, the ORF of the DNA encoding E7-indelin is, for example, pE Such as the 7Mo plasmid (Barbosa et al., EMBO J. 9: 153 (1990)). Metallothionein or Moloney leukemia virus vector in a mammalian expression vector Would be located downstream of a moderately active promoter, such as a promoter . The resulting plasmid expressing E7-indelin, under the same conditions, (See below), two otherwise identical tests differing by the presence of the E7 protein Will be introduced into the cell culture system. Human cultures lacking and expressing E7 Such pairs of systems are described, inter alia, in Barbosa et al. (EMBO J. 9: 153 (1990)). Which also have previously been described as E7 and E6 HPV proteins. Quality as described (eg, Kessis et al., Proc. Natl. Acad. Sci. USA 90: 3988 (1993)), and cloned it with the HPV gene of E7. Any human cell line that lacks HPV by stably transfecting Can be newly produced. High efficacy of mammalian cells with plasmid-based vectors Transient transfection at a rate is a well-established technique. Above all, We are provided by Gibco BRL Inc Lipofectin-mediated transfection protocol To use. This produces this lipophilic, transfection-enhancing reagent. This The protocol is suitable for use in cell culture systems without significant toxicity or cell death at the same time. This results in the introduction of the transfecting plasmid into almost all cells.   This study is expected to be the first "unregulated" design of E7-indine Will immediately point out if it works. Because in this case Most of cells expressing E7 protein express E7-indelin plus While transfection with mid is likely to die, Transcripts (with the same plasmid) of otherwise identical cells lacking the E7 protein This is because it will not be observed during the infection. In fact, the former cells Now, E7 interacts with the incoming E7-binding single-chain antibody domain of E7-indelin. It appears to interact and shields nearby degrons. The result is longer life and And appears to be toxic E7-indine. In contrast, in cells lacking E7, The degradation signal adjacent to the E7 binding single chain antibody domain remains active ( (Not hindered), but also a short-lived and thus non-toxic E7- Sprinkle.   If the above (desired) effects are not observed, use the E7-intralin design Will be implemented. First, to simplify the adjustment procedure, The region of the ORF of the DNA encoding the cytotoxic (lysine) domain is a standard set Using recombinant DNA technology, several of the commercially available monoclonal antibodies One of these epitopes, such as influenza virus hemagglutinin and And Boehringer Ink (B oehringer Inc) from the corresponding anti-ha monoclonal antibody available from a short DNA region encoding a nine residue epitope, designated "ha" Will be replaced). This modification fixes the lysine domain of indelin. Without having to deal with the toxicities of N-degron and E7-indelin Will allow to change the distance between E7 binding single chain antibody domains U.   The protocol for adjustment of this or any other indelin is It will alter the distance between Glon and the E7 binding domain of indelin. Among them And the toxic C-terminal domain of indelin, such as lysine, is like ha (see above) The resulting construct, which has been replaced by a unique epitope tag, is Using the cell lines and expression vectors described, 7 will be expressed in any human cell culture system lacking. These constructs (Exclusively due to the mutual arrangement and proximity of the degron and E7 binding domains Pulse chase analysis of the metabolic stability of In vivo half-life will be determined directly. In the pulse chase assay, the ratio Relatively short time ("pulse") radioactive tracers (eg radioactive amino acids) A series of protein molecules, labeled with, are then "tracked" and this tracking is Immunoprecipitation and electrophoretic analysis of the protein of interest at different times after completion (and Quantification). Pulse chase analysis is a routine assay, The protocols and logic are well known to those skilled in the art (eg, (Bachmair et al., Science 234: 179 (1986)).   The purpose of adjusting E7-indelin was to Short-lived (as possible) in vivo and in the presence of E7 protein (possible To produce long-lived constructs. The final satisfactory structure is detailed Fused to the vesicle toxic domain (see above), reverting the organization of indelin. result E7-indelin, which occurs as follows, contains E7, as described above. And tested again for conditional toxicity in cell culture systems lacking E7. all Even the first, even the first, E7-Inderin seems to possess the required quality Should be emphasized. Adjustment measures are based on the initial metabolic characteristics of E7-indine. Used only if the performance is never satisfactory according to the criteria stated above. It is expected to be.   Exactly the same approach is needed to design, test, and If so, it will be used to readjust. After that, E6 / E7-Indelier Nearly the last stage of the construction of the protein, its E6 binding domain and nearby The DNA fragment encoding the region of E6-indelin contained is The E7 binding domain was excised from the RF and Inserted between the coding region and the C-terminal cytotoxic domain coding region. Will be.   The ORF of the resulting DNA of E6 / E7-indelin from the N-terminus The following protein regions or domains that begin with: 1) Ubiquitin, 2) Bachmair and Varshavsky (Cell 56: 5019 (19) 89)), N-degron, 3) 100201 single strand against E7 protein of HPV16 human papillomavirus Antibodies (see above), 4) Internal degron identified by the cyclin's "destruction box" (see above) , 5) a single-chain antibody against the E6 protein of HPV16 human papillomavirus, and 4) Lysine toxic domain (A chain) Would code   At this time, monoclonal antibodies are available for the E7 protein, However, it is not available for the E6 protein (see above). Therefore, E6 / One preliminary step in the construction of E7-indelin is the E6 protein (E6 A panel of monoclonals against the producing E. coli Producing antibodies, the appropriate (highest affinity) antibody produced And encodes the corresponding single-chain antibody to the E6 protein Use the corresponding hybridoma cells as described above to construct the ORF Will do. Both steps of this protocol (monochrome with the required specificity) Production of a hybridoma secreting a null antibody and a single-chain control PCR-mediated construction of the loading ORF) is a standard procedure for those skilled in the art. You. In addition, many potential targets of indelin are oncoproteins, tumor suppressors And other regulatory proteins in mammals, so many of these proteins Extensive (and rapidly growing) collection of monoclonal antibodies against Exists. Hybridoma cell clones that produce these antibodies Can be used in the construction of chain antibodies and is often It makes it possible to bypass the first step of producing null antibodies. Example 2 Construction and Testing of Intralin   Intralin is a translocation signal, especially a nuclear localization signal ( NLS) (FIG. 2). In the experiment of this example, Intralin specific for cells infected with bisvirus (see below) is constructed Will be. This intraline, designated "nsP2 / 4-intraline" P^*Type domains are the following “early” proteins of Sindbis virus, The word a. nsP2 protein (site-specific protease) b. nsP4 protein (RNA polymerase) Will be designed to combine   Is a + strand RNA virus that infects humans and some other metazoan hosts These cytosolic (cytosolic) proteins of Sindbis virus are One of the preferred (first) intracellular targets for mutoxin design and testing You. Sindbis virus is a member of the alphavirus family. this The family has 26 currently recognized members (Strauss and Strauss, Microbiol. Rev. 58: 491 (1994)). Many strains of the alphavirus family represent a serious threat to human health. For example, eastern and western equine encephalomyelitis virus is deadly in both North and South America. Cause encephalitis. Ross River virus and related alphaviruses are human Cause epidemic polyarthritis, and the disability symptoms of the disease last several years. obtain. Sindbis virus is almost (but not completely) non-toxic to humans, At the same time, of the alphavirus family like the viruses listed above Closely related to toxic members. this Kills cells infected with Sindbis virus, but A first-generation intralin extract designed to save uninfected cells This makes it an attractive experimental target. Because these intralins are Because they can be tested and completed without extensive precautionary measures against You. At the same time, Sindbis virus-specific intralin was designed and optimized. Once optimized, it can be used to address members of the alphavirus family of virulence. It can be easily modified to convert to intraraline.   Sindbis virus and other alphaviruses are exclusively used in infected cells In the cytoplasm (more specifically, the cytosol). Intralin of FIG. 2A Given the logic, the localization of the Sindbis virus life cycle to the cytosol is This is one reason why Lus was chosen as the primary target for intralin. Sindbis The genomic RNA of the virus is 11,703 nucleotides (nt) in length. It is two Including the main areas. That is, non-structural proteins including RNA polymerase Nonstructural Domains Encoding Quality and Three Structural Tampers of Sindbis Virions A structural domain that encodes a protein. Non-structural ("early") protein nsP 1-nsP4 is one or two polyproteins from genomic RNA itself Will be translated as These proteins are nsP1, nsP2, nsP3 and And nsP4. Two of these proteins are constructed Should be a target for intralin. These are (nsP2 first Processing Sindbis polyprotein (including polyprotein) Site-specific proteolytic (cleaving) to produce individual Sindbis virus proteins Is a tease nsP2 and viral RN that replicate genomic RNA of Sindbis virus A polymerase nsP4 (Strauss and Strauss ( Strauss), Microbiol. Rev. 58: 491 (1994)). nsP2 and And nsP4 are located in the cytosol and, unlike normal cellular proteins, Produced early in the infection cycle, and therefore its function is Prior to the formation of significant amounts of mature Sindbis virions in these cells Is a good target for intralin, which appears to selectively kill. (Shin Although the Dobis nsP2 protein is a protease, its highly restricted nature Substrate specificity makes it non-toxic to mammalian cells and it It should be mentioned that it is impossible to cut off. These antibodies are It will be used below as a domain binding sP2 and nsP4. )   Methods and protocols for constructing intralin, construct indelin Highly similar to those used to do so. Therefore, in the description below, we NsP, essentially different from the steps in the construction of E6 / E7-indelin (see above) Only some aspects of the construction of 2 / 4-intraline are considered in some detail, and other relevant The description of the next step will refer to the protocol for Indellin.   The ORF of nsP2 / 4-intraline associated DNA starts at the N-terminus. The following protein regions or domains: a) a single chain antibody against the nsP2 protein of Sindbis virus, b) Nuclear localization signal (NLS) Pro-Ly located close to the domain of item a s-Lys-Lys-Arg-Lys-Val, c) a single-chain antibody against the nsP4 protein of Sindbis virus, d) NLS sequence Pro-Lys-Lys-Lys-Arg-Ly located close to the domain of item C s-Val (same as item b above), and e) Toxic domain of diphtheria toxin (A chain) Would code   Comments on the nsP2 / 4-intraline construction protocol: 1) The NLS to be used in items b and d above is the SV40 virus large It is present on the T antigen. This powerful yet short and simple NLS is widely used Characterized (Dingwall and Laskey, Trends  Biochem. Sci. 16: 478 (1991)). 2) Single-chain antibodies to nsP2 and nsP4 are directed to E6 / E7-indelin Produced exactly as described above, with anti-nsP2 and anti-nsP4 Preparation of Noclonal Antibodies Single-Chain Antibodies Against nsP2 and nsP4, respectively Before constructing the ORF that encodes 3) The regulation strategy for nsP2 / 4-intraline (if they prove necessary) , Performed in a manner similar to the adjustment strategy described above for E6 / E7-indelin Will. In particular, the position of the NLS is determined by the intrans nsP2 or nsP 4 will be varied for positions near the binding domain. 4) E6 / E7-Indelin using lysine-based cytotoxic domain and control In general (see above), nsP2 / 4-intraline is used as its cytotoxic domain. Would utilize the A chain of diphtheria toxin. The reason for this change is illustrated in Figure 2A. Understanding the logic of intralin design Occurs. That is, the cytotoxic domain of this type of intralin is toxic in the nucleus. Should be, but not in the cytosol. In fact, the nuclear and A chain of diphtheria toxin, unlike ricin A chain, which is toxic in both Is an elongation factor 2 (E) that ribosylates (and thereby inactivates) ADP. F2). Most of EF2 is cytosolic, so diphtheria type Cytosolic to nuclear translocation of intralin containing the toxic domain of The solution appears to physically separate the toxin from its substrate. 5) Intermediate design (nsP2-intraline and nsP4-intraline) Or testing the efficacy of any of the final constructs (nsP2 / 4-intraline) Human cells infected and uninfected with Sindbis virus as test targets Trial of E6 / E7-indelin and its precursors, except as would be used Test will be performed as well. Cells and virus strains to be used are i) and Rice (J. Virology 63: 1326 (1989)). Cell SW-13 line and Sindbis virus Toto-1000 strain U.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), OA (BF, BJ, CF, CG , CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, U G), AM, AT, AU, BB, BG, BR, BY, C A, CH, CN, CZ, DE, DK, EE, ES, FI , GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV, MD, M G, MN, MW, MX, NO, NZ, PL, PT, RO , RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, UZ, VN

Claims (1)

[Claims] 1. a) effector domain, b) a first co-dominant cell located in close proximity to the binding domain of the first protein Signal part, and c) a second co-dominant cell located in close proximity to the binding domain of the second protein Signal part inside And co-dominantly mediated toxins. 2. 2. The method of claim 1, wherein the first and second intracellular signal portions are degradation signals. Toxin mediated by co-dominance of 3. The first and second intracellular signal portions are translocation signals. The co-dominantly mediated toxin of claim 1. 4. 4. The method of claim 3, wherein the translocation signal is a nuclear localization signal. Toxins mediated by co-dominance. 5. The first intracellular signal portion is a degradation signal and the second intracellular signal 2. The co-dominantly mediated toxin of claim 1, wherein the moiety is a nuclear localization signal. 6. a) effector domain, b) first codominant degradation located in close proximity to the binding domain of the first protein Signals, and c) Second co-dominant degradation located in close proximity to the binding domain of the second protein signal A ligand-regulated toxin that is dependent on intracellular degradation signals. 7. a) effector domain, b) a first co-dominant located close to the binding domain of the first protein Sexual translocation signal, and c) a second co-dominant tiger located in close proximity to the binding domain of the second protein Location signal Intracellular translocation signal-dependent and regulated by ligands toxin. 8. First and second translocation signals are nuclear localization signals Intracellular translocation signal dependent and ligand regulated as claimed in claim 7 Toxins to be done. 9. 9. The intracellular cell of claim 8, wherein the effector domain is cytosolic. A ligand-regulated toxin that is translocation signal dependent. 10. 9. The intracellular trans of claim 8, wherein the effector domain is nuclear specific. A toxin that is location signal dependent and regulated by a ligand. 11. a) effector domain, b) cell surface binding domain, c) cytosolic transmembrane domain, d) a first co-dominant cell located in close proximity to the binding domain of the first protein Signal part, and e) a second co-dominant cell located in close proximity to the binding domain of the second protein Signal part inside And co-dominantly mediated toxins. 12. The claim wherein the first and second intracellular signal portions are degradation signals. 11 co-dominantly mediated toxins. 13. The first and second intracellular signal parts 12. The co-dominantly mediated toxin of claim 11, which is a gunal. 14． Claim 1 wherein the translocation signal is a nuclear localization signal. 3. Co-dominantly mediated toxin. 15. 15. The method of claim 14, wherein the effector domain is cytosolic. Sex-mediated toxins. 16. 18. The co-dominant mediation of claim 14, wherein the effector domain is nuclear specific Toxins to be done. 17． The first intracellular signal portion is a degradation signal and the second intracellular signal 12. The co-dominantly mediated toxin of claim 11, wherein said moiety is a nuclear localization signal. 18. Known to contain both a first protein and a second protein A method for selectively subduing or eliminating cell division, the method comprising: a) i) effector domain, ii) first co-dominant degradation located close to the binding domain of the first protein Signals, and iii) a second co-dominant component located in close proximity to the binding domain of the second protein Solution signal Encoding a ligand-regulated toxin dependent on intracellular degradation signals, including Providing a NA expression construct; and b) preparing the DNA construct from step a) with a ligand in a manner dependent on the intracellular degradation signal; Introducing into the cells to be killed under conditions suitable for the expression of the toxin being nodulated; A method that includes 19. Cells known to contain the first protein and the second protein 19. The method of claim 18, which is a prokaryotic cell. 20. Cells known to contain the first protein and the second protein 19. The method of claim 18, which is a eukaryotic cell. 21. Contains both the first and second proteins in the cytosol A method of selectively calming or eliminating eukaryotic cell division, which is known to That way a) i) cytosole-specific effector domain, ii) a first co-dominant nuclear station located in close proximity to the binding domain of the first protein Localization signal, and iii) a second co-dominant nucleus located in close proximity to the binding domain of the second protein Localization signal Intracellular translocation signal dependent and ligand regulated toxins, including Providing a DNA expression construct encoding the element; and b) The DNA construct from step a) is ligand-regulated in an intracellular degron-dependent manner. Introducing into the cells to be killed under conditions appropriate for the expression of the toxin A method that includes 22. Lack both first and second proteins in the cytosol A method of selectively calming or eliminating eukaryotic cell division, which is known to That way a) i) a nuclear-specific effector domain, ii) a first co-dominant nuclear station located in close proximity to the binding domain of the first protein Localization signal, and iii) a second co-dominant nucleus located in close proximity to the binding domain of the second protein Localization signal Intracellular translocation signal dependent and ligand regulated toxins, including Providing a DNA expression construct encoding the element; and b) converting the DNA construct from step a) into an intracellular translocation signal Introducing into the cell under conditions suitable for the expression of a sexual, ligand-regulated toxin; A method that includes 23. Known to contain both a first protein and a second protein A method for selectively subduing or eliminating eukaryotic cell division, the method comprising: a) i) effector domain, ii) a cell surface binding domain, iii) cytosolic transmembrane domain, iv) First co-dominant cells located in close proximity to the binding domain of the first protein Signal part, and v) a second co-dominant cell located in close proximity to the binding domain of the second protein Signal part inside Providing a multi-domain protein fusion, comprising: b) contacting the multi-domain fusion of step a) with a target cell; A method that includes 24. The claim wherein the first and second intracellular signal portions are degradation signals. 23 methods. 25. The first and second intracellular signal parts 24. The method of claim 23, which is a signal. 26. Claim 2 wherein the translocation signal is a nuclear localization signal. 3. Co-dominantly mediated toxin. 27. The first intracellular signal portion is a degradation signal and the second intracellular signal 24. The method of claim 23, wherein the moiety is a nuclear localization signal. 28. Conditional, intracellular resources specific for one predetermined intracellular ligand A fusion protein exhibiting Gand-dependent toxicity, wherein the fusion protein is a) effector domains, and b) an intracellular signal portion located in close proximity to the binding domain of the protein; A protein containing. 29. Conditional, intracellular resources specific for one predetermined intracellular ligand A fusion protein exhibiting Gand-dependent toxicity, wherein the fusion protein is a) effector domain, b) cell surface binding domain, c) a cytosolic transmembrane domain, and d) an intracellular signal portion located in close proximity to the binding domain of the protein, A protein containing.