Ricin-like toxin variants for treatment of cancer, viral or parasitic infections Number:6,803,358 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides a protein having an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains. The linker sequence contains a cleavage recognition site for a disease specific protease such as a cancer, fungal, viral or parasitic protease. The invention also relates to a nucleic acid molecule encoding the protein and to expression vectors incorporating the nucleic acid molecule. Also provided is a method of inhibiting or destroying mammalian cancer cells, cells infected with a virus, a fugus, or parasites, or parasites utilizing the nucleic acid molecules and proteins of the invention and pharmaceutical compositions for treating human cancer, viral infection, fugal infection, or parasitic infection.<H infections, parasitic, Ricin, like, toxi, 6803358.html, Patent, pto, 6803358

Title: Ricin-like toxin variants for treatment of cancer, viral or parasitic infections

Abstract: The present invention provides a protein having an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains. The linker sequence contains a cleavage recognition site for a disease specific protease such as a cancer, fungal, viral or parasitic protease. The invention also relates to a nucleic acid molecule encoding the protein and to expression vectors incorporating the nucleic acid molecule. Also provided is a method of inhibiting or destroying mammalian cancer cells, cells infected with a virus, a fugus, or parasites, or parasites utilizing the nucleic acid molecules and proteins of the invention and pharmaceutical compositions for treating human cancer, viral infection, fugal infection, or parasitic infection.

Patent Number: 6,803,358 Issued on 10/12/2004 to Borgford

Inventors: Borgford; Thor (Burnaby, CA)
Assignee: Twinstrand Therapeutics Inc. (Burnaby, CA)

Appl. No.: 09/551,151

Filed: April 14, 2000

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
403752		6593132

Current U.S. Class: 514/8 ; 435/195; 435/252.3; 435/320.1; 435/440; 435/69.1; 435/69.7; 514/2; 530/350; 530/370

Field of Search: 514/2,8,18 530/350,370,378.3 435/69.1,320.1,69.7,195,252.3,440 424/278.1

References Cited [Referenced By]

U.S. Patent Documents


6531125	March 2003	Borgford

Foreign Patent Documents


WO 9741233	Nov., 1997	WO

Other References

Sampson M. T. et al. (2003) Coagulation proteases and human cancer. Biochem. Soc. Trans. vol. 30, pp. 201-207. Review.* .
Westby, M. et al. (1992) Preparation and characterization of recombinant proricin containing an alternative protease-sensitive linker sequence. Bioconjug. Chem. vol. 3, pp. 375-381..

Primary Examiner: Carlson; Karen Cochrane
Assistant Examiner: Liu; Samuel W.
Attorney, Agent or Firm: Bereskin & Parr Gravelle; Micheline

Parent Case Text

This is a continuation-in-part of U.S. patent application Ser. No. 09/403,752 fied Oct. 29, 1999 which is a 371 of PCT/CA98/00394 issued Apr. 30, 1998.

Claims

I claim:

1. A recombinant protein comprising a ricin A chain, a ricin B chain and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for a matrix metalloproteinase.

2. The recombinant protein of claim 1 having the linker amino acid sequence according to SEQ ID NO: 43.

3. The recombinant protein of claim 1, wherein the matrix metalloproteina is matrix metalloproteinase-9.

4. A pharmaceutical composition for treating cancer or a fungal, or viral, or parasitic infection in an animal comprising the recombinant protein of claim 1 and a pharmaceutically acceptable carrier, diluent or exciplent.

Description

FIELD OF THE INVENTION

The invention relates to proteins useful as therapeutics against cancer, viral infections, parasitic and fungal infections. The proteins contain A and B chains of a ricin-like toxin linked by a linker sequence that is specifically cleaved and activated by proteases specific to disease-associated pathogens or cells.

BACKGROUND OF THE INVENTION

Bacteria and plants are known to produce cytotoxic proteins which may consist of one, two or several polypeptides or subunits. Those proteins having a single subunit may be loosely classified as Type I proteins. Many of the cytotoxins which have evolved two subunit structures are referred to as type II proteins (Saelinger, C. B. in Trafficking of Bacterial Toxins (eds. Saelinger, C. B.) 1-13 (CRC Press Inc., Boca Raton, Fla., 1990). One subunit, the A chain, possesses the toxic activity whereas the second subunit, the B chain, binds cell surfaces and mediates entry of the toxin into a target cell. A subset of these toxins kill target cells by inhibiting protein biosynthesis. For example, bacterial toxins such as diphtheria toxin or Pseudomonas exotoxin inhibit protein synthesis by inactivating elongation factor 2. Plant toxins such as ricin, abrin, and bacterial toxin Shiga toxin, inhibit protein synthesis by directly inactivating the ribosomes (Olsnes, S. & Phil, A. in Molecular action of toxins and viruses (eds. Cohen, P. & vanHeyningen, S.) 51-105 Elsevier Biomedical Press, Amsterdam, 1982).

Ricin, derived from the seeds of Ricinus communis (castor oil plant), may be the most potent of the plant toxins. It is estimated that a single ricin A chain is able to inactivate ribosomes at a rate of 1500 ribosomes/minute. Consequently, a single molecule of ricin is enough to kill a cell (Olsnes, S. & Phil, A. in Molecular action of toxins and viruses (eds. Cohen, P. & vanHeyningen, S.) (Elsevier Biomedical Press, Amsterdam, 1982). The ricin toxin is a glycosylated heterodimer consisting of A and B chains with molecular masses of 30,625 Da and 31,431 Da linked by a disulphide bond. The A chain of ricin has an N-glycosidase activity and catalyzes the excision of a specific adenine residue from the 28S rRNA of eukaryotic ribosomes (Endo, Y. & Tsurugi, K. J., Biol. Chem. 262:8128 (1987)). The B chain of ricin, although not toxic in itself, promotes the toxicity of the A chain by binding to galactose residues on the surface of eukaryotic cells and stimulating receptor-mediated endocytosis of the toxin molecule (Simmons et al., Biol. Chem. 261:7912 (1986)). Once the toxin molecule consisting of the A and B chains is internalized into the cell via clathrin-dependent or independent mechanisms, the greater reduction potential within the cell induces a release of the active A chain, eliciting its inhibitory effect on protein synthesis and its cytotoxicity (Emmanuel, F. et al., Anal. Biochem. 173: 134-141 (1988); Blum, J. S. et al., J. Biol. Chem. 266: 22091-22095 (1991); Fiani, M. L. et al., Arch. Biochem. Biophys. 307: 225-230 (1993)). Empirical evidence suggests that activated toxin (e.g. ricin, shiga toxin and others) in the endosomes is transcytosed through the trans-Golgi network to the endoplasmic reticulum by retrograde transport before the A chain is translocated into the cytoplasm to elicit its action (Sandvig, K. & van Deurs, B., FEBS Lett. 346: 99-102 (1994).

Protein toxins are initially produced in an inactive, precursor form. Ricin is initially produced as a single polypeptide (preproricin) with a 35 amino acid N-terminal presequence and 12 amino acid linker between the A and B chains. The pre-sequence is removed during translocation of the ricin precursor into the endoplasmic reticulum (Lord, J. M., Eur. J. Biochem. 146:403-409 (1985) and Lord, J. M., Eur. J. Biochem. 146:411-416 (1985)). The proricin is then translocated into specialized organelles called protein bodies where a plant protease cleaves the protein at a linker region between the A and B chains (Lord, J. M. et al., FASAB Journal 8:201-208 (1994)). The two chains, however, remain covalently attached by an interchain disulfide bond (cysteine 259 in the A chain to cysteine 4 in the B chain) and mature disulfide linked ricin is stored in protein bodies inside the plant cells. The A chain is inactive in proricin (O'Hare, M. et al., FEBS Lett. 273:200-204 (1990)) and it is inactive in the disulfide-linked mature ricin (Richardson, P. T. et al., FEBS Lett. 255:15-20 (1989)). The ribosomes of the castor bean plant are themselves susceptible to inactivation by ricin A chain; however, as there is np cell surface galactose to permit B chain recognition the A chain cannot re-enter the cell. The exact mechanism of A chain release and activation in target cell cytoplasm is not known (Lord, J. M. et al., FASAB Journal 8:201-208 (1994)). However, it is known that for activation to take place the disulfide bond between the A and B chains must be reduced and, hence, the linkage between subunits broken.

Diphtheria toxin is produced by Corynebacterium diphtheriae as a 535 amino acid polypeptide with a molecular weight of approximately 58 kD (Greenfield, L. et al., Proc. Natl. Acad. Sci. USA 80:6853-6857 (1983); Pastan, I. et al., Annu. Rev. Biochem. 61:331-354 (1992); Collier, R. J. & Kandel, J., J. Biol. Chem. 246:1496-1503 (1971)). It is secreted as a single-chain polypeptide consisting of 2 functional domains. Similar to proricin, the N-terminal domain (A-chain) contains the cytotoxic moiety whereas the C-terminal domain (B-chain) is responsible for binding to the cells and facilitates toxin endocytosis. Conversely, the mechanism of cytotoxicity for diphtheria toxin is based on ADP-ribosylation of EF-2 thereby blocking protein synthesis and producing cell death. The 2 functional domains in diphtheria toxin are linked by an arginine-rich peptide sequence as well as a disulphide bond. Once the diphtheria toxin is internalized into the cell, the arginine-rich peptide linker is cleaved by trypsin-like enzymes and the disulphide bond (Cys 186-201) is reduced. The cytotoxic domain is subsequently translocated into the cytosol substantially as described above for ricin and elicits ribosomal inhibition and cytotoxicity.

Pseudomonas exotoxin is also a 66 kD single-chain toxin protein secreted by Pseudomonas aeruginosa with a similar mechanism of cytotoxicity to that of diphtheria toxin (Pastan, I. et al., Annu. Rev. Biochem. 61:331-354 (1992); Ogata, M. et al., J. Biol. Chem. 267:25396-25401 (1992); Vagil, M. L. et al., Infect. Immunol. 16:353-361 (1977)). Pseudomonas exotoxin consists of 3 conjoint functional domains. The first domain Ia (amino acids 1-252) is responsible for cell binding and toxin endocytosis, a second domain II (amino acids 253-364) is responsible for toxin translocation from the endocytic vesicle to the cytosol, and a third domain III (amino acids 400-613) is responsible for protein synthesis inhibition and cytotoxicity. After Pseudomonas exotoxin enters the cell, the liberation of the cytotoxic domain is effected by both proteolytic cleavage of a polypeptide sequence in the second domain (near Arg 279) and the reduction of the disulphide bond (Cys 265-287) in the endocytic vesicles. In essence, the overall pathway to cytotoxicity is analogous to diphtheria toxin with the exception that the toxin translocation domain in Pseudomonas exotoxin is structurally distinct.

Class 2 ribosomal inhibitory proteins (RIP-2) constitute other toxins possessing distinct functional domains for cytotoxicity and cell binding/toxin translocation which include abrin, modeccin, volkensin, (Sandvig, K. et al., Biochem. Soc. Trans. 21:707-711 (1993)) and mistle toe lectin (viscumin) (Olsnes, S. & Phil, A. in Molecular action of toxins and viruses (eds. Cohen, P. & vanHeyningen, S.) 51-105 Elsevier Biomedical Press, Amsterdam, 1982; and Fodstad, et al. Canc. Res. 44:862 (1984)). Some toxins such as Shiga toxin and cholera toxin also have multiple polypeptide chains responsible for receptor binding and endocytosis.

The ricin gene has been cloned and sequenced, and the X-ray crystal structures of the A and B chains have been described (Rutenber, E. et al. Proteins 10:240-250 (1991); Weston et al., Mol. Bio. 244:410-422, 1994; Lamb and Lord, Eur. J. Biochem. 14:265 (1985); Halling, K. et al. Nucleic Acids Res. 13:8019 (1985)). Similarly, the genes for diptheria toxin and Pseudomonas exotoxin have been cloned and sequenced, and the 3-dimensional structures of the toxin proteins have been elucidated and described (Columblatti, M. et al., J. Biol. Chem. 261:3030-3035 (1986); Allured, V. S. et al., Proc. Nati. Acad. Sci. USA 83:1320-1324 (1986); Gray, G. L. et al., Proc. Natl. Acad. Sci. USA 81:2645-2649 (1984); Greenfield, L. et al., Proc. Natl. Acad. Sci. USA 80:6853-6857 (1983); Collier, R. J. et al., J. Biol. Chem. 257:5283-5285 (1982)).

The potential of bacterial and plant toxins for inhibiting mammalian retroviruses, particularly acquired immunodeficiency syndrome (AIDS), has been investigated. Bacterial toxins such as Pseudomonas exotoxin-A and subunit A of diphtheria toxin; dual chain ribosomal inhibitory plant toxins such as ricin, and single chain ribosomal inhibitory proteins such as trichosanthin and pokeweed antiviral protein have been used for the elimination of HIV infected cells (Olson et al., AIDS Res. and Human Retroviruses 7:1025-1030 (1991)). The high toxicity of these toxins for mammalian cells, combined with a lack of specificity of action poses a major problem to the development of pharmaceuticals incorporating the toxins, such as immunotoxins.

Due to their extreme toxicity there has been much interest in making ricin-based immunotoxins as therapeutic agents for specifically destroying or inhibiting infected or tumourous cells or tissues (Vitetta et al., Science 238:1098-1104(1987)). An immunotoxin is a conjugate of a specific cell binding component, such as a monoclonal antibody or growth factor and the toxin in which the two protein components are covalently linked. Generally, the components are chemically coupled. However, the linkage may also be a peptide or disulfide bond. The antibody directs the toxin to cell types presenting a specific antigen thereby providing a specificity of action not possible with the natural toxin. Immunotoxins have been made both with the entire ricin molecule (i.e. both chains) and with the ricin A chain alone (Spooner et al., Mol. Immunol. 31:117-125, (1994)).

Immunotoxins made with the ricin dimer (IT-Rs) are more potent toxins than those made with only the A chain (IT-As). The increased toxicity of IT-Rs is thought to be attributed to the dual role of the B chains in binding to the cell surface and in translocating the A chain to the cytosolic compartment of the target cell (Vitetta et al., Science 238:1098-1104 (1987); Vitetta & Thorpe, Seminars in Cell Biology 2:47-58 (1991)). However, the presence of the B chain in these conjugates also promotes the entry of the immunotoxin into nontarget cells. Even small amounts of B chain may override the specificity of the cell-binding component as the B chain will bind nonspecifically to galactose associated with N-linked carbohydrates, which is present on most cells. IT-As are more specific and safer to use than IT-Rs. However, in the absence of the B chain the A chain has greatly reduced toxicity. Due to the reduced potency of IT-As as compared to IT-Rs, large doses of IT-As must be administered to patients. The large doses frequently cause immune responses and production of neutralizing antibodies in patients (Vitetta et al., Science 238:1098-1104 (1987)). IT-As and IT-Rs both suffer from reduced toxicity as the A chain is not released from the conjugate into the target cell cytoplasm.

A number of immunotoxins have been designed to recognize antigens on the surfaces of tumour cells and cells of the immune system (Pastan et al., Annals New York Academy of Sciences 758:345-353 (1995)). A major problem with the use of such immunotoxins is that the antibody component is its only targeting mechanism and the target antigen is often found on non-target cells (Vitetta et al., Immunology Today 14:252-259 (1993)). Also, the preparation of a suitable specific cell binding component may be problematic. For example, antigens specific for the target cell may not be available and many potential target cells and infective organisms can alter their antigenic make up rapidly to avoid immune recognition. In view of the extreme toxicity of proteins such as ricin, the lack of specificity of the immunotoxins may severely limit their usefulness as therapeutics for the treatment of cancer and infectious diseases.

The insertion of intramolecular protease cleavage sites between the cytotoxic and cell-binding components of a toxin can mimic the way that the natural toxin is activated. European patent application no. 466,222 describes the use of maize-derived pro-proteins which can be converted into active form by cleavage with extracellular blood enzymes such as factor Xa, thrombin or collagenase. Garred, O. et al. (J. Biol. Chem. 270:10817-10821 (1995)) documented the use of a ubiquitous calcium-dependent serine protease, furin, to activate shiga toxin by cleavage of the trypsin-sensitive linkage between the cytotoxic A-chain and the pentamer of cell-binding B-units. Westby et al. (Bioconjugate Chem. 3:375-381 (1992)) documented fusion proteins which have a specific cell binding component and proricin with a protease sensitive cleavage site specific for factor Xa within the linker sequence. O'Hare et al. (FEBS Lett. 273:200-204 (1990)) also described a recombinant fusion protein of RTA and staphylococcal protein A joined by a trypsin-sensitive cleavage site. In view of the ubiquitous nature of the extracellular proteases utilized in these approaches, such artificial activation of the toxin precursor or immunotoxin does not confer a mechanism for intracellular toxin activation and the problems of target specificity and adverse immunological reactions to the cell-binding component of the immunotoxin remain.

In a variation of the approach of insertion of intramolecular protease cleavage sites on proteins which combine a binding chain and a toxic chain, Leppla, S. H. et al. (Bacterial Protein Toxins zbl.bakt.suppl. 24:431-442 (1994)) suggest the replacement of the native cleavage site of the protective antigen (PA) produced by Bacillus anthracis with a cleavage site that is recognized by cells that contain a particular protease. PA, recognizes, binds, and thereby assists in the internalization of lethal factor (LF) and edema toxin (ET). also produced by Bacillus anthracis. However, this approach is wholly dependent on the availability of LF, or ET and PA all being localized to cells wherein the modified PA can be activated by the specific protease. It does not confer a mechanism for intracellular toxin activation and presents a problem of ensuring sufficient quantities of toxin for internalization in target cells.

The in vitro activation of a Staphylococcus-derived pore-forming toxin, .alpha.-hemolysin by extracellular tumour-associated proteases has been documented (Panchel, R. G. et al., Nature Biotechnology 14:852-857 (1996)). Artificial activation of .alpha.-hemolysin in vitro by said proteases was reported but the actual activity and utility of .alpha.-hemolysin in the destruction of target cells were not demonstrated.

Hemolysin does not inhibit protein synthesis but is a heptameric transmembrane pore which acts as a channel to allow leakage of molecules up to 3 kD thereby disrupting the ionic balances of the living cell. The .alpha.-hemolysin activation domain is likely located on the outside of the target cell (for activation by extracellular proteases). The triggering mechanism in the disclosed hemolysin precursor does not involve the intracellular proteolytic cleavage of 2 functionally distinct domains. Also, the proteases used for the .alpha.-hemolysin activation are ubitquitiously secreted extracellular proteases and toxin activation would not be confined to activation in the vicinity of diseased cells. Such widespread activation of the toxin does not confer target specificity and limits the usefulness of said .alpha.-hemolysin toxin as therapeutics due to systemic toxicity.

A variety of proteases specifically associated with malignancy, viral infections and parasitic infections have been identified and described. For example, cathepsin is a family of serine, cysteine or aspartic endopeptidases and exopeptidases which has been implicated to play a primary role in cancer metastasis (Schwartz, M. K., Chim. Clim. Acta 237:67-78 (1995); Spiess, E. et al., J. Histochem. Cytochem. 42:917-929 (1994); Scarborough, P. E. et al., Protein Sci. 2:264-276 (1993); Sloane, B. F. et al., Proc. Natl. Acad. Sci. USA 83:2483-2487 (1986); Mikkelsen, T. et al., J. Neurosurge 83:285-290 (1995)). Matrix metalloproteinases (MMPs or matrixins) are zinc-dependent proteinases consisting of collagenases, matrilysin, stromelysins, gelatinases and macrophage elastase (Krane, S. M., Ann. N.Y. Acad. Sci. 732:1-10 (1994); Woessner, J. F., Ann. N.Y. Acad. Sci. 732:11-21 (1994); Carvalho, K. et al., Biochem. Biophys. Res. Comm. 191:172-179 (1993); Nakano, A. et al. J. of Neurosurge, 83:298-307 (1995); Peng, K-W, et al. Human Gene Therapy, 8:729-738 (1997); More, D. H. et al. Gynaecologic Oncology, 65:78-82 (1997)). These proteases are involved in pathological matrix remodeling. Under normal physiological conditions, regulation of matrixin activity is effected at the level of gene expression. Enzymatic activity is also controlled stringently by tissue inhibitors of metalloproteinases (TIMPs) (Murphy, G. et al., Ann. N.Y. Acad. Sci. 732:31-41 (1994)). The expression of MMP genes is reported to be activated in inflammatory disorders (e.g. rheumatoid arthritis) and malignancy.

In malaria, parasitic serine and aspartic proteases are involved in host erythrocyte invasion by the Plasmodium parasite and in hemoglobin catabolism by intraerythrocytic malaria (O'Dea, K. P. et al., Mol. Biochem. Parasitol. 72:111-119 (1995); Blackman, M. J. et al., Mol. Biochem. Parasitol. 62:103-114 (1993); Cooper, J. A. et al., Mol. Biochem. Parasitol. 56:151-160 (1992); Goldberg, D. E. et al., J. Exp. Med. 173:961-969 (1991)). Schistosoma mansoni is also a pathogenic parasite which causes schistosomiasis or bilharzia. Elastinolytic proteinases have been associated specifically with the virulence of this particular parasite (McKerrow, J. H. et al., J. Biol. Chem. 260:3703-3707 (1985)).

Welch, A. R. et al. (Proc. Natl. Acad. Sci. USA 88:10797-10800 (1991)) has described a series of viral proteases which are specifically associated with human cytomegalovirus, human herpesviruses, Epstein-Barr virus, varicella zoster virus-I. and infectious laryngotracheitis virus. These proteases possess similar substrate specificity and play an integral role in viral scaffold protein restructuring in capsid assembly and virus maturation. Other viral proteases serving similar functions have also been documented for human T-cell leukemia virus (Blaha, I. et al., FEBS Lett. 309:389-393 (1992); Pettit, S. C. et al., J. Biol. Chem. 266:14539-14547 (1991)), hepatitis viruses (Hirowatari, Y. et al., Anal. Biochem. 225:113-120 (1995); Hirowatari, Y. et al., Arch. Virol. 133:349-356 (1993); Jewell, D. A. et al., Biochemistry 31:7862-7869 (1992)), poliomyelitis virus (Weidner, J. R. et al., Arch. Biochem. Biophys. 286:402-408 (1991)), and human rhinovirus (Long, A. C. et al., FEBS Lett. 258:75-78 (1989)).

Candida yeasts are dimorphic fungi which are responsible for a majority of opportunistic infections in AIDS patients (Holmberg, K. and Myer, R., Scand. J. Infect. Dis. 18:179-192 (1986)). Aspartic proteinases have been associated specifically with numerous virulent strains of Candida including Candida albican, Candida tropicalis, and Candida parapsilosis (Abad-Zapatero, C. et al., Protein Sci. 5:640-652 (1996); Cutfield, S. M. et al., Biochemistry 35:398-410 (1995); Ruchel, R. et al, Zentralbl. BakterioL Mikrobiol Hyg. I Abt. Orig. A. 255:537-548 (1983); Remold, H. et al., Biochim. Biophys. Acta 167:399-406 (1968)), and the levels of these enzymes have been correlated with the lethality of the strain (Schreiber, B, et al., Diagn. Microbiol. Infect. Dis. 3:1-5 (1985)).

SUMMARY OF THE INVENTION

The invention relates to novel recombinant toxic proteins which are specifically toxic to diseased cells but do not depend for their specificity of action on a specific cell binding component. The recombinant proteins of the invention have an A chain of a ricin-like toxin linked to a B chain by a synthetic linker sequence which may be cleaved specifically by a protease localised in cells or tissues affected by a specific disease to liberate the toxic A chain thereby selectively inhibiting or destroying the diseased cells or tissues. The term diseased cells as used herein, includes cells affected by cancer, or infected by fungi, or viruses, including retroviruses, or parasites.

Toxin targeting using the recombinant toxic proteins of the invention takes advantage of the fact that many DNA viruses exploit host cellular transport mechanisms to escape immunological destruction. This is achieved by enhancing the retrograde translocation of host major histocompatibility complex (MHC) type I molecules from the endoplasmic reticulum into the cytoplasm (Bonifacino, J. S., Nature 384: 405-406 (1996); Wiertz, E. J. et al., Nature 384: 432-438 (1996)). The facilitation of retrograde transport in diseased cells by the virus can enhance the transcytosis and cytotoxicity of a recombinant toxic protein of the present invention thereby further reducing non-specific cytotoxicity and improving the overall safety of the product.

The recombinant toxic proteins of the present invention may be used to treat diseases including various forms of cancer such as T- and B-cell lymphoproliferative diseases, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, non small cell lung cancer, malaria, and diverse viral disease states associated with infection with human cytomegalovirus, hepatitis virus, herpes virus, human rhinovirus, infectious laryngotracheitis virus, poliomyelitis virus, or varicella zoster virus.

In one aspect, the present invention provides a purified and isolated nucleic acid having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains. The linker sequence is not a native linker sequence of a ricin-like toxin, but rather a synthetic heterologous linker sequence containing a cleavage recognition site for a disease-specific protease. The A and or the B chain may be those of ricin.

In an embodiment, of the invention the cleavage recognition site is the cleavage recognition site for a cancer-associated protease. In particular embodiments, the linker amino acid sequence comprises SLLKSRMVPNFN (SEQ ID NO: 40) or SLLIARRMPNFN (SEQ ID NO: 90) cleaved by cathepsin B; SKLVQASASGVN (SEQ ID NO: 45) or SSYLKASDAPDN (SEQ ID NO: 46) cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ ID NO: 41) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO: 42) cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ ID NO: 43) cleaved by MMP-9; DVDERDVRGFASFL (SEQ ID NO: 44) cleaved by a thermolysin-like MMP; SLPLGLWAPNFN (SEQ ID NO: 87) cleaved by matrix metalloproteinase 2(MMP-2); SLLIFRSWANFN (SEQ ID NO: 93) cleaved by cathespin L; SGVIATVIVIT (SEQ ID NO: 96) cleaved by cathespin D; SLGPQGIWGQFN (SEQ ID NO: 99) cleaved by matrix metalloproteinase 1(MMP-1); KKSPGRVVGGSV (SEQ ID NO: 102) cleaved by urokinase-type plasminogen activator; PQGLLGAPGILG (SEQ ID NO: 105) cleaved by membrane type 1 matrixmetalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQ ID NO: 108) cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase and stromelysin-1; GPQGLAGQRGIV (SEQ ID NO: 111) cleaved by matrix metalloproteinase 13 (collagenase-3); GGSGQRGRKALE (SEQ ID NO: 114) cleaved by tissue-type plasminogen activator(tPA); SLSALLSSDIFN (SEQ ID NO: 117) cleaved by human prostate-specific antigen; SLPRFKIIGGFN (SEQ ID NO: 120) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ ID NO: 123) cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO: 126) cleaved by calpain (calcium activated neutral protease). The nucleic acid sequences for ricin A and B chains with each of the linker sequences are shown in FIGS. 2D, 35C, 3D, 4D, 5D, 6D, 16D, 17D, 34C, 36C, 37C, 38C, 39C, 40C, 41C, 42C, 43C, 44C, 45C, 46C and 47C, respectively.

In another embodiment, the cleavage recognition site is the cleavage recognition site for a protease associated with the malaria parasite, Plasmodium falciparum. In particular embodiments, the linker amino acid sequence comprises QVVQLQNYDEED (SEQ ID NO: 55); LPIFGESEDNDE (SEQ ID NO: 56); QVVTGEAISVTM (SEQ ID NO: 57); ALERTFLSFPTN (SEQ ID NO: 58) or KFQDMLNISQHQ (SEQ ID NO: 59). The nucleic nucleotide sequences for ricin A and B chains with each of the linker sequences are shown in FIGS. 7D, 8D, 9D, 10D, and 11D.

In a another embodiment, the cleavage recognition site is the cleavage recognition site for a viral protease. The linker sequences preferably comprise the sequence Y-X-Y-A-Z wherein X is valine or leucine, Y is a polar amino acid, and Z is serine, asparagine or valine. In particular embodiments, the linker amino acid sequence comprises SGVVNASCRLAN (SEQ ID NO: 63) or SSYVKASVSPEN (SEQ ID NO: 64) cleaved by a human cytomegalovirus protease; SALVNASSAHVN (SEQ ID NO: 60) or STYLQASEKFKN (SEQ ID NO: 61) cleaved by a herpes simplex 1 virus protease; SSILNASVPNFN (SEQ ID NO: 62) cleaved by a human herpes virus 6 protease; SQDVNAVEASSN (SEQ ID NO: 65) or SVYLQASTGYGN (SEQ ID NO: 66) cleaved by a varicella zoster virus protease; or SKYLQANEVITN (SEQ ID NO: 67) cleaved by an infectious laryngotracheitis virus protease. The nucleic nucleotide sequences for ricin A and B chains with each of the linker sequences are shown in FIGS. 12D, 13D, 14D, 15D, 18D, 19D, 20D, and 22D.

In another embodiment, the cleavage recognition site is the cleavage recognition site for a hepatitis A viral protease. In particular embodiments, the linker amino acid sequence comprises SELRTQSFSNWN (SEQ ID NO: 68) or SELWSQGIDDDN (SEQ ID NO: 69) cleaved by a hepatitis A virus protease. The nucleic nucleotide sequences for ricin A and B chains with each of the linker sequences are shown in FIGS. 23D or 24D.

In another embodiment, the cleavage recognition site is the cleavage recognition site for a hepatitis C viral protease. In particular embodiments, the linker amino acid sequence comprises DLEVVTSTWVFN (SEQ ID NO: 75), DEMEECASHLFN (SEQ ID NO: 78), EDVVCCSMSYFN (SEQ ID NO: 81) or KGWRLLAPITAY (SEQ ID NO: 84) cleaved by a hepatitis C virus protease. The nucleic nucleotide sequences for ricin A and B chains with each of the linker sequences are shown in FIGS. 30C, 31C, 32C and 33C.

In another embodiment, the cleavage recognition site is the cleavage recognition site for a Candida fungal protease. In particular embodiments, the linker amino acid sequence is SKPAKFFRLNFN (SEQ ID NO: 70), SKPIEFFRLNFN (SEQ ID NO: 71) or SKPAEFFALNFN (SEQ ID NO: 72) cleaved by Candida aspartic protease. The nucleic nucleotide sequences for ricin A and B chains with the first linker sequence are shown in FIGS. 25D.

The present invention also provides a plasmid incorporating the nucleic acid of the invention. In an embodiment, the plasmid has the restriction map as shown in FIGS. 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A, 14A, 15A, 16A, 17A, 18A, 19A, 20A, 21A, 22A, 23A, 24A, or 25A.

In another embodiment, the present invention provides a baculovirus transfer vector incorporating the nucleic acid of the invention. In particular embodiments, the invention provides a baculovirus transfer vector having the DNA sequence as shown in FIG. 1.

In a further embodiment, the present invention provides a baculovirus transfer vector incorporating the nucleic acid of the invention. In particular embodiments, the invention provides a baculovirus transfer vector having the restriction map as shown in FIGS. 2C, 3C, 4C, 5C, 6C, 7C, 8C, 9C, 10C, 11C, 12C, 13C, 14C, 15C, 16C, 17C, 18C, 19C, 20C, 21C, 22C, 23C, 24C, 25C, 30A, 31A, 32A, 33A, 34A, 35A, 36A, 37A, 38A, 39A, 40A, 41A, 42A, 43A, 44A, 45A, 46A, or 47A. or having the DNA sequence as shown in FIG. 1.

In a further aspect, the present invention provides a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for a disease-specific protease (e.g. a cancer, viral, parasitic, or fungal protease). The A and/or the B chain may be those of ricin. In an embodiment, the cleavage recognition site is the cleavage recognition site for a cancer, viral or parasitic protease substantially as described above. In a particular embodiment, the cancer is T-cell or B-cell lymphoproliferative disease. In another particular embodiment, the virus is human cytomegalovirus, Epstein-Barr virus, hepatitis virus, herpes virus, human rhinovirus, infectious laryngotracheitis virus, poliomyelitis virus, or varicella zoster virus. In a further particular embodiment, the parasite is Plasmodium falciparum.

In a further aspect, the invention provides a pharmaceutical composition for treating a fungal infection, such as Candida, in a mammal comprising the recombinant protein of the invention and a pharmaceutically acceptable carrier, diluent or excipient.

In yet another aspect, the invention provides a method of inhibiting or destroying cells affected by a disease, which cells are associated with a disease specific protease, including cancer or infection with a virus, fungus, or a parasite each of which has a specific protease, comprising the steps of preparing a recombinant protein of the invention having a heterologous linker sequence which contains a cleavage recognition site for the disease-specific protease and administering the recombinant protein to the cells. In an embodiment, the cancer is T-cell or B-cell lymphoproliferative disease, ovarian cancer, pancreatic cancer, head and neck cancer, squamous cell carcinoma, gastrointestinal cancer, breast cancer, prostate cancer, non small cell lung cancer. In another embodiment, the virus is human cytomegalovirus, Epstein-Barr virus, hepatitis virus, herpes virus, human rhinovirus, human T-cell leukemia virus, infectious laryngotracheitis virus, poliomyelitis virus, or varicella zoster virus. In another embodiment, the parasite is Plasmodium falciparum.

The present invention also relates to a method of treating a mammal with disease wherein cells affected by the disease are associated with a disease specific protease, including cancer or infection with a virus, fungus, or a parasite each of which has a specific protease by administering an effective amount of one or more recombinant proteins of the invention to said mammal.

Still further, a process is provided for preparing a pharmaceutical for treating a mammal with disease wherein cells affected by the disease are associated with a disease specific protease, including cancer or infection with a virus, fungus, or a parasite each of which has a specific protease comprising the steps of preparing a purified and isolated nucleic acid having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for the disease-specific protease; introducing the nucleic acid into a host cell; expressing the nucleic acid in the host cell to obtain a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains wherein the linker sequence contains the cleavage recognition site for the disease-specific protease; and suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient.

In an embodiment, a process is provided for preparing a pharmaceutical for treating a mammal with disease wherein cells affected by the disease are associated with a disease specific protease, including cancer or infection with a virus, fungus, or a parasite each of which has a specific protease comprising the steps of identifying a cleavage recognition site for the protease; preparing a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains wherein the linker sequence contains the cleavage recognition site for the protease and suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient.

In a further aspect, the invention provides a pharmaceutical composition for treating for treating a mammal with disease wherein cells affected by the disease are associated with a disease specific protease, including cancer or infection with a virus, fungus, or a parasite comprising the recombinant protein of the invention and a pharmaceutically acceptable carrier, diluent or excipient.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the drawings in which:

FIG. 1 shows the DNA sequence of the baculovirus transfer vector, pVL1393 (SEQ ID NO: 1);

FIG. 2A summarizes the cloning strategy used to generate the pAP-213 construct (SEQ ID NO: 2);

FIG. 2B shows the nucleotide sequence of the Cathepsin B linker regions of pAP-213;

FIG. 2C shows the subcloning of the Cathepsin B linker variant into a baculovirus transfer vector;

FIG. 2D shows the DNA sequence of the pAP-214 insert containing ricin and the Cathepsin B linker (SEQ ID NO: 3);

FIG. 3A summarizes the cloning strategy used to generate the pAP-215 construct;

FIG. 3B shows the nucleotide sequence of the MMP-3 linker regions of pAP-215 (SEQ ID NO: 4);

FIG. 3C shows the subcloning of the MMP-3 linker variant into a baculovirus transfer vector;

FIG. 3D shows the DNA sequence of the pAP-216 insert containing ricin and the MMP-3 linker (SEQ ID NO: 5);

FIG. 4A summarizes the cloning strategy used to generate the pAP-217 construct;

FIG. 4B shows the nucleotide sequence of the MMP-7 linker regions of pAP-217 (SEQ ID NO: 6);

FIG. 4C shows the subcloning of the MMP-7 linker variant into a baculovirus transfer vector;

FIG. 4D shows the DNA sequence of the pAP-218 insert containing ricin and the MMP-7 linker (SEQ ID NO: 7);

FIG. 5A summarizes the cloning strategy used to generate the pAP-219 construct;

FIG. 5B shows the nucleotide sequence of the MMP-9 linker regions of pAP-219 (SEQ ID NO: 8);

FIG. 5C shows the subcloning of the MMP-9 linker variant into a baculovirus transfer vector;

FIG. 5D shows the DNA sequence of the pAP-220 insert containing ricin and the MMP-9 linker (SEQ ID NO: 9);

FIG. 6A summarizes the cloning strategy used to generate the pAP-221 construct;

FIG. 6B shows the nucleotide sequence of the thermolysin-like MMP linker regions of pAP-221 (SE ID NO: 10);

FIG. 6C shows the subcloning of the thermolysin-like MMP linker variant into a baculovirus transfer vector.

FIG. 6D shows the DNA sequence of the pAP-222 insert containing ricin and the thermolysin-like MMP linker (SEQ ID NO: 11);

FIG. 7A summarizes the cloning strategy used to generate the pAP-223 construct;

FIG. 7B shows the nucleotide sequence of the Plasmodium falciparum-A linker regions of pAP-223 (SEQ ID NO: 12);

FIG. 7C shows the subdloning of the Plasmodium falciparum-A linker variant into a baculovirus transfer vector;

FIG. 7D shows the DNA sequence of the pAP-224 insert containing ricin and the Plasmodium falciparum-A linker (SEQ ID NO: 13);

FIG. 8A summarizes the cloning strategy used to generate the pAP-225 construct;

FIG. 8B shows the nucleotide sequence of the Plasmodium falciparum-B linker regions of pAP-225 (SEQ ID NO: 14);

FIG. 8C shows the subcloning of the Plasmodium falciparum-B linker variant into a baculovirus transfer vector;

FIG. 8D shows the DNA sequence of the pAP-226 insert containing ricin and the Plasmodium falciparum-B linker (SEQ ID NO: 15);

FIG. 9A summarizes the cloning strategy used to generate the pAP-227 construct;

FIG. 9B shows the nucleotide sequence of the Plasmodium falciparum-C linker regions of pAP-227 (SEQ ID NO: 16);

FIG. 9C shows the subcloning of the Plasmodium falciparum-C linker variant into a baculovirus transfer vector;

FIG. 9D shows the DNA sequence of the pAP-228 insert containing ricin and the Plasmodium falciparum-C linker (SEQ ID NO: 17);

FIG. 10A summarizes the cloning strategy used to generate the pAP-229 construct;

FIG. 10B shows the nucleotide sequence of the Plasmodium falciparum-D linker regions of pAP-229 (SEQ ID NO: 18);

FIG. 10C shows the subcloning of the Plasmodium falciparum-D linker variant into a baculovirus transfer vector;

FIG. 10D shows the DNA sequence of the pAP-230 insert containing-ricin and the Plasmodium falciparum-D linker (SEQ ID NO: 19);

FIG. 11A summarizes the cloning strategy used to generate the pAP-231 construct;

FIG. 11B shows the nucleotide sequence of the Plasmodium falciparum-E linker regions of pAP-231 (SEQ ID NO: 20);

FIG. 11C shows the subcloning of the Plasmodium falciparum-E linker variant into a baculovirus transfer vector;

FIG. 11D shows the DNA sequence of the pAP-232 insert containing ricin and the Plasmodium falciparum-E linker (SEQ ID NO: 21);

FIG. 12A summarizes the cloning strategy used to generate the pAP-233 construct;

FIG. 12B shows the nucleotide sequence of the HSV-A linker regions of pAP-233 (SEQ ID NO: 22);

FIG. 12C shows the subcloning of the HSV-A linker variant into a baculovirus transfer vector;

FIG. 12D shows the DNA sequence of the pAP-234 insert containing ricin and the HSV-A linker (SEQ ID NO: 23);

FIG. 13A summarizes the cloning strategy used to generate the pAP-235 construct;

FIG. 13B shows the nucleotide sequence of the HSV-B linker regions of pAP-235 (SEQ ID NO: 24);

FIG. 13C shows the subcloning of the HSV-B linker variant into a baculovirus transfer vector;

FIG. 13D shows the DNA sequence of the pAP-236 insert containing ricin and the HSV-B linker (SEQ ID NO: 25);

FIG. 14A summarizes the cloning strategy used to generate the pAP-237 construct;

FIG. 14B shows the nucleotide sequence of the VZV-A linker regions of pAP-237 (SEQ ID NO: 26);

FIG. 14C shows the subcloning of the VZV-A linker variant into a baculovirus transfer vector;

FIG. 14D shows the DNA sequence of the pAP-238 insert containing ricin and the VZV-A linker (SEQ ID NO: 27);

FIG. 15A summarizes the cloning strategy used to generate the pAP-239 construct;

FIG. 15B shows the nucleotide sequence of the VZV-B linker regions of pAP-239 (SEQ ID NO: 28);

FIG. 15C shows the subcloning of the VZV-B linker variant into a baculovirus transfer vector;

FIG. 15D shows the DNA sequence of the pAP-240 insert containing ricin and the VZV-B linker (SEQ ID NO: 29);

FIG. 16A summarizes the cloning strategy used to generate the pAP-241 construct;

FIG. 16B shows the nucleotide sequence of the EBV-A linker regions of pAP-241 (SEQ ID NO: 30);

FIG. 16C shows the subcloning of the EBV-A linker variant into a baculovirus transfer vector;

FIG. 16D shows the DNA sequence of the pAP-242 insert containing ricin and the EBV-A linker (SEQ ID NO: 31);

FIG. 17A summarizes the cloning strategy used to generate the pAP-243 construct;

FIG. 17B shows the nucleotide sequence of the EBV-B linker regions of pAP-243 (SEQ ID NO: 32);

FIG. 17C shows the subcloning of the EBV-B linker variant into a baculovirus transfer vector;

FIG. 17D shows the DNA sequence of the pAP-244 insert containing ricin and the EBV-B linker (SEQ ID NO: 33);

FIG. 18A summarizes the cloning strategy used to generate the pAP-245 construct;

FIG. 18B shows the nucleotide sequence of the CMV-A linker regions of pAP-245 (SEQ ID NO: 34);

FIG. 18C shows the subcloning of the CMV-A linker variant into a baculovirus transfer vector;

FIG. 18D shows the DNA sequence of the pAP-246 insert containing ricin and the CMV-A linker (SEQ ID NO: 35);

FIG. 19A summarizes the cloning strategy used to generate the pAP-247 construct;

FIG. 19B shows the nucleotide sequence of the CMV-B linker regions of pAP-247 (SEQ ID NO: 36);

FIG. 19C shows the subcloning of the CMV-B linker variant into a baculovirus transfer vector;

FIG. 19D shows the DNA sequence of the pAP-248 insert containing ricin and the CMV-B linker (SEQ ID NO: 37);

FIG. 20A summarizes the cloning strategy used to generate the pAP-249 construct;

FIG. 20B shows the nucleotide sequence of the HHV-6 linker regions of pAP-249 (SEQ ID NO: 38);

FIG. 20C shows the subcloning of the HHV-6 linker variant into a baculovirus transfer vector;

FIG. 20D shows the DNA sequence of the pAP-250 insert containing ricin and the HHV-6 linker (SEQ ID NO: 39);

FIG. 21 shows the amino acid sequences of the wild type ricin linker and cancer protease-sensitive amino acid linkers contained in pAP-213 to pAP-222 and linkers pAP-241 to pAP-244 (SEQ ID NOS: 40-46 and 127);

FIG. 22A summarizes the cloning strategy used to generate the pAP-253 construct;

FIG. 22B shows the nucleotide sequence of the ILV linker regions of pAP-253 (SEQ ID NO: 47);

FIG. 22C shows the subcloning of the ILV linker variant into a baculovirus transfer vector;

FIG. 22D shows the DNA sequence of the pAP-254 insert containing ricin and the ILV linker (SEQ ID NO: 48);

FIG. 23A summarizes the cloning strategy used to generate the pAP-257 construct;

FIG. 23B shows the nucleotide sequence of the HAV-A linker regions of pAP-257 (SEQ ID NO: 49);

FIG. 23C shows the subcloning of the HAV-A linker variant into a baculovirus transfer vector;

FIG. 23D shows the DNA sequence of the pAP-258 insert containing ricin and the HAV-A linker (SEQ ID NO: 50);

FIG. 24A summarizes the cloning strategy used to generate the pAP-255 construct;

FIG. 24B shows the nucleotide sequence of the HAV-B linker regions of pAP-255 (SEQ ID NO: 51);

FIG. 24 shows the subcloning of the HAV-B linker variant into a baculovirus transfer vector;

FIG. 24D shows the DNA sequence of the pAP-256 insert containing ricin and the HAV-B linker (SEQ ID NO: 52);

FIG. 25A summarizes the cloning strategy used to generate the pAP-259 construct;

FIG. 25B shows the nucleotide sequence of the CAN linker regions of pAP-259 (SEQ ID NO: 53);

FIG. 25C shows the subcloning of the CAN linker variant into a baculovirus transfer vector;

FIG. 25D shows the DNA sequence of the pAP-260 insert containing ricin and the CAN linker (SEQ ID NO: 54);

FIG. 26 shows the amino acid sequences of the wild type ricin linker and Plasmodium falciparum protease-sensitive amino acid linkers contained in linkers pAP-223 to pAP-232 (SEQ ID NOS: 55-59 and 127);

FIG. 27 shows the amino acid sequences of the wild type ricin linker and the viral protease-sensitive amino acid linkers contained in pAP-233 to pAP-240, pAP-245-pAP-248, pAP-253 to pAP-258 (SEQ ID NOS: 53-64, 60-62, 65-69 and 127);

FIG. 28 shows the amino acid sequences of the wild type ricin linker and the Candida aspartic protease-sensitive amino acid linker contained in pAP-259 to pAP-264 (SEQ ID NOS: 70-72 and 127);

FIG. 29 describes an alternative mutagenesis and subcloning strategy to provide a baculovirus transfer vector containing the ricin-like toxin variant gene; and

FIG. 30A summarizes the cloning strategy used to generate the pAP-262 construct;

FIG. 30B shows the nucleotide sequence of the HCV-A linker region of pAP-262 (SEQ ID NO: 73);

FIG. 30C shows the DNA sequence of the pAP-262 insert (SEQ ID NO: 74);

FIG. 30D shows the aminoaci squence comparison of mutant preproricin linker region HCV-A to wild type (SEQ ID NOS: 75-127);

FIG. 31A summarizes the cloning strategy used to generate the pAP-264 construct;

FIG. 31B shows the nucleotide sequence of the HCV-B linker region of pAP-264 (SEQ ID NO: 76);

FIG. 31C shows the DNA sequence of the pAP-264 insert (SEQ ID NO: 77);

FIG. 31D shows the amino acid sequence comparison of mutant preproricin linker region HCV-B to wild type (SEQ ID NOS: 127, 78);

FIG. 32A summarizes the cloning strategy used to generate the pAP-266 construct;

FIG. 32B shows the nucleotide sequence of the HCV-C linker region of pAP-266 (SEQ ID NO: 79);

FIG. 32C shows the DNA sequence of the pAP-266 insert (SEQ ID NO: 80);

FIG. 32D shows the amino acid sequence comparison of mutant preproricin linker region HCV-C to wild type (SEQ ID NOS: 81and 187);

FIG. 33A summarizes the cloning strategy used to generate the pAP-268 construct;

FIG. 33B shows the nucleotide sequence of the HCV-D linker region of pAP-268 (SEQ ID NO: 82);

FIG. 33C shows the DNA sequence of the pAP-268 insert (SEQ ID NO: 83);

FIG. 33D shows the amino acid sequence comparison of mutant preproricin linker region HCV-D to wild type (SEQ ID NOS: 84 and 127);

FIG. 34A summarizes the cloning strategy used to generate the pAP-270 construct;

FIG. 34B shows the nucleotide sequence of the MMP-2 linker region of pAP-270 (SEQ ID NO: 85);

FIG. 34C shows the DNA sequence of the pAP-270 insert (SEQ ID NO: 86);

FIG. 34D shows the amino acid sequence comparison of mutant preproricin linker region of MMP-2 to wild type (SEQ ID NOS: 87 and 127);

FIG. 35A summarizes the cloning strategy used to generate the pAP-272 construct;

FIG. 35B shows the nucleotide sequence of the Cathepsin B (Site 2) linker region of pAP-272 (SEQ ID NO: 88);

FIG. 35C shows the DNA sequence of the pAP-272 insert (SEQ ID NO: 89);

FIG. 35D shows the amino acid sequence comparison of mutant preproricin linker region of Cathepsin B (Site 2) to wild type (SEQ ID NO: 90);

FIG. 36A summarizes the cloning strategy used to generate the pAP-274 construct;

FIG. 36B shows the nucleotide sequence of the Cathepsin L linker region of pAP-274 (SEQ ID NO: 91);

FIG. 36C shows the DNA sequence of the pAP-274 insert (SEQ ID NO: 92);

FIG. 36D shows the amino acid sequence comparison of mutant preproricin linker region of Cathepsin L to wild type (SEQ ID NOS: 127, 93);

FIG. 37A summarizes the cloning strategy used to generate the pAP-276 construct;

FIG. 37B shows the nucleotide sequence of the Cathepsin D linker region of pAP-276 (SEQ ID NO: 94);

FIG. 37C shows the DNA sequence of the pAP-276 insert (SEQ ID NO: 95);

FIG. 37D shows the amino acid sequence comparison of mutant preproricin linker region of Cathepsin D to wild type (SEQ ID NOS: 96 and 127);

FIG. 38A summarizes the cloning strategy used to generate the pAP-278 construct;

FIG. 38B shows the nucleotide sequence of the MMP-1 linker region of pAP-278 (SEQ ID NO: 97);

FIG. 38C shows the DNA sequence of the pAP-278 insert (SEQ ID NO: 98);

FIG. 38D shows the amino,acid sequence comparison of mutant preproricin linker region of MMP-1 to wild type (SEQ ID NOS: 99 and 127);

FIG. 39A summarizes the cloning strategy used to generate the pAP-280 construct;

FIG. 39B shows the nucleotide sequence of the Urokinase Type Plasminogen Activator linker region of pAP-280 (SEQ ID NO: 100);

FIG. 39C shows the DNA sequence of the pAP-280 insert (SEQ ID NO: 101);

FIG. 39D shows the amino acid sequence comparison of mutant preproricin linker region of Urokinase-Type Plasminogen Activator to wild type (SEQ ID NO: 102);

FIG. 40A summarizes the cloning strategy used to generate the pAP-282 construct;

FIG. 40B shows the nucleotide sequence of the MT-MMP linker region of pAP-282 (SEQ ID NO: 103);

FIG. 40C shows the DNA sequence of the pAP-282 insert (SEQ ID NO: 104);

FIG. 40D shows the amino acid sequence comparisoin of mutant preproricin linker region of MT-MMP to wild type (SEQ ID NOS: 105 and 127);

FIG. 41A summarizes the cloning strategy used to generate the pAP-284 construct;

FIG. 41B shows the nucleotide sequence of the MMP-11 linker region of pAP-284 (SEQ ID NO: 106);

FIG. 41C shows the DNA sequence of the pAP-284 insert (SEQ ID NO: 107);

FIG. 41D shows the amino acid sequence comparison of mutant preproricin linker region of MMP-11 to wild type (SEQ ID NOS: 108 and 127);

FIG. 42A summarizes the cloning strategy used to generate the pAP-286 construct;

FIG. 42B shows the nucleotide sequence of the MMP-13 linker region of pAP-286 (SEQ ID NO: 109);

FIG. 42C shows the DNA sequence of the pAP-286 insert (SEQ ID NO: 110);

FIG. 42D shows the amino acid sequence comparison of mutant preproricin linker region of MMP-13 to wild type (SEQ ID NOS: 111 and 127);

FIG. 43A summarizes the cloning strategy used to generate the pAP-288 construct;

FIG. 43B shows the nucleotide sequence of the Tissue type Plasminogen Activator linker region of pAP-288 (SEQ ID NO: 112);

FIG. 43C shows the DNA sequence of the pAP-288 insert (SEQ ID NO: 113);

FIG. 43D shows the amino acid sequence comparison of mutant preproricin linker region of Tissue-type Plasminogen Activator to wild type (SEQ ID NOS: 114 and 127);

FIG. 44A summarizes the cloning strategy used to generate the pAP-290 construct;

FIG. 44B shows the nucleotide sequence of the human Prostate-Specific Antigen linker region of pAP-290 (SEQ ID NO: 115);

FIG. 44C shows the DNA sequence of the pAP-290 insert (SEQ ID NO: 116);

FIG. 44D shows the amino acid sequence comparison of mutant preproncin linker region of the human Prostate-Specific Antigen to wild type (SEQ ID NOS: 117 and 127);

FIG. 45A summarizes the cloning strategy used to generate the pAP-292 construct;

FIG. 45B shows the nucleotide sequence of the kallikrein linker region of pAP-292 (SEQ ID NO: 118);

FIG. 45C shows the DNA sequence of the pAP-292 insert (SEQ ID NO: 119);

FIG. 45D shows the amino acid sequen comparison of mutant preproricin linker region of the kallikrein to wild type (SEQ ID NOS: 120 and 127);

FIG. 46A summarizes the cloning strategy used to generate the pAP-294 construct;

FIG. 46B shows the nucleotide sequence of the neutrophil elastase linker region of pAP-294 (SEQ ID NO: 121);

FIG. 46C shows the DNA sequence of the pAP-294 insert (SEQ ID NO: 122);

FIG. 46D shows the amino acid sequence comparison of mutant preproricin linker region of neutrophil elastase to wild type (SEQ ID NOS: 123 and 127);

FIG. 47A summarizes the cloning strategy used to generate the pAP-296 construct;

FIG. 47B shows the nucleotide sequence of the calpain linker region of pAP-296 (SEQ ID NO: 124);

FIG. 47C shows the DNA sequence of the pAP-296 insert (SEQ ID NO: 125);

FIG. 47D shows the amino acid sequence comparison of mutant preproricin linker region of calpain to wild type (SEQ ID NOS: 126 and 127);

FIG. 49 is a blot showing cleavage of pAP-220 with MMP-9;

FIG. 50 is a blot showing activation of pAP-214; and

FIG. 51 is a blot showing activation of pAP-220.

FIG. 52 is a blot showing cleavage of pAP-248 with Human Cytomegalovirus (HCMV).

FIG. 53 is a blot showing activation of pAP-248.

FIG. 54 is a blot showing cleavage of pAP-256 by HAV 3C.

FIG. 55 is a blot showing activation of pAP-256.

FIG. 56 is a semi-logithmic graph illustrating the cytotoxicity to COS-1 cells of undigested pAP-214 and pAP-214 digested with Cathepsin B.

FIG. 57 is a semi-logithmic graph illustrating the cytotoxicity of pAP-220 digested with MMP-9 compared to freshly thawed pAP-220 and ricin on COS-1 cells.

FIG. 58 is a blot showing cleavage of pAP-270 with MMP-2.

FIG. 59 is a blot showing activation of pAP-270.

FIG. 60 is a blot showing cleavage of pAP-288 by t-PA.

FIG. 61 is a blot showing activation of pAP-288.

FIG. 62 is a blot showing cleavage of pAP-294 with human neutrophil elastase.

FIG. 63 is a blot showing activation of pAP-294.

FIG. 64 is a blot showing cleavage of pAP-296 with calpain.

FIG. 65 is a blot showing activation of pAP-296.

FIG. 66 is a blot showing cleavage of pAP-222 with MMP-2.

FIG. 67 is a blot showing activation of pAP-222.

DETAILED DESCRIPTION OF THE INVENTION

Nucleic Acid Molecules of the Invention

As mentioned above, the present invention relates to novel nucleic acid molecules comprising a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains. The heterologous linker sequence contains a cleavage recognition site for a disease-specific protease (e.g. a viral protease, parasitic protease, cancer-associated protease, or a fungal protease).

The term "isolated and purified" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An "isolated and purified" nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived. The term "nucleic acid" is intended to include DNA and RNA and can be either double stranded or single stranded.

The term "linker sequence" as used herein refers to an internal amino acid sequence within the protein encoded by the nucleic acid molecule of the invention which contains residues linking the A and B chain so as to render the A chain incapable of exerting its toxic effect, for example catalytically inhibiting translation of a eukaryotic ribosome. By heterologous is meant that the linker sequence is not a sequence native to the A or B chain of a ricin-like toxin or precursor thereof. However, preferably, the linker sequence may be of a similar length to the linker sequence of a ricin-like toxin and should not interfere with the role of the B chain in cell binding and transport into the cytoplasm. When the linker sequence is cleaved the A chain becomes active or toxic.

The nucleic acid molecule of the invention is cloned by subjecting a preproricin cDNA clone to site-directed mutagenesis in order to generate a series of variants differing only in the sequence between the A and B chains (linker region). Oligonucleotides, corresponding to the extreme 5' and 3' ends of the preproricin gene are synthesized and used to PCR amplify the gene. Using the cDNA sequence for preproricin (Lamb et al., Eur. J. Biochem. 145:266-270 (1985)), several oligonucleotide primers are designed to flank the start and stop codons of the preproricin open reading frame.

The preproricin cDNA is amplified using the upstream primer Ricin-99 or Ricin-109 and the downstream primer Ricin1729C with Vent DNA polymerase (New England Biolabs) using standard procedures (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (Cold Spring Harbor Laboratory Press, 1989)). The purified PCR fragment encoding the preproricin cDNA is then ligated into an Eco RI-digested pBluescript II SK plasmid (Stratagene), and is used to transform competent XL1-Blue cells (Stratagene). The cloned PCR product containing the putative preproricin gene is confirmed by DNA sequencing of the entire cDNA clone. The sequences and location of oligonucleotide primers used for sequencing are shown in Table 1.

The preproricin cDNA clone is subjected to site directed mutagenesis in order to generate a series of variants differing only in the sequence between the A and B chains (linker region). The wild-type preproricin linker region is replaced with the heterogenous linker sequences that are cleaved by the various disease-specific proteases as shown in FIGS. 21, 26, 27, 28, and Part D of FIGS. 30-47. Linker identification as used herein in connection with the sequences provided in these figures have been assigned the sequence ID numbers as discussed below.

The linker regions of the variants encode a cleavage recognition sequence for a disease-specific protease associated with for example, cancer, viruses, parasites, or fungii. The mutagenesis and cloning strategy used to generate the disease-specific protease-sensitive linker variants are summarized in Part A of FIGS. 2-20, and Part A of FIGS. 22-25. The first step involves a DNA amplification using a set of mutagenic primers in combination with the two flanking primers Richin-99Eco or Ricin-109Eco and Ricin1729C Pst I. Restriction digested PCR fragments are gel purified and then ligated with PBluescript SK which has been digested with Eco RI and Pst I. Ligation reactions are used to transform competent XL1-Blue cells (Stratagene). Recombinant clones are identified by restriction digests of plasmid miniprep DNA and the mutant linker sequences are confirmed by DNA sequencing. With respect to the nucleotide sequences and amino acid sequences prepared as a result of the implementation of this strategy the following sequences have bee