sign in sign up
<<
Home , Pseudomonas exotoxin, Taipoxin, Tetanospasmin

US 2014.0005362A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0005362 A1 Bujko et al. (43) Pub. Date: Jan. 2, 2014

(54) RECOMBINANT CYTOTOXINAS WELL ASA (30) Foreign Application Priority Data METHOD OF PRODUCING IT Dec. 31, 2010 (PL) ...... PL393.529 (76) Inventors: Anna Bujko, Kalisz (PL); Magdalena Lukasiak, Parzeczew (PL); Jaroslaw PublicationDCOSSO Classificati Dastych, Lodz (PL); Miroslawa (51) Int. Cl. Skupinska, Poznan (PL); Ewelina C07K I4/2 (2006.01) Rodakowska, Kostrzyn (PL); Leszek (52) U.S. Cl. Rychlewski, Poznan (PL) CPC ...... C07K 14/21 (2013.01) USPC ...... S30/371. 530/350, 536/234:435/320.1 (21) Appl. No.: 13/976,914 s s 530370 (22) PCT Filed: Dec. 29, 2011 (57) ABSTRACT The subject of the present invention is a method of modifying (86). PCT No.: PCT/PL2011/050.058 proteinaceous through the addition of an NLS motif. S371 (c)(1), The resulting cytotoxin facilitates the selective elimination of (2), (4) Date: Sep. 16, 2013 proliferating cells, particularly tumour cells. Patent Application Publication Jan. 2, 2014 Sheet 1 of 5 US 2014/0005362 A1

binding translocatin domain domain catalytic domain

Drawing 1 Schematic of the structure of PE of Pseudomonas aeruginosa. According to: Kreitman (2009), modified.

Eissers

Drawing 2 Schematic of PE intoxication. Source: Kreitman (2009), Patent Application Publication Jan. 2, 2014 Sheet 2 of 5 US 2014/0005362 A1

pit is

rix territats: 8 :

EX fissi & 3 &

pa208:2678: REEixi: sag opt safe: SSgs; s-r 52s. & S.

Aspicin

Fig. 1 Map of vector pJ206:26701. Red frames delineate restriction sites for EcoRI and HindIII. Patent Application Publication Jan. 2, 2014 Sheet 3 of 5 US 2014/0005362 A1

Fig. 2 Map of vector UC57. Red frames delineate restriction sites for EcoRI and HindIII. Patent Application Publication Jan. 2, 2014 Sheet 4 of 5 US 2014/0005362 A1

kDa

Fig. 3 Image of X-ray film with visible signals corresponding to the following : GFP (GFP), native exotoxin (PET) and modified exotoxin (PET mod1). Patent Application Publication Jan. 2, 2014 Sheet 5 of 5 US 2014/0005362 A1

Cytotoxicity aSSay

120 ------

‘.....:3:..... GF P (10)

arriers PET (10)

yyy&xxxxPETm1 (10)

... ." GF P (4)

n a w PET (4)

is y PE m1(4) concentration ug/ml)

Fig. 4 The effect of modified exotoxin (PETm1), native exotoxin (PET) and GFP as controls, on 3T3 fibroblast viability, measurement using the neutral red method after 24h. Culture confluence denoted as: (10) - 100% confluence, (4) - 40% confluence. US 2014/0005362 A1 Jan. 2, 2014

RECOMBINANT CYTOTOXINAS WELL ASA bacterial specific for tumour cells is one of the METHOD OF PRODUCING IT targeted therapeutic strategies against cancer. Exotoxins 0001. The subject of the present invention is a method of secreted naturally by the disease-causing bacteria Pseudomo modifying proteinaceous toxins through the addition of an nas aeruginosa and Diphtheriae typhimurium are com NLS motif. The resulting cytotoxins facilitate the selective pounds of very high cytotoxicity. Sometimes many times elimination of proliferating cells, particularly tumour cells. higher than of classic antitumour drugs. In many cases a 0002 A poison is an organic or inorganic Substance single molecule is capable of killing a cell, which makes which, even at low concentrations, has a deleterious effect on them some of the most lethal compounds. An exotoxin fre living organisms. Poisons are divided into two basic catego quently used to construct fusion proteins with antitumour ries. The first consists of natural poisons, produced mainly by activity is exotoxin PE from Pseudomonas aeruginosa pathogenic bacteria, poisonous fungi and , as well as (, PE) 1. A molecule of native PE Venomous animals. The second group of poisons consists of toxin consists of a catalytic domain connected with a domain anthropogenic poisons. Bacterial toxins () are various that binds a receptor through a central translocation domain, chemical compounds produced by bacteria which poison a which facilitates the transfer of the C-terminal catalytic higher organism. They act specifically on various systems domain into the cytosol (drawing 1). So far, the role of the Ib (i.e. on the gastrointestinal tract) or cells of an organism (i.e. domain of PE remains unknown, but it is known that it con ). These are differentiated into exotoxins and tains a disulphide bridge necessary for molecule maturation. endotoxins. Exotoxins, secreted outside of the live cell are Drawing 2 shows the mechanism of PE intoxication. Due to strong venoms and induce specific disease symptoms. They the interaction with the host cell, PE binds to the O2 macro have a proteinaceous structure (metabolic product) and thus globulins. Prior to entering the cell, the toxin is cleaved pro are sensitive to high temperatures (>60° C.) as well as being teolytically. A caroxypeptidase cuts off the terminal lysine degraded by digestive enzymes (with the exception of botu which exposes the REDL motif. Next, the exotoxins are inter linum toxin and Staphylococcus ). They have nalised through endocytosis dependent on the receptor. After strong antygenic properties, and anatoxins made therefrom entering endocytotic vesicles, the toxin is cleaved proteolyti are used to immunise humans and animals. They are made cally by furin inside the translocation domain, and the disul mainly by Gram-positive bacteria. These are some of the phide bridges hold the formed fragments until they are strongest toxic Substances known. Endotoxins are released reduced. The PE migration pathway in the cell is through the only following the degradation of the bacterial cell. They are Golgi apparatus and encompasses retention in the endoplas weak venoms, and the symptoms they induce are not specific. matic reticulum due to the REDL signal at the C-terminus of Chemically, these are glycolipopolypeptide complexes (li the protein. Then, the freed catalytic domain is translocated popolysaccharide) which most often occur in Gram-negative through the reticulum wall into the cytosol. There, the active bacteria in one of the three cell-wall layers. They are poorly protein catalyses ADP-ribosylation of His at position 699 of antygenic. They are not degraded by digestive enzymes but the translation factor eEF2 and thereby inhibits protein syn are thermostable. Exotoxins secreted by bacteria (but also by thesis, thereby quickly leading to cell death 1. The use of PE plants, fungi and some animals) exhibit cytotoxic properties in targeted therapy entails the replacement of the receptor against a host cells, usually due to the enzymatic inhibition of binding domain with an or a portion thereof, a protein synthesis. The essential condition toxin activity is cytokine or growth factor (hence the name immunotoxins). their binding of surface receptors on the target cell and their The most frequently used form of PE is a fractional exotoxin internalisation through endocytsis, and then translocation of 38 kDa composed of amino-acids 253-364 and 381-613. from the endoplasmatic reticulum into the cytosol. Bacterial Chimeric immunotoxins based on PE molecules are most exotoxins are currently produced using genetic engineering often directed against receptors IL2 and IL6 as well as growth methods or chemically conjugated with ligands and antibod factor TGFC. (Tab. 1) 2). The table below lists information ies so as to bind to specific cell types. This facilitates the regarding the use of propharmaceuticals containing immuno selective destruction of disease-altered cell lines. The use of toxins in the treatment of tumours (clinical trials). TABLE 1 Immunotoxins based on PE in clinical trials, 2009 data (2 Immunotoxin Construction Target antigen Tumors References CD19-ETA ScFw fused to PE38KDEL CD19 Lymphoma, leukemia Schwemmlein et al. (2007) Anti-Tac{Fv)-PE38KDEL ScFw fused to PE38KDEL CD25 CD25 positive tumor cells Kreitman et al. (1994) LMB2) Anti-Tac{Fv)-PE40KDEL ScFw fused to PE40KDEL CD25 Chronic lymphocytic leukemia Kreitman et al. (1992) RTF5(scFv)-ETA ScFw fused to PE40 CD25 Lymphoma Barth et al. (1998) RFB(dsFv)-PE38 B1.22) dsFwfused to PE38 CD22 B-cell leukemia Kreitman et al. (2000a) G28-SSFw-PE40 ScFw fused to PE40 CD40 Burkilt's lymphoma Francisco et al. (1997) K14(scFv)-ETA' ScFw fused to PE40 CD30 Hodkin's lymphoma Klimka et al. (1999) CD7-ETA ScFw fused to PE40 CD7 T-lineage acute lymphoblastic Peipp et al. (2002) eukemia OVB3-PE mAb linked via bond Ovary Ovarian Willingham et al. (1987) to PE B3-Lys-PE38 LMB-1 mAb chemically linked to PE38 LeY Various Pastan (1997) B1(dsFv)-PE38 dsFw fused to PE38 LeY LeY positive tumor cells Benhar et al. (1995) B3(dsFv)-PE38 dsFw fused to PE38 LeY LeY positive tumor cells Benhar et al. (1995) BR96sFv-PE40 (SGN-10) ScFw fused to PE40 LeY LeY positive tumor cells Friedman et al. (1993) US 2014/0005362 A1 Jan. 2, 2014

TABLE 1-continued Immunotoxins based on PE in clinical trials, 2009 data (2 Immunotoxin Construction Target antigen Tumors References IL4(38-37)PE38KDEL IL4 fused to PE38KDEL ILA-R Breast, SCCHN, pancreas, Leland et al. (2000); Kawakami NBI-3001) medulloblastoma et al. (2000, 2002); Strome et al. (2002); Joshi et al. (2002) IL13-PE38QQR IL13 fused to PE38QQR IL13-R. Head and neck Kawukumi et al. (2001) scFv(FRP5)-ETA ScFw fused to PE40 erbB2 Ovarian, prostate Wels et al., (1992); Schmidt et al. (2001); Wang et al. (2001) AR209 e23(Fv)PE38KDEL scFv fused to PE38KDEL erbB-2 Lung, prostate Skrepniket al. (1996, 1999); Erb-38 dSFw fused to PE38 erbB2 Epidermoid carcinoma, breast Reiter and Pastan (1996) MR1(Fv)-PE38 ScFw fused to PE38 EGFRVIII Glioblastoma Beers et al. (2000) TP38 TGF-C. fused to PE38 EGFR Glioma Sampson et al. (2003) TP40 TGF-C. fused to PE40 EGFR Glioma, prostate, epidermoid Sarosdy et al., (1993); Paietal. (1991a); Kunwar et al. (1993) 425.3PE mAb chemically linked to PE EGFR Breast Anderssan et al. (2004) AS-PE40 ScFw fused to PE40 PSMA Prostate Wolfetal. (2006, 2008) SS1(dsFv)PE38 SSIP dSFw fused to PE38 Mesothelin Ovarian, cervical Hussan et al. (2002) scFv(MUC1)-ETA ScFw fused to PE40 MUC1 Breast Singh et al. (2007) 9.2.27-PE mAb chemically linked to PE HMW-MAA Gliomblastoma Hartland et al. (2004) TP-3(scFv)-PE38 ScFw fused to PE38 Osteosarcoma Osteosarcoma Onda et al. (2001) antigen TP-3(dsFv)-PE38 dSFw fused to PE38 Osteosarcoma Osteosarcoma Onda et al. (2001) antigen 8H9(dsFv)-PE38 dSFw fused to PE38 Cell surface Breast, osteosarcoma, Onda et al. (2004) glycoprotein neuroblastoma 4D6MOCB-ETA ScFw fused to PE40KDEL Ep-CAM Lung, colon, SCC Di Panlo et al. (2003) HB21(Fv)-PE40 ScFw fused to PE40 TR Colon Shinohara et al. (2000)

0003. Denileukin diftitox (ONTAK) is at present the only fusion protein containing the amino-acid sequence encom available therapeutic which is an immunotoxin. Registered in passing the sequence of bacterial exotoxin as well as the 1999, it is used in the therapy of CTCL, Cutaneous T-Celi sequence of a human NLS motif. Lymphoma. The FDA report of 16.10.2008 gives it a full 0008 For the purposes of this description, “protein toxins' marketing permits. should be understood as natural polypeptides with toxic prop 0004. The distribution of cell surface antigens used in erties. Such as: targeted therapy is very often not limited to tumour cells, but 0009 neurotoxins, which hinder neurotransmission, is only characterised by increased frequency in comparison to 0.010 enterotoxins, which damage the gastrointestinal normal cells. This often causes side effects during the use of muCOSa, the drugs in the form of the destruction of healthy cells, even 0.011 cytotoxins, which destroy cells in tissues and organs with different functions. For example, in 0012 Protein toxins may be of various origins. Known are the therapy of breast cancertargeted against HER2 receptors, the following toxins: one observes the non-specific ingress of immunotoxins into 0013 animal i.e.: Cubozoa contains protein tox hepatocytes or macrophages, which induces liver damage, ins with neurotoxic and cardiotoxic properties, which and the release of cytokines by the macrophages causes Sub also cause tissue necrosis; (Oxyuranus scutel sequent non-specific changes. Newest generation immuno latus), inhibits acetylcholine release from terminal neu toxins are characterised by a higher specificity, Stemming rons and some cholinergic neurons of the autonomous from the fact that their binding-activity requires not one, but nervous system; o- (Latrodectus) binds with two or more factors specific to tumour cells. membrane neurexins and causes the Sudden depletion of 0005. The goal of the present invention is to deliver a synaptic vesicles; compound, whose activity will be dependent on the phase of 0014 fungal i.e.: C.-amanitin (deathcap mushroom), the cell cycle and will be preferably apparent in intensively binds with RNA polymerase II, at higher concentrations proliferating cells, particularly tumour cells. It is desirable also with RNA polymerase III, preventing RNA elonga that the sought Substance, in addition to binding specifically tion during synthesis; C.-sarcin (Aspergillus giganteus) defined epitopes, is subject to specific activation in cancerous inhibits protein synthesis by hydrolyzing phosphodi cells. This type of substance should be fit for use in the ester bonds in 28S RNA in the large ribosomal subunit; production of novel pharmaceutical compositions character 0.015 i.e.: Holotoxins (also called class II ribo ised by increased therapeutic efficiency w the treatment of Some-inactivating proteins) i.e.: (castor oil plant), tumours as well as a lower number of undesirable side effects. (rosary pea), lectin (mistletoe), modecin (Adenia 0006 Unexpectedly, the above stated goal has been digitata); Hemitoxins (also called class I ribosome-in achieved in the present invention. activating proteins) i.e.: PAP (pokeweed antiviral pro 0007. The subject of the present invention is a method of tein), Saporin (Saponaria officinalis), bouganin (Bouga modifying a protein toxin through the addition of an NLS invillea spectabilis) and gelonin (Gelonium motif, which unexpectedly decreases the toxicity of the Multiflorum)3: (Holotoxins are composed of a binding resulting toxin towards non-proliferating cells. In the domain and a catalytic domain whereas hemitoxins con example embodiment of the present invention we design a tain only a catalytic domain. Plant toxins inhibit the US 2014/0005362 A1 Jan. 2, 2014

binding of the elongation factors EF-1 and EF-2 with the intervals. Up to 8 combined cycles have been used. In 12 ribosomal 60S subunit by removing the A residue in patients, reduced leukaemic cells have been observed in position 4324 of 28SRNA. Ricin also removes the the blood of over 80% of the patients, and in 6 a decrease neighbouring G residue at position 43234. The result in lymph node volume. 6 of 22 patients who received at of such toxin activity is cell death via apoptosis. Only the least 2 cycles fulfilled the criteria for full or partial enzymatic domain is translocated into the cytoplasm, remission. and thus the binding domain of holotoxins is cleaved off 0.019 BL22—a recombinant immunotoxins containing through the reduction of the disulphide bond 5-7.); an IgG immunoglobulin fragment, which recognizes 0016 bacterial i.e.: Neurotoxins: botulin ( antigen CD22, conjugated with the exotoxin of botulinum) (the activity is based on Pseudomonas 9. The antibody is highly active in the permanent affixation to the neuromotor plate and dis case of hairy cell leukaemia 10.11). A high efficacy was ruption of muscle contraction. This is done by the frag also observed against CLL, but not against CR 11,12. mentation of the SNAP-25 protein essential to acetyl Currently, a BL22 mutant termed HA22 is undergoing choline secretion from the presynaptic terminus), clinical trials 9. toxin, () (teta 0020 For the purposes of this description the human nospasmin binds to peripheral motor neurons, enters the “NLS' motif (nuclear localization signal or sequence) should axon and from there transfers to neurons of the brain be understood as an amino-acid sequence motif warranting stem and spinal chord. It then migrates through the Syn intracellular transport of a protein into the nucleus. It com apse to the presynaptic terminus where it blocks the prises a sequence of positively charged amino-acids, lysines release of ( and GABA); and arginines (so-called single NLS), meeting the consensus Enterotoxins: Streptolysine O (Streptococcus pyo K-K/R-X-KR with the sequence: KKKRKR 13). genes), listeriolysine O (Listeria monocytogenes), 0021. An example use of the present invention is exotoxin alpha-toxin (Staphylococcus aureus) (these toxins are A of Pseudomonas aeruginosa modified Such that in the capable of integrating into the cell membrane in which amino-acid sequence it contains an additional NLS motif: they form channels. In this way the porous membrane KKKRKR added at position -633, behind proline-632 from can no longer function, and ions begin to egress the cell the amino end (as shown in sequence 1) in relation to the whereas water begins to flow inside and the cell may native protein. Swell and lyse.); Cytotoxins: collagenases, hyalu 0022. The next subject of the present invention are nucle ronidases orphospholipases are enzymes which respec otide sequences of DNA, cDNA and mRNA encoding exo tively degrade collagen (facilitating deep penetration of toxin A of Pseudomonas aeruginsa modified Such that at tissue) and membrane phospholipids; , StX position 1933 in relation to the native sequence the addition (Shigella dysenteriae) this protein is composed of 6 ally contain a motif encoding NLS, taking into account the subunits: 5 B subunits, responsible for binding the toxin degeneration of genetic code, meaning that all DNA encodes to its receptor globotriaosylceramide (Gb3) of a the protein with the amino-acid sequence according to the eukaryotic cell and an A Subunit, which is proteolysed present invention as it has been defined above. Particular following endocytosis to peptides A1 and A2. StXA2 is embodiment of the nucleotide sequence according to the an enzyme which cleaves an adenine off 28S ribosomal present invention is sequence 2. RNA. This inhibits tprotein synthesis in a cell and its 0023 The next subject of the present invention are modi death; (Vibrio cholerae) catalyses the fied proteins, derivatives of exotoxins containing the protein binding of ADP-ribose to a G-protein subunit which lose fragment described above, with the modification described its GTPase activity. It fails to dissociate from adenylate above, as well as the DNA sequences encoding them. cyclase, of which it is an activator. Surplus synthesis of 0024. The next subject of the present invention are recom cyclic AMP causes an increased concentration of elec binant expression vectors as well as expression cassettes con trolytes in the intestinal lumen (storage of chlorides and taining said DNA sequences. inhibited potassium absorption), which causes constant 0025. The next subject of the present invention is the pro water flow into the intestines; dyphtherotoxin (Coryne duction of said proteins through overexpression in cells and in bacterium diphtheriae), a which transfers extracellular systems. ADP-ribose from NAD+ to eEF-2 (ADP-ribosylation) 0026. The next subject of the present invention is the use of and in this way inhibits the translocation and thus the said proteins to treat eukaryotic cells. elongation of a polypeptide chain; exotoxin A 0027. The next subject of the present invention the use of (Pseudomonas). said proteins in the production of pharmaceutical composi 0017 For the purposes of this description, “immunotox tions. ins' should be understood as complexes of or their 0028. The description of the present invention is illus fragments with toxins, chemically bound. The antibody is trated by the attached figures. FIG. 1 represents a schematic directed against structures on the tumour cell Surface. Most representation of the structure of the exoPE toxin 14. FIG.2 often, recombinant immunotoxins produced by E. coli are represents the mechanism of PE intoxication. used. Such as: 0029. To better understand the present invention defined 0018 human interleukin-2 (IL-2) combined with doph above, the present description also contains an example thitoxin (denileukin diftitox)—reacts with the IL-2 embodiment of the present invention. This example, however, receptor. This drug is registered for the treatment of should not be treated as limiting the scope encompassed by dermal T-cell lymphomas. It has also been tested in CLL the present invention. The example embodiments are illus patients resistant to other antileukaemia drugs 8. trated by the attached figures, wherein FIG. 1 shows the map Denileukin diftitox is administered at a rate of 18 g/kg/ of the vectorp.J206, wherein the frame encloses the restriction day in a 60 minute infusion over five days at 21 day sites EcoRI and HindIII. FIG. 2 shows the map of the vector US 2014/0005362 A1 Jan. 2, 2014

pUC57, where the frame encloses the restriction sites for TABLE 3 EcoRI and HindIII. FIG. 3 in turn shows an X-Ray film with Molecular mass of the proteins obtained through in vitro visible signals corresponding to GFP (GFP), native exotoxin transcription translation, evaluated using the Agilent microchip. (PET) as well as modified exotoxin (PET mod 1). FIG. A. mass kDa shows the effect of the selected chimeric toxins on live human GFP 30 fibroblasts, 3T3, neutral red method after 24h, where: GFP PET 74 translation from the vector with GFP (translation control), PET mod1 76 PETm1—translation from the vector with the modified toxin whereas PET is the native toxin. 0034) To confirm that the resulting fusion protein of the desired mass was obtained, we performed Western blot analy EXAMPLE1 sis. Detection was performed using an antibody against the His-tag, conjugated with HRP (horseradish peroxidase). As is 0030 The sequence encoding the modified exotoxin was shown in FIG.3, all resulting proteins bound the anti-His-tag designed with the further addition of elements necessary to antibody. The resulting signals corresponded to a mass of obtain (via in vitro synthesis) proteins containing a tag in the about 30 kDa (GFP) and 75 kDa (native and modified toxins). form of 6 histidine residues. The nucleotide sequence of the 0035. We then tested the effect of the modified toxins on entire expression cassette is shown as sequence No. 3 whereas the growth of NIH/3T3 mouse fibroblasts and its selective the protein it encodes has the amino-acid sequence termed cytotoxic effect on intensively dividing cells. FIG. 4 shows a sequence 4 (in the sequence list). compilation of survivability results of the treated cells. In the case of modified exotoxin, we observed differences depen 0031. The expression cassette containing: a promoter for dent on the stage of development of the culture (confluence of the T7 polymerase, a ribosome binding site, a start codon, a 100% and 40%). Intensively dividing cells (initial confluence linker with the His-tag as well as a sequence encoding the 40%) were more sensitive to the modified exotoxin than modified exotoxin, was obtained through chemical synthesis 100% confluent cells, and the difference was about 11%. performed by the GenScript company. This cassette was then cloned by the producer into the vector p206 between the References: restriction sites EcoRI and HindIII (FIG. 1). From such a 0036 1. Kreitman R.J. Recombinant immunotoxins con vector we digested out the entire expression cassette and taining truncated bacterial toxins for the treatment of transcloned it into the plasmid puC57 (FIG. 2) using the hematologic malignancies. BioDrugs., 2009, 23(1):1-13. EcoRI and HindIII restrictases. In the same way we synthe 0037 2. Wolf P. Elsasser-Beile U. Pseudomonas exotoxin sized and prepared the vectors used to express proteins that A: from to anti-cancer agent. Int J Med were the controls in the experiment: unmodified (native) exo Microbiol. 2009, 299, 161-76. toxin A of Pseudomonas aeruginsa as well as GFP. 0038. 3. Bolognesi A, Polito L, Tazzari PL, et al. In vitro anti-tumour activity of anti-CD80 and anti-CD86 immu 0032. The vector containing the insert, the expression cas notoxins containing type 1 ribosome inactivating proteins. sette for the modified exotoxin as well as vectors for the BrJ Haematol. 2000: 110: 351-361. expression of the native exotoxin and GFP were used for in 0039 4. Endo Y. Mitsui K, Motizuki M., Tsurugi K. The vitro transcription and translation using the commercial "RTS mechanism of action of ricin and related toxic lectins on 100 E. coli HY Kit containing E. coli lysate (5Prime). Pro eukaryotic ribosomes. J Biol Chem. 1987:262:5908-5912. teins synthesized in this fashion were purified on Ni-NTA 0040. 5. Bolognesi A, Tazzari P L, Olivieri F, Polito L, agarose (Qiagen) and dialysed against PBS, and then concen Falini B, Stirpe F. Induction of apoptosis by ribosome trated using Amicon centrifuge filters. The concentration of inactivating proteins and related immunotoxins. IntJ Can the resulting proteins in Subsequent purification steps was cer: 1996; 68: 349-355. estimated using the BCA method (Tab. 2). 0041) 6. Hughes J. N. Lindsay CD, Griffiths G D. Mor phology of ricin and abrin exposed endothelial cells is TABLE 2 consistent with apoptotic cell death. Hum Exp Toxicol. 1996; 15:443-451. Concentrations of proteins obtained via in vitro 0042 7. Bergamaschi G. Perfetti V, Tonon L, et al. transcription translation, estimated using the BCA method. Saporin, a ribosome inactivating protein used to prepare immunotoxins, induces cell death via apoptosis. Br. J Hae protein concentration g/ml matol. 1996: 93: 789-794. preparation cleaned on preparation following 0043 8. Frankel A E, Surendranathan A, Black J. H. White Ni-NTA-agarose concentration on Amicon filters A. Ganjoo, Cripe L. D. Phase II clinical studies of denileu kin diffitox fusion protein in patients with previously GFP 75 227 treated chronic lymphocytic leukemia. Cancer. 2006; 106: PET <10 39 2158-21 64. PET mod1 22 64 0044. 9. Robak T. Novel monoclonal antibodies for the treatment of chronic lymphocytic leukemia. Curr Cancer Drug Targets. 2008; 8: 156-171. 0033. The molecular mass of the resulting proteins was 0045 10. Kreitman RJ, Wilson W H, Bergeron K, et al. evaluated using electrophoresis on Agilent microchips. The Efficacy of the anti CD22 recombinant immunotoxins results are shown below (Tab. 3) and reflect the predicted BL22 in chemotherapy resistant hairy cell leukemia. N mass of the resulting polypeptides. Engl J Med. 2001; 345: 241-247. US 2014/0005362 A1 Jan. 2, 2014

0046) 11. Robak T. New agents in chronic lymphocytic 0048 13. Chelsky D, Ralph R. Jonak G. Sequence require leukemia. Curr Treat Options Oncol. 2006; 7: 200-212. ments for synthetic peptide-mediated translocation to the 0047 12. Kreitman R.J. Squires DR, Stetler-Stevenson M, nucleus. Mol Cell Biol. 1989 Jun; 9(6):2487-92. et al. Phase I trial of recombinant immunotoxins RFB4 (dsRV)-PE38 (BL22) in patients with B-cell malignancies. 0049 14. Kreitman RJ. Immunotoxins for targeted cancer J. Clin Oncol. 2005; 23: 6719-6729. therapy. AAPSJ. 2006 Aug 18; 8(3):E532-51.

SEQUENCE LISTING

NUMBER OF SEO ID NOS: 4.

SEO ID NO 1 LENGTH: 643 TYPE PRT ORGANISM: artificial sequence FEATURE; OTHER INFORMATION: fusion protein

<4 OOs SEQUENCE: 1.

Met His Lell Thir Pro His Trp Ile Pro Lell Wall Ala Ser Tell Gly Lell 1. 5 15

Luell Ala Gly Gly Ser Phe Ala Ser Ala Ala Glu Glu Ala Phe Asp Lell 25 3 O

Trp Asn Glu Cys Ala Ala Cys Wall Lell Asp Luell Lys Asp Gly Wall 35 4 O 45

Arg Ser Ser Arg Met Ser Wall Asp Pro Ala Ile Ala Asp Thir Asn Gly SO 55 60

Glin Gly Wall Luell His Tyr Ser Met Wall Lell Glu Gly Gly ASn Asp Ala 65 70

Luell Lell Ala Ile Asp ASn Ala Lell Ser Ile Thir Ser Asp Gly Lell 85 90 95

Thir Ile Arg Luell Glu Gly Gly Wall Glu Pro Asn Pro Wall Arg 105 110

Ser Thir Arg Glin Ala Arg Gly Ser Trp Ser Luell Asn Trp Lell Wall 115 12O 125

Pro Ile Gly His Glu Pro Ser Asn Ile Wall Phe le His Glu 13 O 135 14 O

Luell Asn Ala Gly Asn Glin Luell Ser His Met Ser Pro Ile Tyr Thir Ile 145 15 O 155 16 O

Glu Met Gly Asp Glu Luell Luell Ala Lell Ala Arg Asp Ala Thir Phe 1.65 17 O 17s

Phe Wall Arg Ala His Glu Ser Asn Glu Met Glin Pro Thir Tell Ala Ile 18O 185 190

Ser His Ala Gly Wall Ser Wall Wall Met Ala Glin Ala Glin Pro Arg Arg 195 2 OO

Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Wall Luell Lell Lell 210 215 22 O

Asp Pro Lell Asp Gly Wall Asn Tyr Lell Ala Glin Glin Asn 225 23 O 235 24 O

Luell Asp Asp Thir Trp Glu Gly Ile Tyr Arg Wall Luell Ala Gly Asn 245 25 O 255

Pro Ala His Asp Luell Asp Ile Lys Pro Thir Wall Ile Ser His Arg 26 O 265 27 O

Luell His Phe Pro Glu Gly Gly Ser Lell Ala Ala Luell Thir Ala His Glin 27s 28O 285 US 2014/0005362 A1 Jan. 2, 2014

- Continued Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg 29 O 295 3 OO Gly Trp Glu Glin Lieu. Glu Gln Cys Gly Tyr Pro Val Glin Arg Lieu Val 3. OS 310 315 32O Ala Lieu. Tyr Lieu Ala Ala Arg Lieu. Ser Trp Asn Glin Val Asp Glin Val 3.25 330 335 Ile Arg Asn Ala Lieu Ala Ser Pro Gly Ser Gly Gly Asp Lieu. Gly Glu 34 O 345 35. O Ala Ile Arg Glu Gln Pro Glu Glin Ala Arg Lieu Ala Lieu. Thir Lieu Ala 355 360 365 Ala Ala Glu Ser Glu Arg Phe Val Arg Glin Gly Thr Gly Asn Asp Glu 37 O 375 38O Ala Gly Ala Ala Ser Ala Asp Val Val Ser Lieu. Thir Cys Pro Val Ala 385 390 395 4 OO Ala Gly Glu. Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Lieu. Lieu. Glu 4 OS 41O 415 Arg Asn Tyr Pro Thr Gly Ala Glu Phe Lieu. Gly Asp Gly Gly Asp Ile 42O 425 43 O Ser Phe Ser Thr Arg Gly Thr Glin Asn Trp Thr Val Glu Arg Lieu. Leu 435 44 O 445 Glin Ala His Arg Glin Lieu. Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr 450 45.5 460 His Gly Thr Phe Lieu. Glu Ala Ala Glin Ser Ile Val Phe Gly Gly Val 465 470 47s 48O Arg Ala Arg Ser Glin Asp Lieu. Asp Ala Ile Trp Arg Gly Phe Tyr Ile 485 490 495 Ala Gly Asp Pro Ala Lieu Ala Tyr Gly Tyr Ala Glin Asp Glin Glu Pro SOO 505 51O Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Lieu. Lieu. Arg Val Tyr Val 515 52O 525 Pro Arg Ser Ser Lieu Pro Gly Phe Tyr Arg Thr Gly Lieu. Thir Lieu Ala 53 O 535 54 O Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Lieu. Ile Gly His Pro Lieu 5.45 550 555 560 Pro Lieu. Arg Lieu. Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg 565 st O sts Lieu. Glu Thir Ile Lieu. Gly Trp Pro Lieu Ala Glu Arg Thr Val Val Ile 58O 585 59 O Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Lieu. Asp 595 6OO 605 Pro Ser Ser Ile Pro Asp Llys Glu Glin Ala Ile Ser Ala Lieu Pro Asp 610 615 62O Tyr Ala Ser Glin Pro Gly Llys Pro Llys Llys Lys Arg Lys Arg Arg Glu 625 630 635 64 O Asp Lieu Lys

<210s, SEQ ID NO 2 &211s LENGTH: 1968 &212s. TYPE: DNA <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: sequence encoding fusion protein

<4 OOs, SEQUENCE: 2

US 2014/0005362 A1 Jan. 2, 2014

- Continued

212. TYPE: PRT <213> ORGANISM: artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: fusion protein <4 OOs, SEQUENCE: 4 Met Ser Gly Ser His His His His His His Ser Ser Gly Ile Glu Gly 1. 5 1O 15 Arg Met His Lieu. Thr Pro His Trp Ile Pro Leu Val Ala Ser Leu Gly 2O 25 3O Lieu. Lieu Ala Gly Gly Ser Phe Ala Ser Ala Ala Glu Glu Ala Phe Asp 35 4 O 45 Lieu. Trp Asn. Glu. Cys Ala Lys Ala Cys Val Lieu. Asp Lieu Lys Asp Gly SO 55 6 O Val Arg Ser Ser Arg Met Ser Val Asp Pro Ala Ile Ala Asp Thir Asn 65 70 7s 8O Gly Glin Gly Val Lieu. His Tyr Ser Met Val Lieu. Glu Gly Gly Asn Asp 85 90 95 Ala Lieu Lys Lieu Ala Ile Asp Asn Ala Lieu. Ser Ile Thir Ser Asp Gly 1OO 105 11 O Lieu. Thir Ile Arg Lieu. Glu Gly Gly Val Glu Pro Asn Llys Pro Val Arg 115 12 O 125 Tyr Ser Tyr Thr Arg Glin Ala Arg Gly Ser Trp Ser Lieu. Asn Trp Lieu. 13 O 135 14 O Val Pro Ile Gly His Glu Lys Pro Ser Asn Ile Llys Val Phe Ile His 145 150 155 160 Glu Lieu. Asn Ala Gly Asn Gln Leu Ser His Met Ser Pro Ile Tyr Thr 1.65 17O 17s Ile Glu Met Gly Asp Glu Lieu. Lieu Ala Lys Lieu Ala Arg Asp Ala Thr 18O 185 19 O Phe Phe Val Arg Ala His Glu Ser Asn Glu Met Gln Pro Thr Lieu Ala 195 2OO 2O5 Ile Ser His Ala Gly Val Ser Val Val Met Ala Glin Ala Glin Pro Arg 21 O 215 22O Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser Gly Llys Val Lieu. Cys Lieu 225 23 O 235 24 O Lieu. Asp Pro Lieu. Asp Gly Val Tyr Asn Tyr Lieu Ala Glin Glin Arg Cys 245 250 255 Asn Lieu. Asp Asp Thir Trp Glu Gly Lys Ile Tyr Arg Val Lieu Ala Gly 26 O 265 27 O Asn Pro Ala Lys His Asp Lieu. Asp Ile Llys Pro Thr Val Ile Ser His 27s 28O 285 Arg Lieu. His Phe Pro Glu Gly Gly Ser Lieu Ala Ala Lieu. Thir Ala His 29 O 295 3 OO Glin Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg His Arg Glin Pro 3. OS 310 315 32O Arg Gly Trp Glu Gln Lieu. Glu Gln Cys Gly Tyr Pro Val Glin Arg Lieu 3.25 330 335 Val Ala Lieu. Tyr Lieu Ala Ala Arg Lieu. Ser Trp Asn Glin Val Asp Glin 34 O 345 35. O Val Ile Arg Asn Ala Lieu Ala Ser Pro Gly Ser Gly Gly Asp Lieu. Gly 355 360 365

Glu Ala Ile Arg Glu Gln Pro Glu Glin Ala Arg Lieu Ala Lieu. Thir Lieu. US 2014/0005362 A1 Jan. 2, 2014 10

- Continued

37 O 375 38O

Ala Ala Ala Glu Ser Glu Arg Phe Wall Arg Glin Gly Thr Gly Asn Asp 385 390 395 4 OO

Glu Ala Gly Ala Ala Ser Ala Asp Wall Wall Ser Lieu. Thir Pro Wall 4 OS 415

Ala Ala Gly Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Lieu. Luell 425 43 O

Glu Arg Asn Pro Thir Gly Ala Glu Phe Lieu. Gly Asp Gly Gly Asp 435 44 O 445

Ile Ser Phe Ser Thir Arg Gly Thr Gin Asn Trp Thir Wall Glu Arg Lieu. 450 45.5 460

Lell Glin Ala His Arg Glin Lieu. Glu Glu Arg Gly Wall Phe Val Gly 465 470

His Gly Thir Phe Lell Glu Ala Ala Glin Ser Ile Wall Phe Gly Gly 485 490 495

Val Arg Ala Arg Ser Glin Asp Luell Asp Ala Ile Trp Arg Gly Phe SOO 505

Ile Ala Gly Asp Pro Ala Lell Ala Tyr Gly Ala Glin Asp Glin Glu 515 525

Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Lieu. Lieu. Arg Wall 53 O 535 54 O

Wall Pro Arg Ser Ser Leul Pro Gly Phe Arg Thir Gly Luell Thir Lieu. 5.45 550 555 560

Ala Ala Pro Glu Ala Ala Gly Glu Wall Glu Arg Lieu. Ile Gly His Pro 565 st O sts

Lieu Pro Lieu. Arg Lieu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly 585 59 O

Arg Lieu. Glu Thir Ile Lieu. Gly Trp Pro Lieu Ala Glu Arg Thir Wall Wall 595 605

Ile Pro Ser Ala Ile Pro Thir Asp Pro Arg ASn Wall Gly Gly Asp Luell 610 615 62O

Asp Pro Ser Ser Ile Pro Asp Glu Glin Ala Ile Ser Ala Luell Pro 625 630 635 64 O

Asp Ala Ser Glin Pro Gly Pro Llys Arg Arg Arg 645 650 655 Glu Asp Lieu Lys 660

1. A method of producing a recombinant cytotoxin, the 6. The fusion protein according to claim 2, wherein the method comprising adding an NLS motif to the amino-acid NLS motif comprises the amino-acid sequence KKKRKR. sequence of a known protein toxin or its derivative, wherein 7. The fusion protein according to claim 2, comprising the the result is a recombinant cytotoxin with a decreased overall amino-acid sequence shown as SEQID NO: 1 or SEQID NO: cytotoxicity in comparison to the cytotoxicity of the known 4. protein toxin or its derivative. 8. A fusion protein according to claim 2 for use in anti 2. A fusion protein comprising an amino-acid sequence of tumour therapy. a protein toxin and a human NLS motif amino acid sequence. 9. A polynucleotide encoding a fusion protein according to 3. The fusion protein according to claim 2, wherein the claim 2. protein toxin is a known protein toxin from animals, fungi, 10. A polynucleotide according to claim 9, comprising the plants or bacteria. nucleotide sequence shown as SEQID NO: 2 or SEQID NO: 4. The fusion protein according to claim 2, wherein the 3. protein toxin is exotoxin A of Pseudomonas aeruginsa. 11. A biologically active vector, comprising a polynucle 5. The fusion protein according to claim 2, wherein the otide according to claim 9. protein toxin is an immunotoxin. k k k k k