Recombinant cytotoxin as well as a method of producing it

#The subject of the present invention is a method of modifying proteinaceous toxins through the addition of an NLS motif. The resulting cytotoxins facilitate the selective elimination of proliferating cells, particularly tumour cells.

#A poison is an organic or inorganic substance which, even at low concentrations, has a deleterious effect on living organisms. Poisons are divided into two basic categories. The first consists of natural poisons, produced mainly by pathogenic bacteria, poisonous fungi and plants, as well as venomous animals. The second group of poisons consists of anthropogenic poisons. Bacterial toxins (venoms) are various chemical compounds produced by bacteria which poison a higher organism. They act specifically on various systems (i.e. on the gastrointestinal tract) or cells of an organism (i.e. neurotoxins). These are differentiated into exotoxins and endotoxins. Exotoxins, secreted outside of the live cell are strong venoms and induce specific disease symptoms. They have a proteinaceous structure (metabolic product) and thus are sensitive to high temperatures (>60° C.) as well as being degraded by digestive enzymes (with the exception of botulinum toxin and Staphylococcus enterotoxins). They have strong antygenic properties, and anatoxins made therefrom are used to immunise humans and animals. They are made mainly by Gram-positive bacteria. These are some of the strongest toxic substances known. Endotoxins are released only following the degradation of the bacterial cell. They are weak venoms, and the symptoms they induce are not specific. Chemically, these are glycolipopolypeptide complexes (lipopolysaccharide) which most often occur in Gram-negative bacteria in one of the three cell-wall layers. They are poorly antygenic. They are not degraded by digestive enzymes but are thermostable. Exotoxins secreted by bacteria (but also by plants, fungi and some animals) exhibit cytotoxic properties against a host cells, usually due to the enzymatic inhibition of protein synthesis. The essential condition toxin activity is their binding of surface receptors on the target cell and their internalisation through endocytsis, and then translocation from the endoplasmatic reticulum into the cytosol. Bacterial exotoxins are currently produced using genetic engineering methods or chemically conjugated with ligands and antibodies so as to bind to specific cell types. This facilitates the selective destruction of disease-altered cell lines. The use of bacterial exotoxins specific for tumour cells is one of the targeted therapeutic strategies against cancer. Exotoxins secreted naturally by the disease-causing bacteria Pseudomonas aeruginosa and Diphtheriae typhimurium are compounds of very high cytotoxicity, sometimes many times higher than of classic antitumour drugs. In many cases a single toxin molecule is capable of killing a cell, which makes them some of the most lethal compounds. An exotoxin frequently used to construct fusion proteins with antitumour activity is exotoxin PE from Pseudomonas aeruginosa (Pseudomonas exotoxin, PE) [1]. A molecule of native PE toxin consists of a catalytic domain connected with a domain that binds a receptor through a central translocation domain, which facilitates the transfer of the C-terminal catalytic domain into the cytosol (FIG. 1). So far, the role of the Ib domain of PE remains unknown, but it is known that it contains a disulphide bridge necessary for molecule maturation. FIG. 2 shows the mechanism of PE intoxication. Due to the interaction with the host cell, PE binds to the α2 macroglobulins. Prior to entering the cell, the toxin is cleaved proteolytically. A caroxypeptidase cuts off the terminal lysine which exposes the REDL motif. Next, the exotoxins are internalised through endocytosis dependent on the receptor. After entering endocytotic vesicles, the toxin is cleaved proteolytically by furin inside the translocation domain, and the disulphide bridges hold the formed fragments until they are reduced. The PE migration pathway in the cell is through the Golgi apparatus and encompasses retention in the endoplasmatic reticulum due to the REDL signal at the C-terminus of the protein. Then, the freed catalytic domain is translocated through the reticulum wall into the cytosol. There, the active protein catalyses ADP-ribosylation of His at position 699 of the translation factor eEF2 and thereby inhibits protein synthesis, thereby quickly leading to cell death [1]. The use of PE in targeted therapy entails the replacement of the receptor-binding domain with an antibody or a portion thereof, a cytokine or growth factor (hence the name immunotoxins). The most frequently used form of PE is a fractional exotoxin of 38 kDa composed of amino-acids 253-364 and 381-613. Chimeric immunotoxins based on PE molecules are most often directed against receptors IL2 and IL6 as well as growth factor TGFα (Tab. 1) [2]. The table below lists information regarding the use of propharmaceuticals containing immunotoxins 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′ scFv fused to PE38KDEL CD19 Lymphoma, leukemia Schwemmlein et al. (2007) Anti-Tac(Fv)-PE38KDEL scFv fused to PE38KDEL CD25 CD25 positive tumor cells Kreitman et al. (1994) [LMB2] Anti-Tac(Fv)-PE40KDEL scFv fused to PE40KDEL CD25 Chronic lymphocytic leukemia Kreitman et al. (1992) RTF5(scFv)-ETA′ scFv fused to PE40 CD25 Lymphoma Barth et al. (1998) RFB(dsFv)-PE38 [B1.22] dsFv fused to PE38 CD22 B-cell leukemia Kreitman et al. (2000a) G28-5 sFv-PE40 scFv fused to PE40 CD40 Burkilt's lymphoma Francisco et al. (1997) Ki4(scFv)-ETA′ scFv fused to PE40 CD30 Hodkin's lymphoma Klimka et al. (1999) CD7-ETA scFv fused to PE40 CD7 T-lineage acute lymphoblastic Peipp et al. (2002) leukemia OVB3-PE mAb linked via disulfide 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 dsFv fused to PE38 LeY LeY positive tumor cells Benhar et al. (1995) B3(dsFv)-PE38 dsFv fused to PE38 LeY LeY positive tumor cells Benhar et al. (1995) BR96sFv-PE40 [SGN-10] scFv fused to PE40 LeY LeY positive tumor cells Friedman et al. (1993) IL4(38-37)PE38KDEL IL4 fused to PE38KDEL IL4-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 scFv 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 Skrepnik et al. (1996, 1999); Erb-38 dsFv fused to PE38 erbB2 Epidermoid carcinoma, breast Reiter and Pastan (1996) MR1(Fv)-PE38 scFv fused to PE38 EGFRvIII Glioblastoma Beers et al. (2000) TP38 TGF-α fused to PE38 EGFR Glioma Sampson et al. (2003) TP40 TGF-α fused to PE40 EGFR Glioma, prostate, epidermoid Sarosdy et al., (1993); Pai et al. (1991a); Kunwar et al. (1993) 425.3PE mAb chemically linked to PE EGFR Breast Andersson et al. (2004) A5-PE40 scFv fused to PE40 PSMA Prostate Wolf et al. (2006, 2008) SS1(dsFv)PE38 [SSIP] dsFv fused to PE38 Mesothelin Ovarian, cervical Hussan et al. (2002) scFv(MUC1)-ETA scFv fused to PE40 MUC1 Breast Singh et al. (2007) 9.2.27-PE mAb chemically linked to PE HMW-MAA Gliomblastoma Hjortland et al. (2004) TP-3(scFv)-PE38 scFv fused to PE38 Osteosarcoma Osteosarcoma Onda et al. (2001) antigen TP-3(dsFv)-PE38 dsFv fused to PE38 Osteosarcoma Osteosarcoma Onda et al. (2001) antigen 8H9(dsFv)-PE38 dsFv fused to PE38 Cell surface Breast, osteosarcoma, Onda et al. (2004) glycoprotein neuroblastoma 4D6MOCB-ETA scFv fused to PE40KDEL Ep-CAM Lung, colon, SCC Di Panlo et al. (2003) HB21(Fv)-PE40 scFv fused to PE40 TfR Colon Shinohara et al. (2000)

#FIG. 1 shows a schematic of the structure of PE exotoxin of Pseudomonas aeruinosa. According to: Kreitman (2009), modified.

#FIG. 2 shows a schematic of PE intoxication. Source: Kreitman (2009).

#FIG. 3 shows a map of vector pJ206:26701. Red frames delineate restriction sites for EcoRl and Hindlll.

#The sequence encoding the modified exotoxin was designed with the further addition of elements necessary to obtain (via in vitro synthesis) proteins containing a tag in the form of 6 histidine residues. The nucleotide sequence of the entire expression cassette is shown as sequence No. 3 whereas the protein it encodes has the amino-acid sequence termed sequence 4 (in the sequence list).

#The expression cassette containing: a promoter for the T7 polymerase, a ribosome binding site, a start codon, a linker with the His-tag as well as a sequence encoding the modified exotoxin; was obtained through chemical synthesis performed by the GenScript company. This cassette was then cloned by the producer into the vector pJ206 between the restriction sites EcoRI and HindIII (FIG. 1). From such a vector we digested out the entire expression cassette and transcloned it into the plasmid pUC57 (FIG. 2) using the EcoRI and Hind III restrictases. In the same way we synthesized and prepared the vectors used to express proteins that were the controls in the experiment: unmodified (native) exotoxin A of Pseudomonas aeruginsa as well as GFP.

#The vector containing the insert, the expression cassette for the modified exotoxin as well as vectors for the expression of the native exotoxin and GFP were used for in vitro transcription and translation using the commercial “RTS 100 E. coli HY Kit containing E. coli lysate (5Prime)”. Proteins synthesized in this fashion were purified on Ni-NTA-agarose (Qiagen) and dialysed against PBS, and then concentrated using Amicon centrifuge filters. The concentration of the resulting proteins in subsequent purification steps was estimated using the BCA method (Tab. 2).