EP4622994A1 - Deimmunized pseudomonas exotoxin a - Google Patents

Abstract

The technology provided herein relates to novel Pseudomonas exotoxin A enzymes (PEs); to nucleic acid molecules encoding said PEs, vectors, host cells containing the nucleic acids and methods for preparation and producing such PEs; compositions and methods for using said PEs for the treatment of diseases, in particular cancers especially including hematological and solid malignancies, chronic inflammatory diseases and allergies including allergic asthma.

Description

DEIMMUNIZED PSEUDOMONAS EXOTOXIN A

FIELD OF THE DISCLOSURE

The technology provided herein relates to novel Pseudomonas exotoxin A enzymes (PEs); to nucleic acid molecules encoding said PEs, vectors, host cells containing the nucleic acids and methods for preparation and producing such PEs; compositions and methods for using said PEs for the treatment of diseases, in particular cancers especially including hematological and solid malignancies, chronic inflammatory diseases and allergies including allergic asthma.

BACKGROUND

Protein toxins are extremely potent cell-killing agents that are responsible for many human diseases. Because of their high activity, some of these agents have been attached to monoclonal antibodies in order to form cytotoxic agents (immunotoxins) which specifically bind to target cells. These immunotoxins are, therefore, most useful in cancer therapy.

Pseudomonas Exotoxin A (PE) is the most toxic virulence factor of the pathogenic bacterium Pseudomonas aeruginosa and an extremely active monomeric protein (molecular weight 66Kd), secreted by the bacterium, which inhibits protein synthesis in eukaryotic cells through the inactivation of elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD onto EF-2). Therefore, Pseudomonas exotoxin A is very effective for destroying or inhibiting the growth of undesirable cells, e.g., cancer cells.

The PE gene was originally cloned from the P. aeruginosa strain PA 103 and analysis of the 5' and 3' flanking regions evidenced that the PE gene is translated from a monocistronic message (Gray et al., 1984). PE is expressed as a protein with a length of 638 amino acids (aa) and can be divided into several structural and functional domains (Wedekind et al., 2001; Figure 1A). Generally, PE belongs to the two-component AB toxin family, composed of an A domain with enzymatic activity and a B domain as cell binding subunit (Odumosu et al., 2010). In detail, PE contains a highly hydrophobic leader peptide of 25 aa at its N-terminus, which is removed during secretion. The leader sequence is followed by the receptor binding domain la (aa 1-252), which is composed of antiparallel R-sheets. Domain II (aa 253-364) with six consecutive a-helices, enables the toxin to translocate across cell membranes. The last four residues (aa 400-404) of domain lb (aa 365-404) together with domain III (aa 405-613) form the catalytic subunit of the toxin with ADP-ribosyltransferase activity (Siegall et al., 1989).

The ADP-ribosylation mechanism of PE was studied in detail and it turned out that it follows an SN1 nucleophilic substitution mechanism (Beattie et al., 1996; Armstrong et al., 2002; Jorgensen et al., 2005; Figure 2). Initially, the PE-fragment binds to NAD+ and interacts via the so-called "active-site loop L4" (aa 483-490 of domain III) with eEF-2 (Yates and Merrill, 2004). Afterward it facilitates the cleavage of the glycosidic bond (C-N) between the nicotinamide and N-ribose of NAD+. This results in a reactive oxacarbenium intermediate, which in turn is stabilized by residue E-553 of the PE-fragment (Li et al., 1996; Jorgensen et al., 2005). This step is followed by a nucleophilic attack of eEF-2, based on its nucleophilic residue diphthamide, a post-translationally modified histidine residue (2-(3-carboxyamido-3- [trimethylammonio]propyl) histidine) (Ortiz and Kinzy, 2005). The ADP-ribose group is subsequently transferred to the N3 atom of the diphthamide imidazole ring, which results in the ADP-ribosylated eEF-2 protein (Armstrong et al., 2002; Jorgensen et al., 2005). The ADP-ribosylation inactivates eEF-2 and the protein biosynthesis of the host cell comes to a standstill. As a consequence, apoptosis is induced, and the host cell irreversibly dies.

As mentioned above, PE may be useful for treating or preventing diseases such as, e.g., cancer. However, PE may be highly immunogenic. Accordingly, PE administration may stimulate an anti-PE immune response including, for example, the production of anti-PE antibodies and/or T-cells, that undesirably neutralizes the cytotoxic activity of PE. Such immunogenicity may reduce the amount of PE that can be given to the patient which may, in turn, reduce the effectiveness of the PE for treating the disease, e.g., cancer. Thus, there is a need for improved PE.

Several engineered variants of deimmunized Pseudomonas exotoxins (PE) are known in art. The domain II deleted versions (for example, PE24) may be less immunogenic and may cause fewer side effects (such as, for example, capillary leak syndrome and hepatotoxicity) as compared to PE38, which contains domain II. Without being bound to a particular theory, it is believed that the reduced immunogenicity and fewer side effects of PE24 could, at least in part, be due to the reduced size of PE24, which disadvantageous results in a shorter serum half-life. Different furin cleavable linkers may be employed in PE24 variants. PE immunoconjugates have mostly used scFv and dsFv fragments as targeting moieties. Such deimmunized Pseudomonas exotoxins (PE) are described in, for example, International Patent Application Publications W02005052006, W02007016150, W02007014743, W02007031741, WO200932954, WO201 132022, WO2012/154530, and WO 2012/170617.

Allergy is defined as the hypersensitivity of the immune system to harmless environmental substances and affects about 30% of the world's population. Asthma for example is a chronic inflammatory disease characterized by narrowing of the airways, hypertrophy of smooth muscles and increased mucus production. Although inhaled corticosteroids are traditionally recommended as the first line of asthma treatment, not all patients achieve asthma control using these drugs. Current therapies for biological intervention in asthma are mostly based on monoclonal antibodies that neutralize the functions of allergen reactive IgE antibodies. Though these approaches showed some success when matched to the appropriate patient group, blocking the functions of circulating IgEs is unlikely to provide a cure for asthma, since the primary source of the IgEs is not eliminated. To maintain clinical benefits, patients are required to stay on anti-lgE medication for the rest of their lives, often with an impaired quality of life, a higher risk of adverse effect, asthma-induced hospitalization, or death.

However, in particular the de-immunization of PE leads often to a reduction of the enzymatic activity of the enzyme. In the publication ALEWINE ET AL: "Efficacy of RG7787, a Next-Generation Mesothelin- Targeted Immunotoxin, against Triple-Negative Breast and Gastric.Cancers", MOLECULAR CANCER THERAPEUTICS, vol. 13, no. 11, 19 September 2014 (2014-09-19) discloses a de-immunized truncated, B- cell epitope silenced, 24 kD fragment of Pseudomonas exotoxin A (PE24) but with less cytotoxicity.

Thus, there is a need for improved Pseudomonas exotoxin variants.

SUMMARY OF THE DISCLOSURE

The present disclosure relates to improved deimmunized recombinant Pseudomonas exotoxin molecules that demonstrate high enzyme activity than prior described molecules. The technical effect of the Pseudomonas exotoxin variations according to the present disclosure is the combination of deimmunized Pseudomonas exotoxins having high cytotoxicity.

In a first aspect, the present disclosure pertains to recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the substitution is R456T or R456C, and wherein the de-immunized PE or the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial Pseudomonas exotoxin of SEQ ID NO: 1.

In a second aspect, the present disclosure pertains to recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising Domain 2 and Domain 3 of the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 11, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the substitution is R456T or R456C, and wherein the de-immunized PE or the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial Pseudomonas exotoxin of SEQ ID NO: 1.

In still another aspect, embodiments of this disclosure provide a recombinant immunotoxin (rIT) suitable to induce apoptosis in a eukaryotic target cell comprising a target cell-specific binding component and an effector domain, wherein the effector domain comprises a recombinant de-immunized bacterial Pseudomonas exotoxin A (PE), wherein the de-immunized PE or the variant contains substitutions at positions that corresponds structurally or by amino acid sequence homology to positions 427, 456, 463, 467, 505 and 538, in particular wherein the substitutions are R427A, R456T, D463A, R467A, R505A, R538A or R427A, R456C, D463A, R467A, R505A, R538A, in particular wherein the de-immunized PE or the variant contains substitutions at positions that corresponds structurally or by amino acid sequence homology to positions 427, 456, 463, 467, 490, 505 and 538, in particular wherein the substitutions are R427A, R456T, D463A, R467A, R490A, R505A, R538A or R427A, R456C, D463A, R467A, R490A, R505A, R538A.

Therefore, the present disclosure pertains to a recombinant de-immunized bacterial pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the substitution is R456T or R456C, and wherein the de-immunized PE or the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1, wherein the deimmunized PE or the variant contains substitutions at positions that corresponds structurally or by amino acid sequence homology to positions 427, 456, 463, 467, 505 and 538, in particular wherein the substitutions are R427A, R456T, D463A, R467A, R505A, R538A or R427A, R456C, D463A, R467A, R505A, R538A, in particular wherein the de-immunized PE or the variant contains substitutions at positions that corresponds structurally or by amino acid sequence homology to positions 427, 456, 463, 467, 490, 505 and 538, in particular wherein the substitutions are R427A, R456T, D463A, R467A, R490A, R505A, R538A or R427A, R456C, D463A, R467A, R490A, R505A, R538A.

In still another aspect, embodiments of this disclosure provide a recombinant immunotoxin (rIT) suitable to induce apoptosis in a eukaryotic target cell comprising a target cell-specific binding component and an effector domain, wherein the effector domain comprises a recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) according to the present disclosure.

In still another aspect, embodiments of this disclosure provide nucleic acids encoding a Pseudomonas exotoxin A according to the present disclosure, in particular a recombinant Pseudomonas exotoxin A as disclosed herein, as well as vectors and host cells comprising such nucleic acids.

In other aspects, the present disclosure relates to compositions comprising the Pseudomonas exotoxin A according to the present disclosure, in particular the recombinant Pseudomonas exotoxin A as described herein, wherein the Pseudomonas exotoxin A may be useful for, or used in therapeutical, cosmetic and/or diagnostic applications. In an advantageous embodiment, the Pseudomonas exotoxins A according to the present disclosure are used as a therapeutical composition for the treatment of diseases, in particular of cancers, chronic inflammatory diseases and allergies including allergic asthma.

In still another aspect, embodiments of this disclosure provide medicaments, drugs or pharmaceutical compositions comprising the Pseudomonas exotoxin A according to the present disclosure in combinations with a pharmacologically acceptable carrier, diluent, stabilizer or formulation.

In a further aspect, embodiments of the present disclosure relate to methods for producing the Pseudomonas exotoxin A according to the present disclosure, in particular for producing a recombinant Pseudomonas exotoxin A in a host cell by transforming the host cell with a DNA construct, advantageously including a promoter having transcriptional activity in the host cell, cultivating the transformed host cell in a suitable culture medium to allow expression of said Pseudomonas exotoxin A and producing said Pseudomonas exotoxin A. The method may also include isolating/purifying the produced protein.

A further aspect pertains to methods of treating a malignant disease comprising administering an effective amount of the Pseudomonas exotoxin A according to the present disclosure to a patient in need thereof.

A further aspect pertains to methods for preparing a recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present disclosure or a recombinant immunotoxin to the present disclosure, wherein the method comprises culturing the host cell of the present disclosure and isolating the PE or the immunotoxin from the cell culture.

A further aspect pertains to a recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present disclosure or a recombinant immunotoxin according to the present disclosure for the use in treating malignant diseases including hematological cancers exemplified by e.g. acute or chronic leukemias, solid cancers as exemplified by e.g. gynecological, skin, intestinal, oesophageal, head & neck as well as brain cancers, or chronic inflammatory diseases, such as, rheumatoid and juvenile arthritis, multiple sclerosis, COPD including emphysema, intrinsic and extrinsic asthma; cutaneous disease including atopic dermatitis, polymorphic light eruption, SLE; autoimmune diseases, including graft versus host, macrophage activation syndrome,; intestinal diseases including Crohn's disease and chronic bowel disease as well as allergies.

Before the disclosure is described in detail, it is to be understood that this disclosure is not limited to the particular the Pseudomonas exotoxin A according to the present disclosure and production methods described in the present disclosure. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic representation of different variants of deimmunized ETA'

Figure 2 is a SDS PAGE and western blot analysis of deimmunized proDer p 1-ETA¹ variants.

Figure 3 is a ADP-Ribosylation assay.

Figure 4 shows amino acid sequences of embodiments of A) WT-ETA sequence comprising Domain 1, II, and III, with a length of 613 amino acids (SEQ ID NO. 1), B) the variant of the prior art RG7787 and C) a new ETA-variant of the present disclosure comprising the R456C without the R490A mutation.

Figure 5 shows further amino acid sequences of embodiments of new ETA-variants of the present disclosure.

Figure 6 shows amino acid sequences of embodiments of new proDer p 1-dETA' variants of the present disclosure.

Figure 7 shows further amino acid sequences of embodiments of new proDer p 1-dETA' variants of the present disclosure.

Figure 8 shows the amino acid sequence of A) ETA WILD TYPE PE38 (PE with Domains 2 and 3 but without Domain 1, B) the WT-ETA sequence with a 25 amino acid signal peptide, C) the RG7787 variant without Domain 1 and D) an ETA-variant of the present disclosure comprising Domain 2 and 3 (without Domain 1) having the R456C mutation without the R490A mutation. Figure 9 shows further amino acid sequences of embodiments of new ETA-variants of the present disclosure.

Figures 10 to 12 show the results of XTT assays performed with the wt ETA constructs.

Figures 13 to 15 show the results of XTT assays performed with the deimmunized ETA constructs (dETA) mSAPl or mSAP3.

DETAILED DESCRIPTION OF THIS DISCLOSURE

Pseudomonas exotoxin A ("PE") is a bacterial toxin (molecular weight 66 kD) secreted by Pseudomonas aeruginosa. The native, wild-type PE sequence (SEQ ID NO: 1) is set forth in U.S. Patent 5,602,095, which is incorporated herein by reference. Native, wild- type PE includes three structural domains that contribute to cytotoxicity. Domain la (amino acids 1 -252) mediates cell binding, domain II (amino acids 253-364) mediates translocation into the cytosol, and domain III (amino acids 400-613) mediates ADP ribosylation of elongation factor 2. While the structural boundary of domain III of PE is considered to start at residue 400, it is contemplated, that domain III may require a segment of domain lb to retain ADP-ribosylating activity. Accordingly, functional domain III is defined as residues 395-613 of PE. The function of domain lb (amino acids 365-399) remains undefined.

Without being bound by a particular theory or mechanism, it is believed that the cytotoxic activity of PE occurs through the inhibition of protein synthesis in eukaryotic cells, e.g., by the inactivation of the ADP- ribosylation of elongation factor 2 (EF-2).

Substitutions of PE are defined herein by reference to the amino acid sequence of PE. Thus, substitutions of PE are described herein by reference to the amino acid residue present at a particular position, followed by the amino acid with which that residue has been replaced in the particular substitution under discussion. In this regard, the positions of the amino acid sequence of a particular embodiment of a PE are referred to herein as the positions of the amino acid sequence of the particular embodiment or as the positions as defined by SEQ ID NO: 1. When the positions are as defined by SEQ ID NO: 1 , then the actual positions of the amino acid sequence of a particular embodiment of a PE are defined relative to the corresponding positions of SEQ ID NO: 1 and may represent different residue position numbers than the residue position numbers of SEQ ID NO: 1. Thus, for example, substitutions refer to a replacement of an amino acid residue in the amino acid sequence of a particular embodiment of a PE corresponding to the indicated position of the 613-amino acid sequence of SEQ ID NO: 1 with the understanding that the actual positions in the respective amino acid sequences may be different.

For example, when the positions are as defined by SEQ ID NO: 1, the term "R490" refers to the arginine normally present at position 490 of SEQ ID NO: 1, "R490A" indicates that the arginine normally present at position 490 of SEQ ID NO: 1 is replaced by an alanine, while "K590Q" indicates that the lysine normally present at position 590 of SEQ ID NO: 1 has been replaced with a glutamine. In the event of multiple substitutions at two or more positions, the two or more substitutions may be the same or different, i.e., each amino acid residue of the two or more amino acid residues being substituted can be substituted with the same or different amino acid residue unless explicitly indicated otherwise.

The terms Pseudomonas exotoxin" and "PE" as used herein include PE that has been modified from the native protein to reduce or to eliminate immunogenicity. Such modifications may include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II, and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus such as DEL and REDL (SEQ ID NO: 7). See Siegall et al., J. Biol. Chem., 264: 14256-14261 (1989). Such modified PEs may be further modified to include any of the inventive substitution(s) for one or more amino acid residues within one or more T-cell and/or B-cell epitopes described herein.

In an embodiment, the modified PE may be a cytotoxic fragment of native, wild-type PE. Cytotoxic fragments of PE may include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell (e.g., as a protein or pre-protein). In a preferred embodiment, the cytotoxic fragment of PE retains at least about 20%, preferably at least about 40%, more preferably about 50%, even more preferably 75%, more preferably at least about 90%, and still more preferably 95% of the cytotoxicity of native PE. In particularly preferred embodiments, the cytotoxic fragment has at least the cytotoxicity of native PE, and preferably has increased cytotoxicity as compared to native PE. [0031] Modified PE that reduces or eliminates immunogenicity includes, for example, PE4E, PE40, PE38, PE25, PE38QQR, PE38KDEL, and PE35. In an embodiment, the PE may be any of PE4E, PE40, PE38, PE25, PE38QQR (in which PE38 has the sequence QQR added at the C-terminus), PE38KDEL (in which PE38 has the sequence KDEL (SEQ ID NO: 5) added at the C-terminus), PE-LR (resistance to lysosomal degradation), and PE35.

In an embodiment, the PE has been modified to reduce immunogenicity by deleting domain la as described in in U.S. Patent 4,892,827, which is incorporated herein by reference. The PE may also be modified by substituting certain residues of domain la. In an embodiment, the PE may be PE4E, which is a substituted PE in which domain la is present but in which the basic residues of domain la at positions 57, 246, 247, and 249 are replaced with acidic residues (e.g., glutamic acid), as disclosed in U.S. Patent 5,512,658, which is incorporated herein by reference.

PE40 is a truncated derivative of PE (Pai et al., Proc. Nat. Acad. Sci. USA, 88: 3358-62 (1991) and Kondo et aL, Biol. Chem., 263: 9470-9475 (1988)). PE35 is a 35 kD carboxyl-terminal fragment of PE in which amino acid residues 1-279 have been deleted and the molecule commences with a Met at position 280 followed by amino acids 281-364 and 381-613 of native PE. PE35 and PE40 are disclosed, for example, in U.S. Patents 5,602,095 and 4,892,827, each of which is incorporated herein by reference. PE25 contains the 1 1 - residue fragment from domain II and all of domain III. In some embodiments, the PE contains only domain III.

Therefore, in a first aspect the present disclosure pertains to a recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%.

In particular, the present disclosure pertains to a recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the substitution is R456T or R456C, and wherein the de-immunized PE or the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial Pseudomonas exotoxin of SEQ ID NO: 1, in particular an ADP-ribosylation activity.

In particular, the present disclosure pertains also to a recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising Domain 2 and Domain 3 of the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 11, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the substitution is R456T or R456C, and wherein the de-immunized PE or the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial Pseudomonas exotoxin of SEQ ID NO: 1, in particular an ADP-ribosylation activity.

The terms "polypeptide", "peptide", or "protein" are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The amino acid residues are preferably in the natural "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. In addition, the amino acids, in addition to the 20 "standard" amino acids, include modified and unusual amino acids.

The terms "recombinant fusion protein" and "fusion protein" are used herein interchangeably to refer for example to a protein produced by recombinant technology which comprises segments i.e. amino acid sequences, from heterologous sources, such as different proteins or different organisms. The segments are joined either directly or indirectly to each other via peptide bonds. By indirect joining it is meant that an intervening amino acid sequence, such as a peptide linker is juxtaposed between segments forming the fusion protein. A recombinant fusion protein is encoded by a nucleotide sequence, which is obtained by genetically joining nucleotide sequences derived from different regions of one gene and/or by joining nucleotide sequences derived from two or more separate genes. Furthermore, the encoding nucleotide sequences may be synthesized in vitro without the need for initial template DNA samples e.g. by oligonucleotide synthesis from digital genetic sequences and subsequent annealing of the resultant fragments. Desired protein sequences can be "reverse translated" e.g. using appropriate software tools. Due to the degeneracy of the universal genetic code, synonymous codons within the open-reading frame (i.e. the recombinant protein coding region) can be exchanged in different ways, e.g. to remove cis-acting instability elements (e.g. AUUUA), to remove, introduce or modify the secondary and tertiary mRNA structures (e.g. pseudoknots, stem-loops,...), to avoid self-complementary regions that might trigger post-transcriptional gene silencing (PGTS), to change the overall AT:GC content, or to adjust the codon-usage to the expression host. Such changes can be designed manually or by using appropriate software tools or through a combination.

Therefore, the recombinant Pseudomonas exotoxin A according to the present disclosure may be prepared by using recombinant DNA methodology and expression in a suitable host cell, as is known in the art (see for example (Sambrook, J. & Russell, D., Molecular Cloning: a Laboratory Manual, 2001' 3rd ed).

In an advantageous embodiment, the recombinant proteins according to the present disclosure, in particular the recombinant Pseudomonas exotoxin A enzymes are isolated. The term "isolated" when used in relation to a nucleic acid or protein refers to a nucleic acid sequence or protein that is identified and separated from at least one contaminant (nucleic acid or protein, respectively) with which it is ordinarily associated in its natural source. Isolated nucleic acid or protein is present in a form or setting that is different from that in which it is found in nature.

The enzyme activity of the ADP ribosylating enzymes according to the present disclosure, e.g the Pseudomonas exotoxin A enzyme activity can be verified by an ADP-ribosylation assay as described in the examples of the present disclosure.

The isolated polypeptides e.g. the Pseudomonas exotoxin A enzymes according to the present disclosure are characterized by specific amino acids and is encoded by specific nucleic acid sequences. It will be understood that such sequences include analogues and variants produced by recombinant or synthetic methods wherein such polypeptide sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptides and the complexes according to the present disclosure still are suitable to bind and kill an allergen-reactive human B lymphocyte or cancer cell. Substitutions are preferably "conservative". Substitutions are preferably silent substitutions in the codon usage, which will not lead to any change in the amino acid sequence but may be introduced to enhance the expression of the protein.

In some advantageous embodiments, the Pseudomonas exotoxin A according to the present disclosure comprises an amino acid sequence of SEQ ID NO: 5 or 6, or homologous polypeptides thereof having an amino acid which is at least 85%, preferably 90%, more preferably 95% identical to the amino acid sequence of SEQ ID NO. 1, which are produced by recombinant or synthetic methods by substitution, insertion, addition or deletion of one or more amino acid residues, or fragments thereof.

In some embodiments, the recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present disclosure or variants thereof comprises at least one or more further variations at positions corresponding to amino acid residues 427, 463, 467, 505 or 538 and wherein the amino acid sequence of said PE has at least a minimum percentage sequence identity and/or percentage homology of at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99% to the amino acid sequence of SEQ ID NO. 1, wherein the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial Pseudomonas exotoxin of SEQ ID NO: 1..

In some embodiments, the recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present disclosure or variants thereof contains a substitution at the positions 427, 456, 463, 467, 505 and 538.

In some embodiments, the recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present disclosure or variants thereof contains further a substitution at a position that corresponds structurally or by amino acid sequence homology to position 490, in particular wherein the substitution is R490A.

In some embodiments, the recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present d isclosure comprises the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6 or variants thereof, wherein said variants have at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, and wherein the variants thereof have about the same or a greater enzymatic activity compared to the wild type bacterial Pseudomonas exotoxin of SEQ ID NO: 1.

In some advantageous embodiments, the recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present disclosure or variants thereof contains no substitution at a position that corresponds structurally or by amino acid sequence homology to position 490.

In some embodiments, the recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present disclosure comprises the amino acid sequence of SEQ ID NO. 3 orSEQ ID NO. 4 or variants thereof, wherein said variants have at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, and wherein the variants thereof have about the same or a greater enzymatic activity compared to the wild type bacterial Pseudomonas exotoxin of SEQ ID NO: 1.

In another aspect, the present disclosure pertains to a recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising Domain 2 and Domain 3 of the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 11, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the substitution is R456T or R456C, and wherein the de-immunized PE or the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial Pseudomonas exotoxin of SEQ ID NO: 1.

Furthermore, the present disclosure pertains to a recombinant immunotoxin (rIT) suitable to induce apoptosis in a eukaryotic target cell comprising a target cell-specific binding component and an effector domain, wherein the effector domain comprises a recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) according to the present disclosure. In particular, the target cell-specific binding component comprises moieties which are affinity moieties from affinity substances or affinity substances in their entirety selected from the group consisting of antibodies, antibody fragments, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotypical antibodies, autoimmune-provoking structures, tissue-rejection-inducing structures, immunoglobulin constant regions and their derivatives, mutants or combinations thereof.

In some embodiment, the target cell-specific binding component is an antibody or an antibody fragment selected from the group consisting of a monoclonal antibody, Fab, scFv; single domain, or a fragment thereof, bis scFv, Fab₂, Fab₃, minibody, diabody, triplebody, tetrabody and tandab.

In an advantageous embodiment, the target cell-specific binding component is a human antibody or human antibody fragment that specifically binds to CD64. Further embodiments as targets for the RIT may be ASPH, CD14, CD30, CD32, CD33, CD44, CD45, CD64, CD89, CLCA2, CSPG4, EGFR, EpCAM, FAPalpha, GPR8, HLA-DR, IL4R, ILY6K, LGR5, LIV1, Melanotransferrin, Mesothelin, MPO, PD-L1, or TROP2.

As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain ( L) and variable heavy chain ( H) refer to these light and heavy chains respectively.

Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'z, a dimer of Fab which itself is a light chain joined to V.sub.H- C.sub.Hl by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')₂ dimer into two Fab' monomers. The Fab' monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA technology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA technologies. Preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked V_H-V heterodimer which may be expressed from a nucleic acid including V_H- and V -encoding sequences either joined directly or joined by a peptide-encoding linker. Huston et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the _H and are connected to each as a single polypeptide chain, the _H and domains associate non-covalently.

The present disclosure explored a different therapeutic approach for the treatment of diseases like cancer or allergies, e.g. represented by house dust mite allergy which might lead to allergic asthma by aiming to selectively eliminate specific population of B-cells responsible for the production of allergen reactive IgEs. In particular, the present disclosure pertains to a recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present disclosure or a recombinant immunotoxin according to the present disclosure for the use in treating malignant diseases including hematological cancers exemplified by e.g. acute or chronic leukemias, solid cancers as exemplified by e.g. gynecological, skin, intestinal, oesophageal, head & neck as well as brain cancers, or chronic inflammatory diseases, such as, rheumatoid and juvenile arthritis, multiple sclerosis, COPD including emphysema, intrinsic and extrinsic asthma; cutaneous disease including atopic dermatitis, polymorphic light eruption, SLE; autoimmune diseases, including graft versus host, macrophage activation syndrome,; intestinal diseases including Crohn's disease and chronic bowel disease as well as allergies.

In some advantageous embodiments, the recombinant immunotoxin according to the present disclosure is an allergen-based fusion protein, in particular wherein the target cell-specific binding component is the Dermatophagoides pteronyssinus Peptidase 1 (Der p 1).

To achieve the aim of the treatment of asthma, at least two different allergen-based fusion proteins were developed and evaluated for the treatment of House dust mite allergen; Dermatophagoides pteronyssinus (Der p 1) induced asthma. The strategy involved the use of the proDer p 1 allergen as a cell specific ligand to selectively deliver an apoptosis inducing toxin (ETA') or small molecule compound (BG-AURIF) into the cytosol of Der p 1-reactive B-cells. Results from this thesis showed that both allergen-fusion proteins (proDer p 1-ETA' and proDer p 1-SNAP) selectively bound to Der p 1-reactive hybridoma cells and primary cells from Der p 1 sensitized mice. The therapeutic potential for these novel fusion proteins was further confirmed by the selective cytotoxic activities of proDer p 1-ETA' and proDer p 1-SNAP-AURIF on 4C1 and 10BP Der p 1 reactive hybridoma cells. Due to the limitation associated with the use of recombinant immunotoxins in patients (immunogenicity), this study also employed a super-computing based molecular dynamics simulation model (in collaboration with partners at the department of computational biophysics, German research school for simulation sciences, Julich Germany) to identify 4 new mutations that could restore the enzymatic activity of a human B-cell deimmunized ETA' toxin (RG7787).

Briefly, the removal of human B-cell epitopes from ETA' toxin was previously accompanied with reduction of enzymatic activity. The results documented in this thesis confirmed an enhanced catalytic activity for the deimmunized ETA' variants bearing the 4 mutations. These results allowed the development of next generation deimmunized proDer p 1-based immunotoxins for asthma therapy. Altogether, the proof-of- concept experiments documented in this thesis provide a promising approach for the specific depletion of allergen reactive B-cells and likewise offer a curative strategy for the potential treatment of allergic asthma.

In some advantageous embodiments, the recombinant immunotoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, or homologous polypeptides thereof, which are produced by recombinant or synthetic methods by substitution, insertion, addition or deletion of one or more amino acid residues, or fragments thereof.

Furthermore, the rIT-complex according to the present disclosure comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, or homologous polypeptides, variants or mutations thereof, wherein the recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) comprised in the rIT-complex having an amino acid which is at least 85%, preferably 90%, more preferably 95% identical to the amino acid sequence of SEQ ID NO. 13, 14, 15 or 16, and that are produced by recombinant or synthetic methods by substitution, insertion, addition or deletion of one or more amino acid residues, or fragments thereof.

"Percent sequence identity", with respect to two amino acid or polynucleotide sequences, refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. Thus, 80% amino acid sequence identity means that 80% of the amino acids in two optimally aligned polypeptide sequences are identical. Percent identity can be determined, for example, by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN14, National Biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman for peptide analysis.15. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters 5 recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which was described before. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Likewise, computer programs for determining percent homology are also readily available.

The term "mutation" refers to the substitution or replacement of single or multiple nucleotide triplets, insertions or deletions of one or more codons, homologous or heterologous recombination between different genes, fusion of additional coding sequences at either end of the encoding sequence, or insertion of additional encoding sequences or any combination of these methods, which result in a polynucleic acid sequence encoding the desired protein.

Thus, the term "mutations" also refers to all of the changes in the polypeptide sequence encoded by the polynucleic acid sequence modified by one or more of the above-described changes. The term "variant" means that the amino acid sequence has been modified but retains the same functional characteristics, in particular the binding and destroying effect on human cancer cells. A variant has a sequence identity of at least 70% or preferably at least 80%, 85%, 90%, 95%, 97% or 99% to the parent amino acid sequence. The variants of the recombinant de-immunized bacterial Pseudomonas exotoxin A (PE) according to the present disclosure show the same or a greater enzymatic activity compared to the wild type bacterial Pseudomonas exotoxin of SEQ ID NO: 1, in particular an ADP-ribosylation activity.

The term "variant" refers further to a polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or Oalkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

The above-mentioned recombinant proteins, e.g. the Pseudomonas exotoxin A enzymes may also include on the N-Terminus prior to the single chain sequence: a pelB leader sequence for periplasmic transport, a Hisio-Tag for affinity purification and enterokinase cleavage site allowing removal of tags after use in purification.

To express a recombinant protein, e.g. a Pseudomonas exotoxin A enzyme according to the present disclosure in a recombinant expression system, a DNA encoding the fusion protein or parts thereof, may be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that a protein gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the protein gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The isolated protein domain sequences are typically inserted into the same expression vector. The protein genes are inserted into the expression vector by standard methods. Additionally, the recombinant expression vector can encode a signal peptide that facilitates co-translational translocation of the nascent polypeptide chain into the endoplasmic reticulum (ER). The folded polypeptide (recombinant fusion protein according to this disclosure) may be secreted from a host cell or may be retained within the host cell. Intracellular retention or targeting can be achieved by the use of an appropriate targeting peptide such as C-terminal KDEL-tag for ER retrieval.

In general, those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press (or later editions of this work) and Current Protocols in Molecular Biology, Second Edition, Ausubel etal. eds., John Wiley & Sons, 1992, which are incorporated herein by reference.

Furthermore, the present disclosure relates to nucleic acid molecules or nucleic acids encoding such a recombinant protein, e.g. a Pseudomonas exotoxin A enzyme as well as to vectors comprising the nucleic acid molecule and host cells comprising a nucleic acid molecule encoding said recombinant protein or a vector comprising said vector. The disclosure pertains also to methods of manufacturing said recombinant proteins in a recombinant expression system.

In particular, the present disclosure relates to an isolated nucleic acid molecule encoding a recombinant de-immunized bacterial Pseudomonas exotoxin A according to the present disclosure or a recombinant immunotoxin according to the present disclosure.

Furthermore, the present disclosure relates to a vector comprising a nucleic acid molecule according to the present disclosure and to a host cell being transformed with a vector of the present disclosure and/or comprising a nucleic acid molecule of the present disclosure.

The term "vector" includes a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression system" or "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g. replication-defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The present disclosure is also directed to a host cell with a vector comprising a recombinant protein, e.g. a Pseudomonas exotoxin A enzyme according to the present disclosure. The phrase "recombinant host cell" (or simply "host cell") includes a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes a cell transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the present disclosure. A host cell, which comprises a recombinant vector of the disclosure, may also be referred to as a "recombinant host cell".

The term "host cell(s)" refers to cell(s), which may be used in a process for purifying a recombinant protein in accordance with the present disclosure. Such host cells carry the protein of interest (POI). A host cell may also be referred to as a protein-expressing cell. A host cell, according to the present invention, may be, but is not limited to, prokaryotic cells, eukaryotic cells, archaebacteria, bacterial cells, insect cells, yeast, mammal cells, and/or plant cells. Bacteria envisioned as host cells can be either gram-negative or gram-positive, e.g. Escherichia coli, Erwinia sp., Klebsellia sp., Lactobacillus sp. or Bacillus subtilis. Typical yeast host cells are selected from the group consisting of Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris. In some advantageous embodiments, the host cell is a HEK293T cell.

The Pseudomonas exotoxin A enzyme of the present disclosure can be used with a "pharmaceutically acceptable carrier" which includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g. antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.

Therefore, ass used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

The Pseudomonas exotoxin A of the present disclosure can be used with a "pharmaceutically acceptable salt" which includes any salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'- dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

The actual dosage amount of a Pseudomonas exotoxin A or composition of the present disclosure administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

The following methods and examples are offered for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way.

Methods and Examples

It should be understood that the following examples are for illustrative purpose only and are not to be construed as limiting this disclosure in any manner. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

In-silico design and molecular cloning of deimmunized variants of proDer p 1 -ETA immunotoxin

In the present disclosure, we aimed to use super-computing molecular dynamic simulations (MDS) to identify silent point mutations that would improve the cytotoxic / enzymatic activities of proteins bearing the RG7787 mutations (Table 1) (in collaboration with the group of Prof. Paolo Carloni (Institute for Advanced Simulation, Forschungszentrum Julich, Germany)). RG7787 is a deimmunized immunotoxin developed by the Ira Pastan group and composed of a humanized antibody fragment against Mesothelin attached to a PE24 variant of ETA with ablated human B-cell epitopes. The mutations introduced by the Ira Pastan group to remove human B-cell epitopes from RG7787 is depicted in table 1. To improve the enzymatic activity of RG7787, the point mutations identified by our collaborators (group of Prof. Paolo Carloni) are listed in table 2. To evaluate improvement in enzymatic activity, these new mutations, together with mutations present on RG7787 were introduced into a proDer p-1 backbone bearing the wildtype PE38 variant of ETA. These new de-immunized ETA variants were termed mSAPl-4 and evaluated in vitro. Table 1: Deimmunized ETA variants from the Ira Pastan group used in this patent together with their genetic characteristics. mRG7787, mLRLOlO, and mLOlO = mutations present on the RG7787, LRLOIO and L010 respectively.

Table 2: Variations according to the present disclosure improving the cytotoxic activity of proteins bearing RG7787 mutations (mSAPl-4).

MDS: Molecular dynamics simulation.

In-Silico and molecular cloning of deimmunized ETA' variants

The SnapGene™ software was used to introduce the four mSAP mutations listed (in Table 2) unto the PE38 version of ETA and fused to the following ligands: 1) proDer p 1, 2) anti-LGR5(scFv), 3) anti-CD90(scFv), 4) anti-EpCAM(scFv). Once the DNA mutagenesis process was completed, the open reading frame (ORF) of the newly design ETA constructs were flanked by Notl and Blpl restriction sites (figure 1) and synthesized in a pUC57 plasmid at GeneScript (USA). As controls, mutations present on the RG7787 construct was also grafted onto the PE38 ETA' moiety without the MDS mutations (Figure 1). The synthesized ORFs (opening reading frames) were thereafter subcloned into the proDer p 1-ETA plasmid via a Notl and Blpl restriction digest to replace the wild-type ETA (wt ETA) sequence with the deimmunized variants. mSAP mutation (1-4) were introduced into the PE38 version of ETA using the SnapGene™ software. As controls, mutations present on RG7787 was also introduced unto the PE38 version of ETA. The deimmunized ETA' open reading framed were flanked with Notl and Blpl restriction site for easy subcloning into the pMT-proDer p 1-ETA plasmid. The anti-LGR5/CD90/EpCAM scFv were used to replace proDer p 1 by Sfil/Notl Coloured triangles represent mutations present on each construct. dETA = deimmunized ETA.

Expression and characterization of deimmunized variants of ETA'

All deimmunized ETA recombinant immunotoxin variants were expressed using the osmotic stress expression protocol by Barth et al., 2000 (1). Briefly, bacterial cultures bearing the pMT-proDer p 1/anti- LGR5(scFv)/anti-CD90(scFv)/anti-EpCAM(scFv)-dETA variants plasmids were grown at 26°C until an optical density; OD600 nm of 1.6 was reached. At this O.D, the cultures were supplemented with 0.5 M sorbitol, 4% NaCI, and 40 mM glycine-betaine monohydrate and cultured for another 30 mins after which protein expression was induced by the addition of Isopropyl 0- d-l-thiogalactopyranoside (IPTG). For purification, the expressed dETA variants were purified by immobilized metal affinity chromatography (IMAC) on an AKTA Explorer system (GE, Healthcare). Afterwards, the proteins were analyzed via SDS-PAGE. The tubes that showed a band size corresponding to the protein of interest (~79.7 kDa) was pooled and concentrated with an Amicon ultracentrifugation column (10K mwco). The page ruler marker (NEB, 7719S) was used as the standard to identify the protein of interest. The IMAC purified samples were thereafter loaded unto the S200 sephadex column to further purify the recombinant immunotoxins by size exclusion chromatography. Figure 2A shows the resulting bands at 79.7kda on the 10% SDS-PAGE gel. To further confirm the expression of the different deimmunized ETA' variants, concentrated protein fractions after size exclusion chromatography were subjected to immunoblotting to detect the histidine tag located at the N-terminus of each fusion protein (Figure 2B). Finally, concentrated protein samples were sterile filtered (using 0.22 pm syringe filters), then quantified by densitometry before aliquoting and storing samples at 4°C. Expression of deimmunized ETA variants was performed in E. coli using the stress expression protocol as described above. For each rIT, two litres of bacterial cell cultures were subjected to affinity chromatography and later to size exclusion chromatography. Structural characterization by SDS-PAGE and western blot confirmed the successful expression of each rIT variant. Each protein sample was sterile filtered and quantified by densitometry against a BSA standard. The total protein yield obtained for each rIT variant as exemplified for proDer pl-ETA was from 1.5 mg - 3 mg /L. M = protein standard; Lane 1 = proDer p 1-ETA; lane 2 = proDer p 1-ETA-mSAPl; lane 3 = proDer p l-ETA-mSAP2; lane 4 = proDer p 1-ETA- mSAP3; lane 5= proDer p l-ETA-mSAP4; lane 8 = proDer p l-ETA-mRG7787.

8.3 ADP-ribosylation activity of deimmunized proDer p 1-ETA variants.

The ADP-ribosylation assay was used to compare the enzymatic activity of the different deimmunized proDer p 1-ETA variants. Briefly, elongation factor-2 (EF-2) from wheat germ extract was incubated with biotinylated NAD⁺ in the presence of 150 nM concentration of deimmunized proDer p 1-ETA variants. After incubating for 1 hour at 37°C, the amount of biotinylated-NAD⁺ incorporated into EF-2 was determined by western blot via a streptavidin-horseradish peroxidase (HRP) labelled antibody. Figure 3 shows that the activity of mRGG778 was improved by the point mutations identified by molecular dynamics simulations and introduced into rIT mSAPl, mSAP2, mSAP3, and mSAP4.

As a result, Figure 3 shows an ADP-Ribosylation assay. To measure and compare the activities of the different proDer p 1- ETA variants, an enzymatic assay in which EF-2 from wheat germ extract was incubated with biotinylated NAD⁺ in the presence of each deimmunized recombinant immunotoxin was performed. After incubating the reaction component for 1 hour, the activity of each rIT was determined by western blot using a streptavidin-HRP conjugate. The resulting immunoblots were quantified with the ImageJ software (https://imagej.nih.gov/ij/).

8.4 Cell viability assays for ETA/dETA-mSAPl variants.

The CaSki, HL60 and MCF7 cancer cells treated with rITs (mSAPl variants) were evaluated by XTT assays using the cell proliferation kit II (XTT)(Roche, CH). The cell lines (5xl0³) were seeded in a 96-well plate and incubated overnight at 37°C and 5% CO2. The following day the cells were treated with varying concentrations of rITs and was incubated at 37°C and 5% CO₂ for 72 hours. After the 72 hours, XTT reagent was added to each well and incubated for 4 hours. As a control Zeocin (lOOpl/mL) was used to achieve 100% cell killing. Results were analysed by measuring the absorbance of the XTT reagent at 450nm as the measurement filter and 650mn as the reference filter on a spectrophotometer (iMark™ Absorbance reader, Bio-Rad, USA). All experiments performed had five replicates. The absorbance readings were normalized in comparison to the control (untreated and Zeocin) and the results represented as a percentage of cell viability. GraphPrism v.8.0 software was used to calculate the concentration of rIT would achieve 50% cell death (IC₅o). Negative controls for these sets of experiments were the HL60 and MCF7 cell lines as well as the use of anti-CD64(scFv)-ETA. HL60 were used to assess the LGR5 and EpCAM rITs for nonspecific killing. MCF7 were used to assess the CD90 rITs for nonspecific killing. H22(scFv)-ETA was the rIT negative control as the H22 antigen is not expressed in CaSki cell line.

Figures 10 to 12 show the results of XTT assays performed with the wt ETA constructs (ETA). The cell lines were treated with serially diluted concentrations of rITs followed by performing an XTT cell viability assay. Controls were untreated (negative control) and cells treated with zeocin(100pg/ml) (positive control). The HL60 cell lines served as an antigen negative control for LGR5 and EpCAM rITs. The MCF7 cell line served as an antigen negative control for the CD90 rIT. Anti-H22(scFv)-ETA served as a rIT negative control for the CaSki cell line.

Figures 13 to 15 show the results of XTT assays performed with the deimmunized ETA mSAPl constructs (dETA). Cell lines were treated with serially diluted concentrations of rITs followed by performing an XTT cell viability assay. Controls were untreated (negative control) and cells treated with zeocin(100pg/ml) (positive control). The HL60 cell lines served as an antigen negative control for LGR5 and 15EpCAM rITs. The MCF7 cell line served as an antigen negative control for the CD90 rIT. Anti-H22(scFv)-ETA served as a rIT negative control for the CaSki cell line. Table 3 is showing an overview of all results.

Table 3: Results of the cell viability assays in example 8.4

In table 1 of the publication Alewine et al., 2014 more than 2-fold reduced IC50 values of RG7787 (including R490A) compared to their internal wt control (SS1-LR): 45.2 vs 19.6 pmol/l on HCC70, 64.8 vs 30.2 pmol/l on SUM149 cells is showed.

The results of Table 3 of the present disclosure demonstrate impressive the recovery of wt cytotoxicity for all dETA-mSAPl constructs targeting LGR5, EpCAM, or CD90 with less than 10% difference in comparison to their wt IC50 values.

References:

Barth S, Huhn M, Matthey B, Klimka A, Galinski E, Engert A. Compatible-solute-supported periplasmic expression of functional recombinant proteins under stress conditions. Applied and environmental microbiology. 2000;66(4):1572-9.

BAUSS FRIEDER ET AL: "Characterization of a re-engineered, mesothelin-targeted Pseudomonas exotoxin fusion protein for lung cancer therapy", MOLECULAR ONCOLOGY, ELSEVIER, vol. 10, no. 8, 14 July 2016 (2016-07-14), pages 1317-1329

C. ALEWINE ET AL: "Efficacy of RG7787, a Next-Generation Mesothelin-Targeted Immunotoxin, against Triple-Negative Breast and Gastric.Cancers", MOLECULAR CANCER THERAPEUTICS, vol. 13, no. 11, 19 September 2014 (2014-09-19), pages 2653-2661

Claims

Claims A recombinant de-immunized bacterial pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the substitution is R456T or R456C, and wherein the deimmunized PE or the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1. The recombinant de-immunized bacterial pseudomonas exotoxin A according to claim 1, wherein the de-immunized PE or the variant comprises at least one or more further variations at positions corresponding to amino acid residues 427, 463, 467, 505 or 538 and wherein the amino acid sequence of said PE has at least a minimum percentage sequence identity and/or percentage homology of at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99% to the amino acid sequence of SEQ ID NO. 1, wherein the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1.. The recombinant bacterial pseudomonas exotoxin A according to any one of claims 1 to 2, wherein the de-immunized PE or the variant contains a substitution at the positions 427, 456, 463, 467, 505 and 538. The recombinant bacterial pseudomonas exotoxin A according to any one of claims 1 to 3, wherein the de-immunized bacterial pseudomonas exotoxin A or the variant thereof contains further a substitution at a position that corresponds structurally or by amino acid sequence homology to position 490, in particular wherein the substitution is R490A. The recombinant de-immunized bacterial pseudomonas exotoxin A according to any one of claims 1 to 4, wherein the de-immunized PE comprises the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6 or variants thereof, wherein said variants have at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, and wherein the variants thereof have about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1. The recombinant de-immunized bacterial pseudomonas exotoxin A according to any one of claims 1 to 3, wherein the de-immunized PE contains no substitution at a position that corresponds structurally or by amino acid sequence homology to position 490. The recombinant de-immunized bacterial pseudomonas exotoxin A according to claim 6, wherein the de-immunized PE comprises the amino acid sequence of SEQ ID NO. 3 or SEQ ID NO. 4 or variants thereof, wherein said variants have at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, and wherein the variants thereof have about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1. A recombinant de-immunized bacterial pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising Domain 2 and Domain 3 of the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 11, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the substitution is R456T or R456C, and wherein the de-immunized PE or the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1. The recombinant de-immunized bacterial pseudomonas exotoxin A according to claim 8, wherein the de-immunized PE or the variant comprises at least one or more further variations at positions corresponding to amino acid residues 427, 463, 467, 505 or 538 and wherein the amino acid sequence of said PE has at least a minimum percentage sequence identity and/or percentage homology of at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99% to the amino acid sequence of SEQ ID NO. 11, wherein the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1.. The recombinant bacterial pseudomonas exotoxin A according to any one of claims 8 to 9, wherein the de-immunized PE or the variant contains a substitution at the positions 427, 456, 463, 467, 505 and 538. The recombinant bacterial pseudomonas exotoxin A according to any one of claims 8 to 10, wherein the de-immunized bacterial pseudomonas exotoxin A or the variant thereof contains further a substitution at a position that corresponds structurally or by amino acid sequence homology to position 490, in particular wherein the substitution is R490A. The recombinant de-immunized bacterial pseudomonas exotoxin A according to any one of claims 8 to 11, wherein the de-immunized PE comprises the amino acid sequence of SEQ ID NO. 16 or SEQ ID NO. 17 or variants thereof, wherein said variants have at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 11, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, and wherein the variants thereof have about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1. The recombinant de-immunized bacterial pseudomonas exotoxin A according to any one of claims 8 to 10, wherein the de-immunized PE contains no substitution at a position that corresponds structurally or by amino acid sequence homology to position 490. The recombinant de-immunized bacterial pseudomonas exotoxin A according to claim 13, wherein the de-immunized PE comprises the amino acid sequence of SEQ ID NO. 14 or SEQ ID NO. 15 or variants thereof, wherein said variants have at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 11, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, and wherein the variants thereof have about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1. A recombinant immunotoxin (RIT) suitable to induce apoptosis in a eukaryotic target cell comprising a target cell-specific binding component and an effector domain, wherein the effector domain comprises a recombinant de-immunized bacterial pseudomonas exotoxin A (PE) according to any one of claims 1 to 14. The recombinant immunotoxin according to claim 15, wherein the target cell-specific binding component comprises moieties which are affinity moieties from affinity substances or affinity substances in their entirety selected from the group consisting of antibodies, antibody fragments, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotypical antibodies, autoimmune-provoking structures, tissue-rejection-inducing structures, immunoglobulin constant regions and their derivatives, mutants or combinations thereof. The recombinant immunotoxin according to claim 16, wherein the recombinant immunotoxin is an allergen-based fusion protein. The recombinant immunotoxin according to claim 17, wherein the recombinant immunotoxin is an allergen-based fusion protein, wherein the target cell-specific binding component is the Dermatophagoides pteronyssinus Peptidase 1 (Der p 1). The recombinant immunotoxin according to claim 18, wherein the recombinant immunotoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10. An isolated nucleic acid molecule encoding a recombinant de-immunized bacterial pseudomonas exotoxin A according to any one of claims 1 to 14 or a recombinant immunotoxin according to any one of claims 15 to 19. A vector comprising a nucleic acid molecule of claim 20. A host cell being transformed with the vector of claim 21 and/or comprising a nucleic acid molecule of claim 20. A method for preparing a recombinant de-immunized bacterial pseudomonas exotoxin A according to any one of claims 1 to 14 or a recombinant immunotoxin according to any one of claims 15 to 19, wherein the method comprises culturing the host cell of claim 22 and isolating the PE or the immunotoxin from the cell culture. A recombinant de-immunized bacterial pseudomonas exotoxin A according to any one of claims 1 to 14 or a recombinant immunotoxin according to any one of claims 15 to 19 for the use in treating malignant diseases, or chronic inflammatory diseases, such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic and extrinsic asthma; cutaneous disease including atopic dermatitis, polymorphic light eruption, SLE; autoimmune diseases, including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases including Crohn's disease and chronic bowel disease. 5. The recombinant human cytolytic fusion protein according to claim 24 for the use in treating asthma. 6. A drug comprising recombinant de-immunized bacterial pseudomonas exotoxin A according to any one of claims 1 to 14 or a recombinant immunotoxin according to any one of claims 15 to 19 in combinations with a pharmacologically acceptable carrier. l. A recombinant de-immunized bacterial pseudomonas exotoxin A (PE) suitable to induce apoptosis in a eukaryotic target cell comprising the PE wild type amino acid sequence of SEQ ID NO. 1 or a variant thereof, wherein the PE or the variant contains a substitution at a position that corresponds structurally or by amino acid sequence homology to position 456, and wherein said variant has at least a minimum percentage sequence identity and/or percent homology to SEQ ID NO. 1, wherein the minimum percent identity and/or homology is at least 75%, at least 80 %, at least 85%, at least 90%, at least 93%, at least 96%, at least 97%, at least 98% or at least 99%, wherein the substitution is R456T or R456C, and wherein the deimmunized PE or the variant thereof has about the same or a greater enzymatic activity compared to the wild type bacterial pseudomonas exotoxin of SEQ ID NO: 1, wherein the de-immunized PE or the variant contains substitutions at positions that corresponds structurally or by amino acid sequence homology to positions 427, 456, 463, 467, 505 and 538, in particular wherein the substitutions are R427A, R456T, D463A, R467A, R505A, R538A or R427A, R456C, D463A, R467A, R505A, R538A, in particular wherein the de-immunized PE or the variant contains substitutions at positions that corresponds structurally or by amino acid sequence homology to positions 427, 456, 463, 467, 490, 505 and 538, in particular wherein the substitutions are R427A, R456T, D463A, R467A, R490A, R505A, R538A or R427A, R456C, D463A, R467A, R490A, R505A, R538A. 8. A recombinant immunotoxin (RIT) suitable to induce apoptosis in a eukaryotic target cell comprising a target cell-specific binding component and an effector domain, wherein the effector domain comprises a recombinant de-immunized bacterial pseudomonas exotoxin A (PE) according to claim 27. The recombinant immunotoxin according to claim 28, wherein the target cell-specific binding component comprises moieties which are affinity moieties from affinity substances or affinity substances in their entirety selected from the group consisting of antibodies, antibody fragments, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotypical antibodies, autoimmune-provoking structures, tissue-rejection-inducing structures, immunoglobulin constant regions and their derivatives, mutants or combinations thereof. The recombinant immunotoxin according to claim 28, wherein the recombinant immunotoxin is an allergen-based fusion protein. The recombinant immunotoxin according to claim 30, wherein the recombinant immunotoxin is an allergen-based fusion protein, wherein the target cell-specific binding component is the Dermatophagoides pteronyssinus Peptidase 1 (Der p 1). The recombinant immunotoxin according to claim 31, wherein the recombinant immunotoxin comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10. An isolated nucleic acid molecule encoding a recombinant de-immunized bacterial pseudomonas exotoxin A according to claim 27 or a recombinant immunotoxin according to any one of claims 28 to 32. A vector comprising a nucleic acid molecule of claim 33. A host cell being transformed with the vector of claim 34 and/or comprising a nucleic acid molecule of claim 33. A method for preparing a recombinant de-immunized bacterial pseudomonas exotoxin A according to claim 27 or a recombinant immunotoxin according to any one of claims 28 to 32, wherein the method comprises culturing the host cell of claim 35 and isolating the PE or the immunotoxin from the cell culture. A recombinant de-immunized bacterial pseudomonas exotoxin A according to claim 27 or a recombinant immunotoxin according to any one of claims 28 to 32 for the use in treating malignant diseases, or chronic inflammatory diseases, such as acute myeloid leukemia, arthritis, COPD including emphysema, intrinsic and extrinsic asthma; cutaneous disease including atopic dermatitis, polymorphic light eruption, SLE; autoimmune diseases, including graft versus host, multiple sclerosis, macrophage activation syndrome, rheumatoid arthritis, juvenile arthritis; intestinal diseases including Crohn's disease and chronic bowel disease. The recombinant human cytolytic fusion protein according to any one of claims 28 to 32 for the use in treating asthma. A drug comprising recombinant de-immunized bacterial pseudomonas exotoxin A according to claim 27 or a recombinant immunotoxin according to any one of claims 28 to 32 in combinations with a pharmacologically acceptable carrier.