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Purpose: Treatment of cancer using standard chemotherapy still offers a poor prognosis combined with severe side effects. Novel antibody-based therapies have been shown to overcome low efficiency and lack of selectivity by targeting cancer-associated antigens, such as aminopeptidase CD13., Methods: We isolated a high-affinity CD13-specific single-chain fragment variable (scFv13) from a phage display library of V-genes from mice immunized with soluble antigen. An immunotoxin comprising the scFv13 and a truncated version of the exotoxin A of Pseudomonas aeruginosa (ETA', scFv13-ETA') and a bispecific scFv targeting CD13 and CD16 simultaneously (bsscFv[13xds16]) was generated and investigated for their therapeutic potential., Results: Both fusion proteins bound specifically to target cells with high affinity. Furthermore, scFv13-ETA' inhibited the proliferation of human cancer cell lines efficiently at low concentrations (IC 50 values of 408 pM-7 nM) and induced apoptosis (40-85% of target cells). The bsscFv triggered dose-dependent antibody-dependent cell-mediated cytotoxicity, resulting in the lysis of up to 23.9% A2058 cells, 18.0% MDA-MB-468 cells and 19.1% HL-60 cells., Conclusion: The provided data demonstrate potent therapeutic activity of the scFv13-ETA' and the bsscFv[13xds16]. The CD13-specific scFv is therefore suitable for the direct and specific delivery of both cytotoxic agents and effector cells to cancer-derived cells, making it ideal for further therapeutic evaluation.

Purpose: Despite great progress in the diagnosis and treatment of localized prostate cancer (PCa), there remains a need for new diagnostic markers that can accurately distinguish indolent and aggressive variants. One promising approach is the antibody-based targeting of prostate stem cell antigen (PSCA), which is frequently overexpressed in PCa. Here, we show the construction of a molecular imaging probe comprising a humanized scFv fragment recognizing PSCA genetically fused to an engineered version of the human DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT), the SNAP-tag, enabling specific covalent coupling to various fluorophores for diagnosis of PCa. Furthermore, the recombinant immunotoxin (IT) PSCA(scFv)-ETA' comprising the PSCA(scFv) and a truncated version of Pseudomonas exotoxin A (PE, ETA') was generated., Methods: We analyzed the specific binding and internalization behavior of the molecular imaging probe PSCA(scFv)-SNAP in vitro by flow cytometry and live cell imaging, compared to the corresponding IT PSCA(scFv)-ETA'. The cytotoxic activity of PSCA(scFv)-ETA' was tested using cell viability assays. Specific binding was confirmed on formalin-fixed paraffin-embedded tissue specimen of early and advanced PCa., Results: Alexa Fluor ® 647 labeling of PSCA(scFv)-SNAP confirmed selective binding to PSCA, leading to rapid internalization into the target cells. The recombinant IT PSCA(scFv)-ETA' showed selective binding leading to internalization and efficient elimination of target cells., Conclusions: Our data demonstrate, for the first time, the specific binding, internalization, and cytotoxicity of a scFv-based fusion protein targeting PSCA. Immunohistochemical staining confirmed the specific ex vivo binding to primary PCa material.

Cattle are reservoirs of enterohemorrhagic Escherichia coli; however, their role in the epidemiology of other pathogenic E. coli remains undefined. A new set of quantitative real-time PCR assays for the direct detection and quantification of nine virulence-associated genes (VAGs) characteristic of the most important human E. coli pathotypes and four serotype-related genes (wzxO104 , fliCH4 , rbfO157 , fliCH7 ) that can be used as a surveillance tool for detection of pathogenic strains was developed. A total of 970 cattle fecal samples were collected in slaughterhouses in Germany and Spain, pooled into 134 samples and analyzed with this tool. stx1, eae and invA were more prevalent in Spanish samples whereas bfpA, stx2, ehxA, elt, est and the rbfO157 /fliCH7 combination were observed in similar proportions in both countries. Genes characteristic of the hybrid O104:H4 strain of the 2011 German outbreak (stx2/aggR/wzxO104 /fliCH4 ) were simultaneously detected in six fecal pools from one German abattoir located near the outbreak epicenter. Although no isolate harboring the full stx2/aggR/wzxO104 /fliCH4 combination was cultured, sequencing of the aggR positive PCR products revealed 100% homology to the aggR from the outbreak strain. Concomitant detection by this direct approach of VAGs from a novel human pathogenic E. coli strain in cattle samples implies that the E. coli gene pool in these animals can be implicated in de novo formation of such highly-virulent strains. The application of this set of qPCRs in surveillance studies could be an efficient early-warning tool for the emergence of zoonotic E. coli in livestock., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)

The current standard treatment for acute myeloid leukemia (AML) is chemotherapy based on cytarabine and daunorubicine (7 + 3), but it discriminates poorly between malignant and benign cells. Dose-limiting off‑target effects and intrinsic drug resistance result in the inefficient eradication of leukemic blast cells and their survival beyond remission. This minimal residual disease is the major cause of relapse and is responsible for a 5-year survival rate of only 24%. More specific and efficient approaches are therefore required to eradicate malignant cells while leaving healthy cells unaffected. In this study, we generated scFv antibodies that bind specifically to the surface of AML blast cells and AML bone marrow biopsy specimens. We isolated the antibodies by phage display, using subtractive whole-cell panning with AML M2‑derived Kasumi‑1 cells. By selecting for internalizing scFv antibody fragments, we focused on potentially novel agents for intracellular drug delivery and tumor modulation. Two independent methods showed that 4 binders were internalized by Kasumi-1 cells. Furthermore, we observed the AML‑selective inhibition of cell proliferation and the induction of apoptosis by a recombinant immunotoxin comprising one scFv fused to a truncated form of Pseudomonas exotoxin A (ETA'). This method may therefore be useful for the selection of novel disease-specific internalizing antibody fragments, providing a novel immunotherapeutic strategy for the treatment of AML patients.

The treatment of rhabdomyosarcoma (RMS) remains challenging, with metastatic and alveolar RMS offering a particularly poor prognosis. Therefore, the identification and evaluation of novel antigens, which are suitable targets for immunotherapy, is one attractive possibility to improve the treatment of this disease. Here we show that chondroitin sulfate proteoglycan 4 (CSPG4) is expressed on RMS cell lines and RMS patient material. We evaluated the immunotoxin (IT) αMCSP-ETA', which specifically recognizes CSPG4 on the RMS cell lines RD, FL-OH1, TE-671 and Rh30. It is internalized rapidly, induces apoptosis and thus kills RMS cells selectively. We also demonstrate the specific binding of this IT to RMS primary tumor material from three different patients., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy., Methods: We transfected A-431 tumour cells with the far red-emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A' (ETA'). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker., Results: The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA' resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health., Conclusions: We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA'.

The distribution of many genes encoding virulence and virulence life-style (VL-S) factors in Brachyspira (B.) hyodysenteriae and other Brachyspira species are largely unknown. Their knowledge is essential e.g. for the improvement of diagnostic methods targeting the detection and differentiation of the species. Thus 121 German Brachyspira field isolates from diarrhoeic pigs were characterized down to the species level by restriction fragment length polymorphism analysis of the nox gene and subsequently subjected to polymerase chain reaction detecting VL-S genes for inner (clpX) and outer membrane proteins (OMPs: bhlp16, bhlp17.6, bhlp29.7, bhmp39f, bhmp39h), hemolysins (hlyA/ACP, tlyA), iron metabolism (ftnA, bitC), and aerotolerance (nox). For comparison, B. hyodysenteriae reference strains from the USA (n=7) and Australia (2) were used. Of all genes tested only nox was detected in all isolates. The simultaneous presence of both the tlyA and hlyA/ACP was restricted to the species B. hyodysenteriae. The hlyA infrequently occurred also in weakly hemolytic Brachyspira. Similarly to tlyA and hlyA all B. hyodysenteriae strains contained the ferritin gene ftnA which was also found in two Brachyspira intermedia isolates. OMP encoding genes were present in B. hyodysenteriae field isolates in rates of 0% (bhlp17.6, bhmp39h), 58.1% (bhlp29.7), and 97.3% (bhmp39f). Since the study revealed a high genetic heterogeneity among German B. hyodysenteriae field isolates differentiating them from USA as well as Australian strains, targets for diagnostic PCR were limited to the nox gene (genus specific PCR) as well as to the species specific nox(hyo) gene and the combination of hlyA and tlyA which allow to specifically detect B. hyodysenteriae., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Testing 1,666 fecal or intestinal samples from healthy and diarrheic pigs, we obtained hemolytic Escherichia coli isolates from 593 samples. Focusing on hemolytic E. coli isolates without virulence-associated genes (VAGs) typical for enteropathogens, we found that such isolates carried a broad variety of VAGs typical for extraintestinal pathogenic E. coli.

Target-specific acute myeloid leukemia (AML) immunotherapy requires selective cell-surface antigens on AML blast cells. CD64 is a promising candidate antigen because it is abundantly expressed on monocytoid differentiated AML subtypes. In previous studies, a chemically linked full-length anti-CD64 immunotoxin based on ricin A showed promising results in several animal models, but further development has been hindered by its substantial, dose-limiting off-target effects. We recently constructed the recombinant immunotoxin H22(scFv)-ETA', comprising a truncated Pseudomonas exotoxin A (PE) and a humanized scFv antibody against CD64. This molecule was shown to kill CD64(+) AML-derived tumor cell lines and primary patient-derived AML cells specifically, both in vitro and ex vivo. Here we describe the in vivo efficiency of H22(scFv)-ETA' in the U937/SCID mouse xenograft model for human AML, by providing immunohistochemical evidence for the elimination of human CD64(+) tumor cells in mouse organs. H22(scFv)-ETA' showed potent antitumor activity against myeloid tumor cells and significantly prolonged the overall survival of AML xenograft animals. In conclusion, H22(scFv)-ETA' is efficacious against AML with monocytoid differentiation in vitro and in animal models in vivo, providing the basis for a novel therapeutic strategy for the treatment of AML patients., (Copyright © 2010 UICC.)

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) gamma-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

CD64, the high affinity receptor for IgG (FcgammaRI) is expressed on acute myeloid leukemia blast cells and has recently been described as a specific target for immunotherapy. To generate a recombinant immunotoxin, the anti-CD64 single chain fragment (scFv) m22 was cloned into the bacterial expression vector pBM1.1 and fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Genetically modified Escherichia coli BL21 Star (DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl beta-D-thiogalactoside induction, the 70-kDa His(10)-tagged m22(scFv)-ETA' was directed into the periplasmic space and purified by a combination of metal-ion affinity and molecular size-chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays, using CD64-positive AML cells. Binding specificity of m22(scFv)-ETA' was verified by competition with the parental anti-CD64 monoclonal antibody m22. The recombinant immunotoxin showed significant toxicity toward the CD64-positive cell lines HL-60 and U937 reaching 50% inhibition of cell proliferation at a concentration (IC(50)) of 11.6 ng/ml against HL-60 cells and 12.9 ng/ml against U937 cells. Approximately 41% of primary leukemia cells from a patient with CD64-positive AML were driven into early apoptosis by m22(scFv)-ETA' as measured by flow cytometric analysis. This is the first article documenting the specific cytotoxicity of a novel recombinant immunotoxin with major implications for immunotherapy of CD64-positive diseases.

Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.

The nucleotide sequence encoding the Salmonella plasmid virulence factor D (SpvD) was determined in 17 Salmonella strains that were different in O and H antigen patterns, animal host and geographical origin, and year of isolation. Nucleotide sequence comparison revealed the existence of at least nine spvD alleles resulting in 8 SpvD protein variants although the nucleotide sequences were highly similar (identity 98.8-100%). The spvD gene products differed from each other in up to 4 amino acid residues only with the exception of the carboxy-terminally truncated SpvD variant of one S. Gallinarum field isolate. The highly conserved primary structure of SpvD in epidemiologically relevant salmonellae suggests that this virulence factor is a promising antigen candidate for diagnostic purposes (i.e. antibody detection in infected animals) but also for immunoprophylaxis in farm animal species.

NeoPharm, under license from the NIH and the FDA, is developing a chimeric human IL-13 fused in frame to a genetically engineered truncated Pseudomonas exotoxin (PE38QQR) molecule, for its potential as an antitumor agent [266296], [281418], [290480]. NeoPharm filed an IND in 1999 for renal cell carcinoma (RCC) and glioma [319690], [325001]; an additional IND was filed in March 2000 for the treatment of glioblastoma. In December 2000, NeoPharm initiated phase I/II trials of IL-13-PE38QQR involving patients with refractory glioblastoma multiforme. This trial was being conducted by the New Approaches to Brain Tumor Therapy, a research consortium sponsored by the NCI. At that time, the first patient with brain cancer had completed treatment with IL-13-PE38QQR [393197]. In October 1999, NeoPharm initiated phase I trials of hIL-13-PE38QQR for the treatment of patients with RCC [343878]. In February 2000, Dirks & Co estimated the potential US market for hIL-13-PE38QQR to be $5.8 billion [414515].

In recent years, substantial experience has been accumulated with tumor-specific immunotherapeutics which seem to be effective against minimal residual disease. The coupling of toxins to monoclonal antibodies has indicated promising results in early clinical trials. Recombinant DNA technology makes it possible to genetically fuse coding regions of V genes or cytokines to modified toxin domains. These recombinant immunotoxins can easily be manipulated to increase the cytotoxic potency or affinity. Binding single-chain variable fragments (scFv) expressed as chimeric fusion proteins on the surface of filamentous bacteriophages were selected on Hodgkin-derived cell lines. This technique was also used to create a new humanized anti-CD30 scFv which exhibits similar binding to the CD30 antigen when compared to its murine predecessor. ScFvs were then inserted into a new bacterial expression vector and thus fused to a deletion mutant of Pseudomonas exotoxin. Anti-CD25(scFv)-ETA' and anti-CD30(scFv)-ETA' were isolated from E. coli periplasm and purified by metal chelate affinity and size exclusion chromatography. All immunotoxins produced showed specific cytotoxicity against Hodgkin lymphoma cell lines as documented by competitive assays. In addition, these constructs were highly efficient in the treatment of disseminated human Hodgkin's disease in SCID mice. These in vivo data indicate a possible clinical impact for patients with relapsed CD25- and/or CD30-positive lymphoma.

Recombinant DNA technology makes it possible to genetically fuse V genes or cytokines to toxin domains, resulting in immunotherapeutics for selective destruction of tumor cells. Since recombinant immunotoxins can be easily manipulated in terms of affinity or cytotoxic potency and produced in large quantities, we have developed a new CD30 ligand-based fusion toxin (CD30L-ETA'). Human CD30L cDNA was ligated into a pET-based expression plasmid and thereby fused to a modified Pseudomonas aeruginosa exotoxin A (ETA') lacking its cell-binding domain I. After IPTG-indiced expression in E. coli strain BL21(DE3), the 60 kDa His-tagged fusion protein (CD30L-ETA') was isolated from inclusion bodies. Denatured protein was renatured in the presence of 0.4 M arginine and a glutathione redox system. Refolded protein was purified and concentrated by ion-exchange chromatography on a HiTrap Q column. The binding properties of CD30L-ETA' were evaluated by competitive ELISA, immunohistochemical staining, and FACS analysis on CD30-expressing cells. The in vitro toxicity of the fusion protein was then tested on the CD30+ Hodgkin-derived cell line L540cy and the Burkitt's lymphoma cell line BL38. CD30L-ETA' exhibited specific cytotoxicity against L540cy cells (IC50 = 24 ng/ml) as determined by [3H]leucine uptake assays. This is the first report on the specificity and cytotoxic potency of a chimeric CD30L fusion toxin against Hodgkin's disease-derived cells.

Recombinant immunotoxins (rITs) are highly specific anti-tumor agents composed of monoclonal antibody fragments or other specific carriers coupled to plant or bacterial toxins. A major problem in the purification of rITs is the low periplasmic yield in currently available expression systems. Thus, the aim of this study was the development of a new bacterial expression system for high-level production of rITs. We constructed a series of pET-based vectors for pelB-directed periplasmic secretion or cytoplasmic production under the control of the T7lac promoter. Expression in Escherichia coli BL21(DE3)pLysS allowed a tightly regulated isopropyl beta-d-thiogalactopyranoside (IPTG) induction of protein synthesis. An enterokinase-cleavable poly-histidine cluster was introduced into this setup for purification by affinity chromatography. A major modification resulted from the insertion of a specifically designed multiple cloning site. It contains only rare restriction enzyme recognition sites used for cloning of immunoglobulin variable region genes, as well as unique SfiI and NotI restriction sites for directed insertion of single-chain variable fragments (scFv) available from established bacteriophage systems. For this purpose, we deleted two naturally occurring internal SfiI consensus sites in a deletion mutant of Pseudomonas aeruginosa exotoxin A (ETA'). Each single structural element of the new vector (promoter, leader sequence, purification tag, scFv sequence, selectable marker, and toxin gene) was flanked by unique restriction sites allowing simple directional substitution. The fidelity of IPTG induction and high-level expression were demonstrated using an anti-CD30 scFv (Ki-4) fused to ETA'. These data confirm a bacterial vector system especially designed for efficient periplasmic expression of ETA'-based fusion toxins.

The data of a closed phase I/II trial in patients with resistant Hodgkin's lymphoma indicate promising results using a chemically linked anti-CD25 ricin-A immunotoxin (IT) (RFT5-SMPT-dgA). This IT is based on the high-affinity moab RFT5. Since recombinant DNA technology permits the readier production of large amounts of ITs, we constructed a new RFT5-based fusion toxin [RFT5(scFv)-ETA']. We isolated mRNA from the hybridoma cell line RFT5, synthesized first strand cDNA and performed RT-PCR. Amplified coding regions of the light and heavy chain variable domains were joined together with a synthetic (Gly4-Ser)3 linker. The resulting single chain variable fragment (scFv) was fused to a modified Pseudomonas aeruginosa exotoxin A (ETA') lacking its cell-binding domain I. After IPTG-induced expression in Escherichia coli, the 70 kDa His-tagged fusion protein [RFT5(scFv)-ETA'] was isolated by osmotic shock and sonication under denaturing conditions. The recombinant toxin was purified on a Ni2+-NTA chelating sepharose and eluted with 250 mM imidazole. Pooled protein was renatured, dialyzed and concentrated by precipitation. Binding properties of RFT5(scFv)-ETA' were assessed on the CD25-expressing cell line L540cy by ELISA, immunohistochemistry and FACS analysis. CD25-specific binding was confirmed by immunoprecipitation experiments with recombinant human IL-2 receptor alpha. The in vitro toxicity of the chimeric protein was tested on the Hodgkin-derived cell lines L540cy, L428, L1236, a monocyte cell line U937 and a Burkitt lymphoma cell line BL38. RFT5(scFv)-ETA' inhibited protein biosynthesis of L540cy and L428 cells by 50% at concentrations (IC50) of 18 and 12 ng/ml, respectively. CD25-specific toxicity was confirmed by competitive toxicity assays. These data confirm for the first time binding specificity and toxicity of a recombinant anti-CD25 immunotoxin, against Hodgkin-derived cell lines; its applicability on Hodgkin's lymphoma needs yet to be evaluated in vivo.

The receptor for human interleukin-9 (hIL-9) might be a target for selective immunotherapy. It is expressed on a variety of malignant cells, including Hodgkin's lymphoma, non-Hodgkin lymphoma and acute myeloid leukemia (AML). We therefore constructed a new chimeric toxin by fusing hIL-9-cDNA to modified Pseudomonas aeruginosa exotoxin A (ETA'). The binding properties of the new recombinant protein, rhIL-9-ETA', were assessed on different cell lines expressing the hIL-9 receptor. The antitumor potency of rhIL-9-ETA' was evaluated against the Hodgkin-derived cell lines L540Cy, KM-H2 and L1236, the Burkitt lymphoma cell line Daudi, the erythroleukemia cell line K562, and the mastocytoma cell line P815-hIL9R, transfected with hIL-9 receptor cDNA. Recombinant hIL-9-ETA' exhibited potent specific cytotoxic effects against P815-hIL9R, K562 and L1236 cells, inhibiting protein synthesis by 50% (IC50) at concentrations of 0.05, 0.58 and 3 micrograms/ml respectively. The cytotoxic effect was abrogated after addition of polyclonal antibodies against the human IL-9. rhIL-9-ETA' might be of potential use against hIL-9R-expressing malignancies.