Maria Serena Fabbrini

THE AMINO-TERMINAL FRAGMENT OF HUMAN UROKINASE DIRECTS A RECOMBINANT CHIMERIC TOXIN TO TARGET CELLS: INTERNALIZATION IS TOXIN MEDIATED

In contrast to two‐chain urokinase (uPA), a chemical conjugate between uPA and native saporin (a cytotoxic plant seed ribosome‐inactivating protein) did not require plasminogen activator inhibitors to be internalized. To dissect this pathway, we constructed a chimera consisting of the amino‐terminal fragment (ATF) of human urokinase fused to a saporin isoform (SAP‐3). The chimeric ATF‐SAP toxin was expressed in Escherichia coli, purified, and characterized for its ribosome‐inactivating activity. Besides being a potent inhibitor of protein synthesis in cell‐free assays, ATF‐SAP was specifically cytotoxic toward cells expressing human uPAR. Competition experiments indicated that both the human uPAR and the LDL‐related receptor protein are involved in mediating the cell killing ability of ATF‐SAP. We conclude that neither plasminogen activator inhibitors nor the catalytic moiety of urokinase are necessary to initiate these internalization pathways. Thus, saporin may play a role similar to plasminogen activator inhibitors in its ability to trigger internalization of uPAR‐bound ligands through endocytic receptors.—Fabbrini, M. S., Carpani, D., Bello‐Rivero, I., Soria, M. R. The amino‐terminal fragment of human urokinase directs a recombinant chimeric toxin to target cells: internalization is toxin‐mediated. FASEB J. 11, 1169–1176 (1997)

Pichia pastoris as a host for secretion of toxic saporin chimeras

Most of the targeting moieties, such as antibody fragments or growth factor domains, used to construct targeted toxins for anticancer therapy derive from secretory proteins. These normally fold in the oxidative environment of the endoplasmic reticulum, and hence their folding in bacterial cells can be quite inefficient. For instance, only low amounts of properly folded antimetastatic chimera constituted by the amino‐terminal fragment of human urokinase (ATF) fused to the plant ribosome‐inactivating protein saporin could be recovered. ATF‐saporin was instead secreted efficiently when expressed in eukaryotic cells protected from autointoxication with neutralizing anti‐saporin antibodies. Pichia pastoris is a microbial eukaryotic host where these domains can fold into a transport‐competent conformation and reach the extracellular medium. We show here that despite some host toxicity codon‐usage optimization greatly increased the expression levels of active saporin but not those of an active‐site mutant SAP‐KQ in GS115 (his4) strain. The lack of any toxicity associated with expression of the latter confirmed that toxicity is due to saporin catalytic activity. Nevertheless, GS115 (his4) cells in flask culture secreted 3.5 mg/L of a histidine‐tagged ATF‐saporin chimera showing an IC50 of 6 X 10−11 M against U937 cells, thus demonstrating the suitability of this expression platform for secretion of toxic saporin‐based chimeras.—Lombardi, A., Bursomanno, S., Lopardo, T., Traini, R., Colombatti, M., Ippoliti, R., Flavell, D. J., Flavell, S. U., Ceriotti, A., Fabbrini, M. S. Pichia pastoris as a host for secretion of toxic saporin chimeras. FASEB J. 24, 253–265 (2010). www.fasebj.org

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism

A fluorescent derivative of a chimeric toxin between human pro‐urokinase and the plant ribo‐some‐inactivating protein saporin (p‐uPA‐SapTRITC), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase‐inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein‐related receptor protein (LRP) family, and involves a macro‐quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two‐step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.—Ippoliti, R., Lendaro, E., Benedetti, P. A., Torrisi, M. R., Belleudi, F., Carpani, D., Soria, M. R., Fabbrini, M. S. Endocytosis of a chimera between human pro‐urokinase and the plant toxin saporin: an unusual internalization mechanism. FASEB J. 14, 1335–1344 (2000)

Distinct cellular responses induced by saporin and a transferrin-saporin conjugate in two different human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ∼12–18 months post‐diagnosis. GBM usually recurs within 12 months post‐resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4‐Pseudomonas aeruginosa exotoxin A (IL4‐PE, NBI‐3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha‐TP‐38, IL13‐PE38, and a transferrin‐C diphtheriae toxin mutant (Tf‐CRM107). In this work, we studied the effects of the plant ribosome‐inactivating saporin and of its chimera transferrin–saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways. J. Cell. Physiol. 227: 939–951, 2012.

The signal peptide of human preproendothelin-1

Synthetic mRNAs were produced using either the complete coding sequence of a human preproendothelin‐1 cDNA clone or a truncated form in which the portion encoding the first 17 amino acids, representing a putative signal peptide for insertion into the endoplasmic reticulum, was replaced with a methionine codon. The mRNAs were translated in vitro in the presence or in the absence of microsomal membranes. Protection from trypsin digestion demonstrated that the full‐length polypeptide, but not the truncated form, could be inserted into the membranes. Sequence analysis revealed that membrane insertion is accompanied by removal of the first 17 amino acids. These results indicate that the first 17 amino acids of human preproendothelin‐1 are a functional signal peptide which allows the protein to enter the secretory pathway.

Signal peptide‐regulated toxicity of a plant ribosome‐inactivating protein during cell stress

The fate of the type I ribosome-inactivating protein (RIP) saporin when initially targeted to the endoplasmic reticulum (ER) in tobacco protoplasts has been examined. We find that saporin expression causes a marked decrease in protein synthesis, indicating that a fraction of the toxin reaches the cytosol and inactivates tobacco ribosomes. We determined that saporin is largely secreted but some is retained intracellularly, most likely in a vacuolar compartment, thus behaving very differently from the prototype RIP ricin A chain. We also find that the signal peptide can interfere with the catalytic activity of saporin when the protein fails to be targeted to the ER membrane, and that saporin toxicity undergoes signal sequence-specific regulation when the host cell is subjected to ER stress. Replacement of the saporin signal peptide with that of the ER chaperone BiP reduces saporin toxicity and makes it independent of cell stress. We propose that this stress-induced toxicity may have a role in pathogen defence.

Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas Exotoxin A or saporin produced in different microbial expression systems

BackgroundAntibodies raised against selected antigens over-expressed at the cell surface of malignant cells have been chemically conjugated to protein toxin domains to obtain immunotoxins (ITs) able to selectively kill cancer cells. Since latest generation immunotoxins are composed of a toxic domain genetically fused to antibody fragment(s) which confer on the IT target selective specificity, we rescued from the hydridoma 4KB128, a recombinant single-chain variable fragment (scFv) targeting CD22, a marker antigen expressed by B-lineage leukaemias and lymphomas. We constructed several ITs using two enzymatic toxins both able to block protein translation, one of bacterial origin (a truncated version of Pseudomonas exotoxin A, PE40) endowed with EF-2 ADP-ribosylation activity, the other being the plant ribosome-inactivating protein saporin, able to specifically depurinate 23/26/28S ribosomal RNA. PE40 was selected because it has been widely used for the construction of recombinant ITs that have already undergone evaluation in clinical trials. Saporin has also been evaluated clinically and has recently been expressed successfully at high levels in a Pichia pastoris expression system. The aim of the present study was to evaluate optimal microbial expression of various IT formats.ResultsAn anti-CD22 scFv termed 4KB was obtained which showed the expected binding activity which was also internalized by CD22+ target cells and was also competed for by the parental monoclonal CD22 antibody. Several fusion constructs were designed and expressed either in E. coli or in Pichia pastoris and the resulting fusion proteins affinity-purified. Protein synthesis inhibition assays were performed on CD22+ human Daudi cells and showed that the selected ITs were active, having IC50 values (concentration inhibiting protein synthesis by 50% relative to controls) in the nanomolar range.ConclusionsWe undertook a systematic comparison between the performance of the different fusion constructs, with respect to yields in E. coli or P. pastoris expression systems and also with regard to each constructs specific killing efficacy. Our results confirm that E. coli is the system of choice for the expression of recombinant fusion toxins of bacterial origin whereas we further demonstrate that saporin-based ITs are best expressed and recovered from P. pastoris cultures after yeast codon-usage optimization.

Engineering a switchable toxin: the potential use of PDZ domains in the expression, targeting and activation of modified saporin variants

A critical problem in studying ribosome-inactivating proteins (RIPs) lies in the very limited possibility to produce them in heterologous systems. In fact, their inherent toxicity for the producing organism nearly always prevents their recombinant expression. In this study, we designed, expressed and characterized an engineered form of the RIP saporin (SapVSAV), bearing a C-terminal extra sequence that is recognized and bound by the second PDZ domain from murine PTP-BL protein (PDZ2). The co-expression of SapVSAV and PDZ2 in Escherichia coli BL21 cells greatly enhances the production of the toxin in a soluble form. The increase of production was surprisingly not due to protection from bacterial intoxication, but may arise from a stabilization effect of PDZ2 on the toxin molecule during biosynthesis. We found that once purified, SapVSAV is stable but is not toxic to free ribosomes, while it is fully active against human cancer cells. This strategy of co-expression of a toxin moiety and a soluble PDZ domain may represent a new system to increase the production of recombinant toxic proteins and could allow the selection of new extra sequences to target PDZ domains inside specific mammalian cellular domains.

Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera

BackgroundThe big challenge in any anti-tumor therapeutic approach is represented by the development of drugs selectively acting on the target with limited side effects, that exploit the unique characteristics of malignant cells. The urokinase (urokinase-type plasminogen activator, uPA) and its receptor uPAR have been identified as preferential target candidates since they play a key role in the evolution of neoplasms and are associated with neoplasm aggressiveness and poor clinical outcome in several different tumor types.ResultsTo selectively target uPAR over-expressing cancer cells, we prepared a set of chimeric proteins (ATF-SAP) formed by the human amino terminal fragments (ATF) of uPA and the plant ribosome inactivating protein saporin (SAP). Codon-usage optimization was used to increase the expression levels of the chimera in the methylotrophic yeast Pichia pastoris. We then moved the bioprocess to bioreactors and demonstrated that the fed-batch production of the recombinant protein can be successfully achieved, obtaining homogeneous discrete batches of the desired constructs. We also determined the cytotoxic activity of the obtained batch of ATF-SAP which was specifically cytotoxic for U937 leukemia cells, while another construct containing a catalytically inactive mutant form of SAP showed no activity.ConclusionOur results demonstrate that the uPAR-targeted, saporin-based recombinant fusion ATF-SAP can be produced in a fed-batch fermentation with full retention of the molecules selective cytotoxicity and hence therapeutic potential.

Human preproendothelin-1 is converted into active endothelin-1 by baculovirus-infected insect cells

To investigate biochemical and biological parameters involved in preproendothelin-1 (preproET-1) maturation we infected Spodoptera frugiperda (Sf21) cells with a suitable engineered baculovirus vector carrying the cDNA encoding the entire human 212 amino acids precursor. Culture supernatants were tested by RIA using an anti-ET-1 serum, ET-1-like immunoreactive material (IRM) was detected in the infected Sf21 cells but not in control, wild-type or mock-infected cells. Fractionation of the culture supernatant by RP-HPLC coupled to an ET-1 specific RIA yielded two main peaks corresponding to the retention times of human bigET-1 and ET-1. Furthermore, culture supernatant of preproET-1 expressing Sf21 cells elicited a characteristic dose-response vasoconstrictive activity on rabbit vena cava, consistent with the amount of ET-1 as estimated by RP-HPLC coupled to RIA. These results suggest that insect cells possess the enzymatic activities necessary for human preproET-1 full maturation even though no such peptide has ever been found in insect cells.