Cloning, expression and characterization of a HER2-alpha luffin fusion protein in Escherichia coli.

In recent decades, immunotoxins have attracted significant attention in treatment of a wide range of diseases including cancers due to their natural origins and their role in blocking crucial pathways within the cells. Ribosome inactivating proteins (RIPs) are efficient molecules in blocking protein synthesis through interactions with ribosomal rRNA molecules. cDNA molecule encoding HER2 scFv antibody fragment originated from trastuzumab attached to the mature alpha luffin gene fragment was subcloned into pET28a expression vector and expressed in different E. coli expression hosts. Identity of the expressed recombinant protein was investigated through western blotting and the fusion protein was purified using Ni-NTA affinity chromatography. The biological activity (toxicity) of the protein was investigated on DNA and RNA samples. A 58 kDa protein was expressed in E. coli. The best protein expression level was achieved in 0.2 mM IPTG at 30 °C in TB medium using E. coli BL21 (DE3) host strain. The fusion protein showed RNase and DNA glycosylase activity on tested RNA and DNA samples. DNA glycosylase activity of the recombinant fusion protein showed that alpha luffin part of this protein is active in conjugation to the scFv molecule and the expressed protein can be further studied in targeted biological in vitro assays.

Expression of novel fusion antiviral proteins ricin a chain-pokeweed antiviral proteins (RTA-PAPs) in Escherichia coli and their inhibition of protein synthesis and of hepatitis B virus in vitro.

BACKGROUND: Ricin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a protein synthesis inhibition activity assay. RESULTS: Results showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with a 50% protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized protein ricin A chain mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance. CONCLUSIONS: Collectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.

Pokeweed antiviral protein alters splicing of HIV-1 RNAs, resulting in reduced virus production.

Processing of HIV-1 transcripts results in three populations in the cytoplasm of infected cells: full-length RNA, singly spliced, and multiply spliced RNAs. Rev, regulator of virion expression, is an essential regulatory protein of HIV-1 required for transporting unspliced and singly spliced viral transcripts from the nucleus to the cytoplasm. Export allows these RNAs to be translated and the full-length RNA to be packaged into virus particles. In our study, we investigate the activity of pokeweed antiviral protein (PAP), a glycosidase isolated from the pokeweed plant Phytolacca americana, on the processing of viral RNAs. We show that coexpression of PAP with a proviral clone alters the splicing ratio of HIV-1 RNAs. Specifically, PAP causes the accumulation of multiply spliced 2-kb RNAs at the expense of full-length 9-kb and singly spliced 4-kb RNAs. The change in splicing ratio is due to a decrease in activity of Rev. We show that PAP depurinates the rev open reading frame and that this damage to the viral RNA inhibits its translation. By decreasing Rev expression, PAP indirectly reduces the availability of full-length 9-kb RNA for packaging and translation of the encoded structural proteins required for synthesis of viral particles. The decline we observe in virus protein expression is not due to cellular toxicity as PAP did not diminish translation rate. Our results describing the reduced activity of a regulatory protein of HIV-1, with resulting change in virus mRNA ratios, provides new insight into the antiviral mechanism of PAP.

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

BACKGROUND: The 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. RESULTS: To 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. CONCLUSION: Our 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.

A small RNA targets pokeweed antiviral protein transcript.

Ribosome-inactivating proteins (RIPs) are a class of plant defense proteins with N-glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad-spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.

Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity.

Saporin L3 from Saponaria officinalis (soapwort) leaves is a type 1 ribosome-inactivating protein. It catalyzes the hydrolysis of oligonucleotide adenylate N-ribosidic bonds to release adenine from rRNA. Depurination sites include both adenines in the GAGA tetraloop of short sarcin-ricin stem-loops and multiple adenines within eukaryotic rRNA, tRNAs, and mRNAs. Multiple Escherichia coli vector designs for saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in E. coli nonexpression strains. Saporin L3 is >10(3) times more efficient at RNA deadenylation on short GAGA stem-loops than saporin S6, the saporin isoform currently used in immunotoxin clinical trials. We engineered a construct for the His-tagged saporin L3 to test for expression in Pichia pastoris when it is linked to the protein export system for the yeast α-mating factor. DNA encoding saporin L3 was cloned into a pPICZαB expression vector and expressed in P. pastoris under the alcohol dehydrogenase AOX1 promoter. A fusion protein of saporin L3 containing the pre-pro-sequence of the α-mating factor, the c-myc epitope, and the His tag was excreted from the P. pastoris cells and isolated from the culture medium. Autoprocessing of the α-mating factor yielded truncated saporin L3 (amino acids 22-280), the c-myc epitope, and the His tag expressed optimally as a 32 kDa construct following methanol induction. Saporin L3 was also expressed with specific alanines and/or serines mutated to cysteine. Native and Cys mutant saporins are kinetically similar. The recombinant expression of saporin L3 and its mutants permits the production and investigation of this high-activity ribosome-inactivating protein.

PDZ Domain in the Engineering and Production of a Saporin Chimeric Toxin as a Tool for targeting Cancer Cells.

In this paper we have studied a PDZ protein domain as a possible tool for cellular targeting of the ribosome inactivating protein Saporin, exploiting the ability of PDZ domains to recognize and bind short peptide sequences located at the C-terminus of a cognate protein. We have focused our attention on the PDZ domain from hCASK (Human calcium/calmodulin-dependent serine protein kinase) that binds extracellular CD98 in epithelial cells, being this antigen recognized as a marker for several human tumors and particularly considered a negative prognostic marker for human glioblastoma. We produced recombinant fusions of one or two hCASK-PDZ domains with the ribosome inactivating protein Saporin and assayed them on two human glioblastoma cell lines (GL15 and U87). These constructs proved to be toxic, with increasing activity as a function of the number of PDZ domains, and induce cell death by apoptotic mechanisms in a dose-dependent and/or time dependent manner.

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

BACKGROUND: Antibodies 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. RESULTS: An 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. CONCLUSIONS: We 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.

Efficient Inhibition of Ovarian Cancer by Gelonin Toxin Gene Delivered by Biodegradable Cationic Heparin-polyethyleneimine Nanogels.

The use of toxins for cancer therapy has great promise. Gelonin, a potent plant toxin, causes cell death by inactivating the 60S ribosomal subunit. Recently, we developed a novel gene delivery system using biodegradable cationic heparin-polyethyleneimine (HPEI) nanogels. In the current study, the antitumor activity of a recombinant plasmid expressing gelonin (pGelonin) on human ovarian cancer was assessed. The application of HPEI nanogels, was also evaluated. Gelonin-cDNA was cloned into the pVAX1 plasmid vector and transfected into SKOV3 human ovarian cancer cells using biodegradable cationic HPEI nanogels. The expression of gelonin in vitro and in vivo was confirmed using RT-PCR and western blot analysis. Cell viability and apoptosis were examined using an MTT assay and flow cytometric analysis. For the in vivo study, an SKOV3 intraperitoneal ovarian carcinomatosis model was established, and nude mice were randomly assigned into four groups receiving i.p. administration of pGelonin/HPEI complexes, pVAX/HPEI complexes, HPEI alone and 5% glucose solution. The tumor weight was monitored, and a TUNEL assay and Ki-67 immunohistochemistry were performed to evaluate apoptosis and cell proliferation in the tumor tissue sections, respectively. Gelonin was efficiently expressed in SKOV3 cancer cells in vitro and in vivo using pGelonin incorporated with HPEI nanogels. The pGelonin/HPEI complexes inhibited cell viability and induced apoptosis in the cell culture. Treatment for intraperitoneal carcinomatosis with pGelonin/HPEI complexes reduced the tumor weight by ~58.55% compared to the control groups (P<0.05). The antitumor effect was accompanied by increased apoptosis and reduced cell proliferation (P<0.05). No significant side effects were observed with i.p. administration of the pGelonin/HPEI complexes. Our data indicate that HPEI nanogel-delivered pGelonin may have promising applications against human ovarian cancer.

Role of lateral hypothalamus in two aspects of attention in associative learning.

Orexin (hypocretin) and melanin-concentrating hormone (MCH) neurons are unique to the lateral hypothalamic (LH) region, but project throughout the brain. These cell groups have been implicated in a variety of functions, including reward learning, responses to stimulants, and the modulation of attention, arousal and the sleep/wakefulness cycle. Here, we examined roles for LH in two aspects of attention in associative learning shown previously to depend on intact function in major targets of orexin and MCH neurons. In experiments 1 and 2, unilateral orexin-saporin lesions of LH impaired the acquisition of conditioned orienting responses (ORs) and bilaterally suppressed FOS expression in the amygdala central nucleus (CeA) normally observed in response to food cues that provoke conditioned ORs. Those cues also induced greater FOS expression than control cues in LH orexin neurons, but not in MCH neurons. In experiment 3, unilateral orexin-saporin lesions of LH eliminated the cue associability enhancements normally produced by the surprising omission of an expected event. The magnitude of that impairment was positively correlated with the amount of LH damage and with the loss of orexin neurons in particular, but not with the loss of MCH neurons. We suggest that the effects of the LH orexin-saporin lesions were mediated by their effect on information processing in the CeA, known to be critical to both behavioral phenomena examined here. The results imply close relations between LH motivational amplification functions and attention, and may inform our understanding of disorders in which motivational and attentional impairments co-occur.

Sugar binding effects on the enzymatic reaction and conformation near the active site of pokeweed antiviral protein revealed by fluorescence spectroscopy.

In various trials for elucidating the physiological function of pokeweed antiviral protein (PAP), studies on the interaction with sugar are essential. The fluorescence titration curves showed that PAP retained the strong affinity against N-acetylglucosamine (NAG) and two sites in one PAP molecule co-operatively participated in the binding. In the complex of PAP with NAG, Trp208 located at the entrance lid site of substrate came closer to Tyr72 about 0.3 Å. Furthermore, the fluorescence anisotropy decay measurement demonstrated that the segmental rotation of Trp208 was enlarged by the binding of PAP with NAG. Such conformational changes around the active site closely correlate with the enzymatic activity of PAP. The N-glycosidase activity of PAP was enhanced more than two times in the presence of NAG. The obtained results consistently suggested the enzymatic activity of PAP would be regulated through the conformation change near the active site induced by the binding with NAG.

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

Biosynthesized tumor acidity and MMP dual-responsive plant toxin gelonin for robust cancer therapy.

Among all kinds of anticancer agents, small molecule drugs produce an unsatisfactory therapeutic effect due to the lack of selectivity, notorious drug resistance and side effects. Therefore, researchers have begun to pay extensive attention to macromolecular drugs with high efficacy and specificity. As a plant toxin, gelonin exerts potent antitumor activity via inhibiting intracellular protein synthesis. However, gelonin lacks a translocation domain, and thus its poor cellular uptake leads to low outcomes of antitumor response. Here, tumor acidity and matrix metalloproteinase (MMP) dual-responsive functional gelonin (Trx-PVGLIG-pHLIP-gelonin, TPpG), composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP), an MMP-responsive motif PVGLIG hexapeptide and gelonin, was innovatively proposed and biologically synthesized by a gene recombination technique. TPpG exhibited good thermal and serum stability, showed MMP responsiveness and could enter tumor cells under weakly acidic conditions, especially for MMP2-overexpressing HT1080 cells. Compared to low MMP2-expressing MCF-7 cells, TPpG displayed enhanced in vitro antitumor efficacy to HT1080 cells at pH 6.5 as determined by different methods. Likewise, TPpG was much more effective in triggering cell apoptosis and inhibiting protein synthesis in HT1080 cells than in MCF-7 cells. Intriguingly, with enhanced stability and pH/MMP dual responsiveness, TPpG notably inhibited subcutaneous HT1080 xenograft growth in mice and no noticeable off-target side effect was observed. This ingeniously designed strategy aims at providing new perspectives for the development of a smart platform that can intelligently respond to a tumor microenvironment for efficient protein delivery.

Inhibition of pokeweed antiviral protein (PAP) by turnip mosaic virus genome-linked protein (VPg).

Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome-inactivating protein (RIP) and an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin loop of large rRNA, arresting protein synthesis at the translocation step. PAP is also a cap-binding protein and is a potent antiviral agent against many plant, animal, and human viruses. To elucidate the mechanism of RNA depurination, and to understand how PAP recognizes and targets various RNAs, the interactions between PAP and turnip mosaic virus genome-linked protein (VPg) were investigated. VPg can function as a cap analog in cap-independent translation and potentially target PAP to uncapped IRES-containing RNA. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe PAP depurination activity and PAP-VPg interactions. PAP binds to VPg with high affinity (29.5 nm); the reaction is enthalpically driven and entropically favored. Further, VPg is a potent inhibitor of PAP depurination of RNA in wheat germ lysate and competes with structured RNA derived from tobacco etch virus for PAP binding. VPg may confer an evolutionary advantage by suppressing one of the plant defense mechanisms and also suggests the possible use of this protein against the cytotoxic activity of ribosome-inactivating proteins.

Production of anti-cancer immunotoxins in algae: ribosome inactivating proteins as fusion partners.

The eukaryotic green algae, Chlamydomonas reinhardtii has been shown to be capable of producing a variety of recombinant proteins, but the true potential of this platform remains largely unexplored. To assess the potential of algae for the production of novel recombinant proteins, we generated a series of chimeric proteins containing a single chain antibody (scFv) targeting the B-cell surface antigen CD22, genetically fused to the eukaryotic ribosome inactivating protein, gelonin, from Gelonium multiflorm. These unique molecules, termed immunotoxins, are encoded as a single gene that produces an antibody--toxin chimeric protein capable of delivering a cytotoxic molecule to targeted B-cells. We show that the addition of an Fc domain of a human IgG1 to these fusion proteins results in the production of assembled dimeric immunotoxins, containing two cell binding scFvs and two gelonin molecules. Additionally, we demonstrate that these algal expressed proteins are capable of binding and reducing the viability of B-cell lymphomas, while treatment of T-cells, that lack the CD22 antigen, had no impact on cell viability. Since other protein expression platforms are incapable of folding and accumulating these complex immunotoxins as soluble and enzymatically active proteins, our studies document a novel and efficient method for immunotoxin production.

Expression of curcin-transferrin receptor binding peptide fusion protein and its anti-tumor activity.

Curcin can inhibit the proliferation of tumor cells and promote tumor cell apoptosis, but the cytotoxicity of curcin is not selective for tumors or normal cells. In order to enhance the targeting of the anti-tumor ability of curcin, a transferrin receptor (TfR) binding peptide, TfRBP9, was fused with curcin. The curcin-TfRBP9 gene was cloned into pQE-30 and the recombinant vector pQE-30-curcin-TfRBP9 was established. Then the recombinant vector pQE-30-curcin-TfRBP9 was transferred into Escherichia coli M15. After being induced by 0.5mM IPTG for 6h at 37°C, the expressed quantity of the recombinant protein was about 30% of the total protein. Recombinant curcin-TfRBP9 was expressed in the form of an inclusion body. After dissolution, purification and renaturation, the purity of the recombinant curcin-TfRBP9 reached 95%. Immunofluorescence analysis showed that the TfRBP9 significantly enhanced the ability of the curcin binding to HepG2, and was enriched in the cytoplasm. The curcin-TfRBP9 fusion protein had significant proliferation inhibition effects on the HepG2 cells that over-expressed transferrin receptors, had lower inhibitory effects on the SKBR-3 cells that expressed low transferrin receptors, and had the lowest inhibitory effects on the LO-2 cells that were normal human liver cells. Compared with curcin, the curcin-TfRBP9 induced higher apoptosis rates in the HepG2 cells.

Small structural differences of targeted anti-tumor toxins result in strong variation of protein expression.

Targeted anti-tumor toxins consist of a toxic functional moiety that is chemically linked or recombinantly fused to a cell-directing ligand. Ribosome-inactivating proteins (RIPs), especially type I RIPs such as saporin or dianthin, are commonly used as toxin components. Although expression of type I RIP-based fusion proteins is well reported, the achievement of higher protein yields in heterologous expression systems through innovative strategies is of major interest. In the present study, the targeted toxins (his)saporin-EGF (SE) and (his)dianthin-EGF (DE) were expressed as fusion proteins under identical expression conditions. However, the total amount of DE was nearly two-times higher than SE. The identity of the heterologously expressed targeted toxins was confirmed by mass spectrometric studies. Their biological specific activity, monitored in real time, was almost equal. Sequence alignment shows 84% identity and a structural comparison revealed five major differences, two of which affect the secondary structure resulting in a loop (SE) to ß-strand (DE) conversion and one introduces a gap in SE (after position 57). In conclusion, these structural variations resulted in different protein expression levels while codon usage and toxicity to bacteria were excluded as a cause. Minor structural differences identified in this study may be considered responsible for the protection of DE from bacterial proteases and therefore may serve as a lead to modify certain domains in type I RIP-based targeted toxins.

Convergent potency of internalized gelonin immunotoxins across varied cell lines, antigens, and targeting moieties.

Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two different tumor antigens (carcinoembryonic antigen and EGF receptor). Constructs were characterized using an antigen-negative cell line (HT-1080), cell lines positive for each antigen (HT-1080(CEA) for carcinoembryonic antigen and A431 for EGF receptor), and a cell line positive for both antigens (HT-29). Immunotoxins exhibited K(d) values between 8 and 15 nm and showed 20-2000-fold enhanced cytotoxicity compared with gelonin (IC(50) ∼ 0.25-30 nM versus 500 nM). Using quantitative fluorescence flow cytometry, we measured internalization of gelonin (via pinocytosis) and gelonin-based immunotoxins (via antigen-dependent, receptor-mediated endocytosis). Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ∼ 5 × 10(6) toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.

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.

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.