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 IC(50) of 6 x 10(-11) M against U937 cells, thus demonstrating the suitability of this expression platform for secretion of toxic saporin-based chimeras.

Ketanserin-induced baroreflex enhancement in spontaneously hypertensive rats depends on central 5-HT(2A) receptors.

1. Ketanserin may influence baroreflex function by blocking 5-HT(2A) receptors and/or alpha(1)-adrenoceptors through central and/or peripheral mechanisms. 2. In the present study, we tested the hypothesis that the baroreflex sensitivity (BRS)-enhancing effects of ketanserin are mediated by central 5-HT(2A) receptors in spontaneously hypertensive rats (SHR). 3. Using a conjugate of a monoclonal antibody to the serotonin reuptake transporter (SERT) and the toxin saporin (anti-SERT-SAP), which specifically eliminates the neurons that express SERT, the effects of ketanserin (0.3 and 3.0 mg/kg, i.g.) on BRS, blood pressure (BP), heart period (HP) and blood pressure variability (BPV) were compared between conscious intact SHR and SHR pretreated with anti-SERT-SAP. 4. Immunochemistry showed that, 2 weeks after intracerebroventricular injection of the toxin, 5-HT expression was strikingly attenuated in the brain, whereas values of BRS, BPV and BP were similar to those in the sham group. In intact SHR, 0.3 mg/kg ketanserin significantly improved BRS (191% control) and reduced BPV without affecting BP; at 3.0 mg/kg, ketanserin significantly increased BRS (197% control) and decreased BPV and BP. In toxin-pretreated SHR, only the high dose of ketanserin improved BRS (132% control), neither of the ketanserin doses reduced BPV, but both significantly decreased BP. 5. We conclude that the BRS-enhancing effects of ketanserin are mediated largely by central 5-HT(2A) receptors, whereas the antihypertensive effect of ketanserin persists even after destruction of serotonergic neurons in the central nervous system.

Characterization of highly toxic type 2 ribosome-inactivating proteins from Adenia lanceolata and Adenia stenodactyla (Passifloraceae).

From the caudices of the Passifloraceae Adenia lanceolata and A. stenodactyla, two lectins called lanceolin and stenodactylin, respectively, were purified by affinity chromatography on CL Sepharose 6B. The lectins are glycoproteins with M(r) 61,243 (lanceolin) and 63,131 (stenodactylin), consisting of an enzymatic A chain linked to a larger B chain with lectin properties, with N-terminal amino acid sequences similar to that of volkensin, the toxic lectin from A. volkensii. The lectins agglutinate red blood cells, inhibit protein synthesis both by a cell-free system and by whole cells, and depurinate ribosomes and DNA, but not tRNA or poly(A). They are highly toxic to cells, in which they induce apoptosis, and to mice, with LD(50)s 8.16 microg/kg (lanceolin) and 2.76 microg/kg (stenodactylin) at 48 h. Thus, lanceolin and stenodactylin have all the properties of the toxic type 2 ribosome-inactivating proteins and are amongst the most potent toxins of plant origin.

Decreased vesicular acetylcholine transporter and alpha(4)beta(2) nicotinic receptor density in the rat brain following 192 IgG-saporin immunolesioning.

Degeneration of cholinergic neurons is a well known characteristic of Alzheimer's disease (AD). Two radioligands were studied in a rat model of cholinergic degeneration to evaluate their potential efficacy for molecular imaging of AD. Following specific cholinergic-cell immunolesioning with 192 IgG-saporin (SAP), ex vivo autoradiography was performed with (123)IBVM, a radioligand which targets the vesicular acetylcholine transporter (VAChT). Following the decay of (123)I, the same animals had in vitro autoradiography performed with (125)I-A-85380, a marker for nicotinic acetylcholine receptors (nAChRs). As expected significant, widespread decreases in (123)IBVM uptake were observed in SAP treated animals. Moderate but significant reductions in (125)I-A-85380 binding in the hippocampus (Hip) and cerebellum (Cbm) were also observed following SAP immunolesioning. The results with (123)IBVM confirm and extend previous work investigating the uptake of radioiodinated IBVM in this animal model. The results with (125)I-A-85380 are unique and are in contrast with work performed in this animal model with other nAChR radioligands, indicating the favourable properties of this radioligand for molecular imaging.

In vitro and in vivo toxicity of type 2 ribosome-inactivating proteins lanceolin and stenodactylin on glial and neuronal cells.

Lanceolin and stenodactylin, new type 2 ribosome-inactivating proteins (RIPs) from Adenia plants were recently isolated and their high cytotoxicity was described. Present experiments were performed to investigate the effect of these toxins on neural cells in culture and their in vivo retrograde transport and neurotoxicity in the central nervous system. The concentrations of lanceolin and stenodactylin inhibiting by 50% protein synthesis were in the 10(-11) and 10(-12) (cerebellar granule neurons), 10(-12) and 10(-13) (astrocytes), and 10(-13) (microglia) molar range, respectively. Both RIPs resulted toxic for glial cells in culture by MTT test, killing 50% of microglia, the most sensitive cell type, at concentrations around 10(-14)M. Stenodactylin was highly neurotoxic in vivo, when injected intracerebrally, and was retrogradely transported through axons projecting to the injected region. Stereotaxic injection of 1.3 ng toxin into the left dorsal hippocampus resulted in loss of cholinergic neurons in the ipsilateral medial septal nucleus, where cell bodies of neurons providing cholinergic input to the hippocampus are located. The retrograde transport of RIPs along neurons allows to perform experiments of target-selective lesioning, and can be exploited also to perform specific experiments of immunolesioning of selected neuronal populations.

Astrocytic reaction to a lesion, under hormonal deprivation.

Gonadal hormones can influence the morphology and function of glial cells, particularly astrocytes. Here we explore the hypothesis that 17beta-estradiol (E2) exerts a positive effect on astrocytes within the region of the cholinergic neurons of the basal forebrain, an area heavily implicated in memory and attentional processes. Female rats were ovariectomized at 3 months of age and lesioned with the immunotoxin 192 IgG-saporin before receiving a subcutaneous pellet containing 0.25mg of estrogen or placebo, released over 60 days. The control, non-ovariectomized group was treated identically. At the end of the treatment, we used image analysis procedures to evaluate changes in the levels of glial fibrillary acidic protein (GFAP) expression in the area of the lesion. Infusion of the immunotoxin induced a slight increase in GFAP expression in some subjects, compared to the contralateral side. However, when differences within animals where factored in, GFAP expression in ovariectomized animals treated with E2 was undistinguishable from intact controls. By contrast, in ovariectomized animals treated with placebo, GFAP expression was significantly higher. These results suggest that E2 deprivation may exacerbate the effects of an immunotoxic lesion, and, more importantly, that E2 administration may contribute to structural recovery of lesioned cholinergic neurons by blocking GFAP expression in the area. These results are particularly relevant in the context of female aging and postmenopausal dementia, and further highlight other potential levels at which to design interventions to preserve an intact cholinergic system, which may be crucial to prevent Alzheimer's disease.

Up-regulation of cation-independent mannose 6-phosphate receptor and endosomal-lysosomal markers in surviving neurons after 192-IgG-saporin administrations into the adult rat brain.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is a single transmembrane domain glycoprotein that plays a major role in the trafficking of lysosomal enzymes from the trans-Golgi network to the endosomal-lysosomal (EL) system. Because dysfunction of EL system is associated with a variety of neurodegenerative disorders, it is possible that the CI-MPR may have a role in regulating neuronal viability after toxicity/injury. In the present study, we report that 192-IgG-saporin-induced loss of basal forebrain cholinergic neurons causes a transient up-regulation of CI-MPR protein levels in surviving neurons of the basal forebrain and frontal cortex but not in the brainstem region, which was relatively spared by the immunotoxin. This was accompanied by a parallel time-dependent increase in other EL markers, ie, cathepsin D, Rab5, and LAMP2 in the basal forebrain region, whereas in the frontal cortex the levels of cathepsin D, and to some extent Rab5, were increased. Given the critical role of the EL system in the clearance of abnormal proteins in response to changing conditions, it is likely that the observed increase in the CI-MPR and components of the EL system in surviving neurons after 192-IgG-saporin treatment represents an adaptive mechanism to restore the metabolic/structural abnormalities induced by the loss of cholin-ergic neurons.

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain.

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.

Ribosome-inactivating proteins: progress and problems.

Ribosome-inactivating proteins (RIPs), mostly from plants, are enzymes which depurinate rRNA, thus inhibiting protein synthesis. They also depurinate other polynucleotide substrates. The biological activity of RIPs is not completely clarified, and sometimes independent of the inhibition of protein synthesis. There are differences in the cytotoxicity of RIPs and, consequently, in their toxicity to animals. Some RIPs are potent toxins, the best known being ricin, a potential biological weapon. New toxins have recently been identified. RIPs cause apoptotic and necrotic lesions, and induce production of cytokines causing inflammation. RIPs are potentially useful in agriculture and medicine because (i) they have antiviral activity and (ii) they are used for the preparation of conjugates with antibodies ('immunotoxins') or other carriers, rendering them specifically toxic to the cell target of the carrier, which may be helpful in therapy. The distribution, mechanism of action and role in nature of RIPs are not completely understood, and we can expect several future developments in their practical application.

Specific dose-dependent damage of Lieberkühn crypts promoted by large doses of type 2 ribosome-inactivating protein nigrin b intravenous injection to mice.

Nigrin b is a non-toxic type 2 ribosome-inactivating protein as active as ricin at ribosomal level but 10(5) and 5 x 10(3) times less toxic for animal cell cultures and mice, respectively, than ricin. The purpose of the present study was to analyze the effects of intravenous injection of large amounts of nigrin b to the mouse. Injection through the tail vein of 16 mg/kg body weight killed all mice studied before 2 days. Analysis of several major tissues by light microscopy did not reveal gross nigrin b-promoted changes, except in the intestines which appeared highly damaged. As a consequence of the injury, the villi and crypt structures of the small intestine disappeared, leading to profuse bleeding and death. In contrast, intravenous injection of 5 mg/kg body weight was not lethal to mice but did trigger reversible toxic effects. In both cases, lethal and sub-lethal doses, the target of nigrin b appeared to be the highly proliferating stem cells of the intestinal crypts, which had undergone apoptotic changes. In contrast to nigrin b, the injection of 3 mug/kg of ricin kills all mice in 5 days but does not trigger apoptosis in the crypts. Therefore, the effect seen with sub-lethal nigrin b concentrations seems to be specific. Nigrin b killed COLO 320 human colon adenocarcinoma cells with an IC(50) of 3.1 x 10(-8) M and the effect was parallel to the extent of DNA fragmentation of these cells. Accordingly, despite the low general toxicity exerted by nigrin b as compared with ricin, intravenous injection of large amounts of nigrin b is able to kill mouse intestinal stem cells without threatening the lives of the animals, thereby opening a door for its use for the targeting of intestinal stem cells.

Mitochondrial DNA D-loop as a new target of Saporin 6 nuclease activity.

The single-chain ribosome-inactivating proteins (RIPs) from plant origin, including Saporin 6 from the seeds of Saponaria officinalis, are ribotoxins known to act as N-glycosidases which depurinate the conserved alpha sarcin loop of large rRNAs. As a consequence, the eukaryotic ribosomes become inactivated, thereby arresting the protein synthesis at the elongation step. RIPs are currently under study as antiviral and antiproliferative agents. Additional in vitro activities of RIPs against either RNA or DNA have been recently described. A specific nuclease activity on plasmidic DNA was demonstrated by either purified or bacterial-recombinant molecules. We report here that human mitochondrial DNA (mtDNA) is a new specific target of Saporin 6 nuclease activity. A unique site of cleavage has been identified and mapped within the most variable part of the D-loop region of the covalently closed circular mtDNA molecule.

Effects of isolectin B4-conjugated saporin, a targeting cytotoxin, on bladder overactivity induced by bladder irritation.

In order to clarify the functional role of the isolectin B4 (IB4)-binding afferent pathway in the micturition reflex, we investigated the effects on bladder activity of intrathecal application of the IB4-saporin conjugate, a targeting cytotoxin that destroys neurons binding IB4. In rats, IB4-saporin (2.5 micro m) or vehicle was administered through an intrathecal catheter implanted at the level of the L6-S1 spinal cord. Three weeks after IB4-saporin administration, cystometry in conscious animals revealed a reduction in bladder overactive responses induced by intravesical capsaicin or ATP infusion without affecting normal voiding function. In histochemical studies, double staining for IB4 and saporin was detected in L6 dorsal root ganglia (DRG) neurons 2 days after the treatment. Three weeks after the treatment, the area in lamina II of the L6 spinal cord stained with IB4 was significantly reduced compared with the area stained in control rats. The staining in the L1 spinal cord was not affected. The percentage of neurons in the L6 DRG intensely labeled with IB4 was also reduced in IB4-saporin-treated rats. These results indicate that intrathecal treatment with the IB4-saporin conjugate at the level of L6-S1 spinal cord, which reduces IB4 afferent nerve terminal staining in lamina II of the L6 spinal cord as well as the number of IB4-binding neurons in L6 DRG, suppressed bladder overactivity induced by bladder irritation without affecting normal micturition. Thus targeting IB4-binding, non-peptidergic afferent pathways sensitive to capsaicin and adenosine 5'-triphosphate may be an effective treatment for overactivity and/or pain responses in the bladder.

Minocycline protects basal forebrain cholinergic neurons from mu p75-saporin immunotoxic lesioning.

Two prominent characteristics of Alzheimer's disease are basal forebrain cholinergic degeneration and neuroinflammation characterized by glial activation and the release of pro-inflammatory cytokines. Mu p75- saporin (SAP) is a novel immunotoxin that mimics the selective loss of basal forebrain cholinergic neurons and induces cognitive impairment in mice. We report that cholinergic cell loss in the medial septal nucleus and ventral diagonal band after i.c.v. injection of mu p75-SAP is accompanied by simultaneous activation of microglia and astrocytes in the basal forebrain region as well as significant memory loss. Consistent with a role of glial cells in the pathology of Alzheimer's disease, minocycline, a second-generation tetracycline with known anti-inflammatory and neuroprotective properties, attenuated mu p75-SAP-induced cholinergic cell loss, glial activation and transcription of downstream pro-inflammatory mediators. In addition to neuroprotection, minocycline treatment mitigated the cognitive impairment that appears to be a functional consequence of mu p75-SAP lesioning. The current study demonstrates that glial-related inflammation plays a significant role in the selective neurotoxicity of mu p75-SAP, and suggests that minocycline may provide a viable therapeutic option for degenerating cholinergic systems.

Cytochrome oxidase activity in the monkey globus pallidus and subthalamic nucleus after ablation of striatal interneurons expressing substance P receptors.

To understand functional roles of striatal interneurons in primate basal ganglia circuitry, we ablated interneurons expressing substance P (SP) receptors (SPR) in the putamen with SP-saporin, a SPR selective neurotoxin. The effect of SP-saporin injection into the putamen was evaluated by examining the loss of cholinergic interneurons and NADPHd-positive (nicotinamide adenine dinucleotide phosphate diaphorase positive) interneurons. We then analyzed regional metabolic changes using cytochrome oxidase (CO) histochemistry. CO activity in some regions of the internal and external segments of the globus pallidus (GP) in the lesioned hemisphere was lower than that in the contralateral or surrounding GP regions. CO activity in the subthalamic nucleus, however, showed no significant change. The present findings suggest that striatopallidal projection neurons exert enhanced inhibitory influence on the GP without modulatory control by the striatal SPR-expressing interneurons.

Extrinsic regulation of injury/growth-related gene expression in the inferior olive of the adult rat.

Successful axon regeneration relies on the capability of the lesioned neurons to up-regulate a specific set of injury/growth-associated genes. In the adult central nervous system, the strength of the cell body response is generally related to the distance of the injury site from the perikaryon, being stronger for proximal lesions. Nevertheless, inferior olive (IO) cells react to injury and regenerate their axons even after distal transections. To investigate the mechanisms that regulate the IO growth properties, we examined the expression of injury/growth markers (nitric oxide synthase, growth-associated protein 43 and c-Jun) after target deletion or axotomy performed at different sites along the olivocerebellar pathway. Both axon injury and target loss disclose two subsets of IO neurons distributed within precise subnuclei: one subset up-regulates all markers in all conditions, whereas the other shows a mild c-Jun expression but remains unresponsive even after a very proximal axotomy. These observations indicate that distinct subpopulations of IO cells respond to different regulatory strategies. Unresponsive neurons appear insensitive to environmental positive or negative cues, suggesting that they are intrinsically unable to set up a cellular reaction to injury. In contrast, cell body changes in reactive neurons are elicited after the removal of retrogradely transported target-derived inhibitory signals. Target loss also induces degeneration of IO cells, whose survival remains partially dependent on Purkinje targets in adulthood. Thus, the intrinsic regenerative potential of a functionally homogeneous population is regulated by multiple mechanisms, specific for distinct neuronal subsets.

Destruction of midbrain dopaminergic neurons by using immunotoxin to dopamine transporter.

1. The ability to target specific neurons can be used to produce selective neural lesions and potentially to deliver therapeutically useful moieties for treatment of disease. In the present study, we sought to determine if a monoclonal antibody to the dopamine transporter (anti-DAT) could be used to target midbrain dopaminergic neurons. 2. The monoclonal antibody recognizes the second, large extracellular loop of DAT. The antibody was conjugated to the "ribosome-inactivating protein"; saporin, and stereotactically pressure microinjected into either the center of the striatum or the left lateral ventricle of adult, male Sprague-Dawley rats. 3. Local intrastriatal injections produced destruction of dopaminergic neurons in the ipsilateral substantia nigra consistent with suicide transport of the immunotoxin. Intraventricular injections (i.c.v.) produced significant loss of dopaminergic neurons in the substantia nigra and ventral tegmental area bilaterally without evident damage to any other aminergic structures such as the locus coeruleus and raphe nuclei. To confirm the anatomic findings, binding of [3-H]mazindol to DAT in the striatum and midbrain was assessed using densitometric analysis of autoradiograms. Anti-DAT-saporin injected i.c.v. at a dose of 21 microg, but not 8 microg, produced highly significant decreases in mazindol binding consistent with loss of the dopaminergic neurons. 4. These results show that anti-DAT can be used to target midbrain dopaminergic neurons and that anti-DAT-saporin may be useful for producing a lesion very similar to the naturally occurring neural degeneration seen in Parkinson's disease. Anti-DAT-saporin joins the growing list of neural lesioning agents based on targeted cytotoxins.

Drosophila S3 ribosomal protein accelerates repair of 8-oxoguanine performed by human and mouse cell extracts.

The S3 ribosomal protein of Drosophila melanogaster possesses various DNA repair activities, including the capacity to incise at apurinic/apyrimidinic (AP) sites and 8-oxo-7,8-dihydroguanine (8-oxoG) residues. We have recently hypothesized that this multifunctional protein may improve the efficiency of DNA base excision repair (BER) in mammalian cells. We have investigated the effect of pure GST-tagged Drosophila S3 on BER of different endogenous lesions performed by human and mouse cell extracts. Drosophila S3 significantly accelerated the BER of 8-oxoG (initiated by the bifunctional glycosylase OGG1). The stimulating effect was linked to the capacity of S3 to remove the 8-oxoG lesion and cleave the resulting AP site, rather than acceleration of downstream steps of the BER pathway (e.g., removal of 3' blocking fragments). No stimulating effect was observed on the BER of uracil, natural AP sites, and beta-lyase-cleaved AP sites. Heterologous expression of Drosophila S3 may be used to enhance 8-oxoG repair in human cells.

Cytotoxicity of ribosome-inactivating protein saporin is not mediated through alpha2-macroglobulin receptor.

Saporin is a single chain ribosome-inactivating protein produced by the plant Saponaria officinalis. Several isoforms of saporin have been isolated from various parts of the plant. In the present study recombinant saporin isoforms 5 and 6 were produced in Escherichia coli. Saporin-6 was found to be more active than saporin-5 in its N-glycosidase, cytotoxic, and genomic DNA fragmentation activities. Earlier, saporin has been shown to bind low-density lipoprotein receptor-related protein (LRP), however, in this study the sensitivities of LRP-negative and LRP-positive cell lines were found to be similar towards saporin-6 toxicity suggesting the internalization of saporin not to be solely dependent on the expression of LRP on eukaryotic cells.

Cytotoxic activity of gonadotropin-releasing hormone (GnRH)-pokeweed antiviral protein conjugates in cell lines expressing GnRH receptors.

Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein isolated from the leaves of Phytolacca americana, has potent cytotoxic activity once it enters the cytoplasm of a cell. It is incapable of entering cells by itself. Therefore, our objective was to determine whether a GnRH analog could be used to deliver PAP specifically to cells expressing GnRH receptors. D-Lys(6)-GnRH-Pro(9)-ethylamide was conjugated to PAP (GnRH-PAP). Chinese hamster ovary cells stably transfected with cDNA for the murine GnRH receptor and a mouse gonadotroph tumor cell line that expresses endogenous GnRH receptors (alphaT3-1 cells) were used to evaluate the cytotoxic effects of GnRH-PAP. We also examined cytotoxicity of GnRH-PAP using human endometrial, breast, and prostate cancer cell lines. Treatment of GnRH receptor-positive cells with GnRH-PAP resulted in dose-dependent cytotoxicity. Cytotoxicity of GnRH-PAP was dependent on number of GnRH receptors (r(2) = 0.871, P < 0.05) and duration of exposure of GnRH-PAP to the cells. In contrast, GnRH-PAP was not cytotoxic to Chinese hamster ovary cells not harboring GnRH receptors. Moreover, the cytotoxic activity of GnRH-PAP could be inhibited by addition of excess GnRH analog. Neither PAP nor GnRH analog alone was cytotoxic. These results suggest that GnRH analogs can be used to specifically deliver toxin molecules to cells that express GnRH receptors. Thus, a new class of biomedicines that act as hormonotoxins against cells expressing GnRH receptors provides a novel approach for inhibiting reproduction and treating cancers that are dependent on reproductive hormones.

A cleavable adapter to reduce nonspecific cytotoxicity of recombinant immunotoxins.

One of the problems associated with the administration of immunotoxins is hypersensitivity reaction such as vascular leak syndrome. This may be prevented by decreasing the plasma half-life. To improve immunotoxins with respect to reduced side effects, we have previously described the development of a cleavable adapter. This adapter links the toxic moiety and ligand that are usually directly coupled. In our study, the cytotoxicity of saporin linked either directly or via the adapter to epidermal growth factor (EGF) was evaluated in vitro. The immunotoxins exhibited similar cytotoxic activity towards A-431 and HER14 cells (IC(50) < 10 nM). The supernatant from 6 hr cultures of HER14 cells incubated in the presence of the adapter-containing immunotoxin exhibited a significantly reduced cytotoxicity as compared to the directly coupled immunotoxin. Western blotting revealed that the adapter was cleaved, thus supporting our proposal that cleavable adapters may reduce nonspecific effects. A similar reduced half-life was detected in platelet-poor plasma. In contrast MCF-7 cells remain unaffected by the immunotoxins. This was shown to be due to the absence of detectable EGF-receptor in comparison to A-431 and HER14 cells as determined by Western blotting. Furthermore, we could show that the adapter does not exert an effect on the N-glycosidase activity of saporin. These results suggest that the use of cleavable adapters may be a useful tool in immunotoxins for reducing the killing of surrounding noncancerous cells due to nonspecific binding.