Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I–truncated Pseudomonas Exotoxin A (PE40/ETA″) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA″ and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETA″ was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a well-defined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETA″, which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy.

Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei.

The procyclic form of Trypanosoma brucei colonises the gut of its insect vector, the tsetse fly. GPEET and EP procyclins constitute the parasite's surface coat at this stage of the life cycle, and the presence or absence of GPEET distinguishes between early and late procyclic forms, respectively. Differentiation from early to late procyclic forms in vivo occurs in the fly midgut and can be mimicked in culture. Our analysis of this transition in vitro delivered new insights into the process of GPEET repression. First, we could show that parasites followed a concrete sequence of events upon triggering differentiation: after undergoing an initial growth arrest, cells lost GPEET protein, and finally late procyclic forms resumed proliferation. Second, we determined the stability of both GPEET and EP mRNA during differentiation. GPEET mRNA is exceptionally stable in early procyclic forms, with a half-life >6h. The GPEET mRNA detected in late procyclic form cultures is a mixture of transcripts from both bona fide late procyclic forms and GPEET-positive 'laggard' parasites present in these cultures. However, its stability was clearly reduced during differentiation and in late procyclic form cultures. Alternatively processed GPEET transcripts were enriched in samples from late procyclic forms, suggesting that altered mRNA processing might contribute to repression of GPEET in this developmental stage. In addition, we detected GPEET transcripts with non-templated oligo(U) tails that were enriched in late procyclic forms. To the best of our knowledge, this is the first study reporting a uridylyl-tailed, nuclear-encoded mRNA species in trypanosomatids or any other protozoa.

Promises of cyclotron-produced 44Sc as a diagnostic match for trivalent β--emitters: in vitro and in vivo study of a 44Sc-DOTA-folate conjugate

In recent years, implementation of 68Ga-radiometalated peptides for PET imaging of cancer has attracted the attention of clinicians. Herein, we propose the use of 44Sc (half-life = 3.97 h, average β+ energy [Eβ+av] = 632 keV) as a valuable alternative to 68Ga (half-life = 68 min, Eβ+av = 830 keV) for imaging and dosimetry before 177Lu-based radionuclide therapy. The aim of the study was the preclinical evaluation of a folate conjugate labeled with cyclotron-produced 44Sc and its in vitro and in vivo comparison with the 177Lu-labeled pendant. Methods: 44Sc was produced via the 44Ca(p,n)44Sc nuclear reaction at a cyclotron (17.6 ± 1.8 MeV, 50 μA, 30 min) using an enriched 44Ca target (10 mg 44CaCO3, 97.00%). Separation from the target material was performed by a semiautomated process using extraction chromatography and cation exchange chromatography. Radiolabeling of a DOTA-folate conjugate (cm09) was performed at 95°C within 10 min. The stability of 44Sc-cm09 was tested in human plasma. 44Sc-cm09 was investigated in vitro using folate receptor–positive KB tumor cells and in vivo by PET/CT imaging of tumor-bearing mice Results: Under the given irradiation conditions, 44Sc was obtained in a maximum yield of 350 MBq at high radionuclide purity (>99%). Semiautomated isolation of 44Sc from 44Ca targets allowed formulation of up to 300 MBq of 44Sc in a volume of 200–400 μL of ammonium acetate/HCl solution (1 M, pH 3.5–4.0) within 10 min. Radiolabeling of cm09 was achieved with a radiochemical yield of greater than 96% at a specific activity of 5.2 MBq/nmol. In vitro, 44Sc-cm09 was stable in human plasma over the whole time of investigation and showed folate receptor–specific binding to KB tumor cells. PET/CT images of mice injected with 44Sc-cm09 allowed excellent visualization of tumor xenografts. Comparison of cm09 labeled with 44Sc and 177Lu revealed almost identical pharmacokinetics. Conclusion: This study presents a high-yield production and efficient separation method of 44Sc at a quality suitable for radiolabeling of DOTA-functionalized biomolecules. An in vivo proof-of-concept study using a DOTA-folate conjugate demonstrated the excellent features of 44Sc for PET imaging. Thus, 44Sc is a valid alternative to 68Ga for imaging and dosimetry before 177Lu-radionuclide tumor therapy.

Future prospects for SPECT imaging using the radiolanthanide terbium-155 — production and preclinical evaluation in tumor-bearing mice

We assessed the suitability of the radiolanthanide 155 Tb (t1/2 = 5.32 days, Eγ = 87 keV (32%), 105 keV (25%)) in combination with variable tumor targeted biomolecules using preclinical SPECT imaging. Methods 155Tb was produced at ISOLDE (CERN, Geneva, Switzerland) by high-energy (~ 1.4 GeV) proton irradiation of a tantalum target followed by ionization and on-line mass separation. 155 Tb was separated from isobar and pseudo-isobar impurities by cation exchange chromatography. Four tumor targeting molecules – a somatostatin analog (DOTATATE), a minigastrin analog (MD), a folate derivative (cm09) and an anti-L1-CAM antibody (chCE7) – were radiolabeled with 155 Tb. Imaging studies were performed in nude mice bearing AR42J, cholecystokinin-2 receptor expressing A431, KB, IGROV-1 and SKOV-3ip tumor xenografts using a dedicated small-animal SPECT/CT scanner. Results The total yield of the two-step separation process of 155 Tb was 86%. 155 Tb was obtained in a physiological l-lactate solution suitable for direct labeling processes. The 155 Tb-labeled tumor targeted biomolecules were obtained at a reasonable specific activity and high purity (> 95%). 155 Tb gave high quality, high resolution tomographic images. SPECT/CT experiments allowed excellent visualization of AR42J and CCK-2 receptor-expressing A431 tumors xenografts in mice after injection of 155 Tb-DOTATATE and 155 Tb-MD, respectively. The relatively long physical half-life of 155 Tb matched in particular the biological half-lives of 155 Tb-cm09 and 155 Tb-DTPA-chCE7 allowing SPECT imaging of KB tumors, IGROV-1 and SKOV-3ip tumors even several days after administration. Conclusions The radiolanthanide 155 Tb may be of particular interest for low-dose SPECT prior to therapy with a therapeutic match such as the β--emitting radiolanthanides 177Lu, 161 Tb, 166Ho, and the pseudo-radiolanthanide 90Y.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

Analysis of ⁸⁵Kr: a comparison at the 10-14 level using micro-liter samples

The isotopic abundance of 85Kr in the atmosphere, currently at the level of 10−11, has increased by orders of magnitude since the dawn of nuclear age. With a half-life of 10.76 years, 85Kr is of great interest as tracers for environmental samples such as air, groundwater and ice. Atom Trap Trace Analysis (ATTA) is an emerging method for the analysis of rare krypton isotopes at isotopic abundance levels as low as 10−14 using krypton gas samples of a few micro-liters. Both the reliability and reproducibility of the method are examined in the present study by an inter-comparison among different instruments. The 85Kr/Kr ratios of 12 samples, in the range of 10−13 to 10−10, are measured independently in three laboratories: a low-level counting laboratory in Bern, Switzerland, and two ATTA laboratories, one in Hefei, China, and another in Argonne, USA. The results are in agreement at the precision level of 5%.

Gold nanoparticle aerosols for rodent inhalation and translocation studies

The intensive use of nano-sized particles in many different applications necessitates studies on their risk assessment as there are still open questions on their safe handling and utilization. For reliable risk assessment, the interaction of nanoparticles (NP) with biological systems after various routes of exposure needs to be investigated using well-characterized NP. We report here on the generation of gold-NP (Au-NP) aerosols for inhalation studies with the spark ignition technique, and their characterization in terms of chemical composition, physical structure, morphology, and specific surface area, and on interaction with lung tissues and lung cells after 1 h inhalation by mice. The originally generated agglomerated Au-NP were converted into compact spherical Au-NP by thermal annealing at 600 °C, providing particles of similar mass, but different size and specific surface area. Since there are currently no translocation data available on inhaled Au-NP in the 10–50 nm diameter range, the emphasis was to generate NP as small as 20 nm for inhalation in rodents. For anticipated in vivo systemic translocation and dosimetry analyses, radiolabeled Au-NP were created by proton irradiating the gold electrodes of the spark generator, thus forming gamma ray emitting 195Au with 186 days half-life, allowing long-term biokinetic studies. The dissolution rate of 195Au from the NP was below detection limits. The highly concentrated, polydisperse Au-NP aerosol (1–2 × 107 NP/cm3) proved to be constant over several hours in terms of its count median mobility diameter, its geometric standard deviation and number concentration. After collection on filters particles can be re-suspended and used for instillation or ingestion studies.

Mechanisms regulating the p120-catenin/Kaiso pathway

The Wnt pathways contribute to many processes in cancer and developmental biology, with β-catenin being a key canonical component. P120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ/ zinc-finger transcription factor Kaiso. In my first project, employing Xenopus embryos and mammalian cell lines, I found that the degradation machinery of the canonical Wnt pathway modulates p120-catenin protein stability, especially p120 isoform-1, through mechanisms shared with b-catenin. Exogenous expression of destruction-complex components such as GSK3b or Axin promotes p120-catenin degradation, and consequently, is able to rescue developmental phenotypes resulting from p120 over-expression during early Xenopus embryonic development. Conversely, as predicted, the in vivo depletion of either Axin or GSK3b coordinately increased p120 and b-catenin levels, while p120 levels decreased upon LRP5/6 depletion, which are positive modulators in the canonical Wnt pathway. At the primary sequence level, I resolved conserved GSK3b phosphorylation sites in p120’s (isoform 1) amino-terminal region. Point-mutagenesis of these residues inhibited the association of destruction complex proteins including those involved in ubiquitination, resulting in p120-catenin stabilization. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and d-catenin, in common with b-catenin and p120, associate with Axin, and are degraded in Axin’s presence. Thus, by similar means, it appears that canonical Wnt signals coordinately modulate multiple catenin proteins having roles in development and conceivably disease states. In my second project, I found that the Dyrk1A kinase exhibits a positive effect upon p120-catenin levels. That is, unlike the negative regulator GSK3b kinase, a candidate screen revealed that Dyrk1A kinase enhances p120-catenin protein levels via increased half-life. Dyrk1A is encoded by a gene located within the trisomy of chromosome 21, which contributes to mental retardation in Down Syndrome patients. I found that Dyrk1A expression results in increased p120 protein levels, and that Dyrk1A specifically associates with p120 as opposed to other p120-catenin family members or b-catenin. Consistently, Dyrk1A depletion in mammalian cell lines and Xenopus embryos decreased p120-catenin levels. I further confirmed that Dyrk overexpression and knock-down modulates both Siamois and Wnt11 gene expression in the expected manner based upon the resulting latered levels of p120-catenin. I determined that Dyrk expression rescues Kaiso depletion effects (gastrulation failure; increased endogenous Wnt11 expression), and vice versa. I then identified a putative Dyrk phosphorylation region within the N-terminus of p120-catenin, which may also be responsible for Dyrk1A association. I went on to make a phosphomimic mutant, which when over-expressed, had the predicted enhanced capacity to positively modulate endogenous Wnt11 and Siamois expression, and thereby generate gastrulation defects. Given that Dyrk1A modulates Siamois expression through stabilization of p120-catenin, I further observed that ectopic expression of Dyrk can positively influence b-catenin’s capacity to generate ectopic dorsal axes when ventrally expressed in early Xenopus embryos. Future work will investigate how Dyrk1A modulates the Wnt signaling pathway through p120-catenin, and possibly begin to address how dysfunction of Dyrk1A with respect to p120-catenin might relate to aspects of Down syndrome. In summary, the second phase of my graduate work appears to have revealed a novel aspect of Dyrk1A/p120-catenin action in embryonic development, with a functional linkage to canonical Wnt signaling. What I have identified as a “Dyrk1A/p120-catenin/Kaiso pathway” may conceivably assist in our larger understanding of the impact of Dyrk1A dosage imbalance in Down syndrome.

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite.

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.

[New oral anticoagulants - influence on coagulation tests].

The new oral anticoagulants (NOACs) represent alternative antithrombotic agents for prophylaxis and therapy of thromboembolic diseases. They act either by inhibition of the clotting factor Xa or IIa (thrombin). As a consequence, they influence several coagulation assays (for example prothrombin time, activated partial thromboplastin time). Because of the short half-life of these new agents, these changes show great variations in the course of 24 hours. Furthermore, there are significant differences of laboratory results depending on the used reagents. We explain the influence of apixaban, rivaroxaban (factor Xa inhibitors) and dabigatran (thrombin inhibitor) on the most commonly used coagulation assays. Besides we show that this influence depends on the way of action of the drug as well as on the principle of the coagulation assay. Being aware of this relationships helps to interpret the results of coagulation assays under influence of NOACs correctly.

Characterization and mechanism of action of suppressor factors derived from human T cell hybridomas

This laboratory developed human T-cell hybridomas which constitutively secrete suppressor factors (SF) capable of inhibiting immune responses (Hybridoma 6:589 (1987). The mechanisms by which human T-cell hybridoma-derived SFs (designated 160 and 169) and Jurkat leukemic T-cell line derived SF inhibit the proliferative response to mitogen by human PBMC were investigated. The Jurkat SF had a pI of 5.2 whereas the 160 and 169 SF had pI of 5.7 and 4.7 (two peaks) and 4.7, respectively. The SF was not transforming growth factor-beta based upon neutralization and iummunoprecipitation experiments with anti-TGF-beta polyclonal antibody. Il-2 production by human PBMC cultured with Con A or OKT3 mAb in the presence of SF was found to be inhibited by greater than 80%. The proliferative responses of SF treated PBMC could not be restored by addition of exogeneous human IL-2. Inhibition of the proliferative responses could not be reversed by addition of exogenous rIL-1, rIL-2 or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on Con A activated cultures time points was not affected by treatment with any SFs. Both the 160 and 169 hybridoma-derived SFs were found to arrest PHA induced cell cycle progression in G$\sb0$/G$\sb1$ phase, whereas SF from the Jurkat T-cell line arrested progression in the S phase. Pretreatment of PBMC with SF prior to the addition of mitogen, followed by washing, did not alter the proliferative response of these PBMC nor their cell cycle progression suggesting that cell activation is necessary for these SF to inhibit proliferative responses. Northern blot analysis of total mRNA from mitogen stimulated PBMC in the presence of SF, revealed a time dependent accumulation of an IL-2 specific mRNA of increased size (2.8 kB) in addition to the expected 1.0 kB mature IL-2 message. Interferon-gamma mRNA was of the appropriate size but its half-life was prolonged in SF treated cultures. IL-2 receptor and IL-1 beta mRNA expression was not altered in these cells. ^

The cyclotron production and nuclear imaging of bromine-77

In this investigation, bromine-77 was produced with a medical cyclotron and imaged with gamma cameras. Br-77 emits a 240 kev photon with a half life of 56 hours. The C-Br bond is stronger than the C-I bond and bromine is not collected in the thyroid. Bromine can be used to label many organic molecules by methods analogous to radioiodination. The only North American source of Br-77 in the 70's and 80's was Los Alamos National Laboratory, but it discontinued production in 1989. In this method, a p,3n reaction on Br-77 produces Kr-77 which decays with a 1.2 hour half life to Br-77. A cyclotron generated 40 MeV proton beam is incident on a nearly saturated NaBr or LiBr solution contained in a copper or titanium target. A cooling chamber through which helium gas is flowed separates the solution from the cyclotron beam line. Helium gas is also flowed through the solution to extract Kr-77 gas. The mixture flows through a nitrogen trap where Kr-77 freezes and is allowed to decay to Br-77. Eight production runs were performed, three with a copper target and five with a titanium target with yields of 40, 104, 180, 679, 1080, 685, 762 and 118 uCi respectively. Gamma ray spectroscopy has shown the product to be very pure, however corrosion has been a major obstacle, causing the premature retirement of the copper target. Phantom and in-vivo rat nuclear images, and an autoradiograph in a rat are presented. The quality of the nuclear scans is reasonable and the autoradiograph reveals high isotope uptake in the renal parenchyma, a more moderate but uniform uptake in pulmonary and hepatic tissue, and low soft tissue uptake. There is no isotope uptake in the brain or the gastric mucosa. ^

SYNTHESIS AND BIOLOGICAL EVALUATION OF ANTITUMOR ALDOPHOSPHAMIDE ACETALS

Although the major metabolic pathways of cyclophosphamide are well established, the mechanism of antitumor drug selectivity is highly controversial. However, it is widely accepted that aldophosphamide, one of the primary metabolites, plays a crucial role in drug selectivity. In an attempt to gain a better understanding of the mechanism of selectivity of cyclophosphamide, a series of aldophosphamide analogs have been synthesized.^ The new analogs, unlike aldophosphamide, are relatively stable in neutral solution; however, they are converted rapidly to aldehydo intermediates in the presence of carboxylate esterase. Due to structural differences, these analogs may be classified into three different groups, arbitrarily designated as A, B, C, depending upon the facility with which the intermediate aldehydes form 4-hydroxy cyclic tautomers. The half-life of the aldehydo/4-hydroxy cyclic tautomeric mixture is longer for bis(acetoxy)aldophosphamide acetal I (a representative of group A), shorter for the n-ethyl analog III (B), and shortest for the N,N-dimethyl analog IV (C). The ratio of aldophosphamide: 4-hydroxycyclophosphamide at pseudoequilibrium is 1: 4 for compound I, 1: 2 for compound III and 0: 1 for compound IV. The therapeutic efficacy of these compounds are group A $>$ group B $>$ group C. It is apparent that the equilibrium position between the aldehydo and 4-hydroxy cyclic tautomers, which determines their stability, is a crucial determinant of both the cytotoxicity and antitumor selectivity. These findings, taken in conjunction with the aldehyde dehydrogenase selectivity hypothesis, may provide an explanation for the unique antitumor activity of cyclophosphamide. ^

The pharmacokinetics of dexmedetomidine administered as a constant rate infusion in horses.

Dexmedetomidine, the most selective α2 -adrenoceptor agonist in clinical use, is increasingly being used in both conscious and anaesthetized horses; however, the pharmacokinetics and sedative effects of this drug administered alone as an infusion are not previously described in horses. Seven horses received an infusion of 8 μg dexmedetomidine/kg/h for 150 min, venous blood samples were collected, and dexmedetomidine concentrations were assayed using liquid chromatography-mass spectrometry (LC/MS) and analyzed using noncompartmental pharmacokinetic analysis. Sedation was scored as the distance from the lower lip of the horse to the ground measured in centimetre. The harmonic mean (SD) plasma elimination half-life (Lambda z half-life) for dexmedetomidine was 20.9 (5.1) min, clearance (Cl) was 0.3 (0.20) L/min/kg, and volume of distribution at steady-state (Vdss ) was 13.7 (7.9) L/kg. There was a considerable individual variation in the concentration of dexmedetomidine vs. time profile. The level of sedation covaried with the plasma concentration of dexmedetomidine. This implies that for clinical use of dexmedetomidine constant rate infusion in conscious horses, infusion rates can be easily adjusted to effect, and this is preferable to an infusion at a predetermined value.

Novel Prodrug-Like Fusion Toxin with Protease-Sensitive Bioorthogonal PEGylation for Tumor Targeting

Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients. Site-specific modification to convert ETA into a prodrug-like toxin which is reactivated specifically in the tumor, and at the same time has a longer circulation half-life and is less immunogenic, is therefore appealing. To engineer a prodrug-like fusion toxin consisting of the anti-EpCAM DARPin Ec1 and a domain I-deleted variant of ETA (ETA″), we used strain-promoted azide alkyne cycloaddition for bioorthogonal conjugation of linear or branched polyethylene glycol (PEG) polymers at defined positions within the toxin moiety. Reversibility of the shielding was provided by a designed peptide linker containing the cleavage site for the rhinovirus 3C model protease. We identified two distinct sites, one within the catalytic domain and one close to the C-terminal KDEL sequence of Ec1-ETA″, simultaneous PEGylation of which resulted in up to 1000-fold lower cytotoxicity in EpCAM-positive tumor cells. Importantly, the potency of the fusion toxin was fully restored by proteolytic unveiling. Upon systemic administration in mice, PEGylated Ec1-ETA″ was much better tolerated than Ec1-ETA″; it showed a longer circulation half-life and an almost 10-fold increased area under the curve (AUC). Our strategy of engineering prodrug-like fusion toxins by bioorthogonal veiling opens new possibilities for targeting tumors with more specificity and efficacy.