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Potent antitumoral activity of TRAIL through generation of tumor-targeted single-chain fusion proteins

Potent antitumoral activity of TRAIL through generation of tumor-targeted single-chain fusion... Citation: Cell Death and Disease (2010) 1, e68; doi:10.1038/cddis.2010.45 & 2010 Macmillan Publishers Limited All rights reserved 2041-4889/10 www.nature.com/cddis Potent antitumoral activity of TRAIL through generation of tumor-targeted single-chain fusion proteins 1 1 2 3 4 5 ,1 1 B Schneider ,S Mu ¨ nkel , A Krippner-Heidenreich , I Grunwald , WS Wels , H Wajant , K Pfizenmaier* and J Gerspach In an attempt to improve TRAIL’s (tumor necrosis factor-related apoptosis-inducing ligand) tumor selective activity a variant was designed, in which the three TRAIL protomers are expressed as a single polypeptide chain (scTRAIL). By genetic fusion with a single-chain antibody fragment (scFv) recognizing the extracellular domain of ErbB2, we further equipped scTRAIL with tumor- targeting properties. We studied tumor targeting and apoptosis induction of scFv–scTRAIL in comparison with non-targeted scTRAIL. Importantly, the tumor antigen-targeted scTRAIL fusion protein showed higher apoptotic activity in vitro, with a predominant action by TRAIL-R2 signaling. Pharmacokinetic studies revealed increased plasma half-life of the targeted scTRAIL fusion protein compared with scTRAIL. In vivo studies in a mouse tumor model with xenotransplanted Colo205 cells confirmed greater response to the ErbB2-specific scTRAIL fusion protein compared with non-targeted scTRAIL both under local and systemic application regimen. Together, in vitro and in vivo data give proof of concept of higher therapeutic activity of tumor-targeted scFv–scTRAIL molecules. Further, we envisage that through targeting of scTRAIL, potential side effects should be minimized. We propose that scFv-mediated tumor targeting of single-chain TRAIL represents a promising strategy to improve TRAIL’s antitumoral action and to minimize potential unwanted actions on normal tissues. Cell Death and Disease (2010) 1, e68; doi:10.1038/cddis.2010.45; published online 26 August 2010 Subject Category: Cancer Although for many malignancies traditional cytotoxic chemo- because preclinical studies revealed that, in contrast to therapy is still the treatment of choice, targeted therapies TNF and CD95 agonists, the application of trimeric, soluble gain increasing importance because they are expected TRAIL is well tolerated. Concomitantly, it demonstrated potent to exert higher tumor-specific activity and less dose-limiting antitumor activity in tumor models based on tumor cell lines 7–10 side effects. The anti-CD20 antibody Rituximab and the and primary tumor samples. Although first clinical trials in Bcr-Abl-selective kinase inhibitor Gleevec, respectively, are various advanced cancers are promising, as stable disease two vivid examples for the success of targeted therapy (for has been reported in many cases, TRAIL monotherapy review, see Gerber ). Targeting the apoptotic machinery of most likely will not result in a sufficient overall therapeutic malignant cells has become an attractive concept, beginning activity. Concurrently, this faces the potential problem with tumor necrosis factor (TNF) and its now established that under combined treatment options normal cells might clinical use for locoregional treatment of limb metastases also be sensitized to TRAIL-induced apoptosis (reviewed 2 12 of soft tissue sarcoma and melanoma and followed by by Falschlehner et al. ). Furthermore, we have recently the current exploitation of other proapoptotic members of the shown that TRAIL-R1 is activated by the soluble as well as TNF ligand family – CD95L and TNF-related apoptosis- the membrane-bound form of the ligand, whereas TRAIL-R2 inducing ligand (TRAIL) – being presently at various stages is preferentially activated by membrane TRAIL or oligo- 13,14 of preclinical and clinical development (for review, see Call merized soluble trimeric TRAIL. As agonistic TRAIL-R2- 3 4 5 et al., Gerspach et al. and Newsom-Davis et al. ). TRAIL, specific antibodies likewise display potent antitumor activity similar to TNF a type II transmembrane protein, is expressed (reviewed by Johnstone et al. ), it is conceivable that on various immune effector cells and appears critically homotrimeric TRAIL could be improved in its activity provided involved in antitumor responses in the body. It is probably efficient TRAIL-R2 signaling is ensured through genetic modi- the best candidate of a death ligand for systemic application fications or other means. On the other hand, the spontaneous 1 2 Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, Stuttgart 70569, Germany; Institute of Cellular Medicine, Newcastle University, 4th Floor Catherine Cookson Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK; Fraunhofer Institute for Manufacturing Technology and Applied Materials Research IFAM, Bonding Technology and Surfaces, Adhesives and Chemistry of Polymers, Wiener Strae 12, Bremen 28359, Germany; 4 5 Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Paul-Ehrlich-Strae 42-44, Frankfurt 60596, Germany and Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital of Wu ¨ rzburg, Ro ¨ ntgenring 11, Wu ¨ rzburg 97070, Germany *Corresponding author: K Pfizenmaier, Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, Stuttgart 70569, Germany. Tel: þ 49 711 685 66986; Fax: þ 49 711 685 67484; E-mail: klaus.pfi[email protected] Keywords: tumor targeting; single-chain TRAIL; scFv–TRAIL fusion proteins; apoptosis; ErbB2 Abbreviations: i.v., intravenous(ly); TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; TRAILR1/2, TRAIL receptor 1/2; scFv, single-chain Fv fragment; scTRAIL, single-chain TRAIL; s.c., subcutaneous(ly) Received 19.4.10; revised 02.7.10; accepted 14.7.10; Edited by R De Maria Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al aggregation of some recombinant TRAIL preparations, Both scTRAIL and scFv–scTRAIL were purified by affinity resembling secondarily crosslinked TRAIL, has been asso- chromatography on monoclonal M2 anti-FLAG agarose from ciated with unwanted actions on normal tissues and the supernatant of stably transfected HEK293 cells with yields thus curtail their use as tumor-selective apoptosis inducers of about 1 mg protein per liter cell culture supernatant. (reviewed by Koschny et al. ). Thus, TRAIL variants Immunoblot analysis and SDS-PAGE (Figure 1b) of the mimicking the full bioactivity of membrane TRAIL could purified protein showed single protein bands with a molecular present powerful cancer therapeutics provided that a tumor- mass of B70 kDa for scTRAIL and B100 kDa for scFv– restricted action can be achieved. A further apparent limitation scTRAIL matching the expected calculated molecular masses of the currently clinically investigated recombinant TRAIL of 71 and 98 kDa, respectively. Gel filtration analysis indicated reagents is their rather short in vivo half-life. Therefore, an a monomeric organization of both scTRAIL variants, corres- improvement of pharmacokinetic properties might also lead to ponding, with respect to the TRAIL part, to a noncovalently increased therapeutic action. A potential solution to increase assembled trimer of a conventional recombinant TRAIL specific activity and bioavailability is the combination of TRAIL molecule (Figure 1c). The molecular masses deduced from function with tumor targeting. Thus, we generated new TRAIL SEC were slightly lower compared with that derived from fusion proteins containing an antibody fragment (single-chain SDS-PAGE (Figure 1b), which did not result from degradation Fv fragment (scFv)) for targeting TRAIL to the tumor to enrich as verified by immunoblot analysis of collected fractions the therapeutic at the tumor site and to enhance its specific (Figure 1c). In the scTRAIL preparation, the minor fraction bioactivity. ErbB2, similar to EGFR/ErbB1, ErbB3 and ErbB4 eluting in SEC at apparent higher molecular weight, thus belonging to the ErbB receptor tyrosine kinase family, potentially comprising aggregated complexes, did not display represents a clinically relevant target antigen. It is highly disproportionately high bioactivity, as revealed from compa- expressed on a variety of human solid tumors (reviewed by rison of cytotoxic activity of fraction 2 and 15, taking relative Holbro and Hynes ) and there is already one humanized protein amounts into consideration (data not shown). monoclonal antibody (mAb) directed against the extracellular domain of ErbB2 in clinical use (Trastuzumab) and approved Target antigen-specific binding of scFv–scTRAIL. To for treatment of breast carcinoma. Several other mAbs are analyze specific binding of scFv–scTRAIL to ErbB2-positive in different stages of clinical evaluation (reviewed by cells, flow cytometry analysis was performed. ErbB2 expres- Baselga and Swain ). Although the clinical data provide sion analysis confirmed a median versus low expression clear evidence that ErbB2 is a relevant target, they also show level for Colo205 colon carcinoma and HT1080 fibrosarcoma that the therapeutic effect of the current antibody reagents cells, respectively (Figure 2a), compared with SKBR3 cells is rather limited. This justifies the search for additional that are well-known to highly express the ErbB2 protein (data ErbB2-targeted strategies. not shown). Incubation of Colo205 and HT1080 cells with Recently, a single-chain TNF molecule, consisting of three the fusion protein revealed a specific, binding to ErbB2- TNF monomers fused by short peptide linkers, was described positive cells (Figure 2a and b) compared with incubation to exert enhanced stability and antitumoral activity. We with the non-targeted scTRAIL, which resulted only in a weak have designed an analogous single polypeptide chain fluorescence signal, either by detection with anti-TRAIL TRAIL variant (scTRAIL), which was used to construct a (Figure 2a) or with anti-FLAG antibodies (data not shown). scFv–scTRAIL fusion protein for tumor targeting. We have As binding of homotrimeric FLAG-tagged TRAIL showed compared the bioactivities of this fusion protein with non- similar weak signals (data not shown), this could reflect targeted scTRAIL in in vitro and in vivo tumor models. We a low level of TRAIL receptor (TRAILR) expression on the show here that the tumor antigen-targeted scTRAIL fusion investigated target cells and/or insufficient sensitivity of protein showed higher apoptotic activity to scTRAIL in vitro, FACS analyses. ErbB2-specific binding was confirmed by with a predominant action by TRAIL-R2 signaling on Colo205 using TRAILR1- and R2-Fc fusion proteins for detection colon carcinoma cells. In vivo studies in a mouse xenograft of scFv–scTRAIL binding to target cells (Figure 2b) and tumor model confirmed significantly higher response to the competition with anti-ErbB2 antibodies (FRP5). This led to tumor-targeted ErbB2-specific scTRAIL fusion protein. a near complete inhibition of scFv–scTRAIL binding to the ErbB2-positive cell line (Figure 2b). Results Target antigen-restricted induction of cell death by Construction and production of TRAIL fusion proteins. scFv–scTRAIL. To address the bioactivity of scFv– To generate a single-chain TRAIL molecule, we followed the scTRAIL in vitro, we first analyzed induction of cell death design principle described for scTNF and covalently fused on ErbB2-negative SKW6.4 cells, which are known to be 23,24 three TRAIL monomers, each consisting of the extracellular TRAIL-R1 and TRAIL-R2 positive (Figure 3a). Although domain of TRAIL (aa 95–281) through two peptide linkers scTRAIL and scFv–scTRAIL did not show cytotoxic activity comprising four repeats of the sequence GGGS (Figure 1a). up to 8 nM, respectively, in the presence of the crosslinking To facilitate purification and analysis an N-terminal FLAG anti-FLAG antibody M2 comparable bioactivities of both tag was added. Furthermore, by additional N-terminal fusion proteins were observed (Figure 3a). In contrast, incu- fusion of a single-chain antibody fragment (scFv) recognizing bation of ErbB2-positive Colo205 and HT1080 cells the FRP5 epitope within the extracellular domain of ErbB2, with scTRAIL or scFv–scTRAIL revealed an approximately we generated an ErbB2-specific scTRAIL fusion protein three- to fivefold higher bioactivity of the ErbB2-specific (Figure 1a). scFv–scTRAIL compared with the non-targeted scTRAIL Cell Death and Disease Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al [GGGS] [GGGS] 4 4 F - C N - L TRAIL TRAIL TRAIL N - F - C L scFv TRAIL TRAIL TRAIL kDa M1 2 kDa M1 2 130 130 55 55 scTRAIL scFv-scTRAIL 443 200 66 29 443 200 66 29 Fraction 2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 kDa kDa Figure 1 Biochemical analysis of scTRAIL and scFv–scTRAIL. (a) Scheme of the scTRAIL fusion proteins used in this study. L, leader peptide; scFv, human ErbB2- specific single-chain antibody fragment; F, FLAG tag; TRAIL, human TRAIL (aa 95–281). (b) Purified scTRAIL (lane 1) and scFv–scTRAIL (lane 2) were analyzed by SDS-PAGE (10%, reducing conditions) followed by immunoblot analysis (left, 250 ng protein/lane) or silver staining (right, 1 mg protein/lane). For immunoblotting, the monoclonal anti-FLAG M2 antibody and an alkaline phosphatase-conjugated secondary antibody was used. (c) The TRAIL variants were separated by gel filtration on a BioSuite250 column. Dotted lines indicate the retention time of the molecular weight standards apoferritin (443 kDa), b-amylase (200 kDa), bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Collected fractions were analyzed by immunoblotting as in b. For scTRAIL fractions 1–9 (every second fraction; first part), fractions 11–16 (second part) and fractions 19–26 (every second fraction; third part) were immunoblotted. For scFv–scTRAIL fractions 8–24 were immunoblotted (Figure 3b; Colo205: EC50 :30 10 pM, EC50 : depended on ErbB2 target binding. Preincubation with FRP5 scFv–scTRAIL scTRAIL ± ± 130 70 pM; HT1080: EC50 :50 5pM, EC50 : or ErbB2 peptides before addition of the ErbB2-specific scFv–scTRAIL scTRAIL 140±10 pM). Furthermore, bioactivity of the targeted scFv–scTRAIL fusion protein restored cell viability of scFv–scTRAIL compared well with that of a commercially Colo205 and HT1080 cells to a significant extent (Figure 3d available high activity TRAIL, the so-called ‘Killer-TRAIL’, and e). Furthermore, as for both tested cell lines TRAIL- 13,25 with about twofold higher (Colo205) or at least comparable induced cell death is primarily mediated by TRAIL-R2, (HT1080) activity of the scFv–scTRAIL reagent (Figure 3c). the higher bioactivity of the scFv–scTRAIL on ErbB2-positive Furthermore, on target-negative SKW6.4 cells, the second- cells pointed to a membrane TRAIL-mimetic activity upon arily crosslinked scTRAIL or scFv–scTRAIL displayed specific cell surface immobilization of the fusion protein (see comparable cytotoxic activity to ‘Killer-TRAIL’ (data not below). Furthermore, inhibition of TRAIL bioactivity of the shown). From blocking studies using anti-ErbB2 anti- scFv–scTRAIL by the use of a TRAIL-neutralizing antibody led bodies (FRP5, Figure 3d) or recombinant ErbB2 peptides to complete restoration of cell viability of Colo205 and HT1080 comprising the epitope of the erbB2-specific scFv cells (Figure 3f). Thus, the ErbB2-specific scFv domain per se (Figure 3e), it became evident that the increased bioactivity did not directly contribute to apoptosis induction. Cell Death and Disease Absorbance Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al scFv–scTRAIL molecule signals apoptosis, neutralization Colo205 HT 1080 experiments with antagonistic, TRAIL-R2-specific monovalent antibody fragments (Fab fragments) were performed. Both ctrl ctrl -ErbB2 -ErbB2 ErbB2-positive cell lines showed dominant TRAIL-R2- mediated cell death at low concentrations of scFv–scTRAIL (Figure 4b), demonstrating that cell surface immobilized scFv– scTRAIL exerted membrane TRAIL-like activity, similar to what has been previously described for conventional scFv–TRAIL 13,26,27 fusion proteins. The concentration of anti-TRAIL-R2 Fab fragments used in these experiments was sufficient to almost completely block cell death of Jurkat cells induced ctrl ctrl scTRAIL scTRAIL by secondary crosslinked, homotrimeric FLAG-tagged TRAIL scFv-scTRAIL scFv-scTRAIL (Figure 4b). In addition, cell surface antigen-bound scFv– scTRAIL also activated the murine TRAILR, which is, similarly to human TRAIL-R2, not or hardly activated by soluble TRAIL. This was shown by mixed cultures using equal numbers of Colo205 cells and L929 mouse fibroblasts (Figure 4c). The latter do not express human ErbB2 and thus, in contrast to log fluorescence intensity Colo205 cells, are not able to immobilize the scFv–scTRAIL by the ErbB2-targeting domain. Thus, if scFv–scTRAIL is able to Colo205 HT1080 induce cell death in L929 cells by mimicking membrane TRAIL 30 after binding to the ErbB2 receptor on Colo205 cells, more than 50% of the cells are expected to die. Indeed, incubation with scFv–scTRAIL induced cell death in over 70% cells of the ** 1 co-culture, whereas equal molar amounts of scTRAIL induced cell death in only about 30% of the mixed cells (Figure 4c). In *** monocultures, the same concentration of scFv–scTRAIL and 0 0 scTRAIL induced cell death in about 90% of Colo205 cells, control scFv- scFv- control scFv- scFv- scTRAIL scTRAIL scTRAIL scTRAIL respectively, but not in L929 cells, confirming that, in the + + soluble form, both proteins are non-toxic to the latter (data not erbB2 erbB2 shown). Furthermore, due to the lack of human ErbB2, the Figure 2 Target antigen-specific binding of scFv–scTRAIL. (a) ErbB2 L929 cells might also be considered as antigen-negative expression of Colo205 and HT1080 cells: 5 10 cells were incubated with bystander cells, potentially occurring in a tumor tissue. Thus, anti-ErbB2 (gray filled) or isotype control antibody (black lines) followed by scFv–scTRAIL bound to antigen is expected to elicit an FITC-conjugated anti-mouse antibody and fluorescence intensity was measured by antitumoral effect also on neighboring antigen-negative cells FACS analysis. Binding of scFv–scTRAIL to ErbB2-positive Colo205 and HT1080 cells compared with non-targeted scTRAIL: 5 10 cells were incubated with within the tumor environment. 50 nM scTRAIL (gray lines) or 50 nM scFv–scTRAIL (gray filled) and, after repeated washes, bound proteins were detected using anti-human TRAIL mAb and Serum half-life of scFv–scTRAIL. To evaluate the pharmaco- FITC-labeled anti-mouse IgG. As control, cells treated with anti-humanTRAIL mAb kinetic characteristics of scFv–scTRAIL in comparison with and FITC-labeled anti-mouse IgG were used (black lines). (b) Binding competition of scTRAIL, serum samples were collected after a single dose scFv–scTRAIL to ErbB2-positive Colo205 cells by ErbB2-specific antibodies: intravenous (i.v.) injection of 400 pmol into Balb/c mice. The 5 10 Colo205 or HT1080 cells were preincubated with FRP5 or mouse IgG1 (10 mg/ml, respectively) before addition of scFv–scTRAIL (50 nM). Detection was relative serum concentrations plotted over time fitted well to carried out by TRAILR-Fc fusion proteins and FITC-labeled anti-human IgG, a two-phase exponential decay regression curve in both Fc-specific antibodies. Shown are X-fold increased mean fluorescence intensities cases. Within 20 min post-injection, both TRAIL variants (MFI) compared with the control (FITC-labeled anti-human Fc) from three displayed similar kinetics with a serum half-life of B35 min independent experiments (mean S.D.). Statistical analysis was carried out with (t a), respectively. At later time points, a slight but significant unpaired t-test, two-tailed test, **P¼ 0.0021, ***Po0.0001 2 difference in bioavailability was detected with half-lifes (t b) of 160 and 124 min and AUCs of 11 191 and 8125 for scFv– scTRAIL and scTRAIL, respectively (Figure 5a). Thus, the TRAIL-R2-mediated apoptosis induction of scFv– ErbB2-specific fusion protein was more efficiently retained in scTRAIL. In absence of the ErbB2 antigen, crosslinking of the bloodstream, which might positively affect therapeutic scFv–scTRAIL by its internal FLAG tag with the FLAG-specific activity. mAb M2 was necessary to induce cell death in SKW (Figure 3a) and in Jurkat cells (Figure 4a). Thus, similar to Tumoricidal effects on human colon carcinomas in nude homotrimeric TRAIL, secondary crosslinking conferred TRAIL- mice. To investigate whether the improved target antigen- R2-stimulating capacity, confirming the nonaggregated state restricted tumoricidal activity of scFv–scTRAIL observed of the single-chain fusion protein with a TRAIL domain resem- in vitro is effective in vivo, in a first experiment a local bling a trimeric soluble TRAIL. The ErbB2-positive Colo205 treatment strategy was applied. NMRI nu/nu mice were and HT1080 cells express both, TRAIL-R1 and -R2. To define injected subcutaneously (s.c.) with Colo205 cells. Treatment through which of the two TRAIL death receptors the commenced 8 days later when vascularized tumor could be Cell Death and Disease x-fold increase in MFI relative cell number x-fold increase in MFI Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al SKW6.4 clearly discerned. Regimen was 4 daily s.c. injections of 1 nmol of scTRAIL and scFv–scTRAIL, respectively, in an area close to the established tumors. Tumor growth was monitored for 22 days. With this treatment protocol, we observed significant tumor regression in the scFv–scTRAIL- scFv-scTRAIL+M2 scTRAIL+M2 treated group compared not only with the control group scFv-scTRAIL (days 1–19; e.g., day 11, **P¼ 0.003) but also with the non- scTRAIL targeted scTRAIL-treated group (days 1–11; e.g., day 11, 0 10 10 10 10 *P¼ 0.04, Figure 5b). These data are in accordance with one concentration [nM] pilot experiment also showing significantly reduced tumor Colo205 HT1080 growth up to 11 days after scFv–scTRAIL treatment started *** 100 (**Po0.003, data not shown). These data prompted studies *** with a systemic treatment protocol to assess the feasibility *** *** 80 of such fusion proteins for potential clinical use. Again *** *** treatment was initiated when a solid, vascularized tumor had been established. Mice received 8 daily i.v. injections scTRAIL scTRAIL 20 20 of 1 nmol of the TRAIL fusion proteins or control injections. scFv-scTRAIL scFv-scTRAIL 0 0 All treatments were well tolerated by the animals, and we 00 10 10 10 10 10 10 10 10 10 10 observed no weight loss or other obvious signs of systemic concentration [nM] concentration [nM] toxicity. Tumor size in control mice increased progressively Colo205 HT1080 over the study period (Figure 5c). Similar to the peritumoral 100 100 treatment, systemic application of the two TRAIL reagents resulted in clear antitumoral activity during treatment. We 60 observed significantly delayed tumor growth on scTRAIL 40 treatment compared with control groups (days 1–19), and an even stronger effect for the tumor-targeted scFv–scTRAIL KillerTRAIL KillerTRAIL fusion protein (days 1–19). Remarkably, during scFv– scFv-scTRAIL scFv-scTRAIL scTRAIL treatment a clear reduction in tumor size was 0 10 10 10 10 10 0 10 10 10 10 10 recorded, which resulted in a significantly enhanced anti- concentration [nM] concentration [nM] tumoral effect compared with non-targeted scTRAIL (days Colo205 HT1080 1–19). Although tumors of both treatment groups began to 100 100 re-grow shortly after discontinuation of treatment, these ** 80 80 differences remained significant during the complete obser- ** ** 60 60 vation period (e.g., day 19 (Figure 5c)). 40 40 20 20 Discussion + ErbB2 + erbB2 0 0 We here show that through genetic engineering and targeting, 0 10 10 10 10 10 010 10 10 10 scFv-scTRAIL [nM] scFv-scTRAIL [nM] TRAIL’s antitumoral activity can be improved. TRAIL is considered as a tumor-selective agent, as cells from normal Colo205 HT1080 100 100 80 80 60 60 Figure 3 Target antigen-dependent bioactivity of scFv–scTRAIL. (a) Bioactivity 40 40 on target antigen-negative cells. 2 10 SKW6.4 cells/well were seeded in 96-well plates. The next day, cells were treated with serial dilutions of the fusion proteins in 20 20 the presence or absence of anti-FLAG M2 antibody (2 mg/ml). (b) Bioactivity of 0 0 4 4 scTRAIL versus scFv–scTRAIL. 5 10 Colo205 or 2 10 HT1080 cells/well scFv-scTRAIL + + – scFv-scTRAIL + + – were seeded in 96-well plates. The following day, cells were treated with 2.5 mg/ml recErbB2–– + recErbB2–– + cycloheximide and challenged in duplicates with increasing concentrations of Colo205 HT1080 scTRAIL and scFv–scTRAIL, respectively. After over night incubation, cell viability was determined by MTT assay or KV staining. Results from three to five 100 100 independent experiments are shown (mean S.D.). (c) Bioactivity of scFv– 80 80 scTRAIL compared to ‘KillerTRAIL’: Same assay as in b but incubation with serial 60 60 dilutions of scFv–scTRAIL or ‘KillerTRAIL’. (d, e) Target antigen-dependent 40 40 apoptosis induction of scFv–scTRAIL: Same assay as in b with scFv–scTRAIL titrated in the presence or absence of anti-ErbB2 (FRP5, 5 mg/ml) (d) or with scFv– 20 20 + TRAIL + TRAIL scTRAIL (0.05 nM) in the presence or absence of recErbB2 (10 mg/ml) (e). 0 0 (f) Contribution of the scFv domain in the scFv–scTRAIL to apoptosis induction. Same 0 10 10 10 10 10 0 10 10 10 10 10 scFv-scTRAIL [nM] scFv-scTRAIL [nM] assayasin b with scFv–scTRAIL titrated in the presence or absence of neutralizing anti-TRAIL antibodies (2E5, 1 mg/ml). (a, c–f) Results from three independent experiments are shown (mean±S.D.). (b, d) Statistical analysis was carried out with unpaired t-test, two-tailed test, **0.0026 oP40.0098, ***Po0.0001 Cell Death and Disease viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al Jurkat + FLAG -3 -2 -1 0 010 10 10 10 scFv-scTRAIL [nM] Colo205 HT1080 Jurkat 100 100 100 80 80 80 60 60 60 40 40 20 20 20 0 0 scFv-scTRAIL +++ – scFv-scTRAIL + – TRAIL + FLAG ++ – TRAILR2 TRAILR2 TRAILR2 –– + ––+– – + Colo205/L929 medium scTRAIL scFv- scTRAIL Figure 4 TRAIL-R2-mediated apoptosis induction of scFv–scTRAIL. (a)1 10 Jurkat cells/well were seeded in 96-well plates. The following day, cells were challenged with the indicated concentration of scFv–scTRAIL in the absence (open symbols) or presence (filled symbols) of 1 mg/ml of the crosslinking anti-FLAG mAb M2. After additional 4 4 5 16 h, cell viability was determined by MTT assay. (b) Cells (5 10 Colo205, 2 10 HT1080, 1 10 Jurkat cells/well) were seeded in 96-well plates. The next day, cells were treated with 2.5 mg/ml cycloheximide (HT1080, Colo205) and challenged with scFv–scTRAIL (1.35 nM) (HT1080, Colo205) or 2 nM TRAILþ 1 mg/ml anti-FLAG mAb M2 (Jurkat) in the presence or absence of TRAIL-R2 Fab (50 mg/ml). After additional 16 h, cell viability was determined by MTT assay or KV staining. (c)1 10 Colo205 and 1 10 L929 cells/well were seeded and co-cultured in 96-well plates. The next day, cells were treated with 2.5 mg/ml cycloheximide and challenged with 1 nM scTRAIL or scFv–scTRAIL. After additional 16 h, cell viability was determined by MTT assay. (a–c) Results from three independent experiments are shown (mean S.D.) tissue appear largely resistant toward apoptosis induction these are subjected to various stress conditions, such 8 12,32–34 by trimeric TRAIL derivatives, and clinical studies so far as infection and chemotherapeutics. Recently, it has 28,29 revealed that homotrimeric TRAIL is well tolerated. also been shown that TRAIL-R2-mediated apoptosis might However, in contrast to many investigated tumor cell lines, substantially contribute to chronic cholestatic disease. diverse primary cancer cells are inherently resistant to Accordingly, an improvement of the tumor-selective action TRAIL-mediated apoptosis (reviewed by Newsom-Davis of TRAIL appears mandatory. Along this line, we and others et al. ). Moreover, it has been demonstrated that TRAIL have already shown earlier that tumor-targeting approaches may under certain conditions even promote survival, proli- through the generation of TNF ligand fusion proteins, which feration, migration and invasion in TRAIL-resistant tumor aim to restrict TNF ligand’s full cytotoxic activity to the 30,31 cells. Thus, combination therapy with other drugs will be cancerous tissue are very promising approaches to improve required, for example, for sensitization of apoptotic pathways therapeutic efficacy of TRAIL and other members of the or suppression of potentially protumoral TRAIL effects, to TNF ligand family. Conventional fusion proteins of TRAIL achieve major and lasting tumor remissions. In this context, and antibody-derived scFv fragments reveal homotrimeric recent data appear of relevance showing that the resistance of proteins with a molecular mass of B150 kDa. This nearly nontransformed cells toward the apoptotic action of TRAIL corresponds in size to complete antibodies that are reported may vanish, at least in some tissues such as the liver, when to penetrate tumor tissues slowly and nonuniformly. Cell Death and Disease viability (%) viability (%) viability (%) viability (%) viability (%) Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al 100 scTRAIL ** scFv-scTRAIL ** *** *** 10 20 0.1 0 100 200 300 400 time post injection (min) 600 ** P=0.003 control scTRAIL * P=0.03 scFv-scTRAIL * P=0.04 0 0 0 5 10 15 20 control scTRAIL scFv- scTRAIL days after first treatment control *** P<0.0001 scTRAIL ** P=0.006 scFv-scTRAIL *** P=0.0009 0 5 10 15 20 control scTRAIL scFv- scTRAIL days after first treatment Figure 5 Pharmacokinetic properties and antitumoral activity of scTRAIL and scFv–scTRAIL. (a) Reduced elimination of scFv–scTRAIL from circulation. scTRAIL and scFv–scTRAIL were detected by ELISA in serum samples obtained at the indicated time points after i.v. injection into Balb/c mice. For each protein three mice were treated. Significant differences between both proteins were marked by asterisks. (b) Antitumoral activity in a xenograft tumor model – local treatment. NMRI nu/nu mice were injected 6 2 s.c. with 3 10 human colon carcinoma Colo205 cells. Treatment started when tumors reached about 25 mm (8 days after inoculation). Mice received four daily s.c. injections of 1 nmol of the corresponding proteins beginning day 0. n¼ 7 tumors per treatment group; symbols, mean of tumor volumes; bars, S.E.M. Individual tumor sizes at day 11 with bars indicating the mean are shown on the right. (c) Antitumoral activity in a xenograft tumor model – systemic treatment. 3 10 Colo205 cells were s.c. inoculated at the left and right dorsal site of NMRI nu/nu mice. Treatment started when tumors reached about 25 mm (5 days after inoculation). Mice received eight daily i.v. injections of 1 nmol of the corresponding proteins beginning at day 0. n¼ 8 tumors per treatment group; symbols, mean of tumor volumes; bars, S.E.M. Individual tumor sizes at day 19 with bars indicating the mean are shown on the right Furthermore, antibody fragments may have an intrinsic a trimeric TRAIL variant. This is of great importance, as tendency to aggregate, thereby potentially leading to higher it became evident that the formation of high order complexes order complexes, which in turn not only results in a further and aggregates of recombinant forms of TRAIL (e.g., increase in size but may also cause target-independent His-TRAIL, crosslinked FLAG–TRAIL) increases the toxicity TRAIL-R2 activity, too. To circumvent or at least minimize of the ligand to normal cells (reviewed by Koschny et al. ). these potential problems, we have developed a new format As expected, the ErbB2-specific scFv–scTRAIL specifically of a TRAIL fusion protein based on scTRAIL, a single bound to ErbB2-positive cells. This could be confirmed by the polypeptide chain composed of three TRAIL monomers. Gel use of TRAILR-Fc fusion proteins to detect the ErbB2 bound filtration and western blot analysis revealed that the ErbB2- scFv–scTRAIL and by blocking of fusion protein binding specific scFv–scTRAIL fusion protein could be expressed with the ErbB2-specific antibody FRP5, thereby indicating in the expected monomeric state corresponding formally to predominant binding of the fusion protein by the antibody Cell Death and Disease relative tumor size (%) relative tumor size (%) % of the 2 min value relative tumor size (%) relative tumor size (%) day 19 day 11 Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al moiety to ErbB2 rather than by the TRAIL domain and growth, as addition of TRAIL-neutralizing antibodies comple- TRAIL-R. Compared with the non-targeted scTRAIL mole- tely restored cell viability on incubation with scFv–scTRAIL. cule, the tumor antigen-targeted scTRAIL fusion protein This is in accordance with previous reports showing that the showed higher apoptotic activity in vitro on ErbB2-positive mAb FRP5 did not affect tumor growth in vivo, despite even cells. This target antigen-dependent cytotoxicity could be partial agonistic activity in vitro, evident from an increase confirmed by preincubation with competing ErbB2-specific in ErbB2 phosphotyrosine level and accelerated receptor antibody FRP5 or with ErbB2 peptide, restoring cell viability to turnover by antibody-mediated receptor crosslinking. In a significant extent. We and others have previously described contrast, a monovalent scFvFRP5–ETA fusion protein did not that, compared with soluble homotrimeric TRAIL, homo- induce ErbB2-tyrosine phosphorylation, indicating that the trimeric scFv–TRAIL fusion proteins gain a higher activity on activity of a monovalent scFv fusion protein might be limited TRAIL-R2 upon cell surface binding by the targeting domain by the intrinsic turnover rate of its target receptor. We have 13,26,27 within the fusion proteins. Of note, although there is own preliminary data confirming an increase in ErbB2- some controversial discussion on this, it has been shown phosphorylation on treatment of Colo205 cells with a that efficient activation of TRAIL-R2 is only reached by conventional, homotrimeric scFvFRP5–TRAIL fusion protein, natural membrane TRAIL, crosslinked soluble or cell whereas treatment with the monovalent scFv–scTRAIL had 13,26,27,37,38 surface immobilized TRAIL. Importantly, the no receptor-activating effect (data not shown). Accordingly, scFv–scTRAIL fusion protein likewise converted into a the single-chain design concept might be of advantage functional equivalent of membrane TRAIL on target-antigen compared with a conventional fusion protein if target-antigen binding, and thus efficiently induced cell death already at low multimerization has to be avoided. Moreover, Stagg concentrations by TRAIL-R2, the predominant TRAIL- et al. could show that combination of ErbB2 blockade and induced apoptosis pathway in the cell lines studied here. TRAIL-R2 activation can elicit potent synergistic antitumor Accordingly, receptor binding and activation could be blocked activity. Thus, modifying the targeting module using an ErB2- by TRAIL-R2-neutralizing monovalent Fab fragments. More- specific scFv antibody fragment also capable of inhibiting over, Jurkat cells, which only express TRAIL-R2, but not ErbB2 signaling might result in further increased ErbB2- ErbB2 and TRAIL-R1 are resistant against soluble scFv– specific antitumor activity of an scFv–TRAIL fusion protein. scTRAIL, whereas crosslinking of the fusion protein led to an The here described antitumoral activity of the scFv–scTRAIL increase in activity. These properties are especially important fusion protein, which resulted in clear reduction in tumor size based on observations that on tumor cells TRAIL-R2 is more under treatment, appears superior to that of published frequently and strongly expressed than TRAIL-R1, that preclinical data with homotrimeric TRAIL, regarding both 7,8,40 apoptotic signaling through TRAIL-R2 may be more potent the applied dose and the treatment periods. However, than through TRAIL-R1 and that, on the other hand, TRAIL- detailed side-by-side comparison of different TRAIL reagents R2 may be a key regulator of cholestatic liver injury by are required to confirm this observation. induction of apoptosis in cholangiocytes. Consequently, the In conclusion, our data provide evidence that bioactive scFv–scTRAIL might be an appropriate therapeutic, which single-chain TRAIL fusion proteins can be generated. Further- meets the criteria of high antitumor and low off-target activity. more, fusion to a target-specific scFv antibody results In vivo analyses in mice revealed that, on i.v. injection, the in target-dependent increase in TRAIL-mediated cytotoxicity scFv–scTRAIL and the scTRAIL displayed biphasic pharma- in vitro, likely due to efficient TRAIL-R2 activation, and cokinetics. In the first phase, the calculated t a of B35 min improves antitumoral activity in vivo. Accordingly, this design was similar for both proteins. In the second phase, the scFv– concept represents a promising strategy to enhance TRAIL’s scTRAIL displayed a slightly increased half-life with t b antitumoral action and to minimize potential unwanted of B160 min compared with 125 min for the scTRAIL. This off-target actions on normal tissues. strongly contrasts to some of the published data for homo- trimeric TRAIL, indicating a half-life of 3–5 min (t a) and Materials and Methods 40,41 20 min (t b) in rodents. Our results rather match with 2 Plasmids and cell lines. The pIRESpuro–scTRAIL expression construct for pharmacokinetic data of LZ-TRAIL, a trimer-stabilized TRAIL human scTRAIL (aa 95–281) was generated by insertion of linker sequences, 1 1 variant (t a: 1.3 h; t b: 4.8 h ). Most important, both single- encoding four repetitions of the GGGS motif, between the three TRAIL modules as 2 2 described previously for the construction of scTNF. chain TRAIL variants significantly reduced growth of estab- The single-chain antigen binding protein scFvFRP5 for the ErbB2-specific lished Colo205 tumors already at rather moderate doses, pIRESpuro–scFv–scTRAIL expression construct was described previously. either on local or systemic treatment. In accordance with the HEK293, Jurkat, Colo205, SKW6.4 and L929 cells were obtained from the in vitro bioactivity, the scFv–scTRAIL displayed superior American Type Culture Collection (Manassas, VA, USA) or DSMZ – Deutsche tumoricidal effects in vivo compared with the scTRAIL. Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, We propose that increased antitumoral activity is largely Germany). The HT1080 cells were a kind gift of W Rettig (Boehringer Ingelheim Pharma, Vienna, Austria). Cells were cultured in RPMI 1640 medium (Invitrogen, related to ErbB2-mediated tumor enrichment and the target- Karlsruhe, Germany) supplemented with 5% or 10% (SKW6.4) fetal calf serum ing-dependent increase in bioactivity rather than the slightly (FCS, PAA Laboratories, Pasching, Austria). increased bioavailability. The observed re-growth of tumors upon cessation of treatment has also been reported with Production and purification of recombinant proteins. The TRAIL homotrimeric TRAIL and might overcome when the combi- fusion proteins were produced in HEK293 cells after stable transfection of the nation treatments with appropriate chemotherapeutics are corresponding expression plasmids using Lipofectamine 2000 (Invitrogen) and 9,10,40 conducted. Furthermore, we can exclude an effect of selection of stable clones by limiting dilution. For protein production, stable clones the ErbB2-specific antibody fragment scFvFRP5 on tumor were expanded and grown in RPMI 1640, 5% FCS, to 90% confluency and Cell Death and Disease Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al The control group received 100 ml concentrated cell-culture supernatant from subsequently cultured in serum-free Optimem (Invitrogen) supplemented with empty vector-transfected cells at the same time intervals. 50 mM ZnCl , replacing media two times every 3 days. Supernatants were pooled and recombinant proteins were purified by affinity chromatography using anti-FLAG Systemic treatment. Colo205 cells (3 10 ) in 100 ml PBS were s.c. inoculated mAb M2 agarose (Sigma-Aldrich, Steinheim, Germany). The bound proteins were at the left and right dorsal sides of 6-week-old female NMRI nu/nu mice. Treatment eluted with 100 mg/ml FLAG peptide (Sigma-Aldrich) and dialyzed against PBS. started 5 days after tumor cell inoculation when tumors reached B25 mm . Mice After concentrating purified proteins by dialysis on PEG35000, they were analyzed received 8 daily i.v. injections of 1 nmol of the affinity purified TRAIL fusion proteins. and quantified by SDS-PAGE, silver gel (Sigma-Aldrich) and gel filtration and stored The control group received 100 ml PBS at the same time intervals. Tumor growth at 41C. was monitored as described. Gel filtration. Protein samples were applied to a BioSuite250 HR SEC (300 7.8) column (Waters, Millipore Corp., Milford, MA, USA) equilibrated in Conflict of interest PBS and eluted at a flow rate of 0.5 ml/min. The authors declare no conflict of interest. Flow cytometry. Cells (5 10 ) were incubated for 2 h at 41C with the indicated concentrations of the TRAIL fusion proteins. After washing the cells three Acknowledgements. We gratefully acknowledge technical support in the times with PBS, 2% FCS, 0.02% sodium azide, bound fusion proteins were detected initial expression of scTRAIL by Sylvia Willy. Our work is supported by grants from by anti-human TRAIL mAb (MAB687) (1 mg/ml, R&D Systems, Wiesbaden, Deutsche Krebshilfe, Grant no. 107551 and 108268 to HW and KP, from BMBF Germany) and fluorescein isothiocyanate-labeled rabbit anti-mouse IgG Ab (Biochance plus, FKz 0315164) to KP and by a career fellowship award by the (1 mg/ml, Sigma-Aldrich). ErbB2 expression was detected by anti-c-neu (AB-5) Peter and Traudl Engelhorn Foundation to JG. (1 mg/ml, Merck KGaA, Darmstadt, Germany) and fluorescein isothiocyanate- labeled rabbit anti-mouse IgG Ab (1 mg/ml, Sigma-Aldrich). Alternatively, cell-bound scFv–scTRAIL was detected by TRAILR–Fc fusion proteins (5 mg/ml TRAILR1-Fc and 3 mg/ml TRAILR2-Fc) and fluorescein isothiocyanate-labeled anti-human IgG, 1. Gerber DE. 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To view a copy of this license, mediated-apoptosis contributes to cholestatic liver disease. Proc Natl Acad Sci USA 2008; 105: 10895–10900. visit http://creativecommons.org/licenses/by-nc-nd/3.0/ Cell Death and Disease http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death & Disease Springer Journals

Potent antitumoral activity of TRAIL through generation of tumor-targeted single-chain fusion proteins

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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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Citation: Cell Death and Disease (2010) 1, e68; doi:10.1038/cddis.2010.45 & 2010 Macmillan Publishers Limited All rights reserved 2041-4889/10 www.nature.com/cddis Potent antitumoral activity of TRAIL through generation of tumor-targeted single-chain fusion proteins 1 1 2 3 4 5 ,1 1 B Schneider ,S Mu ¨ nkel , A Krippner-Heidenreich , I Grunwald , WS Wels , H Wajant , K Pfizenmaier* and J Gerspach In an attempt to improve TRAIL’s (tumor necrosis factor-related apoptosis-inducing ligand) tumor selective activity a variant was designed, in which the three TRAIL protomers are expressed as a single polypeptide chain (scTRAIL). By genetic fusion with a single-chain antibody fragment (scFv) recognizing the extracellular domain of ErbB2, we further equipped scTRAIL with tumor- targeting properties. We studied tumor targeting and apoptosis induction of scFv–scTRAIL in comparison with non-targeted scTRAIL. Importantly, the tumor antigen-targeted scTRAIL fusion protein showed higher apoptotic activity in vitro, with a predominant action by TRAIL-R2 signaling. Pharmacokinetic studies revealed increased plasma half-life of the targeted scTRAIL fusion protein compared with scTRAIL. In vivo studies in a mouse tumor model with xenotransplanted Colo205 cells confirmed greater response to the ErbB2-specific scTRAIL fusion protein compared with non-targeted scTRAIL both under local and systemic application regimen. Together, in vitro and in vivo data give proof of concept of higher therapeutic activity of tumor-targeted scFv–scTRAIL molecules. Further, we envisage that through targeting of scTRAIL, potential side effects should be minimized. We propose that scFv-mediated tumor targeting of single-chain TRAIL represents a promising strategy to improve TRAIL’s antitumoral action and to minimize potential unwanted actions on normal tissues. Cell Death and Disease (2010) 1, e68; doi:10.1038/cddis.2010.45; published online 26 August 2010 Subject Category: Cancer Although for many malignancies traditional cytotoxic chemo- because preclinical studies revealed that, in contrast to therapy is still the treatment of choice, targeted therapies TNF and CD95 agonists, the application of trimeric, soluble gain increasing importance because they are expected TRAIL is well tolerated. Concomitantly, it demonstrated potent to exert higher tumor-specific activity and less dose-limiting antitumor activity in tumor models based on tumor cell lines 7–10 side effects. The anti-CD20 antibody Rituximab and the and primary tumor samples. Although first clinical trials in Bcr-Abl-selective kinase inhibitor Gleevec, respectively, are various advanced cancers are promising, as stable disease two vivid examples for the success of targeted therapy (for has been reported in many cases, TRAIL monotherapy review, see Gerber ). Targeting the apoptotic machinery of most likely will not result in a sufficient overall therapeutic malignant cells has become an attractive concept, beginning activity. Concurrently, this faces the potential problem with tumor necrosis factor (TNF) and its now established that under combined treatment options normal cells might clinical use for locoregional treatment of limb metastases also be sensitized to TRAIL-induced apoptosis (reviewed 2 12 of soft tissue sarcoma and melanoma and followed by by Falschlehner et al. ). Furthermore, we have recently the current exploitation of other proapoptotic members of the shown that TRAIL-R1 is activated by the soluble as well as TNF ligand family – CD95L and TNF-related apoptosis- the membrane-bound form of the ligand, whereas TRAIL-R2 inducing ligand (TRAIL) – being presently at various stages is preferentially activated by membrane TRAIL or oligo- 13,14 of preclinical and clinical development (for review, see Call merized soluble trimeric TRAIL. As agonistic TRAIL-R2- 3 4 5 et al., Gerspach et al. and Newsom-Davis et al. ). TRAIL, specific antibodies likewise display potent antitumor activity similar to TNF a type II transmembrane protein, is expressed (reviewed by Johnstone et al. ), it is conceivable that on various immune effector cells and appears critically homotrimeric TRAIL could be improved in its activity provided involved in antitumor responses in the body. It is probably efficient TRAIL-R2 signaling is ensured through genetic modi- the best candidate of a death ligand for systemic application fications or other means. On the other hand, the spontaneous 1 2 Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, Stuttgart 70569, Germany; Institute of Cellular Medicine, Newcastle University, 4th Floor Catherine Cookson Building, Framlington Place, Newcastle upon Tyne NE2 4HH, UK; Fraunhofer Institute for Manufacturing Technology and Applied Materials Research IFAM, Bonding Technology and Surfaces, Adhesives and Chemistry of Polymers, Wiener Strae 12, Bremen 28359, Germany; 4 5 Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Paul-Ehrlich-Strae 42-44, Frankfurt 60596, Germany and Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital of Wu ¨ rzburg, Ro ¨ ntgenring 11, Wu ¨ rzburg 97070, Germany *Corresponding author: K Pfizenmaier, Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, Stuttgart 70569, Germany. Tel: þ 49 711 685 66986; Fax: þ 49 711 685 67484; E-mail: klaus.pfi[email protected] Keywords: tumor targeting; single-chain TRAIL; scFv–TRAIL fusion proteins; apoptosis; ErbB2 Abbreviations: i.v., intravenous(ly); TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; TRAILR1/2, TRAIL receptor 1/2; scFv, single-chain Fv fragment; scTRAIL, single-chain TRAIL; s.c., subcutaneous(ly) Received 19.4.10; revised 02.7.10; accepted 14.7.10; Edited by R De Maria Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al aggregation of some recombinant TRAIL preparations, Both scTRAIL and scFv–scTRAIL were purified by affinity resembling secondarily crosslinked TRAIL, has been asso- chromatography on monoclonal M2 anti-FLAG agarose from ciated with unwanted actions on normal tissues and the supernatant of stably transfected HEK293 cells with yields thus curtail their use as tumor-selective apoptosis inducers of about 1 mg protein per liter cell culture supernatant. (reviewed by Koschny et al. ). Thus, TRAIL variants Immunoblot analysis and SDS-PAGE (Figure 1b) of the mimicking the full bioactivity of membrane TRAIL could purified protein showed single protein bands with a molecular present powerful cancer therapeutics provided that a tumor- mass of B70 kDa for scTRAIL and B100 kDa for scFv– restricted action can be achieved. A further apparent limitation scTRAIL matching the expected calculated molecular masses of the currently clinically investigated recombinant TRAIL of 71 and 98 kDa, respectively. Gel filtration analysis indicated reagents is their rather short in vivo half-life. Therefore, an a monomeric organization of both scTRAIL variants, corres- improvement of pharmacokinetic properties might also lead to ponding, with respect to the TRAIL part, to a noncovalently increased therapeutic action. A potential solution to increase assembled trimer of a conventional recombinant TRAIL specific activity and bioavailability is the combination of TRAIL molecule (Figure 1c). The molecular masses deduced from function with tumor targeting. Thus, we generated new TRAIL SEC were slightly lower compared with that derived from fusion proteins containing an antibody fragment (single-chain SDS-PAGE (Figure 1b), which did not result from degradation Fv fragment (scFv)) for targeting TRAIL to the tumor to enrich as verified by immunoblot analysis of collected fractions the therapeutic at the tumor site and to enhance its specific (Figure 1c). In the scTRAIL preparation, the minor fraction bioactivity. ErbB2, similar to EGFR/ErbB1, ErbB3 and ErbB4 eluting in SEC at apparent higher molecular weight, thus belonging to the ErbB receptor tyrosine kinase family, potentially comprising aggregated complexes, did not display represents a clinically relevant target antigen. It is highly disproportionately high bioactivity, as revealed from compa- expressed on a variety of human solid tumors (reviewed by rison of cytotoxic activity of fraction 2 and 15, taking relative Holbro and Hynes ) and there is already one humanized protein amounts into consideration (data not shown). monoclonal antibody (mAb) directed against the extracellular domain of ErbB2 in clinical use (Trastuzumab) and approved Target antigen-specific binding of scFv–scTRAIL. To for treatment of breast carcinoma. Several other mAbs are analyze specific binding of scFv–scTRAIL to ErbB2-positive in different stages of clinical evaluation (reviewed by cells, flow cytometry analysis was performed. ErbB2 expres- Baselga and Swain ). Although the clinical data provide sion analysis confirmed a median versus low expression clear evidence that ErbB2 is a relevant target, they also show level for Colo205 colon carcinoma and HT1080 fibrosarcoma that the therapeutic effect of the current antibody reagents cells, respectively (Figure 2a), compared with SKBR3 cells is rather limited. This justifies the search for additional that are well-known to highly express the ErbB2 protein (data ErbB2-targeted strategies. not shown). Incubation of Colo205 and HT1080 cells with Recently, a single-chain TNF molecule, consisting of three the fusion protein revealed a specific, binding to ErbB2- TNF monomers fused by short peptide linkers, was described positive cells (Figure 2a and b) compared with incubation to exert enhanced stability and antitumoral activity. We with the non-targeted scTRAIL, which resulted only in a weak have designed an analogous single polypeptide chain fluorescence signal, either by detection with anti-TRAIL TRAIL variant (scTRAIL), which was used to construct a (Figure 2a) or with anti-FLAG antibodies (data not shown). scFv–scTRAIL fusion protein for tumor targeting. We have As binding of homotrimeric FLAG-tagged TRAIL showed compared the bioactivities of this fusion protein with non- similar weak signals (data not shown), this could reflect targeted scTRAIL in in vitro and in vivo tumor models. We a low level of TRAIL receptor (TRAILR) expression on the show here that the tumor antigen-targeted scTRAIL fusion investigated target cells and/or insufficient sensitivity of protein showed higher apoptotic activity to scTRAIL in vitro, FACS analyses. ErbB2-specific binding was confirmed by with a predominant action by TRAIL-R2 signaling on Colo205 using TRAILR1- and R2-Fc fusion proteins for detection colon carcinoma cells. In vivo studies in a mouse xenograft of scFv–scTRAIL binding to target cells (Figure 2b) and tumor model confirmed significantly higher response to the competition with anti-ErbB2 antibodies (FRP5). This led to tumor-targeted ErbB2-specific scTRAIL fusion protein. a near complete inhibition of scFv–scTRAIL binding to the ErbB2-positive cell line (Figure 2b). Results Target antigen-restricted induction of cell death by Construction and production of TRAIL fusion proteins. scFv–scTRAIL. To address the bioactivity of scFv– To generate a single-chain TRAIL molecule, we followed the scTRAIL in vitro, we first analyzed induction of cell death design principle described for scTNF and covalently fused on ErbB2-negative SKW6.4 cells, which are known to be 23,24 three TRAIL monomers, each consisting of the extracellular TRAIL-R1 and TRAIL-R2 positive (Figure 3a). Although domain of TRAIL (aa 95–281) through two peptide linkers scTRAIL and scFv–scTRAIL did not show cytotoxic activity comprising four repeats of the sequence GGGS (Figure 1a). up to 8 nM, respectively, in the presence of the crosslinking To facilitate purification and analysis an N-terminal FLAG anti-FLAG antibody M2 comparable bioactivities of both tag was added. Furthermore, by additional N-terminal fusion proteins were observed (Figure 3a). In contrast, incu- fusion of a single-chain antibody fragment (scFv) recognizing bation of ErbB2-positive Colo205 and HT1080 cells the FRP5 epitope within the extracellular domain of ErbB2, with scTRAIL or scFv–scTRAIL revealed an approximately we generated an ErbB2-specific scTRAIL fusion protein three- to fivefold higher bioactivity of the ErbB2-specific (Figure 1a). scFv–scTRAIL compared with the non-targeted scTRAIL Cell Death and Disease Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al [GGGS] [GGGS] 4 4 F - C N - L TRAIL TRAIL TRAIL N - F - C L scFv TRAIL TRAIL TRAIL kDa M1 2 kDa M1 2 130 130 55 55 scTRAIL scFv-scTRAIL 443 200 66 29 443 200 66 29 Fraction 2 4 6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 kDa kDa Figure 1 Biochemical analysis of scTRAIL and scFv–scTRAIL. (a) Scheme of the scTRAIL fusion proteins used in this study. L, leader peptide; scFv, human ErbB2- specific single-chain antibody fragment; F, FLAG tag; TRAIL, human TRAIL (aa 95–281). (b) Purified scTRAIL (lane 1) and scFv–scTRAIL (lane 2) were analyzed by SDS-PAGE (10%, reducing conditions) followed by immunoblot analysis (left, 250 ng protein/lane) or silver staining (right, 1 mg protein/lane). For immunoblotting, the monoclonal anti-FLAG M2 antibody and an alkaline phosphatase-conjugated secondary antibody was used. (c) The TRAIL variants were separated by gel filtration on a BioSuite250 column. Dotted lines indicate the retention time of the molecular weight standards apoferritin (443 kDa), b-amylase (200 kDa), bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Collected fractions were analyzed by immunoblotting as in b. For scTRAIL fractions 1–9 (every second fraction; first part), fractions 11–16 (second part) and fractions 19–26 (every second fraction; third part) were immunoblotted. For scFv–scTRAIL fractions 8–24 were immunoblotted (Figure 3b; Colo205: EC50 :30 10 pM, EC50 : depended on ErbB2 target binding. Preincubation with FRP5 scFv–scTRAIL scTRAIL ± ± 130 70 pM; HT1080: EC50 :50 5pM, EC50 : or ErbB2 peptides before addition of the ErbB2-specific scFv–scTRAIL scTRAIL 140±10 pM). Furthermore, bioactivity of the targeted scFv–scTRAIL fusion protein restored cell viability of scFv–scTRAIL compared well with that of a commercially Colo205 and HT1080 cells to a significant extent (Figure 3d available high activity TRAIL, the so-called ‘Killer-TRAIL’, and e). Furthermore, as for both tested cell lines TRAIL- 13,25 with about twofold higher (Colo205) or at least comparable induced cell death is primarily mediated by TRAIL-R2, (HT1080) activity of the scFv–scTRAIL reagent (Figure 3c). the higher bioactivity of the scFv–scTRAIL on ErbB2-positive Furthermore, on target-negative SKW6.4 cells, the second- cells pointed to a membrane TRAIL-mimetic activity upon arily crosslinked scTRAIL or scFv–scTRAIL displayed specific cell surface immobilization of the fusion protein (see comparable cytotoxic activity to ‘Killer-TRAIL’ (data not below). Furthermore, inhibition of TRAIL bioactivity of the shown). From blocking studies using anti-ErbB2 anti- scFv–scTRAIL by the use of a TRAIL-neutralizing antibody led bodies (FRP5, Figure 3d) or recombinant ErbB2 peptides to complete restoration of cell viability of Colo205 and HT1080 comprising the epitope of the erbB2-specific scFv cells (Figure 3f). Thus, the ErbB2-specific scFv domain per se (Figure 3e), it became evident that the increased bioactivity did not directly contribute to apoptosis induction. Cell Death and Disease Absorbance Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al scFv–scTRAIL molecule signals apoptosis, neutralization Colo205 HT 1080 experiments with antagonistic, TRAIL-R2-specific monovalent antibody fragments (Fab fragments) were performed. Both ctrl ctrl -ErbB2 -ErbB2 ErbB2-positive cell lines showed dominant TRAIL-R2- mediated cell death at low concentrations of scFv–scTRAIL (Figure 4b), demonstrating that cell surface immobilized scFv– scTRAIL exerted membrane TRAIL-like activity, similar to what has been previously described for conventional scFv–TRAIL 13,26,27 fusion proteins. The concentration of anti-TRAIL-R2 Fab fragments used in these experiments was sufficient to almost completely block cell death of Jurkat cells induced ctrl ctrl scTRAIL scTRAIL by secondary crosslinked, homotrimeric FLAG-tagged TRAIL scFv-scTRAIL scFv-scTRAIL (Figure 4b). In addition, cell surface antigen-bound scFv– scTRAIL also activated the murine TRAILR, which is, similarly to human TRAIL-R2, not or hardly activated by soluble TRAIL. This was shown by mixed cultures using equal numbers of Colo205 cells and L929 mouse fibroblasts (Figure 4c). The latter do not express human ErbB2 and thus, in contrast to log fluorescence intensity Colo205 cells, are not able to immobilize the scFv–scTRAIL by the ErbB2-targeting domain. Thus, if scFv–scTRAIL is able to Colo205 HT1080 induce cell death in L929 cells by mimicking membrane TRAIL 30 after binding to the ErbB2 receptor on Colo205 cells, more than 50% of the cells are expected to die. Indeed, incubation with scFv–scTRAIL induced cell death in over 70% cells of the ** 1 co-culture, whereas equal molar amounts of scTRAIL induced cell death in only about 30% of the mixed cells (Figure 4c). In *** monocultures, the same concentration of scFv–scTRAIL and 0 0 scTRAIL induced cell death in about 90% of Colo205 cells, control scFv- scFv- control scFv- scFv- scTRAIL scTRAIL scTRAIL scTRAIL respectively, but not in L929 cells, confirming that, in the + + soluble form, both proteins are non-toxic to the latter (data not erbB2 erbB2 shown). Furthermore, due to the lack of human ErbB2, the Figure 2 Target antigen-specific binding of scFv–scTRAIL. (a) ErbB2 L929 cells might also be considered as antigen-negative expression of Colo205 and HT1080 cells: 5 10 cells were incubated with bystander cells, potentially occurring in a tumor tissue. Thus, anti-ErbB2 (gray filled) or isotype control antibody (black lines) followed by scFv–scTRAIL bound to antigen is expected to elicit an FITC-conjugated anti-mouse antibody and fluorescence intensity was measured by antitumoral effect also on neighboring antigen-negative cells FACS analysis. Binding of scFv–scTRAIL to ErbB2-positive Colo205 and HT1080 cells compared with non-targeted scTRAIL: 5 10 cells were incubated with within the tumor environment. 50 nM scTRAIL (gray lines) or 50 nM scFv–scTRAIL (gray filled) and, after repeated washes, bound proteins were detected using anti-human TRAIL mAb and Serum half-life of scFv–scTRAIL. To evaluate the pharmaco- FITC-labeled anti-mouse IgG. As control, cells treated with anti-humanTRAIL mAb kinetic characteristics of scFv–scTRAIL in comparison with and FITC-labeled anti-mouse IgG were used (black lines). (b) Binding competition of scTRAIL, serum samples were collected after a single dose scFv–scTRAIL to ErbB2-positive Colo205 cells by ErbB2-specific antibodies: intravenous (i.v.) injection of 400 pmol into Balb/c mice. The 5 10 Colo205 or HT1080 cells were preincubated with FRP5 or mouse IgG1 (10 mg/ml, respectively) before addition of scFv–scTRAIL (50 nM). Detection was relative serum concentrations plotted over time fitted well to carried out by TRAILR-Fc fusion proteins and FITC-labeled anti-human IgG, a two-phase exponential decay regression curve in both Fc-specific antibodies. Shown are X-fold increased mean fluorescence intensities cases. Within 20 min post-injection, both TRAIL variants (MFI) compared with the control (FITC-labeled anti-human Fc) from three displayed similar kinetics with a serum half-life of B35 min independent experiments (mean S.D.). Statistical analysis was carried out with (t a), respectively. At later time points, a slight but significant unpaired t-test, two-tailed test, **P¼ 0.0021, ***Po0.0001 2 difference in bioavailability was detected with half-lifes (t b) of 160 and 124 min and AUCs of 11 191 and 8125 for scFv– scTRAIL and scTRAIL, respectively (Figure 5a). Thus, the TRAIL-R2-mediated apoptosis induction of scFv– ErbB2-specific fusion protein was more efficiently retained in scTRAIL. In absence of the ErbB2 antigen, crosslinking of the bloodstream, which might positively affect therapeutic scFv–scTRAIL by its internal FLAG tag with the FLAG-specific activity. mAb M2 was necessary to induce cell death in SKW (Figure 3a) and in Jurkat cells (Figure 4a). Thus, similar to Tumoricidal effects on human colon carcinomas in nude homotrimeric TRAIL, secondary crosslinking conferred TRAIL- mice. To investigate whether the improved target antigen- R2-stimulating capacity, confirming the nonaggregated state restricted tumoricidal activity of scFv–scTRAIL observed of the single-chain fusion protein with a TRAIL domain resem- in vitro is effective in vivo, in a first experiment a local bling a trimeric soluble TRAIL. The ErbB2-positive Colo205 treatment strategy was applied. NMRI nu/nu mice were and HT1080 cells express both, TRAIL-R1 and -R2. To define injected subcutaneously (s.c.) with Colo205 cells. Treatment through which of the two TRAIL death receptors the commenced 8 days later when vascularized tumor could be Cell Death and Disease x-fold increase in MFI relative cell number x-fold increase in MFI Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al SKW6.4 clearly discerned. Regimen was 4 daily s.c. injections of 1 nmol of scTRAIL and scFv–scTRAIL, respectively, in an area close to the established tumors. Tumor growth was monitored for 22 days. With this treatment protocol, we observed significant tumor regression in the scFv–scTRAIL- scFv-scTRAIL+M2 scTRAIL+M2 treated group compared not only with the control group scFv-scTRAIL (days 1–19; e.g., day 11, **P¼ 0.003) but also with the non- scTRAIL targeted scTRAIL-treated group (days 1–11; e.g., day 11, 0 10 10 10 10 *P¼ 0.04, Figure 5b). These data are in accordance with one concentration [nM] pilot experiment also showing significantly reduced tumor Colo205 HT1080 growth up to 11 days after scFv–scTRAIL treatment started *** 100 (**Po0.003, data not shown). These data prompted studies *** with a systemic treatment protocol to assess the feasibility *** *** 80 of such fusion proteins for potential clinical use. Again *** *** treatment was initiated when a solid, vascularized tumor had been established. Mice received 8 daily i.v. injections scTRAIL scTRAIL 20 20 of 1 nmol of the TRAIL fusion proteins or control injections. scFv-scTRAIL scFv-scTRAIL 0 0 All treatments were well tolerated by the animals, and we 00 10 10 10 10 10 10 10 10 10 10 observed no weight loss or other obvious signs of systemic concentration [nM] concentration [nM] toxicity. Tumor size in control mice increased progressively Colo205 HT1080 over the study period (Figure 5c). Similar to the peritumoral 100 100 treatment, systemic application of the two TRAIL reagents resulted in clear antitumoral activity during treatment. We 60 observed significantly delayed tumor growth on scTRAIL 40 treatment compared with control groups (days 1–19), and an even stronger effect for the tumor-targeted scFv–scTRAIL KillerTRAIL KillerTRAIL fusion protein (days 1–19). Remarkably, during scFv– scFv-scTRAIL scFv-scTRAIL scTRAIL treatment a clear reduction in tumor size was 0 10 10 10 10 10 0 10 10 10 10 10 recorded, which resulted in a significantly enhanced anti- concentration [nM] concentration [nM] tumoral effect compared with non-targeted scTRAIL (days Colo205 HT1080 1–19). Although tumors of both treatment groups began to 100 100 re-grow shortly after discontinuation of treatment, these ** 80 80 differences remained significant during the complete obser- ** ** 60 60 vation period (e.g., day 19 (Figure 5c)). 40 40 20 20 Discussion + ErbB2 + erbB2 0 0 We here show that through genetic engineering and targeting, 0 10 10 10 10 10 010 10 10 10 scFv-scTRAIL [nM] scFv-scTRAIL [nM] TRAIL’s antitumoral activity can be improved. TRAIL is considered as a tumor-selective agent, as cells from normal Colo205 HT1080 100 100 80 80 60 60 Figure 3 Target antigen-dependent bioactivity of scFv–scTRAIL. (a) Bioactivity 40 40 on target antigen-negative cells. 2 10 SKW6.4 cells/well were seeded in 96-well plates. The next day, cells were treated with serial dilutions of the fusion proteins in 20 20 the presence or absence of anti-FLAG M2 antibody (2 mg/ml). (b) Bioactivity of 0 0 4 4 scTRAIL versus scFv–scTRAIL. 5 10 Colo205 or 2 10 HT1080 cells/well scFv-scTRAIL + + – scFv-scTRAIL + + – were seeded in 96-well plates. The following day, cells were treated with 2.5 mg/ml recErbB2–– + recErbB2–– + cycloheximide and challenged in duplicates with increasing concentrations of Colo205 HT1080 scTRAIL and scFv–scTRAIL, respectively. After over night incubation, cell viability was determined by MTT assay or KV staining. Results from three to five 100 100 independent experiments are shown (mean S.D.). (c) Bioactivity of scFv– 80 80 scTRAIL compared to ‘KillerTRAIL’: Same assay as in b but incubation with serial 60 60 dilutions of scFv–scTRAIL or ‘KillerTRAIL’. (d, e) Target antigen-dependent 40 40 apoptosis induction of scFv–scTRAIL: Same assay as in b with scFv–scTRAIL titrated in the presence or absence of anti-ErbB2 (FRP5, 5 mg/ml) (d) or with scFv– 20 20 + TRAIL + TRAIL scTRAIL (0.05 nM) in the presence or absence of recErbB2 (10 mg/ml) (e). 0 0 (f) Contribution of the scFv domain in the scFv–scTRAIL to apoptosis induction. Same 0 10 10 10 10 10 0 10 10 10 10 10 scFv-scTRAIL [nM] scFv-scTRAIL [nM] assayasin b with scFv–scTRAIL titrated in the presence or absence of neutralizing anti-TRAIL antibodies (2E5, 1 mg/ml). (a, c–f) Results from three independent experiments are shown (mean±S.D.). (b, d) Statistical analysis was carried out with unpaired t-test, two-tailed test, **0.0026 oP40.0098, ***Po0.0001 Cell Death and Disease viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) viability (%) Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al Jurkat + FLAG -3 -2 -1 0 010 10 10 10 scFv-scTRAIL [nM] Colo205 HT1080 Jurkat 100 100 100 80 80 80 60 60 60 40 40 20 20 20 0 0 scFv-scTRAIL +++ – scFv-scTRAIL + – TRAIL + FLAG ++ – TRAILR2 TRAILR2 TRAILR2 –– + ––+– – + Colo205/L929 medium scTRAIL scFv- scTRAIL Figure 4 TRAIL-R2-mediated apoptosis induction of scFv–scTRAIL. (a)1 10 Jurkat cells/well were seeded in 96-well plates. The following day, cells were challenged with the indicated concentration of scFv–scTRAIL in the absence (open symbols) or presence (filled symbols) of 1 mg/ml of the crosslinking anti-FLAG mAb M2. After additional 4 4 5 16 h, cell viability was determined by MTT assay. (b) Cells (5 10 Colo205, 2 10 HT1080, 1 10 Jurkat cells/well) were seeded in 96-well plates. The next day, cells were treated with 2.5 mg/ml cycloheximide (HT1080, Colo205) and challenged with scFv–scTRAIL (1.35 nM) (HT1080, Colo205) or 2 nM TRAILþ 1 mg/ml anti-FLAG mAb M2 (Jurkat) in the presence or absence of TRAIL-R2 Fab (50 mg/ml). After additional 16 h, cell viability was determined by MTT assay or KV staining. (c)1 10 Colo205 and 1 10 L929 cells/well were seeded and co-cultured in 96-well plates. The next day, cells were treated with 2.5 mg/ml cycloheximide and challenged with 1 nM scTRAIL or scFv–scTRAIL. After additional 16 h, cell viability was determined by MTT assay. (a–c) Results from three independent experiments are shown (mean S.D.) tissue appear largely resistant toward apoptosis induction these are subjected to various stress conditions, such 8 12,32–34 by trimeric TRAIL derivatives, and clinical studies so far as infection and chemotherapeutics. Recently, it has 28,29 revealed that homotrimeric TRAIL is well tolerated. also been shown that TRAIL-R2-mediated apoptosis might However, in contrast to many investigated tumor cell lines, substantially contribute to chronic cholestatic disease. diverse primary cancer cells are inherently resistant to Accordingly, an improvement of the tumor-selective action TRAIL-mediated apoptosis (reviewed by Newsom-Davis of TRAIL appears mandatory. Along this line, we and others et al. ). Moreover, it has been demonstrated that TRAIL have already shown earlier that tumor-targeting approaches may under certain conditions even promote survival, proli- through the generation of TNF ligand fusion proteins, which feration, migration and invasion in TRAIL-resistant tumor aim to restrict TNF ligand’s full cytotoxic activity to the 30,31 cells. Thus, combination therapy with other drugs will be cancerous tissue are very promising approaches to improve required, for example, for sensitization of apoptotic pathways therapeutic efficacy of TRAIL and other members of the or suppression of potentially protumoral TRAIL effects, to TNF ligand family. Conventional fusion proteins of TRAIL achieve major and lasting tumor remissions. In this context, and antibody-derived scFv fragments reveal homotrimeric recent data appear of relevance showing that the resistance of proteins with a molecular mass of B150 kDa. This nearly nontransformed cells toward the apoptotic action of TRAIL corresponds in size to complete antibodies that are reported may vanish, at least in some tissues such as the liver, when to penetrate tumor tissues slowly and nonuniformly. Cell Death and Disease viability (%) viability (%) viability (%) viability (%) viability (%) Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al 100 scTRAIL ** scFv-scTRAIL ** *** *** 10 20 0.1 0 100 200 300 400 time post injection (min) 600 ** P=0.003 control scTRAIL * P=0.03 scFv-scTRAIL * P=0.04 0 0 0 5 10 15 20 control scTRAIL scFv- scTRAIL days after first treatment control *** P<0.0001 scTRAIL ** P=0.006 scFv-scTRAIL *** P=0.0009 0 5 10 15 20 control scTRAIL scFv- scTRAIL days after first treatment Figure 5 Pharmacokinetic properties and antitumoral activity of scTRAIL and scFv–scTRAIL. (a) Reduced elimination of scFv–scTRAIL from circulation. scTRAIL and scFv–scTRAIL were detected by ELISA in serum samples obtained at the indicated time points after i.v. injection into Balb/c mice. For each protein three mice were treated. Significant differences between both proteins were marked by asterisks. (b) Antitumoral activity in a xenograft tumor model – local treatment. NMRI nu/nu mice were injected 6 2 s.c. with 3 10 human colon carcinoma Colo205 cells. Treatment started when tumors reached about 25 mm (8 days after inoculation). Mice received four daily s.c. injections of 1 nmol of the corresponding proteins beginning day 0. n¼ 7 tumors per treatment group; symbols, mean of tumor volumes; bars, S.E.M. Individual tumor sizes at day 11 with bars indicating the mean are shown on the right. (c) Antitumoral activity in a xenograft tumor model – systemic treatment. 3 10 Colo205 cells were s.c. inoculated at the left and right dorsal site of NMRI nu/nu mice. Treatment started when tumors reached about 25 mm (5 days after inoculation). Mice received eight daily i.v. injections of 1 nmol of the corresponding proteins beginning at day 0. n¼ 8 tumors per treatment group; symbols, mean of tumor volumes; bars, S.E.M. Individual tumor sizes at day 19 with bars indicating the mean are shown on the right Furthermore, antibody fragments may have an intrinsic a trimeric TRAIL variant. This is of great importance, as tendency to aggregate, thereby potentially leading to higher it became evident that the formation of high order complexes order complexes, which in turn not only results in a further and aggregates of recombinant forms of TRAIL (e.g., increase in size but may also cause target-independent His-TRAIL, crosslinked FLAG–TRAIL) increases the toxicity TRAIL-R2 activity, too. To circumvent or at least minimize of the ligand to normal cells (reviewed by Koschny et al. ). these potential problems, we have developed a new format As expected, the ErbB2-specific scFv–scTRAIL specifically of a TRAIL fusion protein based on scTRAIL, a single bound to ErbB2-positive cells. This could be confirmed by the polypeptide chain composed of three TRAIL monomers. Gel use of TRAILR-Fc fusion proteins to detect the ErbB2 bound filtration and western blot analysis revealed that the ErbB2- scFv–scTRAIL and by blocking of fusion protein binding specific scFv–scTRAIL fusion protein could be expressed with the ErbB2-specific antibody FRP5, thereby indicating in the expected monomeric state corresponding formally to predominant binding of the fusion protein by the antibody Cell Death and Disease relative tumor size (%) relative tumor size (%) % of the 2 min value relative tumor size (%) relative tumor size (%) day 19 day 11 Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al moiety to ErbB2 rather than by the TRAIL domain and growth, as addition of TRAIL-neutralizing antibodies comple- TRAIL-R. Compared with the non-targeted scTRAIL mole- tely restored cell viability on incubation with scFv–scTRAIL. cule, the tumor antigen-targeted scTRAIL fusion protein This is in accordance with previous reports showing that the showed higher apoptotic activity in vitro on ErbB2-positive mAb FRP5 did not affect tumor growth in vivo, despite even cells. This target antigen-dependent cytotoxicity could be partial agonistic activity in vitro, evident from an increase confirmed by preincubation with competing ErbB2-specific in ErbB2 phosphotyrosine level and accelerated receptor antibody FRP5 or with ErbB2 peptide, restoring cell viability to turnover by antibody-mediated receptor crosslinking. In a significant extent. We and others have previously described contrast, a monovalent scFvFRP5–ETA fusion protein did not that, compared with soluble homotrimeric TRAIL, homo- induce ErbB2-tyrosine phosphorylation, indicating that the trimeric scFv–TRAIL fusion proteins gain a higher activity on activity of a monovalent scFv fusion protein might be limited TRAIL-R2 upon cell surface binding by the targeting domain by the intrinsic turnover rate of its target receptor. We have 13,26,27 within the fusion proteins. Of note, although there is own preliminary data confirming an increase in ErbB2- some controversial discussion on this, it has been shown phosphorylation on treatment of Colo205 cells with a that efficient activation of TRAIL-R2 is only reached by conventional, homotrimeric scFvFRP5–TRAIL fusion protein, natural membrane TRAIL, crosslinked soluble or cell whereas treatment with the monovalent scFv–scTRAIL had 13,26,27,37,38 surface immobilized TRAIL. Importantly, the no receptor-activating effect (data not shown). Accordingly, scFv–scTRAIL fusion protein likewise converted into a the single-chain design concept might be of advantage functional equivalent of membrane TRAIL on target-antigen compared with a conventional fusion protein if target-antigen binding, and thus efficiently induced cell death already at low multimerization has to be avoided. Moreover, Stagg concentrations by TRAIL-R2, the predominant TRAIL- et al. could show that combination of ErbB2 blockade and induced apoptosis pathway in the cell lines studied here. TRAIL-R2 activation can elicit potent synergistic antitumor Accordingly, receptor binding and activation could be blocked activity. Thus, modifying the targeting module using an ErB2- by TRAIL-R2-neutralizing monovalent Fab fragments. More- specific scFv antibody fragment also capable of inhibiting over, Jurkat cells, which only express TRAIL-R2, but not ErbB2 signaling might result in further increased ErbB2- ErbB2 and TRAIL-R1 are resistant against soluble scFv– specific antitumor activity of an scFv–TRAIL fusion protein. scTRAIL, whereas crosslinking of the fusion protein led to an The here described antitumoral activity of the scFv–scTRAIL increase in activity. These properties are especially important fusion protein, which resulted in clear reduction in tumor size based on observations that on tumor cells TRAIL-R2 is more under treatment, appears superior to that of published frequently and strongly expressed than TRAIL-R1, that preclinical data with homotrimeric TRAIL, regarding both 7,8,40 apoptotic signaling through TRAIL-R2 may be more potent the applied dose and the treatment periods. However, than through TRAIL-R1 and that, on the other hand, TRAIL- detailed side-by-side comparison of different TRAIL reagents R2 may be a key regulator of cholestatic liver injury by are required to confirm this observation. induction of apoptosis in cholangiocytes. Consequently, the In conclusion, our data provide evidence that bioactive scFv–scTRAIL might be an appropriate therapeutic, which single-chain TRAIL fusion proteins can be generated. Further- meets the criteria of high antitumor and low off-target activity. more, fusion to a target-specific scFv antibody results In vivo analyses in mice revealed that, on i.v. injection, the in target-dependent increase in TRAIL-mediated cytotoxicity scFv–scTRAIL and the scTRAIL displayed biphasic pharma- in vitro, likely due to efficient TRAIL-R2 activation, and cokinetics. In the first phase, the calculated t a of B35 min improves antitumoral activity in vivo. Accordingly, this design was similar for both proteins. In the second phase, the scFv– concept represents a promising strategy to enhance TRAIL’s scTRAIL displayed a slightly increased half-life with t b antitumoral action and to minimize potential unwanted of B160 min compared with 125 min for the scTRAIL. This off-target actions on normal tissues. strongly contrasts to some of the published data for homo- trimeric TRAIL, indicating a half-life of 3–5 min (t a) and Materials and Methods 40,41 20 min (t b) in rodents. Our results rather match with 2 Plasmids and cell lines. The pIRESpuro–scTRAIL expression construct for pharmacokinetic data of LZ-TRAIL, a trimer-stabilized TRAIL human scTRAIL (aa 95–281) was generated by insertion of linker sequences, 1 1 variant (t a: 1.3 h; t b: 4.8 h ). Most important, both single- encoding four repetitions of the GGGS motif, between the three TRAIL modules as 2 2 described previously for the construction of scTNF. chain TRAIL variants significantly reduced growth of estab- The single-chain antigen binding protein scFvFRP5 for the ErbB2-specific lished Colo205 tumors already at rather moderate doses, pIRESpuro–scFv–scTRAIL expression construct was described previously. either on local or systemic treatment. In accordance with the HEK293, Jurkat, Colo205, SKW6.4 and L929 cells were obtained from the in vitro bioactivity, the scFv–scTRAIL displayed superior American Type Culture Collection (Manassas, VA, USA) or DSMZ – Deutsche tumoricidal effects in vivo compared with the scTRAIL. Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, We propose that increased antitumoral activity is largely Germany). The HT1080 cells were a kind gift of W Rettig (Boehringer Ingelheim Pharma, Vienna, Austria). Cells were cultured in RPMI 1640 medium (Invitrogen, related to ErbB2-mediated tumor enrichment and the target- Karlsruhe, Germany) supplemented with 5% or 10% (SKW6.4) fetal calf serum ing-dependent increase in bioactivity rather than the slightly (FCS, PAA Laboratories, Pasching, Austria). increased bioavailability. The observed re-growth of tumors upon cessation of treatment has also been reported with Production and purification of recombinant proteins. The TRAIL homotrimeric TRAIL and might overcome when the combi- fusion proteins were produced in HEK293 cells after stable transfection of the nation treatments with appropriate chemotherapeutics are corresponding expression plasmids using Lipofectamine 2000 (Invitrogen) and 9,10,40 conducted. Furthermore, we can exclude an effect of selection of stable clones by limiting dilution. For protein production, stable clones the ErbB2-specific antibody fragment scFvFRP5 on tumor were expanded and grown in RPMI 1640, 5% FCS, to 90% confluency and Cell Death and Disease Tumor-targeted single-chain TRAIL for cancer therapy B Schneider et al The control group received 100 ml concentrated cell-culture supernatant from subsequently cultured in serum-free Optimem (Invitrogen) supplemented with empty vector-transfected cells at the same time intervals. 50 mM ZnCl , replacing media two times every 3 days. Supernatants were pooled and recombinant proteins were purified by affinity chromatography using anti-FLAG Systemic treatment. Colo205 cells (3 10 ) in 100 ml PBS were s.c. inoculated mAb M2 agarose (Sigma-Aldrich, Steinheim, Germany). The bound proteins were at the left and right dorsal sides of 6-week-old female NMRI nu/nu mice. Treatment eluted with 100 mg/ml FLAG peptide (Sigma-Aldrich) and dialyzed against PBS. started 5 days after tumor cell inoculation when tumors reached B25 mm . Mice After concentrating purified proteins by dialysis on PEG35000, they were analyzed received 8 daily i.v. injections of 1 nmol of the affinity purified TRAIL fusion proteins. and quantified by SDS-PAGE, silver gel (Sigma-Aldrich) and gel filtration and stored The control group received 100 ml PBS at the same time intervals. Tumor growth at 41C. was monitored as described. Gel filtration. Protein samples were applied to a BioSuite250 HR SEC (300 7.8) column (Waters, Millipore Corp., Milford, MA, USA) equilibrated in Conflict of interest PBS and eluted at a flow rate of 0.5 ml/min. The authors declare no conflict of interest. Flow cytometry. Cells (5 10 ) were incubated for 2 h at 41C with the indicated concentrations of the TRAIL fusion proteins. After washing the cells three Acknowledgements. We gratefully acknowledge technical support in the times with PBS, 2% FCS, 0.02% sodium azide, bound fusion proteins were detected initial expression of scTRAIL by Sylvia Willy. Our work is supported by grants from by anti-human TRAIL mAb (MAB687) (1 mg/ml, R&D Systems, Wiesbaden, Deutsche Krebshilfe, Grant no. 107551 and 108268 to HW and KP, from BMBF Germany) and fluorescein isothiocyanate-labeled rabbit anti-mouse IgG Ab (Biochance plus, FKz 0315164) to KP and by a career fellowship award by the (1 mg/ml, Sigma-Aldrich). ErbB2 expression was detected by anti-c-neu (AB-5) Peter and Traudl Engelhorn Foundation to JG. (1 mg/ml, Merck KGaA, Darmstadt, Germany) and fluorescein isothiocyanate- labeled rabbit anti-mouse IgG Ab (1 mg/ml, Sigma-Aldrich). Alternatively, cell-bound scFv–scTRAIL was detected by TRAILR–Fc fusion proteins (5 mg/ml TRAILR1-Fc and 3 mg/ml TRAILR2-Fc) and fluorescein isothiocyanate-labeled anti-human IgG, 1. Gerber DE. 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To view a copy of this license, mediated-apoptosis contributes to cholestatic liver disease. Proc Natl Acad Sci USA 2008; 105: 10895–10900. visit http://creativecommons.org/licenses/by-nc-nd/3.0/ Cell Death and Disease

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