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. 2006 Apr 25;55(12):1590–1600. doi: 10.1007/s00262-006-0162-6

Target-selective activation of a TNF prodrug by urokinase-type plasminogen activator (uPA) mediated proteolytic processing at the cell surface

Jeannette Gerspach 1, Julia Németh 1, Sabine Münkel 1, Harald Wajant 2, Klaus Pfizenmaier 1,✉
PMCID: PMC11029883  PMID: 16636812

Abstract

We have previously developed TNF prodrugs comprised of a N-terminal scFv targeting, a TNF effector and a C-terminal TNFR1-derived inhibitor module linked to TNF via a MMP-2 motif containing peptide, allowing activation by MMP-2-expressing tumor cells. To overcome the known heterogeneity of matrix metalloprotease expression, we developed TNF prodrugs that become processed by other tumor and/or stroma-associated proteases. These TNF prodrugs comprise either an uPA-selective or a dual uPA-MMP-2-specific linker which displayed efficient, target-dependent and cleavage sequence-specific activation by the corresponding tumor cell-expressed proteases. Selective pharmacologic inhibition of endogenous uPA and MMP-2 confirm independent prodrug processing by these two model proteases and indicate the functional superiority of a prodrug containing a multi-specific protease linker. Processing optimised TNF prodrugs should increase the proportion of active therapeutic within the targeted tissue and thus potentially enhance tumor response rate.

Keywords: Targeted TNF activation, Prodrug processing, uPA, MMP-2

Introduction

Targeting death receptors to induce programmed cell death is one of the promising strategies currently being explored in clinical cancer research. In particular, tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL) and monoclonal antibodies specific for the two apoptosis-inducing TRAIL receptors DR4 and DR5 are currently in clinical use or under clinical investigation in phase I and II studies [17]. In contrast, targeting the prototype apoptotic death receptor Fas (CD95) has been excluded recently from clinical evaluation due to severe side effects, specifically acute liver failure, of Fas agonists in pre-clinical animal models [44]. Like Fas agonists, systemic administration of TNF is also associated with severe dose-limiting systemic toxicity [44]. Nevertheless, from a long history of clinical evaluation of TNF, its present, albeit very restricted, use in cancer therapy has emerged. TNF is successfully applied in combination with melphalan as a regional treatment of unresectable extremity sarcoma, transit melanoma and advanced refractory cancers confined to the liver using isolated limb or liver perfusion protocols [16]. In contrast to TRAIL, where the anti-tumoral action is believed to reside largely in its apoptosis-inducing capacity in malignant but not in normal cells [17], TNF’s anti-tumoral potential appears more complex and is effective at different cellular levels (reviewed in [44]). Specifically, the predominant target of TNF’s anti-tumoral action is not the tumor cells themselves but rather the tumor vasculature. Here, TNF exerts multiple dose-dependent actions, including a rise in the vascular permeability to increase local concentration of chemotherapeutics at the tumor site, the induction of endothelial cell apoptosis and blood clotting to abrogate tumor nutrient and oxygen supply. In addition, immune-stimulatory actions of TNF, recruitment and activation of effector cells, is considered to contribute to tumor regression [44].

In order to make a broader use of TNF’s anti-tumoral potential, a useful therapeutic window and strategies to allow systemic TNF application have to be defined [9, 35]. To limit systemic action, specific enrichment of TNF in the tumor area by targeting TNF to tumor-associated antigens is one of the approaches currently exploited in the pre-clinical models. TNF fused to tumor-targeting antibody fragments [3, 5] or peptides [10, 26] provided promising results in various mouse tumor models. The anti-tumoral effect was more pronounced in combination with chemotherapeutics [5, 11, 12], resembling the clinical experience with non-targeted recombinant TNF. Further strategies to reduce TNF’s systemic toxicity include mutagenesis [8, 33] and modification with poly-ethylenglycol or other chemical modifications [24, 46, 48]. In an alternative approach to prevent systemic side effects and at the same time to maintain full biological activity of TNF at the tumor site, we have recently described the development of TNF prodrugs, which are inactive unless specifically bound to the membrane of the target cell, where TNF activity is unmasked by specific proteolytic processing through tumor-associated proteases [19, 45]. Accordingly, TNF prodrugs are comprised of an scFv–TNF fusion protein, targeting a tumor associated marker, to which a TNF receptor fragment is linked C-terminally through a protease sensitive linker. This receptor fragment acts as an intra-molecular inhibitor by blocking TNF binding to cellular TNF receptors ubiquitously expressed on normal tissues. We have recently shown that a TNF prodrug comprising matrix metalloproteinase 2 (MMP-2) selective cleavage sites was activated by proteolytic processing through tumor cell expressed MMPs [19].

MMPs are reported to be expressed both in the malignant cells and the stromal fibroblasts of many types of solid tumors, such as melanoma, breast and colon cancer, and MMP-2 in particular is considered to be involved in tumor progression, angiogenesis and metastasis [14, 15, 22, 29]. However, there is inter- and intra-tumoral heterogeneity in matrix metalloprotease expression. Based on several studies which indicated a negative correlation of MMP-2 with survival time, the abundance of MMP-2 is regarded as a prognostic factor [15]. Due to heterogeneity in MMP-2 expression, a TNF prodrug depending exclusively on MMP-2-mediated activation may have a limited activity in vivo in tumors with low or negative MMP-2 expression. To bypass this potential limitation, we have tested the suitability of other tumor-associated proteases for prodrug activation and investigated the possibility of creating TNF prodrugs comprised of a multi-specific protease linker, thereby allowing the activation by several proteases. As a first step towards this goal, we here describe the feasibility of this approach by generating a prodrug that is comprised of a dual specificity linker and selectively processed by both MMP-2 and urokinase-type plasminogen activator (uPA), which is also reported to be expressed in various tumor tissues [1, 13].

Materials and methods

Cloning of the prodrug constructs

The TNF prodrug protein lacking the protease-sensitive linker was cloned as described previously [19]. The linkers containing cleavage sites for uPA and MMP-2 were introduced via StuI/FseI in the form of two pairs of complementary oligos encoding following protein sequences, uPA (three consecutive uPA cleavage sites with three glycines in between: SGR-SA GGG SGR-SA GGG SGR-SA [25, 31]; MPA (two cleavage sites for MMP-2 followed by two cleavage sites for uPA separated by three or two glycine residues, respectively): HPVG-LLAR GGG HPVG-LLAR GG SGR-SA GG SGR-SA [42]. The final constructs were designated as αFAP-S-uPA and αFAP-S-MPA comprising a serine–glycine linker between scFv and TNF and with cleavage site specificity for either uPA or uPA and MMP-2, respectively. The prodrug constructs were cloned with the leader into the vector pIRESpuro3 (BD Biosciences Clontech, Heidelberg, Germany) via EcoRI. The construction of the FAP-specific control prodrug αFAP-S-PL0 with a protease-insensitive (Ser4Gly)6 linker has already been published [19]. Restriction enzymes were purchased from MBI Fermentas and New England Biolabs.

Production and purification of the prodrug

After stable transfection with Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) and selection with puromycin (EMD Biosciences, Schwalbach, Germany) expression of fusion proteins was performed in HEK293 cells using serum-free media (Optimem, Invitrogen). For this purpose cells were cultured to confluency in growth medium, then the medium was replaced by Optimem and supernatant was harvested and replaced by fresh Optimem three times every second day. Alternatively cells were cultured with growth medium in spinner flasks until they reached a cell densitiy of about 3×104 cells/ml. After centrifugation about 3×107 cells were resuspended in 125 ml Optimem and cultured for 2 days. Supernatant was harvested and the cells transferred to fresh Optimem in order to continue production for further 2 days. The fusion proteins were purified from supernatant by immobilised metalchelate chromatography (IMAC) on Zn2+ loaded Hi-Trap Chelating columns (GE Healthcare Life Sciences, Munich, Germany) or in batch procedure on Chelating Sepharose Fast Flow (GE Healthcare Life Sciences) loaded with Cu2+. Proteins were eluted either with 300 mM Imidazol or with 100 mM EDTA. After dialysis against PBS, protein concentration was determined by the CB-X Protein Assay (Genotech, St. Louis, MO, USA) and purification grade analysed by SDS-PAGE and silver staining (Sigma-Aldrich, Munich, Germany).

Cell lines

The fibrosarcoma cell lines HT1080 and HT1080-FAP have been described previously [38, 43] and were a kind gift of W. Rettig (Boehringer Ingelheim Pharma, Vienna, Austria). The TNF-sensitive human rhabdomyosarcoma cell line Kym-1 was originally supplied by M. Sekiguchi (University of Tokyo, Japan). HEK293 cells were obtained from DSMZ, Braunschweig, Germany. Cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 5% foetal calf serum. HT1080-FAP cells were grown in the presence of 250 μg/ml G418. Stable HEK293 transfectants were cultivated in the presence of 2–3 μg/ml puromycin.

Western blot analyses

Purified proteins or supernatants were separated on a 12% SDS-PAGE under reducing conditions and electroblotted to nitrocellulose membrane (Pall, Dreieich, Germany). Non-specific binding was blocked with 2% Tween 80 and detection performed with rabbit anti-murine TNF-α Ab (HyCult Biotechnology, PB Uden, The Netherlands) and alkaline phosphatase-conjugated goat anti-rabbit IgG Ab (Sigma-Aldrich) or anti-Tubulin alpha mouse mAb (Lab Vision, Newmarket Suffolk, England) and alkaline phosphatase-conjugated goat anti-mouse IgG Ab (Sigma-Aldrich) using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roth, Karlsruhe, Germany) as substrates.

Size exclusion chromatography

Analysis of the apparent molecular weight of αFAP-S-uPA was performed by HPLC on a BioSep-Sec_S 3000 column (Phenomenex, Torrance, USA) under standard conditions with a flow rate of 0.5 ml/min and PBS as running buffer.

Immunofluorescence flow cytometry

HT1080 and HT1080-FAP cells were incubated for 2 h at 4°C with 4–10 μg/ml of purified or supernatant containing αFAP-S-uPA or αFAP-S-MPA. Bound prodrug was detected by anti-c-myc 9E10 mAb followed by fluoresceine isothiocyanate-labelled rabbit anti-mouse IgG Ab (Sigma-Aldrich). Analyses were performed using EPICS-XL (Coulter, Krefeld, Germany) according to standard procedures.

In vitro activation of the TNF prodrugs

For proteolytic activation of the TNF prodrugs by recombinant enzymes, reaction conditions were as follows: 3–10 μg/ml TNF prodrug was incubated for 6 h or over night with 2 μg/ml recombinant MMP-2 (EMD Biosciences) in MMP-2 reaction buffer (200 mM NaCl, 50 mM Tris–HCl pH 7.5, 20 μM ZnSO4, 1 mM CaCl2, 0.05% Brij 35), or over night with 3.5 μg/ml uPA in uPA reaction buffer (150 mM NaCl, 10 mM Tris–HCl pH 7.5) at 37°C. MMP-2 inhibition was assayed in the presence of 25 μM Ilomastat (Chemicon International, Hofheim, Germany) and uPA inhibition in the presence of 200 μM Amiloride (Sigma-Aldrich).

Cytotoxicity assays

Kym-1 cells (1.5×104/well) were grown over night in 100 μl of culture medium in 96-well plates. The following day prodrug (activated or not) was titrated at the indicated concentrations on the cells in duplicates. After over night incubation cell viability was determined using MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) staining as described previously [45]. OD values of untreated control cells have been set as 100% viability. For cytotoxicity assays on HT1080 or HT1080-FAP cells, 2×104 cells/well were seeded 1 day before purified prodrugs or supernatants were titrated in the presence of 2.5 μg/ml cycloheximide. After over night incubation, cell viability was assessed by crystal violet staining as described [43]. For the inhibition of endogenous MMP-2 or uPA cells were pre-incubated with 25 μM Ilomastat or 200 μM Amiloride, respectively. The pan caspase inhibitor z-VAD-fmk (Bachem, Weil am Rhein, Germany) was added at 20 μM where indicated.

Caspase 3 activity assay

2.5×105 HT1080 and HT1080-FAP cells were seeded in 12-well plates and incubated the following day for 7 h with 0.5 nM αFAP-S-uPA, αFAP-S-MPA or TNF in the presence of 2.5 μg/ml cycloheximide. Supernatant and cells, which were harvested by a cell scraper, were collected and centrifuged. Cell pellets were washed in PBS and stored at −20°C. Pellets were lysed for 20 min on ice in 45 μl of lysis buffer containing 50 mM HEPES pH 7.4, 5 mM CHAPS, 5 mM DTT. After centrifugation 35 μl of lysate was transferred and used for caspase 3 activtity assay which was done in triplicates in a total volume of 100 μl in assay buffer (20 mM HEPES pH 7.4, 0.1% CHAPS, 5 mM DTT, 2 mM EDTA). Each reaction contained 5 μl cell lysate and 10 μl of 2 mM Ac-DEVD-pNA substrate (Sigma-Aldrich, 20 mM stock solution in DMSO). For Inhibition of caspase 3 activity 10 μl of 0.2 mM Ac-DEVD-CHO (Sigma-Aldrich, 2 mM stock solution in DMSO) was added. After over night incubation absorbance was measured at 405 nm. OD values resulted from subtraction of blank reaction containing only assay buffer and substrate.

Cell surface biotinylation

HT1080 and HT1080-FAP cells (1×106/well) were grown over night in culture medium in 6-well plates. The following day, medium was removed and 20–30 nM of αFAP-S-uPA, αFAP-S-MPA or αFAP-S-PL0, either as purified protein or culture supernatant from producer cell lines, were added in 1 ml Optimem containing 1% FCS. Upon prodrug incubation for 2 h all further steps were carried out on ice. Cells were washed with ice-cold phosphate buffer and incubated for 20 min with 0.2 mg/ml EZ-Link-Sulfo-NHS-LC-LC-Biotin (Perbio, Bonn, Germany; stock solution of 20 mg/ml was freshly prepared in DMF) in PBS. Cells were washed and incubated for 10 min with 50 mM Glycine in PBS pH 7.4. Again cells were washed and then lysed with 1% Triton, 150 mM NaCl, 20 mM Tris, pH 7.4 containing protease inhibitor cocktail complete (Roche Diagnostics, Mannheim, Germany) for 30 min. After centrifugation lysates were incubated with Streptavidin agarose (EMD Biosciences, Novagen Brand) for 80 min at 4°C. Streptavidin agarose was washed with PBS and bound proteins eluted in 4× SDS loading buffer and analysed in reducing SDS-PAGE followed by Western blot.

Analysis of αFAP-S-uPA incubated on HT1080 or HT1080-FAP cells

HT1080 or HT1080-FAP cells (1.5×105/well) were grown overnight in 24-well plates. The following day, the medium was replaced by 500 μl Optimem with 20 nM purified αFAP-S-uPA. After over night incubation, the supernatant was harvested, centrifuged and further analysed by Western Blot as described above.

Results

Production of recombinant TNF prodrugs comprising uPA- and uPA-MMP-2-sensitive linkers

The TNF prodrugs constructed here have the same domain architecture as described recently [19]. Briefly, a species cross-reactive scFv directed to the tumor stroma marker FAP [18] was coupled via a serine–glycine linker to a murine TNF variant lacking the TACE cleavage site. The ligand-binding domain of human TNFR1 was connected to the scFv–TNF fusion protein via a protease-sensitive linker. This linker either contains three consecutive cleavage sites for uPA or two cleavage sites for MMP-2 followed by two cleavage sites for uPA (Fig. 1a). The corresponding cleavage motifs were HPVG-LLAR (MMP-2, [42]) and SGR-SA (uPA, [25,31]) reported to be preferentially cleaved by the respective enzyme.

Fig. 1.

Fig. 1

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Schematic overview and biochemical analyses of expressed TNF prodrug constructs. a Schematic presentation of the TNF prodrug constructs αFAP-S-uPA and αFAP-S-MPA. αFAP scFv36; S (serine4–glycine)3 linker; muTNF N-terminal deletion mutant of the soluble form of murine TNF; uPA uPA-sensitive linker with three consecutive SGR-SA motifs; MPA protease-sensitive linker with two HPVG-LLAR motifs (cleavage sites for MMP-2) followed by two SGR-SA motifs (cleavage sites for uPA); TNFR1 fragment of the extracellular domain of human TNFR1. b Analysis of αFAP-S-uPA and αFAP-S-MPA in SDS-PAGE. 500–600 ng of IMAC purified, dialysed protein were separated under reducing conditions and subsequently silver stained. M PageRuler™ Prestained Protein Ladder (Fermentas, St. Leon-Rot, Germany). c Elution profile of the TNF prodrug αFAP-S-uPA resulting from size exclution chromatography. Ten micrograms of protein was loaded and run with 0.25 ml/min. d Fractions corresponding to the observed peak were analysed by Western blot using rabbit murine TNF-specific antibodies

SDS-PAGE analysis of the purified TNF prodrugs under reducing conditions showed single bands migrating just above 70 kDa (Fig. 1b), which is somewhat higher than the calculated molecular weight of a prodrug monomer of 65 kDa for αFAP-S-uPA and 66 kDa for αFAP-S-MPA. In gel filtration analysis the TNF prodrug αFAP-S-uPA eluted as a single peak at around 400 kDa (Fig. 1c), which is resolved as a single band migrating at around 70 kDa upon reducing SDS-PAGE (Fig. 1d). Together, the biochemical analyses suggest a hexameric organisation of the fusion protein under native conditions, which is achieved by the intrinsic trimerisation of the TNF domain and the subsequent dimerisation of two TNF prodrug trimers via their TNF and TNFR domains [19].

Antigen-specific binding and specific cleavage of the TNF prodrug by uPA and MMP-2

Immunofluorescence flow cytometry using FAP expressing and parental HT1080 cells showed specific target binding of αFAP-S-uPA (Fig. 2a) and αFAP-S-MPA (Fig. 2b) to FAP antigen confirming that antigen binding was retained in these constructs. Incubation of the TNF prodrugs with recombinant uPA or MMP-2 generated a shortened protein with an apparent molecular weight of ∼45–50 kDa in SDS-PAGE, corresponding in size to the active scFv–TNF part of the TNF prodrug (Fig. 2c, d). A fragment of this size is in accordance with proteolytic cleavage at the protease linker and the removal of the blocking TNFR1 fragment. For αFAP-S-uPA only uPA (Fig. 2c, right panel), but not MMP-2 (Fig. 2c, left panel), was able to remove the inhibitor domain. In contrast, αFAP-S-MPA, which contains cleavage sites for both enzymes, could be processed by both uPA and MMP-2 (Fig. 2d). In the presence of the respective enzyme inhibitor Amiloride (for uPA) or Ilomastat (for MMP-2) cleavage could be blocked, verifying the site-specific processing of the prodrug (Fig. 2c, d).

Fig. 2.

Fig. 2

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Antigen specificity and processing of the TNF prodrugs. Specific binding of TNF prodrugs to FAP antigen. α-FAP-S-uPA (a) or α-FAP-S-MPA (b) were incubated with HT1080 or HT1080-FAP cells. The bound prodrug proteins were detected by indirect immunfluorescence flow cytometry via the myc tag as described in Materials and methods. Ten thousand cells were analysed for each group. Specific processing of αFAP-S-uPA and αFAP-S-MPA by recombinant enzymes. c α-FAP-S-uPA was incubated 6 h (left panel) or over night in the presence or absence of MMP-2 or uPA under respective buffer conditions at 37°C as indicated. For the inhibition of recombinant uPA Amiloride was added to the reaction. d α-FAP-S-MPA was incubated as indicated in a. Inhibition of the respective enzyme was reached by incubation in the presence of Amiloride (uPA inhibitor, 200 μM) or Ilomastat (MMP inhibitor, 25 μM). Proteins were separated by 12% SDS-PAGE (reducing conditions) and detected by Western blot with rabbit murine TNF-specific antibodies

Apoptosis induction by in vitro activated TNF prodrugs

To assess bioactivity of TNF prodrugs specifically processed by uPA or MMP-2, Kym-1 cells, which respond to TNF treatment by apoptosis induction [21], were incubated with non-processed and in vitro processed TNF prodrugs. In this assay only non-targeted TNF bioactivity can be analysed, as Kym-1 cells do not express the FAP antigen. As expected, both TNF prodrugs lacked detectable TNF activity up to a concentration of 5 nM (Fig. 3a–c). At this concentration, complete cell death is induced in Kym-1 cells by standard recombinant TNF (EC50 3±2 pM [19]). Treatment of αFAP-S-uPA (Fig. 3a) or αFAP-S-MPA (Fig. 3b) with purified recombinant uPA led to the restoration of cell death induction in this non-targeted TNF bioassay. For αFAP-S-MPA a similar increase in bioactivity was seen after processing by recombinant active MMP-2 (Fig. 3c). This shows that both recombinant enzymes were able to process αFAP-S-MPA such that an unblocking of TNF activity is achieved. In the presence of the uPA-specific inhibitor Amiloride, prodrug activation by uPA could be substantially diminished for both the αFAP-S-uPA (Fig. 3a) and the αFAP-S-MPA prodrugs (Fig. 3b). Likewise, the MMP inhibitor Ilomastat interfered with MMP-2-mediated (Fig. 3c), but not with uPA-mediated (data not shown) activation of αFAP-S-MPA, confirming the Western blot data on in vitro prodrug processing and the specificity of the applied inhibitors.

Fig. 3.

Fig. 3

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Unmasking of TNF bioactivity after processing of TNF prodrug proteins by recombinant enzymes. Activation of the soluble TNF prodrugs by recombinant uPA. αFAP-S-uPA (a) or αFAP-S-MPA (b) was incubated over night with recombinant uPA (squares) in the absence (filled squares) or presence (open squares) of uPA inhibitor Amiloride or incubated in reaction buffer alone (diamonds). Activity of the TNF prodrug was assayed in a cytotoxicity test on Kym-1 cells. Cell viability was determined as described in Materials and methods. c Activation of the soluble TNF prodrug αFAP-S-MPA by recombinant MMP-2. After over night incubation of αFAP-S-MPA with recombinant MMP-2 (squares) in the absence (filled squares) or presence (open squares) of the inhibitor Ilomastat or in reaction buffer alone (diamonds) activity of the TNF prodrug was assayed as in a, b

αFAP-S-uPA and αFAP-S-MPA are potent apoptosis inducers upon scFv-mediated targeting to cells expressing endogenous MMPs and uPA

HT1080 cells are well known to express the metalloproteinase MMP-2 and the serine proteinase system uPA/uPAR [20, 28, 30, 34, 40]. These cells become highly sensitive towards death receptor induced apoptosis in the presence of low doses of cycloheximide [38, 43]. HT1080 cells stably transfected with FAP are thus a suitable tool to study antigen-dependent targeting of FAP directed TNF prodrugs and their conversion by endogenous proteases into an active cytokine. Viability assays on FAP-positive HT1080 cells revealed that the TNF prodrugs αFAP-S-uPA (Fig. 4a) and αFAP-S-MPA (Fig. 4b) strongly induce apoptosis without addition of exogenous uPA or MMP-2. When compared to recombinant soluble TNF (EC50 of rec muTNF: 0.4±0.2 pM), the TNF prodrugs were found to possess superior (αFAP-S-uPA, 0.1±0.1 pM, Fig. 4a) or equal (αFAP-S-MPA, 0.6±0.2 pM, Fig. 4b) activity on these cells. Efficient induction of apoptosis was verified by caspase-3 activation (Fig. 4c) and by blocking apoptosis with the pan caspase inhibitor z-VAD-fmk (Fig. 4d). The parental, FAP-negative HT1080 cells, which are equally sensitive towards soluble TNF (EC50 of rec muTNF: HT1080 0.7±0.3 pM) that express comparable protein levels and the activity of MMPs and uPA (data not shown), remained unaffected by the treatment with each of the two prodrug proteins over a wide dose range. Induction of cell death in these cells was only observed at concentrations above 1 nM (Fig. 4a, b). To make sure that endogenous MMPs and/or uPA were responsible for the activation of the TNF prodrug αFAP-S-MPA and αFAP-S-uPA, respectively, cytotoxicity was determined in the presence of specific protease inhibitors. The addition of the uPA inhibitor Amiloride led to the inhibition of αFAP-S-uPA-induced cell death on HT1080-FAP cells, whereas the metalloproteinase inhibitor Ilomastat was ineffective (Fig. 4e). In the case of αFAP-S-MPA, comprising a peptide linker sensitive towards both proteases, cytotoxicity could be substantially blocked when a combination of Amiloride and Ilomastat was present (Fig. 4f). In accordance with an independent processing and activation of the prodrug by uPA and MMPs, the addition of each of the inhibitors alone was not sufficient to block prodrug activation and apoptosis induction (Fig. 4f).

Fig. 4.

Fig. 4

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Apoptosis induction after target-dependent TNF prodrug activation by tumor cell expressed proteases. TNF prodrug activation is dependent on antigen expressed on target cells. HT1080-FAP (filled symbols) and HT1080 (open symbols) cells were seeded and incubated the following day with αFAP-S-uPA (a) or αFAP-S-MPA (b) at the indicated concentrations. Cell viability was determined after over night incubation by crystal violet staining as described in Materials and methods. Data shown represent one of at least three independent experiments. Antigen-dependent activation of TNF prodrugs by endogenous proteases induces apoptotic cell death. c HT1080 cells (hatched bars) and HT1080-FAP cells (solid bars) were seeded 1 day before incubation with 0.5 nM αFAP-S-uPA, αFAP-S-MPA or TNF for 7 h. Caspase 3 activity of cell lysates was measured by a conversion of the specific chromogenic substrate Ac-DEVD-pNA in the presence (white) or absence (grey) of caspase 3 specific inhibitor Ac-DEVD-CHO. d HT1080-FAP cells (solid bars) were incubated over night with 10 pM αFAP-S-uPA, αFAP-S-MPA or TNF in the presence of cycloheximide with (white) or without (grey) caspase inhibitor z-VAD-fmk. As a control HT1080 cells (hatched bars) were incubated with prodrugs as above in the absence of z-VAD-fmk. z-VAD-fmk inhibits TNF-induced cell death in both cell lines. TNF prodrug activation is blocked by specific inhibition of endogenous proteases. e HT1080-FAP cells were seeded and pre-incubated for 1 h with MMP-inhibitor Ilomastat or uPA inhibitor Amiloride before adding TNF prodrug αFAP-S-uPA (0.4 pM, e), αFAP-S-MPA (2.5 pM, f) or αFAP-S-PL0 (2.5 pM, TNF prodrug with a protease insensitive SG-linker, f). Cell viability was determined using cristal violet staining (a, b, d–f)

Processing of FAP targeted αFAP-S-uPA and αFAP-S-MPA on the surface of HT1080 cells

To verify that TNF prodrugs are processed on the cell surface of target-positive cells, FAP expressing and control HT1080 cells were incubated with TNF prodrugs, non-bound prodrug was removed by subsequent washings and cell surface biotinylation was performed and labelled membrane proteins isolated from whole cell lysates by Streptavidin agarose. Western blot analyses of biotinylated proteins isolated from HT1080-FAP treated with αFAP-S-uPA (Fig. 5a, left panel) and αFAP-S-MPA (Fig. 5a, middle panel) detected a protein corresponding in size to the respective active scFv–TNF fragment. These data show that the prodrugs were indeed processed and that the active TNF containing fragment of these constructs remained largely attached to the cell surface of FAP-expressing HT1080 cells. The targeting dependence of this membrane-bound processing step was evident from the lack of detectable bands corresponding to either non-processed or processed prodrug from target-negative control cells (Fig. 5a). Equal band intensity of tubulin alpha subunit verified that for both cell lines comparable amounts of cell lysates were applied. As an additional control for the membrane-target-dependent processing, the presence of processed prodrug in the supernatant of prodrug-treated tumor cell cultures was also analysed. To this, αFAP-S-uPA prodrug was incubated at 37°C for a prolonged period (overnight) with HT1080 or HT1080-FAP cells without removing excess amounts of prodrug by washing and culture supernatants were analysed by Western blotting. As already shown previously for an MMP-2-sensitive prodrug [19], under these conditions, a band corresponding in size to the processed αFAP-S-uPA prodrug could be revealed in the culture supernatant of FAP expressing, but not of parental HT1080 cells (Fig. 5b), which is in full accordance with a membrane-target dependence of prodrug processing. Further, we have previously shown for an MMP-2-sensitive prodrug that the processed, bioactivate form could be detected in supernatants from FAP expressing, but not FAP-negative HT1080 cell cultures [19]. Incubation of HT1080-FAP cells with the uPA inhibitor Amiloride largely prevented cell-associated processing of the prodrug αFAP-S-uPA, as revealed by Western Blotting from the recovery of a band corresponding to the full-length protein (Fig. 5, left panel), whereas for the αFAP-S-MPA prodrug, carrying a dual specificity cleavage site, the addition of both inhibitors (for uPA and MMP) was required to inhibit processing by HT1080-FAP cells (Fig. 5, middle panel). The appearance of a double band of the cell processed αFAP-S-MPA prodrug and the differential sensitivity towards the two protease inhibitors further underlines the independent and cleavage site-selective action of the cellular proteases. The prodrug αFAP-S-PL0, which contained a protease-insensitive linker showed binding to FAP, but remained intact at the surface of HT1080-FAP cells (Fig. 5, right panel). Taken together, these findings indicate enzyme and cleavage site-specific processing, and support the view that the targeted tumor cells studied here are capable of prodrug activation through alternative proteolytic systems.

Fig. 5.

Fig. 5

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TNF prodrug activation occurs at the cell surface of FAP antigen-expressing HT1080 cells. a Biochemical analysis of TNF prodrug processing by endogenous proteases. HT1080 and HT1080-FAP cells were seeded and incubated the following day for 2 h with TNF prodrug αFAP-S-uPA, αFAP-S-MPA or αFAP-S-PL0 (20–30 nM) in the presence or absence of Amiloride (uPA inhibitor) and Ilomastat (MMP inhibitor) as indicated. After cell surface biotinylation biotin-labelled proteins were captured by Streptavidin agarose and analysed by Western blot (12% SDS-PAGE, reducing conditions). Detection was performed via rabbit anti-murine TNF antibodies. To control equal protein concentrations of lysates before incubation with Streptavidin agarose, cell lysates were analysed for tubulin alpha subunit by mouse anti-tubulin alpha Ab. FAP HT1080-FAP cells, HT HT1080 wildtype cells. b Biochemical analysis of TNF prodrug in supernatants of FAP-positive and negative HT1080 cells. HT1080 or HT1080-FAP cells (1.5×105 per well) were seeded in 24-well plates. The following day, the medium was replaced by 500 μl Optimem with 20 nM αFAP-S-uPA. After over night incubation, the supernatant was harvested, centrifuged and further analysed by Western Blot as described above. FAP HT1080-FAP cells; HT HT1080 wildtype cells; – incubation of TNF prodrug at 37°C without cells

Discussion

With the data described here, we corroborate the principle of genetic engineering of cytokine prodrugs and their target-specific activation by proteolytic processing. We extend our previous findings by showing that target cell-dependent proteolytic activation (1) can be achieved independently by different tumor cell-associated proteases, (2) is confined to the inserted protease linker, (3) is protease cleavage-site specific and thus amenable to modifications that ultimately will allow the generation of prodrugs tailored to match the inter- and intra-tumoral diversities in protease expression.

Our data reveal that in the cell system studied uPA is equally suitable for TNF prodrug processing as MMP-2 and that the introduction of multi-cleavage site, containing peptide linkers into a prodrug, is possible. Although the two TNF prodrugs, which both contain the uPA cleavage site, can be processed by soluble recombinant, active forms of uPA, activation by cell-associated endogeneous uPA was only observed on target antigen-expressing cells. Prodrug activation by uPA, like MMP-2-mediated processing [19], therefore, is supposed to be a membrane-restricted process too. The important role of cell surface presentation for an efficient conversion of the prodrug by endogenous proteases is further supported by our recent data comparing prodrugs with different target specificity, but the same MMP-2-sensitive linker, for activity on MMP-2-expressing tumor cells; only the prodrug targeted to the cell surface was processed and displayed bioactivity [19]. The strict dependence on membrane targeting suggests a similar mechanism of prodrug activation for both, MMP-2- and uPA-dependent processing and implies that both proteases act on this substrate only in the context of their membrane expressed co-factors, i.e. the MT1-MMP-TIMP complex and the uPAR, respectively [4, 27]. In accordance with this reasoning, uPAR expression has been shown for the HT1080 cells studied here [20, 28, 30]. The confinement of prodrug processing to the cell surface bears the advantage of restricting action to the targeted area and thus, in vivo, could potentially reduce the risk of systemic side effects. However, at the targeted site, membrane-presented TNF can signal towards the targeted and juxtaposed cells [19]. Nevertheless, as suggested from our data, cell-processed prodrug may dissociate to some extent from its target cell and, through diffusion, may therefore also reach targets beyond the directly adjacent cells. In any case, it is expected that the selective target antigen expression will limit the spreading of the bioactive reagent within the tumor micro-environment. A conceptional advantage of membrane targeting and activation of TNF ligands is the fact that they display enhanced signalling capacity in their membrane-bound form, whether presented as the natural type 2 transmembrane cytokine or membrane targeted as a soluble fusion protein via the antibody domain [6, 19, 21, 38]. As underlying mechanisms optimum activation of the cognate receptors and persisting receptor signal complex formation are discussed [44]. More recent data of the functionality and signal capacity of TNF nanoparticles, from our laboratory, provide a first quantitative assessment of the signal capacity of surface-immobilised TNF as compared to soluble TNF, revealing an approximately 20-fold higher apoptosis-inducing capacity of the former ([7], and P. Scheurich, personal communication). The data presented here showing the superior activity of the membrane protease processed TNF prodrugs as compared to soluble TNF, are in accordance with this reasoning.

A prodrug containing a peptide linker comprised of cleavage sites for several proteases, such as the linker with dual specificity for MMP-2 and uPA constructed here, potentially exerts a broader applicability for tumors with unclear or a heterogeneous pattern of protease expression. Although designed as a model to show the feasibility of a multi-specific protease linker for prodrug activation, a TNF prodrug activated by both MMP-2 and uPA appears to be a promising candidate and of potential usefulness. These two enzymes belong to two complementary protease systems, both of which have been associated with tumor progression and metastasis and serve as prognostic markers for a number of different malignancies [14, 23, 32, 36, 37, 41]. Interestingly, clinical studies assessing the frequency and distribution of several members of the matrix metalloproteases and the uPA/uPAR system have recently shown that expression of MMP-2 and uPAR segregate into distinct cohorts [32, 39, 47]. In addition, from immunohistochemical studies, it is apparent that the expression of a given protease is rather heterogeneous between tumors of the same type [2, 15]. Therefore, a prodrug concept based on proteolytic processing demands reagents that are equally sensitive towards two or more of the tumor/stroma-associated proteases, in order to increase the probability of sufficient prodrug activation and measurable anti-tumoral responses.

Acknowledgements

This work was supported by grants from Wilhelm Sander-Stiftung, grant No. 2003.120.1, Deutsche Krebshilfe, grant No. 10–1751 and Schering AG. We like to thank Peter Scheurich for critical reading of the manuscript and for communicating unpublished data. Elke Gerlach’s skilled technical assistance in cell culture of stable producer lines is gratefully acknowledged.

Abbreviations

Ab

Antibody

FAP

Fibroblast activation protein

MMP

Matrix-metalloproteinase

TNF

Tumor necrosis factor

TNFR

TNF receptor

scFv 36

FAP-specific single chain variable fragment

uPA

Urokinase-type plasminogen activator

Footnotes

Authors Jeannette Gerspach and Julia Németh have contributed equally to this work

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