TNF-Selectokine exerts high bioactivity towards the targeted and adjacent, antigen negative cells. The prodrug exerts minimal TNF activity, but can be activated in vitro several thousand-fold by proteolytic digest, showing the principal feasibility of this approach. Choice of cleavage site s recognized by protease s typically associated with a given carcinoma should allow high dose systemic application of the respective TNF prodrug that unveils its specific bioactivity only in targeted tissues.
The use of TNF as a widely applicable tumor therapeutic is hampered by its severe systemic toxicity, allowing successful clinical application only with isolated limb perfusion techniques Lejeune et al. Indeed, tumor endothelial cells are considered as a key target for TNF mediated induction of tumor necrosis Ruegg et al. Like tumor endothelial cells, reactive fibroblasts of the tumor stroma represent a potentially useful, because general, target that is present in the vast majority of all carcinomas.
Antibody mediated targeting of TNF to FAP should not only allow a high tumor enrichment of the cytokine, but also its presentation to adjacent endothelial and tumor cells in a membrane TNF like manner, i. The TNF domain of TNF-fusion proteins described so far is, while in circulation, capable to interact with and signal to non-targeted cells, thus at least partially blunting the selective targeting strategy and potentially resulting in significant systemic toxicity.
In order to prevent targeting-independent action of TNF fusion proteins, we considered a prodrug strategy in analogy to chemotherapeutic prodrug systems, which have been shown to increase drug efficacy by reducing systemic toxicity.
For the activation of chemical prodrugs, tumor targeted as well as intra-tumoral enzymes have been successfully used DeFeo-Jones et al. We reasoned that it should be possible to provide TNF in an inactive proform, which upon target binding, can be proteolytically processed to a bioactive, still target bound TNF. The data described here show the principal feasibility of this approach.
The TNFR1 fragment in the prodrug is separated from TNF by a flexible linker module that carries several consensus cleavage sites for model proteases see Materials and methods to test proteolytic processing and conversion into an active TNF-Selectokine.
For efficient production in mammalian cells, the vectors pDB1 TNF-Selectokine and pW32 prodrug also contain the selectable marker dihydrofolate reductase.
This material could be purified to near homogeneity by affinity chromatography Figure 2. Schematic drawing of the order of the different domains of the TNF-Selectokine and the TNF-Selectokine-Prodrug constructs bottom panel and the trimeric forms derived thereof from mammalian expression systems upper left and upper right, respectively.
Biochemical characterization and specific antigen binding capacity of TNF-Selectokine and prodrug. For the latter, TNF activity was determined by serial dilutions of each of the fractions and activity revealed is shown as the dilution at which EC 80 was obtained rel.
FAP-positive HT 33 open circles and for control antigen negative HT filled circle were incubated with serial dilutions of the TNF-Selectokine DB1 or the prodrug W32 and the bound reagent was detected by indirect immunofluorescence as described in Materials and methods. Reagent concentration is plotted against mean fluorescence intensity. K D app was calculated from the concentration yielding half maximal signal. One representative experiment out of three is shown.
On FAP-positive cells, a concentration dependent increase of the mean fluorescence intensity was measured Figure 2c. The calculated K D app of 4.
Induction of cell death was concentration dependent with an EC 50 of 3. These data show i full bioactivity of the TNF moiety fused the scFv module and ii an almost four orders of magnitude lower activity of the prodrug, indicating a very efficient neutralization of TNF activity by C-terminal fusion of the extracellular TNFR1 fragment.
Short treatment of prodrug W32 with trypsin resulted in appearance of several fragments in Coomassie stained gels, with one band migrating in a size range expected from cleavage at the linker downstream of the TNF module Figure 4a , insert. As only the undigested monomer could be detected in Western-blotting via the c- myc -specific antibody not shown , proteolytic cleavage apparently occurred at multiple sites but always included a removal of the C-terminal tag.
Analyses for the presence of bioactive TNF-Selectokine in the crude tryptic digest of the prodrug revealed a surprisingly high apoptosis inducing activity in the KYM-1 assay, with a greater than fold rise compared to the nondigested prodrug Figure 4a. Appropriate controls with mock digested prodrug as well as treatment of target cells with trypsin containing buffers and neutralization by TNF specific antibodies verified that induction of cell death is mediated by the tryptically activated TNF-Selectokine data not shown.
Proteolytic activation of the prodrug. Open squares, untreated prodrug; filled squares: IMAC purified prodrug W32; right lane: Open squares, untreated prodrug; filled squares, W24 incubated with tPA. A fold activation was revealed in the depicted experiment.
Left lane W24 prodrug, undigested; right lane tPA treated W The arrow indicates a band corresponding to the correct size of a specifically cleaved TNFR-fragment. The demonstration of proteolytic activation of the prodrug by the rather nonselective trypsin cleavage encouraged further studies with more selective and patho-physiologically relevant proteases.
The prodrug contains a linker with a consensus cleavage sites for the proteases thrombin, factor VIIa, uPA and tPA, the latter two as examples of proteases overexpressed in a number of solid tumors Paciucci et al. Whereas thrombin treatment failed to process the prodrug, a partial processing was achieved by in vitro tPa digest. The partial tPA digest of the prodrug gave rise to a —fold activation Figure 4b , indicating correct processing of the prodrug to an active TNF-Selectokine.
The activity of cell bound TNF-Selectokine and of the prodrug either preactivated by trypsin treatment in solution before cell binding or activated subsequent to binding to antigen positive cells was determined. Basically the same result EC 50 4.
Together, these results show, in addition to direct action on the targeted cell, a juxtatropic TNF action. One representative of three experiments is shown. Further, pretreatment of HT 33 and subsequent removal of unbound TNF-Selectokine was sufficient to induce cell death of Colo cells data not shown. Efficient targeting and site specific actions of tumor therapeutics represent a major challenge in oncology, in particular for cytokine based therapeutic strategies because of their typically pleiotropic actions throughout the body.
Antibody-directed targeting of cytokines is a promising first step to improve therapeutic efficacy, when structural prerequisites for assembly of monomeric subunits into functional cytokines can be reliably met. Cytokine fusion proteins, targeted by antibody-domains or other means, however, carry an active cytokine module capable of signaling before the target has been reached, and unwanted systemic side effects have still to be considered.
The prodrug showed the same antigen binding characteristics, which indicates that the fused TNFR domain does not interfere with this activity. Provided these in vitro results translate to the in vivo situation, i. In support of this, we show that the prodrug can be processed in vitro , with respect to TNF activity, into a fully functional molecule, subsequent to cell targeting through the antibody module via proteolytic removal of the inhibitory domain. In vitro activation of the prodrug could be achieved with a rather nonselective tryptic digest, but more relevant, was also obtained by site-specific cleavage with tPA.
Accordingly, the introduction of specific cleavage sites recognized by this and other proteases frequently associated with and overexpressed in solid tumors, such as MMPs and plasminogen activating proteases Paciucci et al. The functionally silent TNF in the prodrug form should highly increase the systemically applicable dose and lead to a better tumor enrichment because of the dual feature of the TNF-Selectokine prodrug, target antigen dependent recruitment via the scFv domain and prevention of non target directed TNF signaling en route to its tumor target due to interaction with ubiquitously expressed TNFRs of normal tissues.
The TNF-Selectokine prodrug represents a prototype that can be readily modified to minimize the inhibitory fragment, e. Further, optimizing the linker both with respect to type of proteolytic cleavage sites and good accessibility of these sites for tumor-associated proteases is amenable and should not only enable a controlled, tumor selective processing, but also allow, by construction of selective linkers, a specific adaptation of the TNF-Selectokine to different tumor entities.
Because the trimeric organization of TNF is essential for bioactivity, a further unique feature of the TNF-Selectokine is the introduction of the Tenascin domain forming a coiled-coil structure to create a linker between the single chain antibody module. This enforces the generation of a homotrimeric fusion protein, which is further stabilized by spontaneous formation of intermolecular disulphide bonds between cysteine residues of the Tenascin domain.
This structural organization of the TNF-Selectokine bears several advantages: First, the four-fold increase in affinity in comparison to the parental scFv Brooks et al. Second, because the size of the trimeric TNF-Selectokine is in the range of normal immunoglobulins and the covalent linkage prevents spontaneous dissociation into monomers, functional inactivation and rapid renal clearance, as known to occur for TNF at low concentrations and also reported for some of the TNF fusion proteins Gasparri et al.
In the prodrug form, which completely lacks TNF activity up to very high concentrations, a prolonged serum half-life should not increase the risk of systemic reactions. Third, the Tenascin stabilized trimeric state of the TNF-Selectokine ensues upon targeting and proteolytic activation in a cell bound state, i. TNF activity is maintained independent of biophysical parameters and thus should lead to a prolonged TNF signal capacity at the site of prodrug activation in vivo.
Our in vitro studies show that, on a molar basis, active TNF-Selectokine exerted as nontargeted molecule the same specific activity as soluble recombinant TNF, indicating that the construction of a rather complex fusion protein had no negative influence on TNF function. In addition, TNF-Selectokine exhibited superior activity in a target bound state towards juxtaposed cells.
The high signal capacity of activated TNF-Selectokine was evident from the coculture assays identifying the juxtatropic action: In contrast to soluble TNF, which typically needs to be present at the effective concentration for several hours in order to induce apoptosis, a decoration of target cells with the TNF-Selectokine and subsequent removal of unbound reagent was sufficient to induce cell death in added tumor cells. This juxtatropic signal capacity via both TNFRs appears of particular interest in the context of the target specificity of the antibody used here for construction of the TNF-Selectokine.
The antigen recognized, FAP, is a dominant marker of stromal fibroblasts within solid tumors Garin-Chesa et al. Stromal cells always contribute to a significant amount to total tumor mass and are dispersed throughout tumor tissue, surrounding malignant cells and tumor vasculature Garin-Chesa et al. Stromal markers like FAP appear therefore ideal candidate targets for recruitment of therapeutics such as cytokines exerting their action not or not only on the targeted cells themselves, but also or predominantly on the surrounding cells.
In the case of TNF, special emphasis is on tumor endothelial cells, considered as main target cells for TNF induced tumor necrosis Ruegg et al.
For all three potential target cells of TNF action at the tumor site, several previous studies have provided evidence for an important role of coactivation of both TNFRs to induce optimum responses for review see Wajant and Scheurich, ; Locksley et al. In conclusion, the TNF-Selectokine prodrug represents the prototype of a novel class of cytokine fusion proteins combining features that potentially allow a high degree of tumor selective action.
It can be envisaged that the principle of site-specific activation will be applicable not only to other members of the TNF family but should also be adaptable to other protein therapeutics with known protein or peptide inhibitors. KYM-1 human rhabdomyosarcoma cells and the Colo human colon carcinoma cells have been described before Grell et al.
The construct carries a C-terminal myc - and His 6 -tag Kontermann et al. Amino acids 58—77 of TNF are used as a linker, because these aa are thought to have no secondary structure. Sequencing of the PCR amplified fragments showed no point mutations. Gels were shared with Coomassie Blue.
All cells were stained for FACS analyses following standard protocols of indirect immunofluorescence. The IMAC purified prodrug was activated with a 0. HT viability was determined by crystal violet staining 0. Assays were set up in quadruplicates.
FAP-negative HT served as controls. The viability of the cells was measured after incubation for 16 Kym-1 or 48 Colo h as described above. For the activation of the prodrug in cell bound form, the assay was performed essentially in the same way but the prodrug was activated by trypsin treatment of prodrug decorated cells 0. In a second assay, HT and HT 33 were preincubated with DB1 in suspension, washed, fixed as above and plated at a 1: DB1 was then added to the coculture at the same concentrations as used during preincubation of HT to identify a potential targeting independent action of soluble TNF-Selectokine on Colo Huang PS , Oliff A.
Wajant H , Scheurich P.