Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug

Gerspach, Jeannette; Müller, Dafne; Münkel, Sabine; Selchow, Olaf; Németh, Júlia; Noack, Melanie; Petrul, Heike M.; Menrad, Andreas; Wajant, Harald; Pfizenmaier, Klaus

doi:10.1038/sj.cdd.4401735

Cited by 39 publications

(26 citation statements)

References 35 publications

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“…HT1080 cells and its transfected derivative HT1080FAP display comparable levels of endogeneous MMP activity, 7 confirmed by gelatin zymography of wholecell lysates (data not shown). In accordance with the expected targeting dependent proteolytic activation, processed SC40-CD95L-PD was detectable in HT1080FAP cell cultures, but not in HT1080 cell cultures ( Figure 5a (Figure 5b).…”

Section: Resultsmentioning

confidence: 58%

“…In a first attempt to construct a CD95L prodrug that becomes selectively activated by tumor-associated proteases, we used the domain architecture of recently published TNF prodrugs. 7,8 In the corresponding CD95L prodrug, named SC40-CD95L-PL-CD95, an amino-terminal scFv (SC40), recognizing the tumor stroma marker fibroblast activation protein (FAP), 9,10 is followed by the extracellular domain of CD95L, a protease-sensitive linker containing repeats of the consensus recognition sequence for MMP2 and related proteases (PL) as well as a carboxy-terminal inhibitory domain composed of the extracellular domain of CD95. To ease purification and analysis, we introduced a Flag tag between the single chain and the CD95L domain of this and all other prodrug-related molecules investigated in this study ( Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…SC40-CD95L-PL-CD95 showed no apoptotic activity, neither on FAP-negative HT1080 cells nor on FAP-positive HT1080 cells (Figure 2c), on which the corresponding TNF prodrug was activated with high efficiency. 7 A CD95L fusion protein (SC40-CD95L-PL) which corresponded in structure to the completely processed CD95L prodrug, that is comprising a Cterminal addition of four amino acids corresponding to a proteolytically cleaved MMP linker, 7 was found to possess only marginal bioactivity after secondary crosslinking (data not shown) suggesting that an authentic carboxy-terminus is more important for CD95L function than for TNF function. We therefore constructed a CD95L prodrug in which the CD95L carboxy-terminus was preserved.…”

Section: Resultsmentioning

confidence: 99%

“…As the inhibitory domain was linked by a protease-sensitive linker to the antibody-TNF domain, the inhibitory domain was released upon proteolytic processing by membrane-associated MMP2 or related proteases when the construct was targeted to tumor cells via its specific antibody module. 7 As even small carboxy-terminal extensions of CD95L, comprising just a few amino acids, strongly reduced CD95L activity in CD95L fusion proteins, we developed a prodrug format in which the authentic CD95L carboxy-terminus is preserved. In this prodrug format, the inhibitory domain was placed aminoterminally to the antibody-CD95L module, separated by a protease sensitive motif.…”

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confidence: 99%

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Activation of CD95L fusion protein prodrugs by tumor-associated proteases

et al. 2006

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To achieve tumor cell-restricted activation of CD95, we developed a CD95L fusion protein format, in which CD95L activity is only unmasked upon antibody-mediated binding to tumor cells and subsequent processing by tumor-associated proteases, such as matrix metalloproteases (MMPs) and urokinase plasminogen activator (uPA). On target-negative, but MMP-and uPA-expressing HT1080 tumor cells, the CD95L prodrugs were virtually inactive. On target antigen-expressing HT1080 cells, however, the CD95L prodrugs showed an apoptotic activity comparable to soluble CD95L artificially activated by crosslinking. CD95 activation by the CD95L prodrugs was preceded by prodrug processing. Apoptosis was blocked by inhibitors of MMPs or uPA and by neutralizing antibodies recognizing the targeted cell surface antigen or the CD95L moiety of the prodrugs. In a xenotransplantation tumor model, local application of the prodrug reduced the growth of target antigen-expressing, but not antigen-negative tumor cells, verifying targeted CD95L prodrug activation in vivo. The CD95L-CD95 system has a complex role in tumor biology. For example, it has been shown that upregulation of CD95 and CD95L by chemotherapeutic drugs can significantly contribute to the antitumoral effects of such reagents and in several experimental models, tumor-localized expression of CD95L boosts antitumoral immunity. 1 Vice versa, there is evidence that CD95-resistant tumors use CD95L to fight tumor infiltrating T cells. 2 Moreover, recent studies revealed a strong capacity of CD95 to engage proinflammatory pathways such as the NF-kB pathway and the MAP kinase cascades. 3 These pathways regulate cell survival, proliferation, angiogenesis and cell migration. It is therefore conceivable that non-apoptotic CD95 signaling exerts protumoral effects in apoptosis-resistant tumor cells. Exploitation of the antitumoral effects of CD95 by exogenous delivery of CD95L or agonistic antibodies is hampered so far by severe systemic toxicity, foremost owing to massive apoptosis induction in hepatocytes, resulting in acute liver failure. 4 To overcome this limitation, solutions have to be found that allow tumorlocalized activation of CD95. The most common way to achieve tumor-restricted action is the use of antibodies, especially recombinant antibody fragments, as targeting devices. The tumor-associated activity of antibody conjugates of effector molecules, ranging from cytokines over toxins to radioactive isotopes, primarily relies on the enrichment of these conjugates in the tumor by antibody-mediated binding to the corresponding tumor-associated antigen. As the effector molecules typically used for the construction of antitumoral antibody conjugates are active independent of antigen recognition, such compounds still may exert considerable systemic activity. A notable exception from this are antibody-CD95L fusion proteins, because soluble, homotrimeric forms of CD95L are poorly active. 5 Therefore, the high tumorselective activity of trimeric antibody-CD95L fusion proteins primarily is no...

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Section: Resultsmentioning

confidence: 58%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Activation of CD95L fusion protein prodrugs by tumor-associated proteases

et al. 2006

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“…The pro-TNF molecule, or ''TNF-selectokine'' prodrug, demonstrated a >1000-fold increase in TNF receptor binding after proteolysis. 8,15,16 These previous studies demonstrate that tethering of a known inhibitor domain to a binding domain can potently block binding function, and that binding function can be restored by sitespecific cleavage of an appropriately positioned linker.…”

Section: Introductionmentioning

confidence: 99%

Proligands with protease‐regulated binding activity identified from cell‐displayed prodomain libraries

Thomas

Daugherty

2009

Protein Science

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A general method was developed for the discovery of protease-activated binding ligands, or proligands, from combinatorial prodomain libraries displayed on the surface of E. coli. Peptide libraries of candidate prodomains were fused with a matrix metalloprotease-2 substrate linker to a vascular endothelial growth factor-binding peptide and sorted using a two-stage flow cytometry screening procedure to isolate proligands that required protease treatment for binding activity. Prodomains that imparted protease-mediated switching activity were identified after three sorting cycles using two unique library design strategies. The best performing proligand exhibited a 100-fold improvement in apparent binding affinity after exposure to protease. This method may prove useful for developing therapeutic and diagnostic ligands with improved systemic targeting specificity.

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Improving TNF as a cancer therapeutic: Tailor‐made TNF fusion proteins with conserved antitumor activity and reduced systemic side effects

2009

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Tumor necrosis factor (TNF) is highly pleiotropic cytokine regulating diverse cellular processes such as proliferation, cell migration, angiogenesis, differentiation, apoptosis, necrosis, but also survival. Because of its name-giving tumor necrosis-inducing capabilities, TNF has attracted attention very early for antitumor therapy. Although TNF is in clinical use for treatment of soft tissue sarcoma in isolated limb perfusion, its broad use in tumor therapy is prevented so far by its strong systemic proinflammatory effects. Nevertheless, over the past decade, a variety of tailor-made TNF variants have been developed with the aim to reduce TNFs systemic activity without losing its antitumoral effects. Here, we review the progress made toward improving the efficacy of TNF by genetic engineering, tumor targeting, and introduction of prodrug concepts.

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Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug

Cited by 39 publications

References 35 publications

Activation of CD95L fusion protein prodrugs by tumor-associated proteases

Activation of CD95L fusion protein prodrugs by tumor-associated proteases

Proligands with protease‐regulated binding activity identified from cell‐displayed prodomain libraries

Improving TNF as a cancer therapeutic: Tailor‐made TNF fusion proteins with conserved antitumor activity and reduced systemic side effects

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