Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity. Ligands of the tumor necrosis factor (TNF) family fulfill crucial roles in the immune system, but have also been implicated in the development of epithelial and endothelial structures. 1 TNF family ligands are primarily expressed as trimeric type II transmembrane proteins and are often processed into soluble variants that are also organized as trimers. 1,2 Although shedding of some TNF ligands does not interfere with their capability to activate their corresponding receptors and might be even important for their physiological function, other TNF ligands become inactivated by proteolytic processing. 2 Soluble TNF ligands that are not or only poorly active still interact with their cognate receptors. For example, the soluble forms of TNF, CD95L, TRAIL, and CD40L interact with TNFR2, CD95, TRAILR2, and CD40, respectively, but do not or only poorly activate signaling by these receptors. [3][4][5][6] Notably, inactive or poorly active soluble TNF ligands can be converted into highly active molecules by artificially increasing their avidity. For example, soluble flag-tagged variants of TNF, CD95L, TRAIL, and CD40L stimulate robust signaling by TNFR2, CD95, TRAILR2, and CD40, respectively, provided they were cross-linked with the Flag-specific mAb M2. Likewise, hexameric and dodecameric fusion proteins of soluble CD95L and soluble CD40L as well as non-specifically aggregated preparations of TNF ligands produced in E. coli display high activity. [6][7][8] The structural hallmark of the ligands of the TNF family is the carboxy-terminal 'TNF homology domain', which is part of both the transmembrane and s...

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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Depletion of donor T cells (TCD) from the graft effectively ameliorates graft-vs-host (GvH) disease (GvHD) in allogeneic stem cell transplantation (SCT). TCD, however, is associated with impaired engraftment, immunosuppression, and abrogation of the beneficial graft-vs-leukemia (GvL) effect. Thus, based on previous results (Hartwig et al., Blood 2002) to eliminate alloreactive specificities but preserve oligoclonal antiviral- and GvL- immunity we explored proapoptotic chimeric molecules composed of the extracellular domains of human CD178 (FasL) and CD28 or RANK to selectively deplete GvH-reactive T cells by AICD in vitro and in vivo. As these fusion proteins are expressed as soluble Flag-tagged trimers and reconstitute bioactivity only upon binding to well defined cell-surface molecules (Samel et al., J. Biol. Chem., 2003), they can be specifically targeted onto e.g. recipient derived DC or donor T cells via the molecules CD80/86 or RANKL/TRANCE, respectively. Using an MHC-mismatched murine BMT model proliferative responses of purified CD90+ alloreactive T cells from C57BL/6 (H-2b) mice were strongly reduced following stimulation with DC from F1 (C57BL/6xBalb/c; H-2bxd) mice in vitro in the presence of CD178-CD28 or CD178-RANK to levels comparably obtained with agonistic anti-CD95 mAb (Jo2). Depletion was specific to alloantigen since H-2d restricted Hemagglutinin (HA)-peptide specific T cells titrated into the system persisted among retained T cells. In addition, adoptive transfer experiments of H-2b derived CD90+ T cells depleted of alloreactivity in vitro prior to injection into irradiated F1 recipients confirmed the in vitro results as these mice did not develop clinical signs of GvHD as compared to controls. Moreover, in current studies the CD178-fusion proteins are applied in vivo in our BMT model by injecting 2.5 to 5.0 x 106 syngeneic (H-2bxd) DC preloaded with CD178-CD28 into lethally irradiated F1 mice reconstituted with 1 x 107 TCD H-2b donor marrow cells. Alternatively, soluble CD178-fusion proteins are applied i.v. following BMT. Preliminary results obtained in first experiments suggest that host DC expressing cell surface bound CD178 fusion proteins confer protection to acute GvH-reactions in recipients challenged with purified CD90+ T cells of H-2b origin as these mice show prolonged survival compared to controls. Furthermore, studies on evaluating GvL-reactivity in this model using donor T cells from HA-primed Balb/c mice and A20 cells expressing HA as a defined tumor surrogate antigen are in progress, and their results will be discussed. Taken together, these results suggest that CD178-based proapoptotic fusion proteins devoid of systemic toxicity due to cell-surface antigen-restricted activation might provide a promising tool to separate GvH- and GvL-responses in vivo without inducing severe side effects on CD95-mediated apoptosis in sensitive tissues.