An optimized antibody-single-chain TRAIL fusion protein for cancer therapy.

Fusion proteins combining oligomeric assemblies of a genetically obtained single-chain (sc) variant of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with antibodies directed against tumor-associated antigens represent a promising strategy to overcome the limited therapeutic activity of conventional soluble TRAIL. To further improve the scTRAIL module in order to obtain a robust, thermostable molecule of high activity, we performed a comprehensive analysis of the minimal TNF homology domain (THD) and optimized linkers between the 3 TRAIL subunits constituting a scTRAIL. Through a stepwise mutagenesis of the N- and C-terminal region and the joining linker sequences, we generated bioactive scTRAIL molecules comprising a covalent linkage of the C-terminal Val280 and the N-terminal position 122 by only 2 amino acid residues in combination with conservative exchanges at positions 122 and 279. The increased thermal stability and solubility of such optimized scTRAIL molecules translated into increased bioactivity in the diabody-scTRAIL (Db-scTRAIL) format, exemplified here for an epidermal growth factor receptor-specific Db-scTRAIL. Additional modifications within the diabody linkers resulted in a fusion protein exerting high, target-dependent apoptosis induction in tumor cell lines in vitro and potent antitumor activity in vivo. Our results illustrate that protein engineering of scTRAIL and associated peptide linkers provides a promising strategy to develop antibody-scTRAIL fusion proteins as effective antitumor therapeutics.

ATROSAB, a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody.

Tumor necrosis factor (TNF) signals through two membrane receptors, TNFR1 and TNFR2, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB, produced in CHO cells, showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1-70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates.

Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand.

For many ligands of the TNF family, trimer stability and oligomerization status are crucial determinants of receptor activation. However, for the immunostimulatory ligands CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand (GITRL) detailed information regarding these requirements is lacking. Here, we comprehensively evaluated the effect of trimer stability and oligomerization on receptor activation by these ligands. Treatment with soluble Flag-tagged CD27L, 41BBL, and GITRL minimally activated receptor signaling, while Flag-CD40L was highly active. Oligomerization with anti-Flag Abs further enhanced the specific activity of Flag-CD40L 10-fold and of Flag-41BBL more than 200-fold, but it failed to activate Flag-CD27L and Flag-GITRL. We next investigated the relevance of trimer stability by introducing the tenascin-C (TNC) trimerization domain, yielding stabilized Flag-TNC-ligand trimers. Oligomerization with anti-Flag Ab potently activated signaling by Flag-TNC-CD27L and Flag-TNC-GITRL and, albeit to a lesser extent, Flag-TNC-CD40L and Flag-TNC-41BBL. Forced hexamerization, by introducing an Ig Fc domain, revealed that hexameric derivatives of Flag-TNC-41BBL, Flag-CD40L, and Flag-TNC-GITRL all activate receptor signaling with high efficiency, whereas hexameric Flag-CD27L variant left inactive. Finally, we attempted to selectively activate receptor signaling on targeted cells, by using Ab fragment (single-chain fragment variable region, scFv)-ligand fusion proteins, an approach previously applied to other TNF ligands. Target cell surface Ag-selective activation was achieved for scFv-41BBL, scFv-CD40L, and scFv-GITRL, although the latter two displayed already significant activity toward Ag-negative cells. In conclusion, our data establish that trimeric CD40L is active, 41BBL requires hexamerization, GITRL requires trimer stabilization, and CD27L requires trimer stabilization and oligomerization. Furthermore, surface immobilization might be exploited to gain locally enhanced ligand activity.

Activity of soluble OX40 ligand is enhanced by oligomerization and cell surface immobilization.

OX40 ligand (OX40L) and OX40 are typical members of the tumor necrosis factor ligand family and the tumor necrosis factor receptor superfamily, respectively, and are involved in the costimulation and differentiation of T cells. Like other tumor necrosis factor ligands, OX40L is a type II transmembrane protein. Recombinant soluble human OX40L assembles into trimers and is practically inactive despite binding to OX40. However, oligomerization of soluble OX40L trimers by cross-linking with antibodies or by expression as a hexameric fusion protein strongly increased the activity of the ligand. Moreover, a fusion protein of OX40L with a single chain fragment recognizing the tumor stroma antigen fibroblast activation protein showed a cell surface antigen-dependent increase in the activity of the ligand domain of the molecule and thus mimicked the activity of membrane OX40L upon antigen binding. Trimeric single chain OX40L fusion proteins therefore represent a novel type of OX40L-derived immunostimulatory molecule with potentially reduced systemic side effects.

A humanized tumor necrosis factor receptor 1 (TNFR1)-specific antagonistic antibody for selective inhibition of tumor necrosis factor (TNF) action.

Tumor necrosis factor (TNF) is a recognized pathogenic mediator in a number of chronic and acute inflammatory diseases. Antibodies targeting TNF have significantly improved therapy of chronic inflammatory diseases, in particular rheumatoid arthritis. Despite this success, anti-TNF treatment shows clinical efficacy only in part of the patients and is often transient, necessitating the development of alternative reagents to combat TNF action. We here describe humanization and functional properties of a TNFR1-specific, monovalent antibody fragment, designated IZI-06.1, which binds to the cysteine-rich domain 1 of TNFR1 with high affinity and competes ligand binding. IZI-06.1 serves as a receptor-selective inhibitor of proapoptotic and antiapoptotic TNF actions, revealed from complete blockage of TNFR1-dependent apoptosis and interleukin-6 induction in Kym 1 and HeLa cells, respectively, whereas TNFR2-mediated signals remained intact, evident from TNF and interleukin-2-mediated costimulation of interferon-gamma production in T cells. Accordingly, IZI-06.1 is a TNFR1-selective TNF antagonist and holds great promise to be developed into a clinically applicable therapeutic. IZI-06.1 could be a useful therapeutic alternative in all diseases already known to clinically respond to anti-TNF treatment and particularly in those diseases, where anti-TNF treatment has failed because of complete blockade of TNF action.

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

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.