Antibody-based soluble and membrane-bound TWEAK mimetic agonists with FcγR-independent activity

Abstract

The present invention relates to multivalent anti-Fn14 antibody constructs that exhibit memTWEAK mimetic agonism. More specifically, the present invention relates to tetravalent, hexavalent, and octavalent antibody constructs composed of Fn14-specific Fab and scFv domains having FcγR-independent activity. The present invention also relates to methods for producing such multivalent anti-Fn14 antibody constructs, pharmaceutical compositions containing the same, and their use for treating cancer.

Description

Technical Field

[0001] Technical Field of the Invention The present invention relates to multivalent anti-Fn14 antibody constructs that exhibit memTWEAK mimetic agonism. More specifically, the present invention relates to tetravalent, hexavalent, and octavalent antibody constructs composed of Fn14-specific Fab and scFv domains having FcγR-independent activity. The present invention also relates to methods for producing such multivalent anti-Fn14 antibody constructs, pharmaceutical compositions containing them, and their use for treating cancer.

[0002] Background Fibroblast growth factor (FGF)-inducible 14 (Fn14) is a small, unusual member of the tumor necrosis factor receptor superfamily (TNFRSF). The extracellular domain contains only a single cysteine-rich domain, and the 28-amino acid intracellular tail contains binding sites for proteins of the TNF receptor-associated factor (TRAF) family (Meighan-Manthaet et al., 1999; Wiley et al., 2001). Fn14 is dynamically and highly expressed during development, but in healthy adult organisms, Fn14 expression is almost limited to the heart, ovary, and mesenchymal progenitor cells (Meighan-Manthaet et al., 1999; Girgenrath et al., 2010). However, Fn14 expression is strongly upregulated after tissue injury in non-hematopoietic cells, regardless of the underlying reason (Wajant, 2013). Since tumor development is inevitably associated with tissue injury and tissue remodeling, Fn14 expression is often high in non-hematopoietic tumor cells and non-transformed non-hematopoietic cells of the tumor microenvironment (Wajant, 2013). Therefore, Fn14 expression can be considered a true marker of tissue remodeling and tissue injury. Fn14 signaling can be induced by tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a ligand of the TNF superfamily (TNFSF). This ligand occurs in two forms, namely as transmembrane TWEAK (memTWEAK) and as soluble TWEAK (sTWEAK) released from memTWEAK by proteolytic processing (Chicheportiche et al., 1997). Similar to other ligands of the TNFSF, memTWEAK and sTWEAK form homotrimeric molecules that can bind to three receptor molecules (Wajant, 2013). TWEAK expression has been shown in various cell lines and cell types by immunohistochemistry and RT-PCR, but memTWEAK expression has only been demonstrated unequivocally in monocytes, dendritic cells, NK cells, and a very small number of tumor cell lines (Wajant, 2013).

[0003] Importantly, sTWEAK and memTWEAK cause different states of Fn14 activity. In response to sTWEAK, Fn14 efficiently stimulates the non-canonical NFκB signaling pathway and primes for TNF-induced cell death (Wajant 2013). Transmembrane TWEAK causes Fn14 signaling events in the same way as sTWEAK, but also enables activation of the classical NFκB pathway by Fn14 (Wajant 2013).

[0004] The diverse and complex functions of the TWEAK / Fn14 system have been described in tissue repair and regeneration. For example, the TWEAK / Fn14 system has been demonstrated to promote the regenerative response after injury in muscle, pancreas, and liver (Girgenrath et al., 2006; Wu et al., 2013; Karaca et al., 2014). However, excessive and / or chronic involvement of the TWEAK / Fn14 system can also cause harmful effects associated with tissue repair such as fibrosis and inflammation (Kuramitsu et al., 2014; Mittal et al., 2010a,b). Therefore, depending on the considered situation and disease, not only inhibition but also stimulation of Fn14 can elicit beneficial therapeutic effects (Wajant 2013).

[0005] Inhibition of the TWEAK / Fn14 axis can be easily achieved with the help of soluble Fn14-Fc fusion proteins, TWEAK neutralizing antibodies or blocking, effector function-deficient Fn14 antibody variants (Wajant 2013). However, specific stimulation of Fn14 signaling is more difficult. Conventional sTWEAK has an extremely short serum half-life (<20 minutes) (Mueller et al., 2010), and oligomeric sTWEAK variants that exhibit memTWEAK-like activity are produced slowly and are more difficult to develop translationally than antibodies. Therefore, the reagent of choice for stimulating Fn14 in vivo is an agonist antibody, but here two fundamental problems arise: First, some anti-Fn14 IgG antibodies can to some extent promote p100 processing, which is characteristic of the non-canonical NFκB pathway, in certain cell lines, but they are mostly not agonistic and require anchoring to Fcγ receptors (FcγR) or oligomerization, such as oligomerization by protein G or antibody cross-linking, to be fully and potently agonistic (Salzmann et al., 2013a, Trebing, Medler). However, antibody oligomerization by protein G or secondary antibodies is not a practical translational option, and the FcγR-binding-dependent mode of anti-Fn14 agonism is necessarily associated with the induction of FcγR effector functions and may interfere with the expected therapeutic effects. Second, when Fn14 antibodies become agonistic by the aforementioned means, they mimic memTWEAK, and thus mimicking sTWEAK with Fn14 antibodies seems almost impossible.

[0006] For tumor treatment, preclinical studies targeting Fn14 have been extensively conducted and are still ongoing. These studies involve using Fn14 as a target for antibodies with cytotoxic activity (ADCC-inducing IgG variants, antibody-drug conjugates), or using blocking antibodies aimed at interfering with tumor-promoting Fn14 activity. Notably, attempts to target Fn14 using agonist Fc effector function-silencing antibodies to elicit the receptor's pro-inflammatory and cell death-promoting activities for tumor treatment have not yet been carried out. This is surely not due to the fact that the anti-Fn14 antibody acts as an effective agonist only when presented by binding to FcγR.

[0007] Therefore, there is still a need for means that can reliably and selectively participate in Fn14 signaling without inducing unwanted FcγR-mediated activity and can be used in cancer treatment.

[0008] Description of the Invention The inventors have identified antibody constructs, including oligovalent variants of the Fn14-specific antibody 18D1, that mimic the activity of sTWEAK or memTWEAK independently of FcγR binding (see, e.g., FIGS. 2 and 5). Surprisingly, the antibody constructs of the present invention have good anti-tumor activity (see, e.g., FIG. 6). In contrast to conventional anti-Fn14 antibodies, the agonism of these novel Fn14 agonist constructs is not restricted by the availability of FcγR-expressing immune cells or competition with endogenous irrelevant antibodies for FcγR binding. This is expected to be advantageous for therapeutic applications such as use in the treatment of cancer. Unexpectedly, all oligovalent anti-Fn14 constructs efficiently induced p100 processing and enhanced TNF-induced cell death, but there was a significant difference in the induction of IL8, a target of the classical NFκB pathway. Constructs having only multiple copies of the scFv18D1 domain were unable to activate IL8 production to any significant extent, while constructs of equivalent valency composed of Fab and scFv domains did so very efficiently (see, e.g., FIG. 2C). Thus, it is expected that the number and type of Fn14-binding domains within the oligovalent 18D1 constructs will determine whether sTWEAK- or memTWEAK-like activity is mimicked. Pathway-selective agonism reflects the activity of sTWEAK and memTWEAK, the natural ligands of Fn14, and there are no such examples yet with anti-Fn14 antibodies. The antibody constructs of the present invention are advantageous because they enable the antibody constructs to selectively exert effects (e.g., pharmacological effects) similar to sTWEAK or memTWEAK without the need for additional features such as FcγR binding.

[0009] According to the present invention, these novel potent agonist antibody-based Fn14 constructs stimulate Fn14 with an antibody in an FcγR-independent manner, and this advantage is expected to lead to the development of new application fields including clinical applications such as cancer treatment.

[0010] Accordingly, the present invention relates to the following preferred embodiments: 1. A multivalent anti-Fn14 antibody construct comprising at least four antigen-binding sites against Fn14. 2. The construct according to item 1, comprising an IgG molecule containing two antigen-binding sites against Fn14. 3. The construct according to item 1, comprising a modified IgG molecule modified by replacing each of the two variable domains of the heavy chain with a scFv containing one of the antigen-binding sites against Fn14, and by replacing each of the two variable domains of the light chain with a scFv containing one of the antigen-binding sites against Fn14. 4. The construct according to item 2 or 3, wherein the construct further comprises a scFv covalently bound to the C-terminus of one of the two heavy chains of the IgG molecule, and the scFv contains one of the antigen-binding sites against Fn14. 5. The construct according to item 4, wherein the construct further comprises a scFv covalently bound to the C-terminus of the other of the two heavy chains of the IgG molecule, and the scFv contains one of the antigen-binding sites against Fn14. 6. The construct according to any one of items 2 to 5, wherein the construct further comprises a scFv covalently bound to the C-terminus of one of the two light chains of the IgG molecule, and the scFv contains one of the antigen-binding sites against Fn14. 7. The construct according to item 6, wherein the construct further comprises a scFv covalently bound to the C-terminus of the other of the two light chains of the IgG molecule, and the scFv contains one of the antigen-binding sites against Fn14. 8. The construct according to any one of items 2 to 7, wherein the IgG is IgG1, IgG2, IgG3 or IgG4. 9. The construct according to any one of items 2 to 7, wherein the IgG is IgG2 or IgG4. 10. The construct according to any one of items 2 to 9, wherein the Fc domain of the IgG contains a mutation that reduces FcγR binding. 11. The construct according to item 10, wherein the mutation is an N297A mutation. 12. The construct according to any one of items 1 to 11, which is a Fn14 agonist. 13. The construct according to item 12, which is capable of activating IL8 production in cells expressing Fn14, preferably HT-1080 cells expressing Fn14. 14. The construct according to any one of items 1 to 13, comprising at least 5 antigen-binding sites for Fn14. 15. The construct according to any one of items 1 to 14, comprising at least 6 antigen-binding sites for Fn14. 16. The construct according to any one of items 1 to 15, comprising 6 antigen-binding sites for Fn14. 17. The construct according to any one of items 1 to 2, 4 to 5, and 8 to 16, comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 3, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, and most preferably comprising the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 3. 18. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 4, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 4, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4, and most preferably an amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 4, the construct according to any one of items 1 to 2 and 6 to 16. 19. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 4, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 4, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4, and most preferably an amino acid sequence of SEQ ID NO: 2 and an amino acid sequence of SEQ ID NO: 4, the construct according to any one of items 1 to 2 and 4 to 16. 20. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 5, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 5, and most preferably an amino acid sequence of SEQ ID NO: 6 and an amino acid sequence of SEQ ID NO: 5, the construct according to any one of items 1, 3, and 8 to 16. 21. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 5, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 5, and most preferably an amino acid sequence of SEQ ID NO: 8 and an amino acid sequence of SEQ ID NO: 5, the construct according to any one of items 1, 3 to 5, and 8 to 16. 22. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 7, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 7, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7, and most preferably an amino acid sequence of SEQ ID NO: 6 and an amino acid sequence of SEQ ID NO: 7, the construct according to any one of items 1, 3, and 6 to 16. 23. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 7, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 7, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7, and most preferably an amino acid sequence of SEQ ID NO: 8 and an amino acid sequence of SEQ ID NO: 7, the construct according to any one of items 1 and 3 to 16. 24. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 11, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 11, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 11, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 11, and most preferably an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 11, the construct according to any one of items 1 to 2, 4 to 5, and 8 to 16. 25. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 9 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 9 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 9 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 12, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 9 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12, and most preferably an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO: 12, the construct according to any one of items 1 to 2 and 6 to 16. 26. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 12, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 12, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 12, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 10 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 12, and most preferably an amino acid sequence that includes the amino acid sequence of SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 12, the construct according to any one of items 1 to 2 and 4 to 16. 27. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 14 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 13, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 14 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 13, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 14 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 13, and most preferably an amino acid sequence that includes the amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 13, the construct according to any one of items 1, 3 and 8 to 16. 28. The construct according to any one of items 1 to 27, comprising a trimerization domain. 29. The construct according to item 28, wherein the trimerization domain is the trimerization domain of tenascin C. 30. The construct according to any one of items 28 and 29, wherein the trimerization domain comprises the amino acid sequence of SEQ ID NO: 21 or a sequence that is at least 70% identical thereto, preferably at least 80% identical thereto, more preferably at least 85% identical thereto, more preferably at least 90% identical thereto, more preferably at least 93% identical thereto, more preferably at least 96% identical thereto. 31. The construct according to any one of items 28 to 30, wherein the construct is the construct according to any one of items 2 to 3, and the trimerization domain is linked to the C-terminus of the constant domain of the heavy chain of IgG. 32. The construct according to any one of items 1, 2, 8, 10 to 16, and 28 to 31, comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 3, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, and most preferably the amino acid sequences of SEQ ID NO: 15 and SEQ ID NO: 3. 33. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 3, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, and most preferably an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 16 and the amino acid sequence of SEQ ID NO: 3, the construct according to any one of items 1, 2, 8, and 10 to 16. 34. An amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 5, preferably an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, more preferably an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 5, and most preferably an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 17 and the amino acid sequence of SEQ ID NO: 5, the construct according to any one of items 1, 3, 8, 10 to 16, and 28 to 31. 35. The construct according to any one of items 1, 3, 8 to 16 and 28 to 30, comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 18, preferably comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 18, more preferably comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 18, more preferably comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 18, more preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 18, and most preferably comprising the amino acid sequence of SEQ ID NO: 18. 36. The construct according to any one of items 1 to 35, further comprising an N-terminal amino acid sequence comprising one or two of the following ((a) and (b)): (a) a leader sequence comprising the amino acid sequence of SEQ ID NO: 19, optionally followed by a first linker sequence, and (b) a FLAG tag comprising the amino acid sequence of SEQ ID NO: 20, optionally followed by a second linker sequence. 37. A pharmaceutical composition comprising the construct according to any one of items 1 to 36. 38. The pharmaceutical composition according to item 37 or the construct according to any one of items 1 to 36 for use in the treatment of cancer. 39. The pharmaceutical composition or construct for use according to item 38, wherein the cancer is a cancer that expresses Fn14. 40. The pharmaceutical composition or construct for use according to any one of items 38 to 39, wherein the cancer is a solid cancer. 41. A nucleic acid or set of nucleic acids encoding the construct according to any one of items 1 to 36. 42. A recombinant cell comprising the nucleic acid or set of nucleic acids according to item 41 and expressing the construct according to any one of items 1 to 36. 43. A method for producing the construct according to any one of items 1 to 36, comprising expressing the construct from the nucleic acid or set of nucleic acids according to item 41 in the recombinant cell according to item 42, and optionally further comprising purifying the construct and formulating it into the pharmaceutical composition according to item 37.

Brief Description of the Drawings

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[0012] Detailed Description of the Invention Definitions and General Methods Unless otherwise specifically defined herein, the terms used in the present invention are to be understood according to their general meanings known to those skilled in the art. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Publications referred to herein may be cited by specifying the complete reference in the text, or by specifying the author and publication year (e.g., "Kuramitsu et al., 2014") and by specifying the corresponding complete reference in the "References" section.

[0013] All proteins according to the present invention, including the antibody constructs of the present invention, can be obtained by methods known in the art. Such methods include methods for producing recombinant proteins. The antibody constructs of the present invention can be expressed in recombinant host cells according to the present invention. The recombinant host cells of the present invention are preferably mammalian cells such as CHO cells and HEK cells. It will be understood that the antibody constructs of the present invention optionally include a secretion signal peptide sequence. Similarly, the antibody constructs of the present invention optionally include an affinity tag, for example to facilitate purification, and also optionally include any protease cleavage site between the tag and the antibody construct, for example to facilitate removal of the tag by protease cleavage. Similarly, it will be understood that the antibody constructs of the present invention optionally include respective propeptides.

[0014] Also, references to amino acid sequences herein include not only the unmodified amino acid sequences, but also typical post-translational modifications of these amino acid sequences (e.g., glycosylation or deamidation of amino acids, cleavage of specific amino acids or other post-translational modifications), and it is also understood that such post-translational modifications occur in cell expression systems known in the art (including insect cells and mammalian cells such as CHO cells and HEK cells).

[0015] As used herein, the term "antibody" is generally described in Paul, W.E. (Ed.).: Fundamental Immunology 2nd Ed. Raven Press, Ltd., New York 1989, which is incorporated herein by reference. Without particular limitation, the term "antibody" encompasses antibodies derived from any suitable source species, including chickens and mammals such as mice, goats, non-human primates, and humans. Preferably, the antibody is a humanized antibody or a human antibody. The antibody is preferably a monoclonal antibody that can be prepared by methods well known in the art. The term "antibody" encompasses isotype antibodies of IgG-1, -2, -3, or -4, IgE, IgA, IgM, or IgD. The term "antibody" encompasses monomeric antibodies (e.g., IgD, IgE, IgG) or oligomeric antibodies (e.g., IgA or IgM). The term "antibody" also encompasses, without particular limitation, isolated antibodies and modified antibodies, e.g., genetically engineered antibodies, e.g., chimeric antibodies, humanized antibodies, or human antibodies. For example, as used in accordance with the present invention, the terms "IgG1", "IgG2", "IgG3", or "IgG4" encompass chimeric, humanized, and human IgG1, IgG2, IgG3, and IgG4, respectively.

[0016] As used herein, "IgG" can be naturally occurring IgG or variant IgG, as is known in the art.

[0017] For IgG1 such as chimeric IgG1, humanized IgG1 or human IgG1, known mutations include, but are not limited to, N297A, N297D, N297Q, N297G, E233P, L234A, L234F, L235A, L235E, P331S, P329A, P329G, P331S, P238S, LALA (LALA = mutation of leucine 234 to alanine and mutation of leucine 235 to alanine), A330S, G237A, M252Y, S254T, S228P, T256E, M252F, M252S, M252W, M252T, T256S, T256R, T256Q, T256D, H268A, and combinations thereof. Known IgG1 mutations also include E345K or E430G that promote hexamerization of IgG1.

[0018] For IgG2 such as chimeric IgG2, humanized IgG2 or human IgG2, known mutations include, but are not limited to, V234A, G237A, P238S, H268A, H268Q, A330S, P331S, P233S, V309L, and combinations thereof.

[0019] For IgG3 such as chimeric IgG3, humanized IgG3 or human IgG3, known mutations include, but are not limited to, substitution of the CH2 domain of IgG3 with the CH2 domain of IgG2, removal of Fc glycosylation, mutations of L322, L276, N297, and combinations thereof.

[0020] For IgG4 such as chimeric IgG4, humanized IgG4 or human IgG4, known mutations include, but are not limited to, V234A, L235E, LALA (mutation from leucine 234 to alanine and mutation from leucine 235 to alanine), F234A, P329G, S228P, G237A, P238S, G236 deletion, and combinations thereof.

[0021] The nomenclature of antibodies, antibody fragments, and their variants follows terms known in the art. According to the present invention, it will be understood by those skilled in the art that this known nomenclature is also applicable to the antibody constructs of the present invention. For example, it will be understood that the known nomenclature is also applicable to the antibodies that form part of the antibody constructs of the present invention. For example, when terms related to antibody mutations such as "N297A" are used herein in relation to IgG1 that forms part of the antibody constructs of the present invention, it will be understood that these terms are based on the known antibody nomenclature.

[0022] Each monomer of an antibody contains two heavy chains and two light chains, as is generally known in the art. Among these, each heavy chain and light chain contains a variable domain (referred to as VH for the heavy chain and VL for the light chain) that is important for antigen binding. The variable domains of these heavy and light chains contain regions of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 (FR, framework region; CDR, complementarity-determining region, also known as the hypervariable region) in order from the N-terminus to the C-terminus. The identification and assignment of the above-described antibody regions within an antibody sequence are generally carried out according to Kabat et al. (Sequences of proteins of immunological interest, U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, Md. 1983), or Chothia et al. (Conformations of immunoglobulin hypervariable regions. Nature. 1989 Dec 21-28;342(6252):877-83.), or can be performed using the IMGT / V-QUEST software described by Giudicelli et al. (IMGT / V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W435-40.), which is incorporated herein by reference. Preferably, the above-described antibody regions are identified and assigned using the IMGT / V-QUEST software.

[0023] A "monoclonal antibody" is an antibody from a substantially homogeneous antibody population, where the antibodies are substantially identical in sequence (i.e., identical except for a few antibodies with naturally occurring sequence modifications such as amino acid modifications at their N- or C-termini). Unlike polyclonal antibodies, which contain a mixture of different antibodies against either a single epitope or a number of different epitopes, monoclonal antibodies are against the same epitope and are thus highly specific. The term "monoclonal antibody" includes (but is not limited to) antibodies obtained from a monoclonal cell population derived from a single cell clone, such as an antibody produced by the hybridoma method described, for example, by Köhler and Milstein (Nature, 1975 Aug 7;256(5517):495-7) or Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988). Monoclonal antibodies can also be obtained from other suitable methods, including phage display techniques such as those described by Clackson et al. (Nature. 1991 Aug 15;352(6336):624-8) or Marks et al. (J Mol Biol. 1991 Dec 5;222(3):581-97). Monoclonal antibodies can be antibodies optimized for antigen-binding properties such as reduced Kd values, optimized association and dissociation kinetics by methods known in the art. For example, Kd values can be optimized by display methods including phage display, resulting in affinity-matured monoclonal antibodies. The term "monoclonal antibody" is not limited to antibodies from a particular origin species or antibodies with a single origin species. Thus, the meaning of the term "monoclonal antibody" encompasses chimeric monoclonal antibodies such as humanized monoclonal antibodies and human antibodies.

[0024] A "humanized antibody" is an antibody that contains human sequences and a very small part of non-human sequences that confer binding specificity to the antigen of interest. Typically, a humanized antibody is produced by replacing the hypervariable region sequences derived from a human acceptor antibody with hypervariable region sequences derived from a non-human donor antibody (e.g., a mouse, rabbit, or rat donor antibody) that binds to the antigen of interest. In some cases, the framework region sequences of the acceptor antibody can also be replaced with the corresponding sequences of the donor antibody. In addition to the sequences derived from the donor and acceptor antibodies, a "humanized antibody" may or may not contain other (additional or alternative) residues or sequences. Such other residues or sequences can help further improve antibody properties such as binding properties (e.g., reducing the Kd value) and / or immunogenic properties (e.g., reducing antigenicity in humans). Non-limiting examples of methods for producing humanized antibodies are known in the art and are described, for example, by Riechmann et al. (Nature. March 24, 1988; 332(6162):323-7) or Jones et al. (Nature. May 29 - June 4, 1986; 321(6069):522-5).

[0025] The term "human antibody" relates to an antibody that contains human variable domain sequences and constant domain sequences. This definition includes antibodies having human sequences with single amino acid substitutions or modifications that can help further improve antibody properties such as binding properties (e.g., for reducing the Kd value) and / or immunogenic properties (e.g., reducing antigenicity in humans). The term "human antibody" excludes humanized antibodies in which a part of the non-human sequence confers binding specificity to the antigen of interest.

[0026] The "antigen-binding portion" of an antibody as used herein refers to the portion of the antibody that retains the ability of the antibody to specifically bind to an antigen. This ability can be determined, for example, by determining the ability of the antigen-binding portion to compete with the antibody for specific binding to the antigen by methods known in the art. The antigen-binding portion can contain one or more fragments of the antibody. Without particular limitation, the antigen-binding portion can be made by any suitable method known in the art, including preparation by recombinant DNA methods and chemical or enzymatic fragmentation of the antibody. The antigen-binding portion can be a Fab fragment, F(ab’) fragment, Fab2 fragment, single-chain variable fragment (scFv), single-domain antibody, diabody, or any other part(s) of the antibody that retains the ability of the antibody to specifically bind to an antigen. It will be understood that the term "Fab2" is synonymous with the terms "Fab2", "Fab2", and "FAB2" according to the meaning of the term "Fab2" known in the art. Thus, for the purposes of this application, the term "Fab2" is used interchangeably with the terms "Fab2", "Fab2", and "FAB2".

[0027] As defined herein, the "antibody construct" according to the present invention is a protein that contains at least four antigen-binding sites for Fn14. The antibody construct can bind to its specific target antigen, i.e., Fn14. It is understood that the antibody construct according to the present invention may be a protein consisting of a single polypeptide chain, or a protein in which two or more polypeptide chains are linked to each other. These can be covalently linked, for example, by a covalent bond. Such a covalent bond can be one or more disulfide bonds. Alternatively, the covalent bond can be obtained by chemical conjugation (preferably by chemical conjugation using click chemistry), and / or they can be any other covalent bond known in the art as a suitable linkage for proteins. Alternatively, the antibody construct according to the present invention may be a protein in which two or more polypeptide chains are non-covalently linked to each other (e.g., by non-covalent interactions). For example, IgG1 mutants E345K and / or E430G promote hexamerization of IgG1. According to the present invention, such IgG1 mutants can be included in the antibody construct of the present invention to obtain a hexameric antibody construct.

[0028] Generally, in relation to all antibody constructs of the present invention, it is understood that portions of the antibody construct can be fused using a linker sequence. In that case, the antibody construct of the present invention will include such a linker sequence. Suitable linker sequences are known in the art and include, for example, but are not limited to, peptide linkers.

[0029] The "antibody construct" according to the present invention may be an antibody construct derivatized or linked to a different molecule. For example, molecules that can be linked to the antibody construct are molecular labels (e.g., fluorescent, luminescent, colored, or radioactive molecules) and / or drugs.

[0030] As used herein, the terms "antigen-binding site(s)" have meanings that are known in the art. Typically, an antigen-binding site comprises six complementarity-determining regions (CDRs). The six complementarity-determining regions (CDRs) are typically located in the VH and VL domains, i.e., CDR1, CDR2, and CDR3 in VH (i.e., the variable domain of the heavy chain), and CDR1, CDR2, and CDR3 in VL (i.e., the variable domain of the light chain). Various forms of antigen-binding sites are known in the art and include, but are not limited to, the antigen-binding site contained in VH and the VH of an antibody, or the antigen-binding site contained in antibody fragments thereof such as scFv.

[0031] As used herein, the term "Fcγ receptor" or "FcγR" refers to any member of a family of proteins that bind to the Fc domain of an IgG antibody and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64) including the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32) including the isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16) including the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2). FcγR can be derived from any organism including, but not limited to, humans, mice, rats, rabbits, and monkeys, and is preferably derived from humans. Mouse FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγR or FcγR isoform or allotype.

[0032] As used herein, the term "mutation that reduces FcγR binding" in relation to the Fc domain of the IgG of the antibody construct of the present invention encompasses any mutation that reduces the binding of the antibody construct to at least one of the Fcγ receptors as compared to the corresponding reference antibody construct in which the mutation is not present in the Fc domain. Whether a mutation reduces FcγR binding to the Fcγ receptor can be determined by methods known in the art, such as comparative surface plasmon resonance measurements of the binding of the antibody construct and the reference antibody construct to immobilized recombinant Fcγ receptors, respectively.

[0033] Mutations that reduce FcγR binding are well known in the art.

[0034] For IgG1 such as chimeric, humanized or human IgG1, known mutations that reduce FcγR binding include, but are not limited to, N297A, N297D, N297Q, N297G, E233P, L234A, L234F, L235A, L235E, P331S, P329A, P329G, P331S, P238S, LALA (LALA = mutation of leucine 234 to alanine and mutation of leucine 235 to alanine), A330S, G237A, M252Y, S254T, S228P, T256E, M252F, M252S, M252W, M252T, T256S, T256R, T256Q, T256D, H268A, and combinations thereof.

[0035] For IgG2 such as chimeric, humanized or human IgG2, known mutations that reduce FcγR binding include, but are not limited to, V234A, G237A, P238S, H268A, H268Q, A330S, P331S, P233S, V309L, and combinations thereof.

[0036] For IgG3 such as chimeric, humanized or human IgG3, known mutations that reduce FcγR binding include substitution of the CH2 domain of IgG3 with the CH2 domain of IgG2, removal of Fc glycosylation, mutations at L322, L276, N297, and combinations thereof, but are not limited thereto.

[0037] For IgG4 such as chimeric, humanized or human IgG4, known mutations that reduce FcγR binding include V234A, L235E, LALA (mutation of leucine 234 to alanine and mutation of leucine 235 to alanine), F234A, P329G, S228P, G237A, P238S, G236 deletion, and combinations thereof, but are not limited thereto.

[0038] The term "Fn14 agonist" refers to a molecule that can induce Fn14 signaling. Fn14 signaling can be induced by the apoptosis tumor necrosis factor (TNF)-like weak inducer (TWEAK), a TNF superfamily (TNFSF) ligand that exists in two forms, namely transmembrane TWEAK (memTWEAK) and soluble TWEAK (sTWEAK) released from memTWEAK by proteolytic processing (Chicheportiche et al., 1997). sTWEAK and memTWEAK induce different states of Fn14 activity. In response to sTWEAK, Fn14 stimulates an alternative NFκB signaling pathway and primes for TNF-induced cell death (Wajant 2013). Transmembrane TWEAK induces the same Fn14 signaling events as sTWEAK, but in addition enables Fn14 to activate the classical NFκB pathway (Wajant 2013). Thus, whether an antibody construct is an Fn14 agonist can be determined by methods known in the art, including, but not limited to, methods for measuring IL8 secretion in cells expressing Fn14 (e.g., by ELISA), methods for determining activation of the alternative NFkB pathway in cells expressing Fn14 (e.g., Western blot for NIK accumulation or Western blot showing increased processing of p100 to p52), and methods for measuring TNF-induced cell death in cells expressing Fn14. Cells expressing Fn14 are known and available in the art and include, for example, HT-1080 cells.

[0039] The cancer to be treated according to the present invention is preferably a solid cancer. A "solid cancer" is a cancer that forms one or more solid tumors. Solid cancers that form such solid tumors are generally known in the art. The term "solid cancer" encompasses both the primary tumor formed by the cancer and secondary tumors that may also be known as metastases. Known solid cancers to be treated according to the present invention include, but are not limited to, melanoma, colorectal cancer, prostate cancer, head and neck cancer, urothelial cancer, gastric cancer, pancreatic cancer, liver cancer, testicular cancer, ovarian cancer, endometrial cancer, cervical cancer, brain tumor, breast cancer, gastric cancer, renal cell carcinoma, Ewing's sarcoma, non-small cell lung cancer, and small cell lung cancer.

[0040] Terms such as "treatment of cancer" or "treating cancer" according to the present invention refer to therapeutic treatments. Evaluation of whether a therapeutic treatment is effective can be performed, for example, by evaluating whether cancer growth in the patient(s) to whom the treatment has been administered is inhibited. Preferably, the inhibition is statistically significant, as evaluated by appropriate statistical tests known in the art. Inhibition of cancer growth can be compared by comparing cancer growth in a group of patients treated according to the present invention with a control group of untreated patients, or by comparing a group of patients who have received treatment according to the present invention in addition to standard cancer treatment in the art with a control group of patients who have received only standard cancer treatment in the art. Such tests for evaluating inhibition of cancer growth are designed according to recognized criteria for clinical trials, for example, double-blind randomized trials having sufficient statistical power. The term "treating cancer" includes inhibition of cancer growth in which cancer growth is partially inhibited (i.e., cancer growth in the patient is delayed compared to patients in the control group), inhibition in which cancer growth is completely inhibited (i.e., cancer growth in the patient stops), and inhibition in which cancer growth is reversed (i.e., the cancer shrinks). Preferably, evaluation of whether a therapeutic treatment is effective can be performed based on classification of response and non-response using Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) (Eisenhauer et al.: New response evaluation criteria in solid tumours: RECIST guideline updated version (version 1.1). In: Eur. J. Cancer. 45, No. 2, January 2009, pp. 228-47). Alternatively, or in addition, evaluation of whether a therapeutic treatment is effective can also be performed based on known clinical indicators of cancer progression.

[0041] The treatment of cancer according to the present invention can be a first-line therapy, second-line therapy, or third-line therapy, or a therapy beyond third-line therapy. The meanings of these terms are known in the art and follow the specialized terms generally used by the US National Cancer Institute.

[0042] Whether the cancer is a cancer that expresses Fn14 can be determined by methods known in the art, for example, immunohistochemical methods using anti-Fn14 antibodies to analyze cancer biopsy samples, binding assays using TWEAK, methods including but not limited to qPCR.

[0043] According to the present invention, each occurrence of the term "comprising" may optionally be replaced by the term "consisting of".

[0044] Methods and Techniques Generally, unless otherwise defined herein, the methods used in the present invention (for example, cloning methods or methods related to antibodies) are procedures known in the art, for example, those described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual.", 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology.", Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988) (all of which are incorporated herein by reference) and are carried out according to the procedures described therein.

[0045] Protein-protein binding, for example, the binding of antibodies to their respective target proteins, can be evaluated by methods known in the art. Protein-protein binding is preferably evaluated by surface plasmon resonance spectroscopy.

[0046] The sequence alignment of the sequences according to the present invention is performed using the BLAST algorithm (see Altschul et al. (1990) "Basic local alignment search tool." Journal of Molecular Biology 215: 403-410; Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402). Appropriate parameters for the sequence alignment of short peptides by the BLAST algorithm, suitable for the peptide antigens according to the present invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters for the sequence alignment of short input sequences. In one embodiment, the following parameters are used: maximum target sequences 10; word size 3; BLOSUM 62 matrix; gap cost: existence 11, extension 1; conditional composition score matrix adjustment. Thus, when used in connection with sequences, terms such as "identity" or "identical" preferably refer to the identity value obtained by using the BLAST algorithm.

[0047] Preparation of Pharmaceutical Compositions of the Invention The pharmaceutical composition of the present invention is prepared in accordance with known criteria for the preparation of pharmaceutical compositions. For example, the pharmaceutical composition is prepared so that it can be properly stored and administered. Thus, the pharmaceutical composition of the present invention may contain pharmaceutically acceptable ingredients such as carriers, excipients and / or stabilizers. Such pharmaceutically acceptable ingredients are not toxic in the amounts used when the pharmaceutical composition is administered to a human patient. The pharmaceutically acceptable ingredients added to the pharmaceutical composition may depend on the chemical nature of the active ingredient present in the composition, the particular intended use of the pharmaceutical composition, and the route of administration. Generally, the pharmaceutically acceptable ingredients used in connection with the present invention are used in accordance with knowledge available in the art, for example, knowledge from Remington’s Pharmaceutical Sciences, Ed. AR Gennaro, 20th Edition, 2000, Williams & Wilkins, Pennsylvania, USA.

[0048] Sequence The preferred amino acid sequences referred to in this application may be independently selected from the following sequences. The sequences are represented in the order from the N-terminus to the C-terminus; they are represented by the one-letter amino acid code.

[0049] The following non-limiting exemplary sequences were used in the experimental examples of this application: Table 1: Amino acid sequences of heavy and light chain variants of the Fn14-specific antibody construct. Legend: XXX linker sequence or internal FLAG tag XXX Tenascin C (TNC) trimerization domain (DIACGCAAAPDIKDLLSRLEELEGLVSSLREQGTG; SEQ ID NO: 21) XXX Constant Light Chain or Constant Heavy Chain XXX VH (variable heavy chain) or VL (variable light chain) domain XXX scFv (single-chain variable fragment) domain XXX linker sequence of the scFv (single-chain variable fragment) domain

Table 1

[0050] For each of Plasmids No. 1 to 17, the encoded amino acid sequence is as follows: Before the amino acid sequences shown in SEQ ID NOs: 1 to 17, there is an N-terminal amino acid sequence consisting of a leader sequence (SEQ ID NO: 19: MNFGFSLIFLVLVLKGVQCEVKLVPR), a first linker consisting of the amino acids QL, a FLAG tag (SEQ ID NO: 20: DYKDDDDK), and a second linker consisting of the amino acids EF, in the order from the N-terminus to the C-terminus.

[0051] For Plasmid No. 18, the encoded amino acid sequence is as follows: Before the amino acid sequence shown in SEQ ID NO: 18, there is an N-terminal amino acid sequence consisting of a leader sequence (SEQ ID NO: 19: MNFGFSLIFLVLVLKGVQCEVKLVPR) and a linker consisting of the amino acids EF, in the order from the N-terminus to the C-terminus. The amino acid sequence of SEQ ID NO: 18 has an internal FLAG tag (SEQ ID NO: 20: DYKDDDDK).

[0052] According to the present invention, the above-mentioned designated linker sequences GS, LE, and EF are cloning-related amino acid linker sequences, which may be independently removed from the amino acid sequences of SEQ ID NOs: 1 to 18, or they may be independently replaced by another linker sequence containing 1 to 20 amino acids. It should be noted that this is the case.

[0053] According to the present invention, the scFv domain linker sequence RSSTKGPKLEEGEFSEAQL may be independently removed from the amino acid sequences of SEQ ID NOs: 2, 4, 5, 6, 7, 8, 10, 12, 13, 14, 17, and 18, or they may be independently replaced by another linker sequence containing 1 to 20 amino acids. According to the present invention, the linker sequence GGSGGRG may be removed from the amino acid sequence of SEQ ID NO: 18.

Table 2

[0054] Examples The present invention is further illustrated by the following non-limiting examples.

[0055] Materials and Methods Cell Lines and Reagents HEK293T cells and HT1080 cells were obtained from the American Type Culture Collection (ATCC) (Rockville, Maryland, USA) or the German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany). HeLa-RIPK3-FADD KOThe cells have been described in another publication (Fuellsack et al., 2019), and the Kym-1 cells (Sekiguchi et al., 1985) were a gift from M. Sekiguchi (University of Tokyo). All cell lines were cultured in RPMI 1640 medium (Thermo Fischer Scientific, UK) supplemented with 10% fetal bovine serum (FBS) (Thermo Fischer Scientific, UK). Expression plasmids encoding the heavy and light chains of various recombinant proteins (Table 1) were generated in pCR3 (Invitrogen, Germany) by standard cloning techniques. The antibodies used in this study were purchased from the following suppliers: Sigma-Aldrich, Germany (anti-FLAG® M2, F3165; anti-β-actin, A1978; anti-NFκB p52, 05-361), Cell Signaling, GB (anti-TRAF-1, 70745), LI-COR Biosciences, Lincoln, USA (IRDye® 800CW anti-mouse IgG, 926-32210), Dako, Glostrup, Denmark (rabbit anti-mouse IgG with horseradish peroxidase (HRP) P0260, goat anti-rabbit IgG with HRP, P0448). The production and characterization of the soluble TWEAK variants Flag-sTWEAK (TWEAK) and Fc-Flag-sTWEAK (Fc-TWEAK) have been described previously (Roos et al., 2010), and TNF was a gift from Prof. Daniela Maennel (University of Regensburg, Germany).

[0056] Production and purification of recombinant proteins HEK293T cells were transiently transfected with expression plasmids encoding the heavy and light chain variants of the antibody variant of interest (ratio 1:1) using polyethyleneimine (PEI, Polyscience Inc., Warrington, USA) as described in a different document (Kucka et al., 2021). One day after adding the medium containing the PEI / DNA mixture, the latter was replaced with RPMI 1640 medium supplemented with 2% FBS containing 100 U / ml penicillin and 100 μg / ml streptomycin (Sigma-Aldrich, Germany). After an additional 5 - 7 days, the supernatant was collected, clarified by centrifugation (10 minutes, 4600×g), and initially assayed for the presence of the recombinant protein by Western blot detection (primary antibody: anti-FLAG M2; secondary antibody anti-mouse IgG IRDye 800CW). Various antibody variants were purified by anti-Flag affinity chromatography as described in a different document (Kucka et al., 2021). The concentration and purity of the purified protein were analyzed by SDS-PAGE, silver staining of the protein gel using the Pierce Silver Stain Kit (Thermo Fischer Scientific, USA), and comparison with protein standards of the LMW Calibration Kit for SDS electrophoresis from Amersham (GE Healthcare). The purity and integrity of the purified recombinant antibody were further analyzed using high-performance liquid chromatography (HPLC) (UltiMate 3000, Thermo Fischer Scientific, USA).

[0057] Analysis of p100 processing and TRAF1 induction HT1080 cells were seeded in 12-well cell culture plates (GIBCO) (2×10 5(Cells / well). The next day, the medium was replaced with fresh medium supplemented with the antibody of interest. As a positive control, cells were treated with 200 ng / ml of Flag-TWEAK. After 20 - 24 hours, cell pellets were suspended in Laemmli sample buffer containing 5% β-mercaptoethanol, sonicated for 25 seconds, and heated at 95°C for 5 minutes to prepare whole cell lysates. The lysates were separated by SDS-PAGE, proteins were transferred to nitrocellulose, and Western blot analysis of p100 - p52 processing (primary antibody: anti-NFκB p52; secondary antibody: HRP-labeled goat anti-mouse IgG) and TRAF1 induction (primary antibody: rabbit anti-TRAF1; secondary antibody: HRP-labeled goat anti-rabbit IgG) was performed. Protein amounts were adjusted by detection of β-actin (primary antibody: anti-β-actin; secondary antibody: HRP-labeled anti-mouse IgG).

[0058] Analysis of IL8 induction HT1080 cells were seeded in 96-well cell culture plates (GIBCO) (2×10 4 (Cells / well), and the next day, the cells were further treated with the reagent of interest for 24 hours. As a positive control, cells were stimulated with anti-Flag antibody M2 oligomerized Flag-TWEAK that mimics the activity of membrane TWEAK (Roos et al., 2010). Cell culture supernatants were collected and analyzed for IL8 content using a human IL8 ELISA kit (BD Biosciences, Heidelberg, Germany).

[0059] Promotion of TNF-induced cell death To analyze the ability of various Fn14-specific antibody variants to enhance TNF-induced toxicity, HeLa-RIPK3-FADD KO cells were seeded in 96-well cell culture plates (GIBCO) (2.5×10 4Cells / well). The next day, the medium was replaced with fresh medium containing the Fn14-specific reagent of interest, with or without 1 ng / ml TNF (Maennel, University of Regensburg, Germany). The next day, cell viability was finally analyzed by crystal violet staining. The viability values were normalized according to untreated cells (viability 100%) and cells incubated with the toxic reagent mixture (viability 0%).

[0060] Example 1: Construction of tetravalent, hexavalent, and octavalent anti-Fn14 variants. In view of the fact that anti-Fn14 antibodies can acquire memTWEAK-like activity upon cross-linking (e.g., Salzmann et al., 2013a), various multivalent variants of anti-Fn14 antibody 18D1 (Trebing et al., 2014) were generated and their ability to stimulate Fn14 in vitro was analyzed. To obtain tetravalent 18D1 variants, the scFv domain derived from 18D1 (construct 18D1-(1) in Figure 1A) and 18D1-IgG1 variants with point mutations that disrupt / decrease FcγR binding were genetically fused to the C-terminus of the heavy or light chain of 18D1-IgG1(N297A), yielding constructs 18D1-(2) and 18D1-(3) shown in Figure 1A. Alternatively, to obtain tetravalent variants with four similarly oriented Fn14 binding sites on the same side of the antibody scaffold, the variable domains of the heavy and light chains of the parental 18D1-IgG1 antibody were replaced with scFv:18D1 domains (Figure 1A, construct 18D1-(5)). Furthermore, hexameric 18D1 variants were generated by fusing the scFv:18D1 domain to the C-terminus of the heavy and light chains of 18D1-(1) (Figure 1A, construct 18D1-(4)), and by fusing this domain to the C-terminus of the heavy or light chain of construct 18D1-(5) (Figure 1A, constructs 18D1-(6) and 18D1-(7)). Finally, octameric variants were obtained by fusing the scFv:18D1 domain to the C-terminus of both the heavy and light chains of construct 18D1-(5) (Figure 1A, construct 18D1-(8)). All antibody constructs were produced by transient co-transfection of HEK293 cells using expression plasmids encoding the corresponding Flag-tagged LC and HC variants. The productivity of the parental 18D1 antibody and all variants derived therefrom was approximately equivalent (Figure 1B).

[0061] Example 2: Oligovalent 18D1 variants enhance TNF-induced toxicity and induce the non-canonical NFκB pathway, but vary greatly in their ability to induce IL8. With the exception of the 18D1-(1) antibody variant, all anti-Fn14 constructs enhanced TNF-induced toxicity to a level comparable to sTWEAK, with ED50 values less than 100 ng / ml (Figure 2A). All oligovalent 18D1 constructs induced more potent p100 processing, starting from approximately 20 ng / ml for 18D1-(2) to 18D1-(4) and approximately 200 ng / ml for 18D1-(5) to 18D1-(8). However, the conventional antibody variant (1) remained inactive in this regard and did not show p100 processing even at the highest concentration of 2 μg / ml (Figure 2B). Similarly, with the exception of 18D1-(1), all constructs upregulated the expression of TRAF1, which is controlled by the NFκB non-canonical pathway (Figure 2B). However, there were distinct differences among the constructs with respect to IL8 induction. Since IL8 is a prototypical target of the classical NFκB pathway, it is not induced or is insufficiently induced by sTWEAK, but is efficiently induced by oligomerized sTWEAK and memTWEAK (Roos et al., 2010). Constructs 18D1-(2) and especially 18D1-(4) showed variable but significant and robust IL8 induction, and in the case of the hexameric construct 18D1-(4), reached the maximum response induced by anti-Flag oligomerized Flag-sTWEAK (Figure 2C). In contrast, all 18D1-derived constructs (18D1-(5) to 18D1-(8)) in which the VH and VL domains were replaced with the scFv:18D1 domain remained mostly inactive (Figure 2C). These variants also showed only weak IL8 induction after protein G crosslinking, whereas the parental antibody was able to elicit this response by this treatment (Figure 7). Furthermore, 18D1-(5) to 18D1-(8) inhibited IL8 induction by memTWEAK-expressing transfectants and hexameric Fc-sTWEAK with memTWEAK-like activity, despite their ability to cause alternative NFκB signaling and promotion of TNF-induced cell death (Figure 2A, B) (Figure 3A, B). In this regard, these constructs are similar to soluble TWEAK and also inhibit the memTWEAK-induced IL8 response (Figure 8).The IKK2-specific inhibitor TPCA-1 efficiently inhibited IL8 induction by oligomerized sTWEAK and 18D1-(4), but had no effect on p100 processing (Figure 3C, D), confirming that IL8 induction via Fn14 reflects activation of the classical NFκB pathway.

[0062] The potential agonist activity of the anti-Fn14 antibody can be liberated by antibody crosslinking / oligomerization reagents such as Protein G (Salzmann et al., 2013a). Therefore, to confirm that the agonist properties observed with the oligomeric 18D1 variants are not due to molecular aggregation but actually reflect intrinsic activity, the parental antibody was purified together with the prototype variants 18D-(2) and 18D1-(4) and analyzed by gel filtration (Figure 4A, B). All three proteins eluted mostly as a single molecular species with an MW well corresponding to the Fc domain dimerized molecule by gel filtration (Figure 4B). Functional analysis further revealed that purification did not affect the functional properties of the molecules (Figure 4C, D, E).

[0063] Example 3: Format (2) and (4) variants of anti-Fn14 mAb PDL192 also exhibit memTWEAK-like activity. To demonstrate that genetic fusion of the Fn14-specific scFv domain with an anti-Fn14 antibody is generally advantageous for achieving memTWEAK mimetic agonism, constructs of format (2) and (4) of a second anti-Fn14 antibody, namely PDL192, were generated and evaluated. Previous studies have shown that 18D1 and PDL192 recognize different epitopes on Fn14 (Trebing et al., 2014). Furthermore, the two antibodies also differ in their ability to inhibit TWEAK binding. 18D1 inhibits TWEAK binding, while PDL192 does not compete with ligand binding (Trebing et al., 2014). Similar to the parental antibody 18D1-(1) and 18D1-(2) variants, the parental form of PDL192 and its PDL192-(2) variant were efficiently expressed. In contrast, the expression level of the hexameric construct of the (4) type was somewhat low.

[0064] The IgG1 (N297A) version of PDL192 did not show agonism, but its tetravalent and hexavalent derivatives stimulated p100 processing and promoted TNF-induced cell death (Figures 5A, B). Both constructs also strongly stimulated IL8 induction (Figure 5C). These data suggest that type (2) and type (4) antibody variants generally confer FcγR-independent memTWEAK-like agonism. To further rule out that the observed agonist activities of the type (2) and type (4) constructs of PDL192 are not due to non-specific aggregation, both constructs were purified by anti-Flag agarose affinity chromatography (Figure 5D). All PDL192 variant proteins were efficiently purified and eluted without evidence of aggregated high molecular weight species (Figure 5D). Thus, the observed agonism of the type (2) and type (4) anti-Fn14 variants is also intrinsic to the molecule.

[0065] Example 4: Antitumor activity of 18D1-(2). Apc ko / ko , Kras LSL-G12D , Tgfbr2 ko / ko and Trp53 ko / ko Mouse tumor organoids (MTOs) derived from tumors from mice with intestinal stem cells (Tauriello et al., 2018) were grown in vitro and injected into the cecal wall of syngeneic C57BL / 6J recipients. Two weeks after tumors were first established, mice were treated with 200 μg of 18D1-(2) three times a week for 2 weeks. For comparison, mice were treated with MSA-sTWEAK, a fusion protein of sTWEAK and serum albumin that extends serum retention, and Fc(DANA)-sTWEAK. In mice treated with 18D1-(2), tumor weight and tumor size were significantly reduced, and tumor weight was also significantly reduced after treatment with Fc(DANA)-sTWEAK, with a tendency for tumor volume to decrease (Figure 6). No significant antitumor effect was obtained in MSA-sTWEA-treated mice (Figure 6).

[0066] Industrial applicability The pharmaceutical compositions, polypeptides, nucleic acids, cells, and products for use in the present invention are industrially applicable. For example, they can be used in the manufacture of pharmaceuticals or as pharmaceuticals.

[0067] References Meighan-Mantha RL, Hsu DK, Guo Y, Brown SA, Feng SL, Peifley KA, Alberts GF, Copeland NG, Gilbert DJ, Jenkins NA, Richards CM, Winkles JA. The mitogen-inducible Fn14 gene encodes a type I transmembrane protein that modulates fibroblast adhesion and migration. J Biol Chem. 1999 Nov 12;274(46):33166-76. Wiley SR, Cassiano L, Lofton T, Davis-Smith T, Winkles JA, Lindner V, Liu H, Daniel TO, Smith CA, Fanslow WC. A novel TNF receptor family member binds TWEAK and is implicated in angiogenesis. Immunity. 2001 Nov;15(5):837-46. Girgenrath M, Weng S, Kostek CA et al. TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration. EMBO J. 2006; 25:5826-39. Wajant H. The TWEAK-Fn14 system as a potential drug target. Br J Pharmacol. 2013 Oct;170(4):748-64. Chicheportiche Y, Bourdon PR, Xu H, Hsu YM, Scott H, Hession C, Garcia I, Browning JL. 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Zhou H, Hittelman WN, Yagita H, Cheung LH, Martin SS, Winkles JA, Rosenblum MG. Antitumor activity of a humanized, bivalent immunotoxin targeting fn14-positive solid tumors. Cancer Res. 2013 Jul 15;73(14):4439-50. Zhou H, Mohamedali KA, Gonzalez-Angulo AM, Cao Y, Migliorini M, Cheung LH, LoBello J, Lei X, Qi Y, Hittelman WN, Winkles JA, Tran NL, Rosenblum MG. Development of human serine protease-based therapeutics targeting Fn14 and identification of Fn14 as a new target overexpressed in TNBC. Mol Cancer Ther. 2014 Nov;13(11):2688-705. Lerchen HG, Wittrock S, Stelte-Ludwig B, Sommer A, Berndt S, Griebenow N, Rebstock AS, Johannes S, Cancho-Grande Y, Mahlert C, Greven S, Terjung C. Antibody-Drug Conjugates with Pyrrole-Based KSP Inhibitors as the Payload Class. Angew Chem Int Ed Engl. 2018 Nov 12;57(46):15243-15247 Alvarez de Cienfuegos A, Cheung LH, Mohamedali KA, Whitsett TG, Winkles JA, Hittelman WN, Rosenblum MG. Therapeutic efficacy and safety of a human fusion construct targeting the TWEAK receptor Fn14 and containing a modified granzyme B. J Immunother Cancer. 2020 Sep;8(2):e001138 Culp PA, Choi D, Zhang Y, Yin J, Seto P, Ybarra SE, Su M, Sho M, Steinle R, Wong MH, Evangelista F, Grove J, Cardenas M, James M, Hsi ED, Chao DT, Powers DB, Ramakrishnan V, Dubridge R. Antibodies to TWEAK receptor inhibit human tumor growth through dual mechanisms. Clin Cancer Res. 2010 Jan 15;16(2):497-508. Michaelson JS, Amatucci A, Kelly R, Su L, Garber E, Day ES, Berquist L, Cho S, Li Y, Parr M, Wille L, Schneider P, Wortham K, Burkly LC, Hsu YM, Joseph IB. Development of an Fn14 agonistic antibody as an anti-tumor agent. MAbs. 2011 Jul-Aug;3(4):362-75. Michaelson JS, Kelly R, Yang L, Zhang X, Wortham K, Joseph IB. The anti-Fn14 antibody BIIB036 inhibits tumor growth in xenografts and patient derived primary tumor models and enhances efficacy of chemotherapeutic agents in multiple xenograft models. 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Claims

1. A polyvalent anti-Fn14 antibody construct comprising at least four antigen-binding sites for Fn14, wherein the construct comprises, (a) An IgG molecule containing two antigen-binding sites for Fn14, or (b) A modified IgG molecule, modified by replacing each of the two variable domains of the heavy chain with an scFv containing one of the antigen-binding sites for Fn14, and by replacing each of the two variable domains of the light chain with an scFv containing one of the antigen-binding sites for Fn14. Includes, A construct comprising a mutation in the Fc domain of the IgG molecule of (a) or (b) that reduces FcγR binding.

2. (i) further comprising an scFv covalently bonded to at least one C-terminus of the two heavy chains of the IgG molecule, wherein the scFv contains one of the antigen-binding sites for Fn14; and / or (ii) The construct according to claim 1, further comprising an scFv covalently bonded to the C-terminus of at least one of the two light chains of the IgG molecule, wherein the scFv contains one of the antigen-binding sites for Fn14.

3. The construct according to claim 1, wherein the IgG molecule is IgG1, IgG2, IgG3, or IgG4, preferably IgG2 or IgG4.

4. The construct according to claim 1, wherein the mutation is the N297A mutation.

5. An Fn14 agonist, The construct according to claim 1, which can optionally activate IL8 production in cells expressing Fn14, preferably HT-1080 cells expressing Fn14.

6. The construct according to claim 1, comprising at least five antigen-binding sites for Fn14, and optionally comprising at least six antigen-binding sites for Fn14.

7. A construct comprising the IgG molecule described in (a), (i) comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 3, preferably comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, more preferably comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, more preferably comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, more preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, most preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, or (ii) The construct according to claim 1, comprising at least 85% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 85% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 11, preferably comprising at least 90% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 90% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 11, more preferably comprising at least 95% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 95% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 11, more preferably comprising at least 98% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 98% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 11, more preferably comprising at least 99% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 99% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 11, most preferably comprising the amino acid sequence of SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO:

11.

8. A construct comprising the IgG molecule described in (a), (i) Consists of an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 1 and at least 85% identical to the amino acid sequence of SEQ ID NO: 4, preferably contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 and at least 90% identical to the amino acid sequence of SEQ ID NO: 4, more preferably contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 and at least 95% identical to the amino acid sequence of SEQ ID NO: 4, more preferably contains an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 1 and at least 98% identical to the amino acid sequence of SEQ ID NO: 4, more preferably contains an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1 and at least 99% identical to the amino acid sequence of SEQ ID NO: 4, most preferably contains an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1 and at least 99% identical to the amino acid sequence of SEQ ID NO: 4, or (ii) The construct according to claim 1, comprising at least 85% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 9 and at least 85% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 12, preferably comprising at least 90% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 9 and at least 90% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 12, more preferably comprising at least 95% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 9 and at least 95% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 12, more preferably comprising at least 98% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 9 and at least 98% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 12, and most preferably comprising the amino acid sequence of SEQ ID NO: 9 and the amino acid sequence of SEQ ID NO:

12.

9. A construct comprising the IgG molecule described in (a), (i) comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 4, preferably comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 4, more preferably comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4, more preferably comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 4, more preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4, most preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 4, or (ii) The construct according to claim 1, comprising at least 85% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 85% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 12, preferably comprising at least 90% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 90% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 12, more preferably comprising at least 95% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 95% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 12, more preferably comprising at least 98% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 98% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 12, most preferably comprising at least 99% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 10 and at least 99% the same amino acid sequence as the amino acid sequence of SEQ ID NO:

12.

10. A construct comprising the IgG molecule described in (b), (i) comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 5, preferably comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5, more preferably comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, more preferably comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, more preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 5, most preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 6 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 5, or (ii) The construct according to claim 1, comprising at least 85% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 14 and at least 85% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 13, preferably comprising at least 90% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 14 and at least 90% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 13, more preferably comprising at least 95% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 14 and at least 95% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 13, more preferably comprising at least 98% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 14 and at least 98% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 13, most preferably comprising at least 99% the same amino acid sequence as the amino acid sequence of SEQ ID NO: 14 and at least 99% the same amino acid sequence as the amino acid sequence of SEQ ID NO:

13.

11. A construct comprising the IgG molecule described in (b), The construct according to claim 1, comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 5, preferably comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5, more preferably comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, more preferably comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, and most preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:

5.

12. A construct comprising the IgG molecule described in (b), The construct according to claim 1, comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 7, preferably comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, more preferably comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7, more preferably comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 7, more preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7, and most preferably comprising an amino acid sequence of SEQ ID NO: 8 and an amino acid sequence of SEQ ID NO:

7.

13. comprising a trimerizing domain, preferably, (i) The trimerizing domain is the trimerizing domain of tenacin C, and / or (ii) The trimerizing domain comprises the amino acid sequence of SEQ ID NO: 21 or a sequence that is at least 70% identical thereto, preferably at least 80% identical thereto, more preferably at least 85% identical thereto, more preferably at least 90% identical thereto, more preferably at least 93% identical thereto, and / or (iii) The trimerizing domain is linked to the C-terminus of the constant domain of the heavy chain of IgG. The structure according to claim 1.

14. The construct according to claim 10, comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 3, preferably comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, more preferably comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, more preferably comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, more preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, and most preferably comprising an amino acid sequence of SEQ ID NO: 15 and an amino acid sequence of SEQ ID NO:

3.

15. A construct comprising the IgG molecule described in (a), The construct according to claim 1, comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 3, preferably comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 3, more preferably comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, more preferably comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 3, more preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 16 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 3, and most preferably comprising an amino acid sequence of SEQ ID NO: 16 and an amino acid sequence of SEQ ID NO:

3.

16. The construct according to claim 10, comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 5, preferably comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 5, more preferably comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, more preferably comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 5, most preferably comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 5, and most preferably comprising an amino acid sequence of SEQ ID NO: 17 and an amino acid sequence of SEQ ID NO:

5.

17. The construct according to claim 10, comprising an amino acid sequence identical to at least 85% of the amino acid sequence of SEQ ID NO: 18, preferably comprising an amino acid sequence identical to at least 90% of the amino acid sequence of SEQ ID NO: 18, more preferably comprising an amino acid sequence identical to at least 95% of the amino acid sequence of SEQ ID NO: 18, more preferably comprising an amino acid sequence identical to at least 98% of the amino acid sequence of SEQ ID NO: 18, more preferably comprising an amino acid sequence identical to at least 99% of the amino acid sequence of SEQ ID NO: 18, and most preferably comprising the amino acid sequence of SEQ ID NO:

18.

18. The construct according to claim 1, further comprising (a) and (b) below: (a) a leader sequence comprising the amino acid sequence of SEQ ID NO: 19, optionally followed by a first linker sequence; and (b) an N-terminal amino acid sequence comprising one or two FLAG tags comprising the amino acid sequence of SEQ ID NO: 20, optionally followed by a second linker sequence.

19. A pharmaceutical composition comprising the structure described in any one of claims 1 to 18.

20. The pharmaceutical composition according to claim 19 for use in the treatment of cancer.

21. The cancer is a cancer that expresses Fn14, The pharmaceutical composition according to claim 20, wherein the cancer is optionally a solid tumor.

22. A nucleic acid or set of nucleic acids that encodes a construct according to any one of claims 1 to 18.

23. A recombinant cell comprising a nucleic acid or set of nucleic acids encoding the construct according to any one of claims 1 to 18, and expressing the construct according to any one of claims 1 to 18.

24. A method for producing a construct according to any one of claims 1 to 18, comprising expressing the construct in recombinant cells from nucleic acids or sets of nucleic acids encoding the construct according to any one of claims 1 to 18, and optionally further comprising purifying the construct and formulating it into a pharmaceutical composition.

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