AU2017201232B2 - TNF superfamily collectin fusion proteins - Google Patents

Abstract

TNF SUPERFAMILY COLLECTIN FUSION PROTEINS The present invention refers to a fusion protein comprising a TNF- superfamily (TNFSF) cytokine or a receptor binding thereof fused a collectin trimerization domain, to a nucleic acid molecule encoding the fusion protein, and to a cell comprising the nucleic acid molecule. The fusion protein is present as a trimeric complex or as an oligomer thereof. The fusion protein, the nucleic acid, and the cell is suitable as pharmaceutical composition or for therapeutic, diagnostic and/or research applications.

Description

The present invention refers to a fusion protein comprising a TNF- superfamily (TNFSF) cytokine or a receptor binding thereof fused a collectin trimerization domain, to a nucleic acid molecule encoding the fusion protein, and to a cell comprising the nucleic acid molecule. The fusion protein is present as a trimeric complex or as an oligomer thereof. The fusion protein, the nucleic acid, and the cell is suitable as pharmaceutical composition or for therapeutic, diagnostic and/or research applications.

2017201232 23 Feb 2017

TNF Superfamily Collectin Fusion Proteins

Description

Cross Reference to Related Applications

This application is a divisional of Australian Patent Application No. 2014202645 (filed 15 May 2014) which in turn was a divisional of Australian Patent No. 2008274490 (filed 10 July 2008; national phase of PCT/EP2008/005644), claiming convention from Europe Patent No. 07013506.6, the entire contents of each of which is incorporated herein by reference.

Field of Invention

The present invention refers to a fusion protein comprising a TNF-superfamily (TNFSF) cytokine or a receptor binding domain thereof fused to a collectin trimerization domain, to a nucleic acid molecule encoding the fusion protein, and to a cell comprising the nucleic acid molecule. The fusion protein is present as a trimeric complex or as an oligomer thereof. The fusion protein, the nucleic acid, and the cell is suitable as pharmaceutical composition or for therapeutic, diagnostic and/or research applications as described herein.

State of the Art

Figands of the tumor necrosis factor (TNF) family fulfill crucial roles in the immune system, but have also been implicated in the development of epithelial and endothelial structures.¹ TNF family ligands are primarily expressed as trimeric type II transmembrane proteins and are often processed into soluble variants that are also organized as trimers. ’ While shedding of some TNF ligands does not interfere with their capability to activate their corresponding receptors and might be even important for their physiological function, other TNF ligands become inactivated by proteolytic processing. Soluble TNF ligands that are not or only poorly active still interact with their cognate receptors. For example, the soluble forms of TNF, CD95F, TRAIF and CD40F interact with TNFR2, CD95, TRAIFR2 and CD40, respectively, but do not or only poorly activate signaling by these receptors. ^3-6 Notably, inactive or poorly active soluble TNF ligands can be converted into highly active molecules by artificially increasing their avidity. For example, soluble Flag-tagged variants of TNF, CD95F, TRAIF and CD40F (12711687_1):GGG

2017201232 23 Feb 2017

-2stimulate robust signaling by TNFR2, CD95, TRAILR2 and CD40, respectively, provided they were crosslinked with the Flag-specific mAb M2. Likewise, hexameric and dodecameric fusion proteins of soluble CD95L and soluble CD40L as well as non-specifically aggregated preparations of TNF ligands produced in E. coli display high activity.^6-8

The structural hall mark of the ligands of the TNF family is the carboxyterminal TNF 2 homology domain (THD) or “receptor binding domain” (RBD), both terms are equally used herein, which is part of both the transmembrane and soluble forms of TNF ligands.¹'² The THDs of the various TNF ligands are composed of a framework of aromatic and hydrophobic residues that adopt an almost identical tertiary fold and cause self association into trimers.¹'² The THD also mediates receptor binding. In general, trimeric ligands of the TNF family bind to three molecules of their is corresponding receptor(s). This interaction alone is not necessarily sufficient to activate receptor-associated intracellular signaling pathways. Several lines of evidence suggest that the initial formation of trimeric signaling competent ligand receptor complexes is followed by secondary multimerization into supramolecular clusters.⁹·¹¹ These two steps in TNF receptor activation (1.

ligand binding; 2. secondary aggregation of receptor ligand complexes) depend to a varying extent on several factors including lipid raft localization, cytoskeleton support, receptor autoaggregation, receptor associated adapter proteins, but also on affinity and avidity of the ligand receptor interaction and the way how the ligand is presented to the receptor (membrane ligand or immobilized ligand versus soluble ligand, trimers versus higher aggregates).

It is known that trimeric complexes of TNF superfamily cytokines are difficult to prepare from recombinant monomeric units.

For example, WO 01/49866 discloses recombinant fusion proteins comprising a TNF cytokine and a multimerization component. A disadvantage of these fusion proteins is, however, that the trimerization domain usually has a large molecular weight and/or that the trimerization is

2017201232 23 Feb 2017

-3rather inefficient.

Schneider et al. (J Exp Med 187 (1989), 1205-1213) describes that trimers of TNF cytokines are stabilized by N-terminally positioned stabilization motifs.

In CD95L, the stabilization of the CD95L-receptor binding domain trimer is presumably caused by N-terminal amino acid domains which are located near the cytoplasmic membrane.

Shiraishi et al. (Biochem Biophys Res Commun 322 (2004), 197-202) io describes that the receptor binding domain of CD95L may be stabilized by

N-terminally positioned artificial α-helical coiled-coil (leucine zipper) motifs. It was found, however, that the orientation of the polypeptide chains to each other, e.g. parallel or antiparallel orientation, can hardly be predicted. Further, the optimal number of hepta-d-repeats in the coiled-coil zipper motif is are difficult to determine. In addition, coiled-coil structures have the tendency to form macromolecular aggregates after alteration of pH and/or ionic strength.

Me Aiinden et al. (J of Biol Chem, 2002, 277(43):41274-41281) discloses the

2o preparation of a fusion protein between a human type IIA procollagen amino acid sequence and a 14 amino acid sequence corresponding to the first two heptad repeats of the rat surfactant protein's (SP-D) neck domain.

WO 01/42298 discloses the preparation of a fusion protein between surfactant protein-D comprising the signal sequence, the collagen domain and the neck domain and CD40L. The disadvantage of those fusion proteins is that they lead to multimeric aggregates that are highly immunogenic and that they do not produce functionally defined trimeric ligands.

It was an object of the present invention to provide fusion proteins comprising a TNF cytokine or a receptor binding domain, which allow efficient recombinant manufacture combined with good trimerization properties and improved pharmaceutical properties.

2017201232 06 Feb 2018

Summary of the Invention

According to a first aspect, the present invention provides a fusion protein comprising:

(i) TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or a receptor binding domain thereof, and (ii) a collectin trimerization domain comprising a neck domain or a neck and carbohydrate binding domain of surfactant protein-D, wherein (ii) is located C-terminally of (i).

According to a second aspect, the present invention provides a nucleric acid molecule encoding the fusion protein of the first aspect.

According to a third aspect, the present invention provides a vector comprising the nucleic acid molecule of the second aspect.

According to a fourth aspect, the present invention provides an isolated cell transformed or transfected with the nucleic acid molecule of the second aspect, or the vector of the third aspect.

The present invention relates to a fusion protein comprising (i) a TNF-superfamily cytokine or a receptor binding domain thereof, and (ii) a collectin trimerization domain.

The invention further relates to a nucleic acid molecule encoding a fusion protein as described herein and to a cell or a non-human organism transformed or transfected with a nucleic acid molecule as described herein.

The invention also relates to a pharmaceutical or diagnostic composition comprising as an active agent a fusion protein, a nucleic acid molecule, or a cell as described herein.

The invention also relates to a fusion protein, a nucleic acid molecule, or a cell as described herein for use in therapy, e.g., the use of a fusion protein, a nucleic acid molecule, or a cell as described herein for the preparation of a pharmaceutical composition in the prophylaxis and/or treatment of proliferative disorders, particularly disorders caused by, associated with and/ or accompanied by dysfunction of TNF cytokines, such as tumors, e.g. solid or (14240575_l):GGG

4a

2017201232 06 Feb 2018 lymphatic tumors, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune disorders, e.g. rheumatoid and/or arthritic diseases, degenerative diseases, e.g. neurodegenerative diseases such as multiple sclerosis, apoptosis-associated diseases and transplant rejections.

The invention also relates to the following:

[1] A fusion protein comprising:

(i) TRAIL (SEQ ID NO: 10) or a receptor binding domain thereof: and (ii) a collectin trimerization domain.

[2] The fusion protein- according to [1] additionally comprising a flexible linker element between (i) and (ii).

[3] The fusion protein according to [2], wherein the flexible linker element has a length of 3-20 amino acids.

[4] The fusion protein according to [2] or [3], wherein the flexible linker element has a length of 3, 6, 9, 10, 12, 15 or 18 amino acids.

[5] The fusion protein according to any one of [2-4], wherein the flexible linker element is a glycine/serine linker.

[6] The fusion protein according to [5], wherein the flexible linker element has the amino acid sequence (GSS)_a (SSG)b(GSG)_c wherein a, b, c is each 0, 1,2, 3, 4, 5 or 6.

[7] The fusion protein according to any one of [2-6], wherein the flexible linker element has a length of 9-15 amino acids.

[8] The fusion protein according to any one of [1-7], wherein (i) comprises amino acids 95-281, 116-281, 117-281, 118-281, 119-281 or 120-281 of human TRAIL (SEQ ID NO: 10).

[9] The fusion protein according to any one of [1-8], wherein (i) comprises a mutant of TRAIL or of a receptor binding domain thereof which binds and/or activates TRAILR1 and/or TRAILR2.

[10] The fusion protein according to any one of [1-9], wherein (i) comprises at least one to six amino acid substitutions.

[11] The fusion protein according to [9] or [10], wherein the amino acid substitution affects at least one of the following amino acid positions of human TRAIL (SEQ ID NO: 10): R130, G160, Y189, R191, Q193, E195, N199, K201, Y213, T214, S215, H264,1266, D267, or D269.

(14240575_l):GGG

4b

2017201232 06 Feb 2018 [12] The fusion protein according to [11], wherein the amino acid substitution is at least one of the following: R130E, G160M, Y189A, Y189Q, R191K, Q193S, Q193R, E195R, N199V, N199R, K201R, Y213W, T214R, S215D, H264R, I266L, D267Q, D269H, D269R, or D269K.

[13] The fusion protein according to any one of [1-12], wherein (ii) is surfactant protein-D, surfactant protein-A, mannan-binding protein-A, mannan-binding-protein-C, collectin liver 1, collectin placenta 1, or collectin-11.

[14] The fusion protein according to any one of [1-13], wherein (ii) comprises the neck and carbohydrate binding domain (CRD) domain of the surfactant protein-D.

[15] The fusion protein according to any one of [1-14], wherein (a) (ii) comprises amino acids 217-375, 218-375, 219-375, 220-375, 221-375, 222-375, 223-375, 224-375, 225-375 of human surfactant protein-D of SEQ ID NO:21; and/or (b) (ii) comprises amino acids 217-257, 218-257, 219-257, 220-257, 221-257, 222-257, 223-257, 224-257, or 225-257 of human surfactant protein-D of SEQ ID NO:21.

[16] The fusion protein according to any one of [1-15], wherein (ii) comprises at least one to six amino acid substitutions.

[17] The fusion protein according to [16], wherein the amino acid substitution affects amino acid position F355 of human surfactant protein-D of SEQ ID NO: 21.

[18] The fusion protein according to [17], wherein the amino acid substitution is one of the following: F355A, F355S, F355T, F355E, F355D, F355K, or F355R.

[19] The fusion protein according to any one of [1-18], wherein (ii) comprises a mutant which does not bind to mannose.

[20] The fusion protein according to any one of [1-13] or [19], wherein (ii) comprises amino acids 110-271, 116-271, or 121-271 of human collectin-11 of SEQ ID NO:22.

[21] The fusion protein according to any one of [1-13] or [19], wherein (ii) comprises amino acids 110-147, 110-148, 110-149, 110-150, 110-151, 116-147, 116-148, 116-149, 116-150, 116-151, 121-147, 121-148, 121-149, 121-150, or 121-151 of human collectin-11 of SEQ ID NO:22.

[22] The fusion protein according to any one of [1-21], wherein (ii) is located C-terminally of (i)· [23] The fusion protein according to any one of [1-22], wherein (ii) is located N-terminally of (i)· (14240575_l):GGG

4c

2017201232 06 Feb 2018 [24] The fusion protein according to any one of [1-23] which additionally comprises an Nterminal signal peptide domain.

[25] The fusion protein according to [24], wherein the N-terminal signal peptide domain comprises a protease cleavage site.

[26] The fusion protein according to [24] or [25], wherein the N-terminal signal peptide domain comprises the sequence SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

[27] The fusion protein according to any one of [1-26] which comprises the sequence of SEQ ID NO:26-SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:40-41 or SEQ ID NO:43-51.

[28] The fusion protein according to any one of [1-26], wherein the fusion protein further comprises a recognition/purification domain.

[29] The fusion protein according to [28], wherein the recognition/purification domain is located at the N-terminus or at the C-terminus.

[30] The fusion protein according to [28] or [29], wherein the recognition/purification domain is a Strep-tag or a poly His-domain.

[31] The fusion protein according to any one of [1-30] which additionally comprises a terminal flexible element.

[32] The fusion protein according to [31], wherein the terminal flexible element includes and/or connects to the recognition/purification domain.

[33] The fusion protein according to any of [1-32] which is present as a trimeric complex or as an oligomer of the trimeric complex.

[34] The fusion protein according to [33], wherein the complex is formed by covalent linkage between three fusion proteins.

[35] The fusion protein according to [34], wherein the covalent linkage preferably consists of disulfide bridges between cysteines of (ii).

[36] The fusion protein according to any one of [33] or [34], wherein the complex consists of three identical fusion proteins.

[37] A nucleic acid molecule encoding a fusion protein according to any one of [1-32], [38] The nucleic acid molecule according to [37] which is operatively linked to an expression control sequence.

[39] The nucleic acid molecule according to [37] or [38] which is located on a vector.

[40] A cell transformed or transfected with a nucleic acid molecule according to any one of [37-39], [41] The cell according to [40] which is a prokaryotic cell, or a eukaryotic cell.

[42] The cell according to [41], wherein said eukaryotic cell is a mammalian cell.

(14240575_l):GGG

4d

2017201232 06 Feb 2018 [43] The cell according to [42], wherein said mammalian cell is a human cell.

[44] A non-human organism transformed or transfected with a nucleic acid molecule according to any one of [37-39].

[45] A pharmaceutical or diagnostic composition comprising as an active agent a fusion protein according to any one of [1-36], a nucleic acid molecule according to any of [37-39], or a cell according to any one of [40-43].

[46] A therapeutic method, the method comprising administering to a subject in need thereof, a fusion protein according to any one of [1-36], a nucleic acid molecule according to any of [37-39], or a cell according to any one of [40-43].

[47] Use of a fusion protein according to any one of [1-36], a nucleic acid molecule according to any of [37-39], or a cell according to any one of [40-43] for the preparation of a medicament for the prophylaxis and/or treatment of proliferative disorders.

[48] A method for prophylaxis and/or treatment of proliferative disorders the method comprising administering an effective amount of the pharmaceutical composition according to [45] to a subject in need thereof.

[49] The use according to [47] or the method according to [48], wherein the disorders are selected from disorders caused by, associated with and/or accompanied by dysfunction of TNF cytokines, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune disorders, degenerative diseases, apoptosis-associated diseases and transplant rejections.

[50] The use or method according to [49], wherein said disorders caused by, associated with and/or accompanied by dysfunction of TNF cytokines are tumors.

[51] The use or method according to [49], wherein said autoimmune disorder is rheumatoid and/or arthritic disease.

[52] The use or method according to [49], wherein said degenerative disease is multiple sclerosis.

[53] The use according to any one of [47] or [49-52] or a method according to any one of [48-52], wherein said medicament or composition is administered in combination with an apoptosis-sensitizing and/or inducing agent.

Detailed Description of the Invention

The fusion protein may be a monomeric protein or a multimeric protein. Preferably, the fusion protein is present as a trimeric complex consisting of three monomeric units which may be identical or different. Preferably, a trimeric complex consists of three identical fusion proteins. In a further (14240575_l):GGG

-52017201232 23 Feb 2017 preferred embodiment, the complex is formed by covalent linkage between three of the fusion proteins described herein, e.g., a covalent linkage of disulfide bridges between cysteines of the collectin trimerization domain (ii) as described herein. The trimeric complex as such shows biological activity.

It was found, however, that oligomers of the trimeric complex, e.g. defined complexes wherein the basic trimeric structure is present 2, 3 or 4 times, also have biological activity. Thus, also preferred is an oligomer of the trimeric complex.

io One component (i) of the fusion protein is a cytokine of the TNF superfamily or a receptor binding domain thereof. Preferably, component (i) is a mammalian, particularly human cytokine or a receptor binding domain thereof including allelic variants and/or derivatives thereof. Further, it is preferred that the TNF cytokine is a receptor binding domain thereof capable is of binding to the corresponding cytokine receptor and preferably capable of receptor activation, whereby apoptotic or proliferative activity may be caused. The cytokine may e.g. be selected from TNF superfamily members, e.g. human TNFSF-1 to -18 as indicated in Table 1, preferably from LTA (SEQ ID NO:1), TNFa (SEQ ID NO:2), LTB (SEQ ID NO:3), OX40L (SEQ ID

NO:4), CD40L (SEQ ID NO:5), CD95L (SEQ ID NO:6), CD27L (SEQ ID NO:

7), CD30L (SEQ ID NO:8), CD137L (SEQ ID NO:9), TRAIL (SEQ ID ΝΟ.Ί0), RANKL (SEQ ID NO:11), TWEAK (SEQ ID NO: 12), APRIL 1 (SEQ ID NO: 13), APRIL 2 (SEQ ID NO:14), BAFF (SEQ ID NO:15), LIGHT (SEQ ID NO: 16), TL1A (SEQ ID NO:17), GITRL (SEQ ID NO:18), EDA-A1 (SEQ ID NO:

19), EDA-A2 (SEQ ID NO:20), or a receptor binding domain thereof.

Preferred receptor binding domains of the respective proteins are indicated in Table 1 (NH₂-aa to COOH-aa) and comprise, e.g., comprises amino acids 59-205 or 60-205 of LTA (SEQ ID NO:1), 86-233 of TNFa (SEQ ID NO:2), 82-244 or 86-244 of LTB (SEQ ID NO:3), 52-183 or 55-183 of OX40L (SEQ

ID NO:4), 112-261 or 117-261 of CD40L (SEQ ID NO:5), 51-193 or 56-193 of CD27L (SEQ ID NO:7), 97-234, 98-234 or 102-234 of CD30L (SEQ ID NO:8), 86-254 of CD137L (SEQ ID NO:9), 161-317 of RANKL (SEQ ID NO: 11), 103-249, 104-249 or 105-249 of TWEAK (SEQ ID NO: 12), 112-247 or

-62017201232 23 Feb 2017

113-247 of APRIL 1 (SEQ ID NO:13), 112-250 or 113-250 of APRIL 2 (SEQ ID NO:14), 140-285 of BAFF (SEQ ID NO:15), 91-240 of LIGHT (SEQ ID NO:16), 91-251 or 93-251 of TL1A (SEQ ID NO:17), 52-177 of GITRL (SEQ ID NO:18), 245-391 of EDA-A1 (SEQ ID NO;19), 245-389 of EDA-A2 (SEQ s ID NO:20).

More preferably, the cytokine of the TNF superfamily or a receptor binding domain thereof is selected from CD95L or TRAIL or a receptor binding domain thereof. In an especially preferred embodiment, the cytokine of the io TNF superfamily or a receptor binding domain thereof comprises the extracellular portion of a TNF cytokine including the receptor binding domain without membrane located domains.

In a preferred embodiment, the cytokine of the TNF superfamily or a is receptor binding domain thereof of the fusion protein is selected from human

CD95L (SEQ ID NO:6), particularly amino acids 142-281 or 144-281 of human CD95L.

In a further preferred embodiment, the cytokine of the TNF superfamily or a receptor binding domain thereof of the fusion protein is selected from human TRAIL (SEQ ID ΝΟ.Ί0), particularly amino acids 95-281, 116-281, 117-281, 118-281, 119-281 or 120-281 of human TRAIL. In another preferred embodiment human TRAIL comprise any amino acid from 95-120 as initial amino acid - amino acid 281 of SEQ ID NO:10.

In a further preferred embodiment of the invention, the cytokine of the TNF superfamily or a receptor binding domain thereof of the fusion protein as described herein comprises a mutant of the cytokine of the TNF superfamily or a receptor binding domain thereof which binds and/or activates TRAIL30 receptor 1 (TRAILR1) and/or TRAIL-receptor 2 (TRAILR2). The binding and/ or activity of the mutant may be, e.g., determined by the assays as disclosed herein, e.g., in the Examples or by the assays disclosed in van der Sloot et al. (PNAS, 2006, 103:8634-8639), Kelley et al. (J. Biol. Chem., 2005,

2017201232 23 Feb 2017

-7280:2205-2215), or MacFarlane et al. (Cancer Res., 2005, 65:

11265-11270).

The mutant may be generated by any technique and is known by the skilled 5 person, e.g., the techniques disclosed in an der Sloot et al. (PNAS, 2006,

103:8634-8639), Kelley et al. (J. Biol. Chem., 2005, 280:2205-2215), or MacFarlane et al. (Cancer Res., 2005, 65: 11265-11270) any may comprise any type of structural mutations, e.g., substitution, deletion, duplication and/or insertion of an amino acid. A preferred embodiment is the generation io of substitutions. The substitution may affect at least one amino acid of the cytokine of the TNF superfamily or a receptor binding domain thereof as described herein. In a preferred embodiment, the substitution may affect at least one of the amino acids of TRAIL, e.g., human TRAIL (e.g., SEQ ID NO:

10). Preferred substitutions in this regard affect at least one of the following is amino acids of human TRAIL of SEQ ID NO:10: R130, G160, Y189, R191,

Q193, E195, N199, K201, Y213, T214, S215, H264, I266, D267, D269. Preferred amino acid substitutions of human TRAIL of SEQ ID NO:10 are at least one of the following substitutions: R130E, G160M, Y189A, Y189Q, R191K, Q193S, Q193R, E195R, N199V, N199R, K201R, Y213W, T214R,

S215D, H264R, I266L, D267Q, D269H, D269R, or D269K.

The amino acid substitution(s) may affect the binding and/or activity of TRAIL, e.g., human TRAIL, to or on either the TRAILR1 or the TRAILR2. Alternatively, the amino acid substitution(s) may affect the binding and/or activity of TRAIL, e.g., human TRAIL, to or on both, the TRAILR1 and the TRAILR2. The binding and/or activity of the TRAILR1 and/or TRAILR2 may be affected positively, i.e., stronger, more selective or specific binding and/or more activation of the receptor. Alternatively, the binding and/or activity of the TRAILR1 and/or TRAILR2 may be affected negatively, i.e., weaker, less

3o selective or specific binding and/or less or no activation of the receptor.

Examples of mutants of TRAIL with amino acid substitution(s) that affect binding and/or activity of both TRAILR1 and TRAILR2 may be found, e.g., in

-82017201232 23 Feb 2017

Table 1 of MacFarlane et al. (cf. above) and may comprise human TRAIL mutants with the following two amino acid substitutions of SEQ ID NO: 10 Y213W and S215D or the following single amino acid substitution Y189A.

Examples of mutants of TRAIL with amino acid substitution(s) that affect binding and/or activity of TRAILR1 may be found, e.g., in Table 1 of MacFarlane et al. (cf. above) and may comprise human TRAIL mutants with the following four amino acid substitutions of SEQ ID NO:10 N199V, K201R, Y213W and S215D or the following five amino acid substitutions Q193S, io N199V, K201R, Y213W and S215D or in Table 2 of Kelley et al. (cf. above) and may comprise human TRAIL mutants with the following six amino acid substitutions Y213W, S215D, Y189A, Q193S, N199V, and K201R or

Y213W, S215D, Y189A, Q193S, N199R, and K201R.

is Examples of mutants of TRAIL with amino acid substitution(s) that affect binding and/or activity of TRAILR2 may be found, e.g., in Table 1 of MacFarlane et al. (cf. above) or in Table 2 of Kelley et al. (cf. above) and may comprise human TRAIL mutants with the following six amino acid substitutions of SEQ ID NO: 14 Y189Q, R191K, Q193R, H264R, I266L, and

D267Q or in Table 2 of van der Sloot et al. (cf. above) and may comprise human TRAIL mutants with the following single amino acid substitution D269H, the following two amino acid substitutions D269H and E195R or D269H and T214R.

In a further preferred embodiment, the cytokine portion of the fusion protein is derived from human LIGHT (SEQ ID NO: 16), particularly amino acids 91-240 of SEQ ID NO: 16.

In a still further preferred embodiment, the cytokine portion of the fusion protein is derived from human APRIL (SEQ ID NO: 13 or 14), particularly amino acids 112-247 or 113-247 of SEQ ID NO:13, or 112-250 or 113-250 of SEQ ID NO:14.

-92017201232 23 Feb 2017

A flexible linker element may additionally located between the cytokine of the TNF superfamily or a receptor binding domain thereof (i) and the collectin trimerization domain as described herein (ii). The flexible linker element preferably has a length of 3-20 amino acids, particularly a length of 3, 6, 9,

10, 12, 15 or 18 amino acids. More preferably, the length of the linker is 9-15 amino acids. The linker element is preferably a glycine/serine linker, i.e., a peptide linker substantially consisting of the amino acids glycine and serine. In an especially preferred embodiment, the linker has the amino acid sequence (GSS)_a(SSG)_b(GSG)_c wherein a, b, c is each 0,1,2, 3, 4, 5 or 6. It io is clear to the skilled person that in cases in which the cytokine of the TNF superfamily or a receptor binding domain thereof already terminates with a G, e.g. human TRAIL (SEQ ID NO:10) such a G may form the first G of the linker in the linker sequence (GSS)_a(SSG)_b(GSG)_c.

is The collectin trimerization domain (ii) may comprise any collectin family member. Such members and their structures are summarized in, e.g., Hakansson et al. (Protein Science, 2000, 9:1607-1617) and may comprise surfactant protein-D, surfactant protein-A, mannan-binding protein-A, mannan-binding-protein-C, collectin liver 1, collectin placenta 1, or collectin-11. The collectin trimerization domain as described herein may be from a different species than the cytokine of the TNF superfamily or a receptor binding domain thereof as described herein. Alternatively, the collectin trimerization domain as described herein may be from the same species than the cytokine of the TNF superfamily or a receptor binding domain thereof described herein. In a preferred embodiment, the collectin domain as described herein is from human and the cytokine of the TNF superfamily or a receptor binding domain thereof as described herein is from human. In a preferred embodiment, the collectin trimerization domain comprises the neck and carbohydrate binding domain (CRD) domain of the surfactant protein-D, particularly amino acids 217-375, 218-375, 219-375, 220-375, 221-375, 222-375, 223-375, 224-375, 225-375 from human surfactant protein-D of SEQ ID NO:21. In another preferred embodiment, the collectin trimerization domain comprises the neck domain of the surfactant

-102017201232 23 Feb 2017 protein-D, particularly amino acids 217-257, 218-257, 219-257, 220-257, 221-257, 222-257, 223-257, 224-257, or 225-257 from human surfactant protein-D of SEQ ID NO:21. In another preferred embodiment, the collectin trimerization domain comprises the neck and carbohydrate binding domain (CRD) domain of collectin-11, particularly amino acids 110-271, 116-271, or 121-271 of human collectin-11 of SEQ ID NO:22. In another preferred embodiment, the collectin trimerization domain comprises the neck domain of collectin-11, particularly amino acids 110-147, 110-148, 110-149, 110-150, 110-151, 116-147, 116-148, 116-149, 116-150, 116-151, 121-147, io 121-148, 121-149, 121-150, or 121-151 of human collectin-11 of SEQ ID

NO:22.

The collectin trimerization domain (ii) may comprise a mutant, e.g., a mutant of surfactant protein-D or collectin-11, which does not bind to mannose, is Such mutants may be identified by methods known to the skilled person, e.g., the methods disclosed in Crouch et al. (J Biol Chem, 2006, 281(26):

18008-18014). The collectin trimerization domain (ii) may further comprise a mutant which comprise at least one amino acid substitution as is described herein and may be generated as described herein. Such amino acid substitutions may modify the binding of the collectin trimerization domain to its ligand mannose and lead to an alteration of the clearance rate of a fusion protein as described herein when used in therapy and/or as pharmaceutical composition. The modification may result in a decreased or no binding to mannose and a low clearance rate. Such modifications may be achieved by,

e.g., amino acid substitution that affect amino acid position F355 of human surfactant protein-D of SEQ ID NO:21, particularly by the amino acid substitutions F355A, F355S, F355T, F355E, F355D, F355K, or F355R. Especially preferred is the substitution F355D. Alternatively, the modification may result in an increased binding to mannose and a high clearance rate.

Such modifications may be achieved by, e.g., amino acid substitution that affect amino acid position F355 of human surfactant protein-D of SEQ ID NO:21, particularly by the amino acid substitutions F355L, F355Y, or F355W.

2017201232 23 Feb 2017

- 11 In the fusion protein of the invention as described herein, the collectin trimerization domain (ii) may be located C-terminally of the cytokine of the TNF superfamily or a receptor binding domain thereof (i). Thus, the fusion protein may comprise a cytokine of the TNF superfamily or a receptor binding domain thereof as described herein and a collectin trimerization domain that comprises the neck domain alone or the neck and the CRD domain, e.g., the neck domain and the CRD and/or neck domain of surfactant protein-D or the neck domain and the CRD and/or neck domain of io collectin-11 both as described herein wherein those domains are located Cterminally of the TNF superfamily or a receptor binding domain thereof (i). In this embodiment, it is preferred that the collectin trimerization domain comprises the neck domain and the CRD.

is In the fusion protein of the invention as described herein, the collectin trimerization domain (ii) may be located N-terminally of the cytokine of the TNF superfamily or a receptor binding domain thereof (i). Thus, the fusion protein may comprise a cytokine of the TNF superfamily or a receptor binding domain thereof as described herein and a collectin trimerization domain that comprises the neck domain, e.g., the neck domain of surfactant protein-D or the neck domain of collectin-11 both as described herein wherein those domains are located N-terminally of the TNF superfamily or a receptor binding domain thereof (i).

In a preferred embodiment, the fusion protein comprises TRAIL, particularly human TRAIL or a receptor binding domain thereof or a mutant of TRAIL as described herein, preferably 95-281, 116-281, 117-281, 118-281, 119-281 or 120-281 of human TRAIL (SEQ ID NO;10) and a collectin trimerization domain or mutant thereof as described herein, particularly the CRD and neck domain of surfactant protein-D, preferably amino acids 217-375, 218-375, 219-375, 220-375, 221-375, 222-375, 223-375, 224-375, 225-375 of human surfactant protein-D of SEQ ID NO:21 wherein the collectin trimerization domain is located C-terminally of TRAIL or mutant TRAIL as described

-122017201232 23 Feb 2017 herein. Preferred fusion proteins in this regard are SEQ ID Nos:26 or 27. Alternatively, the above fusion protein may additionally comprise a linker as described herein, e.g., a linker with the amino acid sequence (GSS)a(SSG)b(GSG)_c wherein a, b, c is each 0, 1, 2, 3, 4, 5 or 6. Preferably, the linker has a length of 9-15 amino acids.

In a preferred embodiment, the fusion protein comprises TRAIL, particularly human TRAIL or a receptor binding domain thereof or a mutant of TRAIL as described herein, preferably 95-281, 116-281, 117-281, 118-281, 119-281 or io 120-281 of human TRAIL (SEQ ID ΝΟ.Ί0) and a collectin trimerization domain or mutant thereof as described herein, particularly the neck domain of surfactant protein-D, preferably amino acids 217-257, 218-257, 219-257,

220-257, 221-257, 222-257, 223-257, 224-257, or 225-257 of human surfactant protein-D of SEQ ID NO:21 wherein the collectin trimerization is domain is located C-terminally of TRAIL or mutant TRAIL as described herein. A preferred fusion protein in this regard is SEQ ID NO:28.

Alternatively, the above fusion protein may additionally comprise a linker as described herein, e.g., a linker with the amino acid sequence (GSS)a(SSG)b(GSG)₀ wherein a, b, c is each 0, 1,2, 3, 4, 5 or 6. Preferably, the linker has a length of 9-15 amino acids.

In another preferred embodiment, the fusion protein comprises TRAIL, particularly human TRAIL or a receptor binding domain thereof or a mutant of TRAIL as described herein, preferably 95-281, 116-281, 117-281,

118-281, 119-281 or 120-281 of human TRAIL (SEQ ID NO: 10) and a collectin trimerization domain or mutant thereof as described herein, particularly the CRD and neck domain of collectin-11, preferably amino acids 110-271, 116-271, or 121-271 of human collectin-11 of SEQ ID NO:22 wherein the collectin trimerization domain is located C-terminally of TRAIL or mutant TRAIL as described herein. Preferred fusion proteins in this regard are SEQ ID Nos:29 or 30. Alternatively, the above fusion protein may additionally comprise a linker as described herein, e.g., a linker with the amino acid sequence (GSS)_a(SSG)_b(GSG)_c wherein a, b, c is each 0,1,2, 3,

- 132017201232 23 Feb 2017

4, 5 or 6. Preferably, the linker has a length of 9-15 amino acids.

In another preferred embodiment, the fusion protein comprises TRAIL, particularly human TRAIL or a receptor binding domain thereof or a mutant of TRAIL as described herein, preferably 95-281, 116-281, 117-281, 118-281, 119-281 or 120-281 of human TRAIL (SEQ ID NO: 10) and a collectin trimerization domain or mutant thereof as described herein, particularly the neck domain of collectin-11, preferably amino acids 110-147, 110-148, 110-149, 110-150, 110-151, 116-147, 116-148, 116-149, 116-150, io 116-151, 121-147, 121-148, 121-149, 121-150, or 121-151 of human collectin-11 of SEQ ID NO:22 wherein the collectin trimerization domain is located C-terminally of TRAIL or mutant TRAIL as described herein. A preferred fusion protein in this regard is SEQ ID NO:31. Alternatively, the above fusion protein may additionally comprise a linker as described herein, is e.g., a linker with the amino acid sequence (GSS)_a(SSG)_b(GSG)_c wherein a, b, c is each 0, 1, 2, 3, 4, 5 or 6. Preferably, the linker has a length of 9-15 amino acids. Preferred fusion proteins in this regard are SEQ ID Nos:36 or

37.

In a preferred embodiment, the fusion protein comprises TRAIL, particularly human TRAIL or a receptor binding domain thereof or a mutant of TRAIL as described herein, preferably 95-281, 116-281, 117-281, 118-281, 119-281 or 120-281 of human TRAIL (SEQ ID NO:10) and a collectin trimerization domain or mutant thereof as described herein, particularly the neck domain of surfactant protein-D, preferably amino acids 217-257, 218-257, 219-257, 220-257, 221-257, 222-257, 223-257, 224-257, or 225-257 of human surfactant protein-D of SEQ ID NO:21 wherein the collectin trimerization domain is located N-terminally of TRAIL or mutant TRAIL as described herein. Alternatively, the above fusion protein may additionally comprise a linker as described herein, e.g., a linker with the amino acid sequence (GSS)_a(SSG)b(GSG)c wherein a, b, c is each 0,1,2, 3, 4, 5 or 6. Preferably, the linker has a length of 9-15 amino acids.

-142017201232 23 Feb 2017 ln another preferred embodiment, the fusion protein comprises TRAIL, particularly human TRAIL or a receptor binding domain thereof or a mutant of TRAIL as described herein, preferably 95-281, 116-281, 117-281, 118-281, 119-281 or 120-281 of human TRAIL (SEQ ID NO: 10) and a collectin trimerization domain or mutant thereof as described herein, particularly the neck domain of collectin-11, preferably amino acids 110-147, 110-148, 110-149, 110-150, 110-151, 116-147, 116-148, 116-149, 116-150, 116-151, 121-147, 121-148, 121-149, 121-150, or 121-151 of human collectin-11 of SEQ ID NO:22 wherein the collectin trimerization domain is io located N-terminally of TRAIL or mutant TRAIL as described herein. Preferred fusion proteins in this regard are SEQ ID Nos:32-34. Alternatively, the above fusion protein may additionally comprise a linker as described herein, e.g., a linker with the amino acid sequence (GSS)_a(SSG)_b(GSG)_c wherein a, b, c is each 0, 1, 2, 3, 4, 5 or 6. Preferably, the linker has a length is of 9-15 amino acids. Preferred fusion proteins in this regard is SEQ ID NO:

35.

In another preferred embodiment, the fusion protein comprises CD95L, particularly human CD95L, or a receptor binding domain thereof as described herein, e.g. amino acids 21-160 of SEQ ID NO:40, and a collectin trimerization domain comprising the neck domain and optionally the CRD of human SP-D, e.g. amino acids 172-209 and 210-327 of SEQ ID NO:40, respectively, or a mutant thereof as described herein. Preferably, the fusion protein may comprise a linker, e.g. a flexible linker, more preferably a glycine/serine linker as described herein having a length of preferably 9-15 amino acids. A preferred fusion protein in this regard comprises SEQ ID NO: 40, particularly amino acids 21-327 of SEQ ID NO:40.

In another preferred embodiment, the fusion protein comprises LIGHT, particularly human LIGHT or a receptor binding domain thereof as described herein, preferably amino acids 21-170 of SEQ ID NO:41, and a collectin trimerization domain comprising the neck domain and optionally the CRD of human SP-D, e.g. amino acids 182-219, and 220-337 of SEQ ID NO:41,

2017201232 23 Feb 2017

- 15respectively, or a mutant thereof as described herein. Preferably, the cytokine and the collectin domain are connected by a linker, e.g. a glycine/serine linker as described herein, having a length of preferably 9-15 amino acids. A preferred fusion protein in this regard comprises SEQ ID NO:

41, particularly amino acids 21-327 of SEQ ID NO:41.

In another preferred embodiment, the fusion protein comprises TRAIL, particularly human TRAIL or a receptor binding domain thereof or mutant of TRAIL as described herein, e.g. amino acids 21-181 of SEQ ID NO:43 (wild io type TRAIL), amino acids 21-181 of SEQ ID NO:47 (TRAILRImut) or amino acids 21-181 of SEQ ID NO:48 (TRAILR2mut). Further, the fusion protein comprises a collectin trimerization domain selected from the neck domain and optionally the CRD of human SP-D, e.g. amino acids 193-230, and

231-384 of SEQ ID NO:43, respectively, or a mutant thereof as described is herein, e.g. mutants as shown in SEQ ID NO:49 or 50. Preferably, the fusion polypeptide comprises both the neck region and the CRD of human SP-D.

The cytokine and collectin domain are preferably connected by a linker, e.g. a glycine/serine linker as described herein. Preferably, the linker has a length of 9-15 amino acids. Preferred fusion proteins in this regard comprise (i) SEQ ID NO:43, particularly amino acids 21-348 of SEQ ID NO:43, (ii) SEQ

ID NO:44, particularly amino acids 21-230 of SEQ ID NO:44, (iii) SEQ ID NO: 47, particularly amino acids 21-348 of SEQ ID NO:47, (iv) SEQ ID NO:48, particularly amino acids 21-348 of SEQ ID NO:48, (v) SEQ ID NO: 49, particularly amino acids 21-348 of SEQ ID NO:49 or (vi) SEQ ID NO:50, particularly amino acids 21-348 of SEQ ID NO:50.

In another preferred embodiment, the fusion protein comprises TRAIL, particularly human TRAIL or receptor-binding domain thereof or a mutant of TRAIL as described herein above, and a collectin trimerization domain, which is the neck domain of human collectin 11, and optionally the CRD of human collectin 11, e.g. amino acids 193-224 and 225-347 of SEQ ID NO: 45, respectively. Preferably, the CRD is present. Preferably, the cytokine and the collectin domain are connected by a linker, e.g. a glycine/serine linker as

2017201232 23 Feb 2017

- 16described above herein, preferably having a length of 9-15 amino acids. Preferred fusion proteins in this regard comprise SEQ ID NO:45 and SEQ ID NO:46, particularly, amino acids 21-347 of SEQ ID NO:45 or amino acids 21-229 of SEQ ID NO:46.

In another preferred embodiment, the fusion protein comprises APRIL, particularly human APRIL or a receptor binding domain thereof as described herein, e.g. amino acids 21-158 of SEQ ID NO:51 and a collectin trimerization domain as described herein, particularly the neck domain and io optionally the CRD of human SP-D or a mutant thereof, as described herein, e.g. amino acids 170-207 and 208-325 of SEQ ID NO:51, respectively. The cytokine and the collectin domain are preferably connected by a linker, e.g. a glycine/serine linker as described herein, preferably having a length of 9-15 amino acids. The preferred fusion protein in this regard comprises SEQ ID is NO:51, particularly amino acids 21-325 of SEQ ID NO:51.

The fusion protein as described herein may additionally comprise an Nterminal signal peptide domain, which allows processing, e.g., extracellular secretion, in a suitable host cell. Preferably, the N-terminal signal peptide domain comprises a protease, e.g., a signal peptidase cleavage site and thus may be removed after or during expression to obtain the mature protein. In a preferred embodiment, the N-terminal signal peptide domain comprises the sequence SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25.

Further, the fusion protein may comprise comprises a recognition/purification domain, e.g., a Strep-tag domain and/or a poly-His domain, which may be located at the N-terminus or at the C-terminus.

The fusion protein may additionally comprise a C-terminal flexible element, having a length of, e.g., 1-50, preferably 10-30 amino acids which may include and/or connect to a recognition/purification domain as described herein.

-172017201232 23 Feb 2017

A further aspect of the present invention relates to a nucleic acid molecule encoding a fusion protein as described herein. The nucleic acid molecule may be a DNA molecule, e.g., a double-stranded or single-stranded DNA molecule, or an RNA molecule. The nucleic acid molecule may encode the fusion protein or a precursor thereof, e.g., a pro- or pre-proform of the fusion protein which may comprise a signal sequence as described herein or other heterologous amino acid portions for secretion or purification which are preferably located at the N- and/or C-terminus of the fusion protein as described herein. The nucleic acid molecule may encode the fusion protein io wherein the heterologous amino acid portions may be linked to the first and/ or second domain via a protease cleavage site, e.g., a Factor X_a, thrombin or

IgA protease cleavage site.

Examples of nucleic acids that comprise the coding sequence of a fusion is protein as described herein are SEQ ID Nos:38, 39 or 42.

The nucleic acid molecule may be operatively linked to an expression control sequence, e.g. an expression control sequence which allows expression of the nucleic acid molecule in a desired host cell. The nucleic acid molecule may be located on a vector, e.g. a plasmid, a bacteriophage, a viral vector, a chromosal integration vector, etc. Examples of suitable expression control sequences and vectors are described for example by Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, and Ausubel et al. (1989), Current Protocols in Molecular Biology, John Wiley &

Sons or more recent editions thereof.

Various expression vector/host cell systems may be used to express the nucleic acid sequences encoding the fusion proteins of the present invention. Suitable host cells include, but are not limited to, prokaryotic cells

3o such as bacteria, e.g. E.coli, eukaryotic host cells such as yeast cells, insect cells, plant cells or animal cells, preferably mammalian cells and, more preferably, human cells. The nucleic acid molecule encoding the fusion protein as described herein may be optimized in view of its codon-usage for

-182017201232 23 Feb 2017 the expression in suitable host cells, e.g. E.coli, yeast cells, plant cells, insect cells, animal cells, e.g., mammalian cells or human cells.

Further, the invention relates to a non-human organism, e.g., mouse or rat, transformed or transfected with a nucleic acid molecule as described herein. Such organisms may be comprise knock-out organisms, generated by known methods of genetic transfer including homologous recombination. Alternatively, such organisms may comprise transgenic organisms which comprise several copies of the nucleic acid molecule as described herein.

io The generation of transgenic organisms is known in the art.

The fusion protein, the nucleic acid coding therefore, the transformed or transfected cell as well as the trimeric complexes or oligomers of the trimeric complexes, all as described herein may be used for pharmaceutical, is diagnostic and/or research applications. For these applications it is preferred to use fusion proteins in which both the TNF-superfamily cytokine or receptor binding domain thereof as described herein and the collectin trimerization domain as described herein are from the same species in order to minimize immunological effects, e.g., from human when applying such proteins to humans. In addition, the fusion of a TNF-superfamily cytokine or receptor binding domain thereof as described herein to a neck-collectin trimerization domain as described herein, e.g., neck domain from surfactant protein-D or collectin-11, may lead to fast clearance. Alternatively, the fusion of a TNFsuperfamily cytokine or receptor binding domain thereof as described herein to a neck and CRD-collectin trimerization domain as described herein, e.g., neck and CRD domain from surfactant protein-D or collectin-11, may lead to low clearance. The use of mutants of the collectin trimerization domain as described herein may modify the clearance rate of the fusion protein in a way as described herein.

A further aspect of the present invention relates to a pharmaceutical or diagnostic composition comprising as an active agent at least one fusion protein, the nucleic acid coding therefore, the transformed or transfected cell

2017201232 23 Feb 2017

- 19as well as the trimeric complexes or oligomers of the trimeric complexes, all as described herein.

At least one fusion protein, the nucleic acid coding therefor, the transformed or transfected cell as well as the trimeric complexes or oligomers of the trimeric complexes, all as described herein may be used in therapy, e.g., in the prophylaxis and/or treatment of disorders selected from proliferative disorders, particularly disorders caused by, associated with and/or accompanied by dysfunction of TNF cytokines, such as tumors, e.g. solid or io lymphatic tumors, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune disorders, e.g. rheumatoid and/or arthritic diseases, degenerative diseases, e.g. neurodegenerative diseases such as multiple sclerosis, apoptosis-associated diseases and transplant rejections.

is The composition may be administered as monotherapy or as combination therapy with further medicaments, e.g. cytostatic or chemotherapeutic agents, corticosteroids and/or antibiotics. Preferably, the composition is administered together with tumor-selective apoptosis sensitizing and/or inducing agents, e.g. as described in Example 2.8.

The fusion protein is administered to a subject in need thereof, particularly a human patient, in a sufficient dose for the treatment of the specific conditions by suitable means. For example, the fusion protein may be formulated as a pharmaceutical composition together with pharmaceutically acceptable carriers, diluents and/or adjuvants. Therapeutic efficacy and toxicity may be determined according to standard protocols. The pharmaceutical composition may be administered systemically, e.g. intraperitoneally, intramuscularly or intravenously or locally, e.g. intranasally, subcutaneously or intrathecally. Preferred is intravenous administration.

The dose of the fusion protein administered will of course be dependent on the subject to be treated, on the subject's weight, the type and severity of the disease, the manner of administration and the judgement of the prescribing

-202017201232 23 Feb 2017 physician. For the administration of fusion proteins, a daily dose of 0.001 to 100 mg/kg is suitable.

Table 1 shows a list of cytokines of the TNF super family which may be used 5 in the present invention.

2017201232 23 Feb 2017

Table 1

In a different aspect, the present invention refers to novel amino acid

-222017201232 23 Feb 2017 substitution variants of human surfactant protein-D (SP-D) comprising a carbohydrate recognition domain with reduced carbohydrate binding capacity, optionally fused to at least one heterologous polypeptide or polypeptide domain as well as nucleic acid molecules encoding such fusion polypeptides. Preferably, the mutated SP-D polypeptides of the present invention have an amino acid substitutions at position F355 of human surfactant protein-D of SEQ ID NO:21, particularly an amino acid substitution by hydrophilic or charged amino acid, e.g. F355S, F355T, F355E, F355D, F355H or F355R, particularly F355D. The heterologous polypeptide or io polypeptide domain is preferably of mammalian, e.g. human origin, e.g. a TNSF cytokine domain as described above. The mutated SP-D polypeptides preferably comprise an SP-D neck domain as described above. The heterologous polypeptide may be fused to N- and/or C-terminus of the SP-D domain. Preferably, a linker, e.g. a linker as described herein above, is is present between the SP-D and heterologous polypeptide domain.

Basic Structure of a Fusion Protein

In the following, the basic structure of the recombinant proteins of the invention is shown exemplified for the TNF-superfamily cytokines as described herein.

1.1 Sequences of the Signal Peptides MNFGFSLIFLVLVLKGVQC (SEQ ID NO :23)

METDTLLLWVLLLWVPGSTG (SEQ ID NO :24)

METDTLLLWVLLLWVPAGNG (SEQ ID NO:25)

1.2 Flaq-epitope/enterokinase-processinq site

DYKDDDDKD

1.3 Human Collectins

Surfactant Protein-D (SEQ ID NO:21)

-232017201232 23 Feb 2017

MLLFLLSALV LLTQPLGYLE AEMKTYSHRT TPSACTLVMC SSVESGLPGR

DGRDGREGPR

GEKGDPGLPG AAGQAGMPGQ AGPVGPKGDN GSVGEPGPKG DTGPSGPPGP

PGVPGPAGRE

121 GPLGKQGNIG PQGKPGPKGE AGPKGEVGAP GMQGSAGARG LAGPKGERGV

PGERGVPGNA

181 GAAGSAGAMG PQGSPGARGP PGLKGDKGIP GDKGAKGESG LPDVASLRQQ

VEALQGQVQH

241 LQAAFSQYKK VELFPNGQSV GEKIFKTAGF VKPFTEAQLL CTQAGGQLAS

PRSAAENAAL

301 QQLWAKNEA AFLSMTDSKT EGKFTYPTGE SLVYSNWAPG EPNDDGGSED

CVEIFTNGKW

361 NDRACGEKRL WCEF is Collectin-11 (SEQ ID NO:22)

MRGNLALVGV LISLAFLSLL PSGHPQPAGD DACSVQILVP GLKGDAGEKG

DKGAPGRPGR

VGPTGEKGDM GDKGQKGSVG RHGKIGPIGS KGEKGDSGDI GPPGPNGEPG

LPCECSQLRK

121 AIGEMDNQVS QLTSELKFIK NAVAGVRETE SKIYLLVKEE KRYADAQLSC

QGRGGTLSMP

181 KDEAANGLMA AYLAQAGLAR VFIGINDLEK EGAFVYSDHS PMRTFNKWRS

GEPNNAYDEE

241 DCVEMVASGG WNDVACHTTM YFMCEFDKEN M

Various fragments of the human collectins Surfactant protein-D and collectin-11 are conceivable as trimerization domains as described herein.

1.4 Flexible Linker Element (GSS)_a(SSG)_b (GSG)_c wherein a, b, c is each 0, 1, 2, 3, 4, 5 or 6

1.5 TNF-Superfamily Cytokine/ Receptor Binding Domain thereof (see also

Table 1)

SEQ-ID-01

SEQ NP_000586_TNFSFl_LTA

KEYWORD PROTEIN

FEATURES

ORIGIN

MTPPERLFLP RVCGTTLHLL LLGLLLVLLP GAQGLPGVGL TPSAAQTARQ

HPKMHLAHST

61 LKPAAHLIGD PSKQNSLLWR ANTDRAFLQD GFSLSNNSLL VPTSGIYFVY

SQWFSGKAY

-242017201232 23 Feb 2017

121 SPKATSSPLY LAHEVQLFSS QYPFHVPLLS SQKMVYPGLQ EPWLHSMYHG

AAFQLTQGDQ

181 LSTHTDGIPH LVLSPSTVFF GAFAL

SEQ-ID-02

SEQ NP_000585_TNFSF2_TNFa

KEYWORD PROTEIN

ORIGIN

MSTESMIRDV ELAEEALPKK

LHFGVIGPQR

EEFPRDLSLI SPLAQAVRSS

ALLANGVELR

121 DNQLWPSEG LYLIYSQVLF

SAIKSPCQRE

181 TPEGAEAKPW YEPIYLGGVF

TGGPQGSRRC LFLSLFSFLI VAGATTLFCL

SRTPSDKPVA HWANPQAEG QLQWLNRRAN

KGQGCPSTHV LLTHTISRIA VSYQTKVNLL

QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL

SEQ-ID-03

SEQ NP_002332_TNFSF3_LTB

KEYWORD PROTEIN

ORIGIN

MGALGLEGRG GRLQGRGSLL LAVAGATSLV TLLLAVPITV LAVLALVPQD

QGGLVTETAD

PGAQAQQGLG FQKLPEEEPE TDLSPGLPAA HLIGAPLKGQ GLGWETTKEQ

AFLTSGTQFS

121 DAEGLALPQD GLYYLYCLVG YRGRAPPGGG DPQGRSVTLR SSLYRAGGAY

GPGTPELLLE

181 GAETVTPVLD PARRQGYGPL WYTSVGFGGL VQLRRGERVY VNISHPDMVD

FARGKTFFGA

241 VMVG

SEQ-ID-04

SEQ NP_003317_TNFSF4_0X40L

KEYWORD PROTEIN

ORIGIN

MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSAL

QVSHRYPRIQ

SIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFS

QEVNISLHYQ

121 KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGEL

ILIHQNPGEF

181 CVL

SEQ-ID-05

SEQ NP_000065_TNFSF5_CD40L

KEYWORD PROTEIN

ORIGIN

-252017201232 23 Feb 2017

MIETYNQTSP RSAATGLPIS MKIFMYLLTV FLITQMIGSA LFAVYLHRRL

DKIEDERNLH

EDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ FEGFVKDIML NKEETKKENS

FEMQKGDQNP

121 QIAAHVISEA SSKTTSVLQW AEKGYYTMSN NLVTLENGKQ LTVKRQGLYY

IYAQVTFCSN

181 REASSQAPFI ASLCLKSPGR FERILLRAAN THSSAKPCGQ QSIHLGGVFE

LQPGASVFVN

241 VTDPSQVSHG TGFTSFGLLK L

SEQ-ID-06

SEQ

KEYWORD

NP_000630_TNFSF6_CD95L

PROTEIN

ORIGIN

MQQPFNYPYP QIYWVDSSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPP

PPPLPPPPPP

61 PPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG MFQLFHLQKE

LAELRESTSQ

121 MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL

LSGVKYKKGG

181 LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM

SYCTTGQMWA

241 RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L

SEQ-ID-07

SEQ NP_001243_TNFSF7_CD27L

KEYWORD PROTEIN

ORIGIN

1 MPEEGSGCSV RRRPYGCVLR AALVPLVAGL VICLWCIQR FAQAQQQLPL

ESLGWDVAEL

QLNHTGPQQD PRLYWQGGPA LGRSFLHGPE LDKGQLRIHR DGIYMVHIQV

TLAICSSTTA

121 SRHHPTTLAV GICSPASRSI SLLRLSFHQG CTIASQRLTP LARGDTLCTN

LTGTLLPSRN

181 TDETFFGVQW VRP

SEQ-ID-08

SEQ NP_001235_TNFSF8_CD30L

KEYWORD PROTEIN

ORIGIN

1 MDPGLQQALN GMAPPGDTAM HVPAGSVASH LGTTSRSYFY LTTATLALCL

VFTVATIMVL

WQRTDSIPN SPDNVPLKGG NCSEDLLCIL KRAPFKKSWA YLQVAKHLNK

TKLSWNKDGI

121 LHGVRYQDGN LVIQFPGLYF IICQLQFLVQ CPNNSVDLKL ELLINKHIKK

QALVTVCESG

181 MQTKHVYQNL SQFLLDYLQV NTTISVNVDT FQYIDTSTFP LENVLSIFLY SNSD

-262017201232 23 Feb 2017

SEQ-ID-09

SEQ NP_003802_TNFSF9_CD137L

KEYWORD PROTEIN

ORIGIN

MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA

CPWAVSGARA

SPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY

SDPGLAGVSL

121 TGGLSYKEDT KELWAKAGV YYVFFQLELR RWAGEGSGS VSLALHLQPL

RSAAGAAALA

181 LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ

GATVLGLFRV

241 TPEIPAGLPS PRSE

SEQ-ID-10

SEQ NP_003801_TNFSF10_TRAIL

KEYWORD PROTEIN

ORIGIN

MAMMEVQGGP SLGQTCVLIV

KSGIACFLKE

DDSYWDPNDE ESMNSPCWQV

SPLVRERGPQ

121 RVAAHITGTR GRSNTLSSPN

NGELVIHEKG

181 FYYIYSQTYF RFQEEIKENT

WSKDAEYGLY

241 SIYQGGIFEL KENDRIFVSV

IFTVLLQSLC VAVTYVYFTN ELKQMQDKYS

KWQLRQLVRK MILRTSEETI STVQEKQQNI

SKNEKALGRK INSWESSRSG HSFLSNLHLR

KNDKQMVQYI YKYTSYPDPI LLMKSARNSC

TNEHLIDMDH EASFFGAFLV G

SEQ-ID-11

SEQ NP_003692_TNFSFll_a_RANKL

KEYWORD PROTEIN

ORIGIN

MRRASRDYTK YLRGSEEMGG GPGAPHEGPL HAPPPPAPHQ PPAASRSMFV

ALLGLGLGQV

VCSVALFFYF RAQMDPNRIS EDGTHCIYRI LRLHENADFQ DTTLESQDTK

LIPDSCRRIK

121 QAFQGAVQKE LQHIVGSQHI RAEKAMVDGS WLDLAKRSKL EAQPFAHLTI

NATDIPSGSH

181 KVSLSSWYHD RGWAKISNMT FSNGKLIVNQ DGFYYLYANI CFRHHETSGD

LATEYLQLMV

241 YVTKTSIKIP SSHTLMKGGS TKYWSGNSEF HFYSINVGGF FKLRSGEEIS

IEVSNPSLLD

301 PDQDATYFGA FKVRDID

SEQ-ID-12

SEQ NP_003800_TNFSF12_TWEAK

KEYWORD PROTEIN

ORIGIN

-272017201232 23 Feb 2017

MAARRSQRRR GRRGEPGTAL LVPLALGLGL ALACLGLLLA WSLGSRASL

SAQEPAQEEL

VAEEDQDPSE LNPQTEESQD PAPFLNRLVR PRRSAPKGRK TRARRAIAAH

YEVHPRPGQD

121 GAQAGVDGTV SGWEEARINS SSPLRYNRQI GEFIVTRAGL YYLYCQVHFD

EGKAVYLKLD

181 LLVDGVLALR CLEEFSATAA SSLGPQLRLC QVSGLLALRP GSSLRIRTLP

WAHLKAAPFL

241 TYFGLFQVH

SEQ-ID-13

SEQ NP_742085_TNFSF13_APRIL_verl

KEYWORD PROTEIN

ORIGIN

MPASSPFLLA PKGPPGNMGG PVREPALSVA

QQTELQSLRR

EVSRLQGTGG PSQNGEGYPW QSLPEQSSDA

QKKQHSVLHL

121 VPINATSKDD SDVTEVMWQP ALRRGRGLQA

FQDVTFTMGQ

181 WSREGQGRQ ETLFRCIRSM PSHPDRAYNS

RARAKLNLSP

241 HGTFLGL

LWLSWGAALG AVACAMALLT

LEAWENGERS RKRRAVLTQK

QGYGVRIQDA GVYLLYSQVL

CYSAGVFHLH QGDILSVIIP

SEQ-ID-14

SEQ NP_003799_TNFSF13_APRIL_ver2

KEYWORD PROTEIN

ORIGIN

MPASSPFLLA PKGPPGNMGG PVREPALSVA LWLSWGAALG AVACAMALLT

QQTELQSLRR

EVSRLQGTGG PSQNGEGYPW QSLPEQSSDA LEAWENGERS RKRRAVLTQK

QKKQHSVLHL

121 VPINATSKDD SDVTEVMWQP ALRRGRGLQA QGYGVRIQDA GVYLLYSQVL

FQDVTFTMGQ

181 WSREGQGRQ ETLFRCIRSM PSHPDRAYNS CYSAGVFHLH QGDILSVIIP

RARAKLNLSP 241 HGTFLGFVKL

SEQ-ID-15

SEQ NP_006564_TNFSF13b_BAFF

KEYWORD PROTEIN

ORIGIN

MDDSTEREQS RLTSCLKKRE EMKLKECVSI LPRKESPSVR SSKDGKLLAA

TLLLALLSCC

LTWSFYQVA ALQGDLASLR AELQGHHAEK LPAGAGAPKA GLEEAPAVTA

GLKIFEPPAP

121 GEGNSSQNSR NKRAVQGPEE TVTQDCLQLI ADSETPTIQK GSYTFVPWLL

SFKRGSALEE

181 KENKILVKET GYFFIYGQVL YTDKTYAMGH LIQRKKVHVF GDELSLVTLF

RCIQNMPETL

-282017201232 23 Feb 2017

241

PNNSCYSAGI AKLEEGDELQ LAIPRENAQI

SEQ-ID-16

SEQ NP_003798_TNFSF14_LIGHT

KEYWORD PROTEIN

SLDGDVTFFG ALKLL

ORIGIN

1 MEESWRPSV FWDGQTDIP FTRLGRSHRR QSCSVARVGL GLLLLLMGAG

LAVQGWFLLQ

LHWRLGEMVT RLPDGPAGSW EQLIQERRSH EVNPAAHLTG ANSSLTGSGG

PLLWETQLGL

121 AFLRGLSYHD GALWTKAGY YYIYSKVQLG GVGCPLGLAS TITHGLYKRT

PRYPEELELL

181 VSQQSPCGRA TSSSRVWWDS SFLGGWHLE AGEKVWRVL DERLVRLRDG

TRSYFGAFMV

SEQ-ID-17

SEQ NP_OO51O9_TNFSF15_TL1A

KEYWORD PROTEIN

ORIGIN

MAEDLGLSFG ETASVEMLPE HGSCRPKARS SSARWALTCC LVLLPFLAGL

TTYLLVSQLR

AQGEACVQFQ ALKGQEFAPS HQQVYAPLRA DGDKPRAHLT WRQTPTQHF

KNQFPALHWE

121 HELGLAFTKN RMNYTNKFLL IPESGDYFIY SQVTFRGMTS ECSEIRQAGR

PNKPDSITW

181 ITKVTDSYPE PTQLLMGTKS VCEVGSNWFQ PIYLGAMFSL QEGDKLMVNV

SDISLVDYTK

241 EDKTFFGAFL L

SEQ-ID-18

SEQ NP_005083_TNFSF18_GITRL

KEYWORD PROTEIN

ORIGIN

MCLSHLENMP LSHSRTQGAQ RSSWKLWLFC SIVMLLFLCS FSWLIFIFLQ

LETAKEPCMA

61 KFGPLPSKWQ MASSEPPCVN KVSDWKLEIL QNGLYLIYGQ VAPNANYNDV

APFEVRLYKN

121 KDMIQTLTNK SKIQNVGGTY ELHVGDTIDL IFNSEHQVLK NNTYWGIILL

ANPQFIS

SEQ-ID-19

SEQ

KEYWORD ORIGIN 1

NP_001390_EDA-A1

PROTEIN

MGYPEVERRE LLPAAAPRER

LALHLLTLCC

YLELRSELRR ERGAESRLGG

GSQGCGCGGA PARAGEGNSC LLFLGFFGLS

SGTPGTSGTL SSLGGLDPDS PITSHLGQPS

-292017201232 23 Feb 2017

PKQQPLEPGE
	121	AALHSDSQDG	HQMALLNFFF
	KNKKKGKKAG
	181	PPGPNGPPGP	PGPPGPQGPP
5	LQGPSGAADK
	241	AGTRENQPAV	VHLQGQGSAI

GELEVLVDGT

301	YFIYSQVEVY	YINFTDFASY
VCLLKARQKI
361	AVKMVHADIS	INMSKHTTFF

PDEKPYSEEE SRRVRRNKRS KSNEGADGPV

GIPGIPGIPG TTVMGPPGPP GPPGPQGPPG

QVKNDLSGGV LNDWSRITMN PKVFKLHPRS

EVWDEKPFL QCTRSIETGK TNYNTCYTAG

GAIRLGEAPA S

SEQ-ID-20

SEQ NP_001005609_EDA-A2

KEYWORD PROTEIN

ORIGIN

MGYPEVERRE LLPAAAPRER GSQGCGCGGA PARAGEGNSC LLFLGFFGLS

LALHLLTLCC

YLELRSELRR ERGAESRLGG SGTPGTSGTL SSLGGLDPDS PITSHLGQPS

PKQQPLEPGE

121 AALHSDSQDG HQMALLNFFF PDEKPYSEEE SRRVRRNKRS KSNEGADGPV

KNKKKGKKAG

181 PPGPNGPPGP PGPPGPQGPP GIPGIPGIPG TTVMGPPGPP GPPGPQGPPG

LQGPSGAADK

241 AGTRENQPAV VHLQGQGSAI QVKNDLSGGV LNDWSRITMN PKVFKLHPRS

GELEVLVDGT

301 YFIYSQVYYI NFTDFASYEV WDEKPFLQC TRSIETGKTN YNTCYTAGVC

LLKARQKIAV

361 KMVHADISIN MSKHTTFFGA IRLGEAPAS

Various fragments, e.g., receptor binding domains, of TNF-superfamily cytokines are conceivable as described herein.

1.6 Examples of Fusion Proteins

SEQ ID NO:26 SP-hsTrailsyn-SPD-Konstrukt-l_PRO.PRO

KEYWORD PROTEIN

ORIGIN

METDTLLLWV LLLWVPAGNG QRVAAHITGT RGRSNTLSSP NSKNEKALGR

KINSWESSRS

61 GHSFLSNLHL RNGELVIHEK GFYYIYSQTY FRFQEEIKEN TKNDKQMVQY

IYKYTSYPDP

121 ILLMKSARNS CWSKDAEYGL YSIYQGGIFE LKENDRIFVS VTNEHLIDMD

HEASFFGAFL

181 VGSGLPDVAS LRQQVEALQG QVQHLQAAFS QYKKVELFPN GOSVGEKIFK

TAGFVKPFTE

241 AQLLCTQAGG QLASPRSAAE NAALQQLWA KNEAAFLSMT DSKTEGKFTY

PTGESLVYSN

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301 WAPGEPNDDG GSEDCVEIFT NGKWNDRACG EKRLWCEF

SEQ ID NO:27 SP-hsTrailsyn-SPD-Konstrukt-2_PRO. PRO

KEYWORD PROTEIN

ORIGIN

METDTLLLWV LLLWVPGSTG ERGPQRVAAH ITGTRGRSNT LSSPNSKNEK

ALGRKINSWE

61 SSRSGHSFLS NLHLRNGELV IHEKGFYYIY SQTYFRFQEE IKENTKNDKQ

MVQYIYKYTS

121 YPDPILLMKS ARNSCWSKDA EYGLYSIYQG GIFELKENDR IFVSVTNEHL

IDMDHEASFF

181 GAFLVGSGLP DVASLRQQVE ALQGQVQHLQ AAFSQYKKVE LFPNGOSVGE

KIFKTAGFVK

241 PFTEAQLLCT QAGGQLASPR SAAENAALQQ LWAKNEAAF LSMTDSKTEG

KFTYPTGESL

301 VYSNWAPGEP NDDGGSEDCV EIFTNGKWND RACGEKRLW CEF

SEQ ID NO:28 ORIGIN

METDTLLLWV LLLWVPGSTG ERGPQRVAAH ITGTRGRSNT LSSPNSKNEK

ALGRKINSWE

SSRSGHSFLS NLHLRNGELV IHEKGFYYIY SQTYFRFQEE IKENTKNDKQ

MVQYIYKYTS

121 YPDPILLMKS ARNSCWSKDA EYGLYSIYQG GIFELKENDR IFVSVTNEHL

IDMDHEASFF

181 GAFLVGSGLP DVASLRQQVE ALQGQVQHLQ AAFSQYKKVE LFPNG

SEQ ID NO :29 SP-hsTrailsyn-collll-Konstrukt-1. pro

KEYWORD PROTEIN

ORIGIN

METDTLLLWV LLLWVPAGNG QRVAAHITGT RGRSNTLSSP NSKNEKALGR

KINSWESSRS

GHSFLSNLHL RNGELVIHEK GFYYIYSQTY FRFQEEIKEN TKNDKQMVQY

IYKYTSYPDP

121 ILLMKSARNS CWSKDAEYGL YSIYQGGIFE LKENDRIFVS VTNEHLIDMD

HEASFFGAFL

181 VGSQLRKAIG EMDNQVSQLT SELKFIKNAV AGVRETESKI YLLVKEEKRY

ADAQLSCQGR

241 GGTLSMPKDE AANGLMAAYL AQAGLARVFI GINDLEKEGA FVYSDHSPMR

TFNKWRSGEP

301 NNAYDEEDCV EMVASGGWND VACHTTMYFM CEFDKENM

SEQ ID NO:30 SP-hsTrailsyn-coll-ll-Konstrukt-2. pro

KEYWORD PROTEIN

ORIGIN

1 METDTLLLWV LLLWVPGSTG ERGPQRVAAH ITGTRGRSNT LSSPNSKNEK

ALGRKINSWE

-31 2017201232 23 Feb 2017

SSRSGHSFLS NLHLRNGELV IHEKGFYYIY SQTYFRFQEE IKENTKNDKQ

MVQYIYKYTS

121 YPDPILLMKS ARNSCWSKDA EYGLYSIYQG GIFELKENDR IFVSVTNEHL

IDMDHEASFF

181 GAFLVGSQLR KAIGEMDNQV SQLTSELKFI KNAVAGVRET ESKIYLLVKE

EKRYADAQLS

241 CQGRGGTLSM PKDEAANGLM AAYLAQAGLA RVFIGINDLE KEGAFVYSDH

SPMRTFNKWR

301 SGEPNNAYDE EDCVEMVASG GWNDVACHTT MYFMCEFDKE NM

	SEQ ID NO:31 SP-hsTrailsyn-coll-ll-Konstrukt-3.pro
KEYWORD	PROTEIN
15	ORIGIN 1	METDTLLLWV LLLWVPGSTG ERGPQRVAAH ITGTRGRSNT LSSPNSKNEK
	61	ALGRKINSWE SSRSGHSFLS NLHLRNGELV IHEKGFYYIY SQTYFRFQEE IKENTKNDKQ
	121	MVQYIYKYTS YPDPILLMKS ARNSCWSKDA EYGLYSIYQG GIFELKENDR IFVSVTNEHL
20	181	IDMDHEASFF GAFLVGSQLR KAIGEMDNQV SOLTSELKFI KNAVAGVRET ES

SEQ ID N0:32 FLAG-hColll-hTRAIL_Glull6_Gly281.pro

KEYWORD PROTEIN

ORIGIN

MNFGFSLIFL VLVLKGVQCD YKDDDDKGLP CECSQLRKAI GEMDNQVSQL

TSELKFIKNA

VAGVRETESE RGPQRVAAHI TGTRGRSNTL SSPNSKNEKA LGRKINSWES

SRSGHSFLSN

121 LHLRNGELVI HEKGFYYIYS QTYFRFQEEI KENTKNDKQM VQYIYKYTSY

PDPILLMKSA

181 RNSCWSKDAE YGLYSIYQGG IFELKENDRI FVSVTNEHLI DMDHEASFFG

AFLVG

SEQ ID NO:33 FLAG-hCollls-hTRAIL_Glull6_Gly281.pro

KEYWORD PROTEIN

ORIGIN

MNFGFSLIFL VLVLKGVQCD YKDDDDKGLP CECSQLRKAI GEMDNQVSQL

TSELKFIKNA

61 VAGVRETERG PQRVAAHITG TRGRSNTLSS PNSKNEKALG RKINSWESSR

SGHSFLSNLH

121 LRNGELVIHE KGFYYIYSQT YFRFQEEIKE NTKNDKQMVQ YIYKYTSYPD

PILLMKSARN

181_SCWSKDAEYG LYSIYQGGIF ELKENDRIFV SVTNEHLIDM DHEASFFGAF LVG

SEQ ID NO:34 hCollls-hTRAIL_Glull6_Gly281.pro

KEYWORD PROTEIN

ORIGIN

MNFGFSLIFL VLVLKGVQCG LPCECSQLRK AIGEMDNQVS QLTSELKFIK

NAVAGVRETE

RGPQRVAAHI TGTRGRSNTL SSPNSKNEKA LGRKINSWES SRSGHSFLSN

LHLRNGELVI

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121 HEKGFYYIYS QTYFRFQEEI KENTKNDKQM VQYIYKYTSY PDPILLMKSA

RNSCWSKDAE

181_YGLYSIYQGG IFELKENDRI FVSVTNEHLI DMDHEASFFG AFLVG

SEQ ID NO:35 FLAG-hColll-GSS-hTRAIL_Glull6_Gly281.pro

KEYWORD PROTEIN

ORIGIN

MNFGFSLIFL VLVLKGVQCD YKDDDDKGLP CECSQLRKAI GEMDNQVSQL

TSELKFIKNA

61 VAGVRETESG SSGSSGSSGS GERGPQRVAA HITGTRGRSN TLSSPNSKNE

KALGRKINSW

121 ESSRSGHSFL SNLHLRNGEL VIHEKGFYYI YSQTYFRFQE EIKENTKNDK

QMVQYIYKYT

181 SYPDPILLMK SARNSCWSKD AEYGLYSIYQ GGIFELKEND RIFVSVTNEH

LIDMDHEASF

241 FGAFLVG

SEQ ID NO:36 Spl-hTRAIL_Glull6_Gly281-GSS-collll.pro

KEYWORD PROTEIN

ORIGIN

MNFGFSLIFL VLVLKGVQCE RGPQRVAAHI TGTRGRSNTL SSPNSKNEKA

LGRKINSWES

SRSGHSFLSN LHLRNGELVI HEKGFYYIYS QTYFRFQEEI KENTKNDKQM

VQYIYKYTSY

121 PDPILLMKSA RNSCWSKDAE YGLYSIYQGG IFELKENDRI FVSVTNEHLI

DMDHEASFFG

181 AFLVGSSGSS GSSGSGLPCE CSQLRKAIGE MDNQVSQLTS ELKFIKNAVA

GVRETES

SEQ ID NO:37 Sp3-hTRAIL_Glull6_Gly281-GSS-collll.pro

KEYWORD PROTEIN

ORIGIN

METDTLLLWV LLLWVPAGNG ERGPQRVAAH ITGTRGRSNT LSSPNSKNEK

ALGRKINSWE

SSRSGHSFLS NLHLRNGELV IHEKGFYYIY SQTYFRFQEE IKENTKNDKQ

MVQYIYKYTS

121 YPDPILLMKS ARNSCWSKDA EYGLYSIYQG GIFELKENDR IFVSVTNEHL

IDMDHEASFF

181 GAFLVGSSGS SGSSGSGLPC ECSQLRKAIG EMDNQVSQLT SELKFIKNAV

AGVRETES

SEQ ID NO:38 SP-hsTrailsyn-SPD-Konstrukt-l_DNA.seq: 1045 bp KEYWORD DNA (DNA coding sequence corresponding to SEQ ID NO :26 starts at base position 16)

ORIGIN

AAGCTTGCCG CCACCATGGA GACCGATACA CTGCTCTTGT GGGTGCTCTT

GCTGTGGGTT

61 CCTGCAGGTA ATGGTCAAAG AGTCGCAGCT CACATCACTG GGACTAGAGG

CAGGAGTAAC

121 ACCCTGAGTT CTCCCAATTC CAAGAACGAG AAAGCCCTGG GTAGGAAGAT

CAACTCCTGG

181 GAAAGCTCCA GAAGCGGCCA TAGCTTTCTT AGCAACCTCC ACTTGAGGAA

-332017201232 23 Feb 2017

TGGCGAACTT

241 GTGATCCATG AGAAGGGCTT CTACTACATC TACAGCCAGA CGTACTTCAG

GTTCCAGGAG

301 GAAATCAAGG AGAACACCAA GAACGACAAG CAGATGGTGC AATACATCTA

CAAGTACACG

361 TCATACCCTG ATCCTATACT GCTGATGAAG TCCGCCAGAA ACAGTTGCTG

GAGCAAAGAC

421 GCTGAATACG GCCTGTATTC CATCTATCAG GGCGGTATCT TTGAACTCAA

GGAGAACGAC

481 AGGATCTTCG TGTCTGTGAC AAACGAGCAT CTGATCGACA TGGACCATGA

AGCGTCTTTC

541 TTCGGTGCCT TCTTGGTGGG ATCCGGTTTG CCAGATGTTG CTTCTTTGAG

ACAACAGGTT

601 GAGGCTTTGC AGGGTCAAGT CCAGCACTTG CAGGCTGCTT TCTCTCAATA

CAAGAAGGTT

661 GAGTTGTTCC CAAATGGTCA ATCTGTTGGC GAAAAGATTT TCAAGACTGC

TGGTTTCGTC

721 AAACCATTCA CGGAGGCACA ATTATTGTGT ACTCAGGCTG GTGGACAGTT

GGCCTCTCCA

781 CGTTCTGCCG CTGAGAACGC CGCCTTGCAA CAATTAGTCG TAGCTAAGAA

CGAGGCTGCT

841 TTCTTGAGCA TGACTGATTC CAAGACAGAG GGCAAGTTCA CCTACCCAAC

AGGAGAATCC

901 TTGGTCTATT CTAATTGGGC ACCTGGAGAG CCCAACGATG ATGGCGGCTC

AGAGGACTGT

961 GTGGAAATCT TCACCAATGG CAAGTGGAAT GACAGAGCTT GTGGAGAGAA

GCGTTTGGTG

1021_GTCTGTGAGT TCTAATAGCG GCCGC

SEQ ID NO:39 SP-hsTrailsyn-SPD-Konstrukt-2_DNA.seq: 1057 bp

KEYWORD DNA (DNA coding sequence corresponding to SEQ ID NO:27 starts at base position 16)

ORIGIN

1 AAGCTTGCCG CCACCATGGA GACCGATACA CTGCTCTTGT GGGTACTCTT

GCTGTGGGTT

CCGGGATCTA CCGGTGAACG TGGTCCTCAA AGAGTCGCAG CTCACATCAC

TGGGACTAGA

121 GGCAGGAGTA ACACCCTGAG TTCTCCCAAT TCCAAGAACG AGAAAGCCCT

GGGTAGGAAG

181 ATCAACTCCT GGGAAAGCTC CAGAAGCGGC CATAGCTTTC TTAGCAACCT

CCACTTGAGG

241 AATGGCGAAC TTGTGATCCA TGAGAAGGGC TTCTACTACA TCTACAGCCA

GACGTACTTC

301 AGGTTCCAGG AGGAAATCAA GGAGAACACC AAGAACGACA AGCAGATGGT

GCAATACATC

361 TACAAGTACA CGTCATACCC TGATCCTATA CTGCTGATGA AGTCCGCCAG

AAACAGTTGC

421 TGGAGCAAAG ACGCTGAATA CGGCCTGTAT TCCATCTATC AGGGCGGTAT

CTTTGAACTC

481 AAGGAGAACG ACAGGATCTT CGTGTCTGTG ACAAACGAGC ATCTGATCGA

CATGGACCAT

541 GAAGCGTCTT TCTTCGGTGC CTTCTTGGTG GGATCCGGTT TGCCAGATGT

TGCTTCTTTG

601 AGACAACAGG TTGAGGCTTT GCAGGGTCAA GTCCAGCACT TGCAGGCTGC

TTTCTCTCAA

-342017201232 23 Feb 2017

661 TACAAGAAGG TTGAGTTGTT CCCAAATGGT CAATCTGTTG GCGAAAAGAT

TTTCAAGACT

721 GCTGGTTTCG TCAAACCATT CACGGAGGCA CAATTATTGT GTACTCAGGC

TGGTGGACAG

781 TTGGCCTCTC CACGTTCTGC CGCTGAGAAC GCCGCCTTGC AACAATTAGT

CGTAGCTAAG

841 AACGAGGCTG CTTTCTTGAG CATGACTGAT TCCAAGACAG AGGGCAAGTT

CACCTACCCA

901 ACAGGAGAAT CCTTGGTCTA TTCTAATTGG GCACCTGGAG AGCCCAACGA

TGATGGCGGC

961 TCAGAGGACT GTGTGGAAAT CTTCACCAAT GGCAAGTGGA ATGACAGAGC

TTGTGGAGAG

1021 AAGCGTTTGG TGGTCTGTGA GTTCTAATAG CGGCCGC is Examples

1. Materials and methods

1.1 Construction of TNF-SF-proteins stabilised by a C-terminal 20 positioned Collectin derived trimerization domain

The trimerization motifs (Tables 2 and 3) derived from human Collectin-11 (Col11), the “coiled coil” of Collectin-11 (CC11), human pulmonary surfactant protein-D (SP-D), the coiled coil” of SP-D (CCSPD) were fused C-terminally to the human receptor binding domain (RBD) of CD95L (“CD95L-RBD”; Glu142-Leu281), human TRAIL-RBD (Gln120-Gly281), human LIGHT-RBD (Glu91-Val240) and human APRIL-RBD (Lys113-Leu250), respectively.

Trimerization motif	Amino acids of the unprocessed wt sequences used for motif construction	Swiss-Prot entry
SPD	220-375	P35247
SPD F335A	220 - 375; Phe355 -> Ala355	P35247
SPD F335D	220 - 375; Phe355 -> Asp355	P35247
CCSPD	220 - 257	P35247
Col11	117-271	Q9BWP8
CC11	116-151	Q9BWP8

Table 2: List of the used regions from wild type (wt) sequences for the construction of trimerizing motifs.

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T rimerization motif	Explanation
SPD	human Surfactant protein-D (coiled-coiled “neck“ + Carbohydrate Recognition Domain, CRD)
SPD_F335A	as in 1, but with the mutation Phe -> Ala at position 335 (numbering referring to processed wild type SP-D)
SPD_F335D	as in 1, but with the mutation Phe -> Asp at position 335 (numbering referring to processed wild type SP-D)
CCSPD	coiled-coiled “neck“ of human SP-D
Col11	human Collectin-11 (coiled-coiled “neck + CRD of human Collectin-11)
CC11	coiled-coiled “neck of human Collectin-11
T4	Bacteriophage T4 Whisker protein (W02008025516)
69	Bacteriophage 69 Whisker protein (W02008025516)

Table 3: Explanation of C-terminal trimerization motifs used to generate stable TNFSF fusion proteins.

Between the TNFSF-RBD and the trimerization domain, a flexible linker element was placed with varying lengths (Table 4):

Linker name	Amino-acid sequence
A	GSS GSS GSS GS
B	GSS GSS GS
C	GSS GS
D	GS

Table 4: Linker names and amino acid sequence (G = glycine; S = serine)

1.2 Generation of Expression Constructs

The nucleic acid molecule encoding the fusion protein as described herein is may be cloned into a suitable vector for expressing the fusion protein. The molecular tools necessary in order to generate such a vector are known to the skilled person and comprise restriction enzymes, vectors, and suitable host for propagating the vectors.

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For purification and analytical strategies, a Strep-tag II (amino acid sequence WSHPQFEK) was added C-terminally. This affinity tag was linked to the trimerization domain by a flexible linker element (amino acid sequence PSSSSSSA). To allow for secretory based expression, signal peptides derived from human Igx were fused to the N-termini of said proteins. The amino acid sequences of the fusion proteins were backtranslated and their codon usage optimised for mammalian cell-based expression. Gene synthesis was done by ENTELECHON GmbH (Regensburg, Germany). The final expression cassettes were subcloned into pCDNA4-HisMax-backbone, io using unique Hind-Ill- and Not-l-sites of the plasmid. All expression cassettes were routinely verified by DNA sequencing.

Data will be presented herein for the following constructs (Table 5a and 5b):

TRAIL (wild-type)

TRAIL Mutein (R1-specific)

TRAIL Mutein (R2-specific)

Linker: Motif

A

B

c

D

A

B

C

D

A

B

C

D

SPD

•

•

•

•

•

n.s.

n.s.

•

•

n.s.

n.s.

•

SPD F335A

•

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

SPD F335D

•

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

CCSPD

•

•

•

•

•

n.s.

n.s.

•

•

n.s.

n.s.

•

Col11

•

•

•

' ·

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

CC11

•

• '

•

•

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

T4

•

• ’

•

•

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

69

•

•

•

•

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

Table 5a: Overview of TRAIL fusion proteins with shown data. Filled circles indicate that data are presented. N.s., not shown.

	LIGHT	APRIL	CD95L
^***-»^Linker: Motif '	A	A	A
SPD	•	•	•
CCSPD	•	•	n.s.
Col11	•	•	n.s.
69	•	•	n.s.

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Table 5b: Overview of LIGHT-, APRIL-, and CD95L-constructs with shown data. Filled circles indicate that data are presented. N.s., not shown.

1.3 Expression and purification of engineered ligands of the TNF

Superfamily

Hek 293T cells grown in DMEM + GlutaMAX (GibCo) supplemented with 10% FBS, 100 units/ml Penicillin and 100 pg/ml Streptomycin were transiently transfected with plasmids encoding a fusion protein as described io herein. Cell culture supernatant containing recombinant proteins were harvested three days post transfection and clarified by centrifugation at

300xg followed by filtration through a 0.22 pm sterile filter. For affinity purification, 4 ml of 50% Streptactin Sepharose (IBA GmbH, Gottingen, Germany) were packed to a 2 ml column and equilibrated with 30 ml is phosphate buffered saline, pH 7.4 (PBS; Invitrogen Cat. 10010) or buffer W (100 mM Tris-HCI, 150 mM NaCI pH 8.0). The cell culture supernatant was applied to the column at 4°C with a flow rate of 2 ml/min. Subsequently, the column was washed with PBS or buffer W and specifically bound proteins were eluted stepwise by addition of 5 x 2 ml buffer E (PBS or buffer W with

2.5 mM Desthiobiotin, pH 7.4). The protein content of the eluate fractions was analysed by absorption spectroscopy and by silver-stained SDS-PAGE. Postitive fractions were subsequently concentrated by ultrafiltration (Sartorius, Vivaspin, 10,000 Da cut-off) and further analysed by size exclusion chromatography (SEC).

SEC was performed on a Superdex 200 column using an Akta chromatography system (GE-Healthcare). The column was equilibrated with PBS (Invitrogen Cat. 10010) and the concentrated, streptactin purified proteins were loaded onto the SEC column at a flow rate of 0.5 ml/min. The elution of was monitored by absorbance at 280 nm. The apparent molecular weight of purified proteins were determined based on calibration of the Superdex 200 column with gel filtration standard proteins (Bio-Rad GmbH, Munchen, Germany).

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1.4. Cell death assays

To analyze caspase activation, a cellular assay with the Jurkat A3 permanent human T-cell line (cat. no. CRL2570, ATCC) was used. Jurkat cells were grown in flasks with RPMI 1640-medium + GlutaMAX (GibCo) supplemented with 10 % FBS (Biochrom), 100 units/ml Penicillin and 100 pg/ ml Streptomycin (GibCo). Prior to the assay, 100,000 cells were seeded per well into a 96-well microtiterplate. The addition of different solutions containing the protein with or without a crosslinking antibody to the wells io (final volume: 200 pi) was followed by a 3 hour incubation at 37°C. Cells were lysed by adding 20 pi lysis buffer (250 mM HEPES, 50 mM MgCI₂, 10 mM EGTA, 5 % Triton-X-100, 100 mM DTT, 10 mM AEBSF, pH 7.5) and plates were incubated on ice for 30 minutes to 2 hours. Apoptosis is paralleled by an increased activity of Caspases. Hence, cleavage of the is specific Caspase substrate Ac-DEVD-AFC (Biomol) was used to determine the extent of apoptosis. For the Caspase activity assay, 20 pi cell lysate was transferred to a black 96-well microtiterplate. After the addition of 80 pi buffer containing 50 mM HEPES, 1 % Sucrose, 0.1 % CHAPS, 50 pM Ac-DEVDAFC, and 25 mM DTT, pH 7.5, the plate was transferred to a Tecan Infinite

F500 microtiterplate reader and the increase in fluorescence intensity was monitored (excitation wavelength 400 nm, emission wavelength 505 nm).

For the determination of cell death in HT1080 fibrosarcoma, HeLa cervix carcinoma and WM35 melanoma cells, 15,000 cells were plated in 96-well plates over night in RPMI 1640-medium + GlutaMAX (GibCo) supplemented with 10 % FBS (Biochrom). For Colo205 cells, 50,000 cells were plated over night. Cells were stimulated the following day with indicated ligand and incubated for an additional 18 hours. For HeLa and HT1080 cells, cycloheximide (Sigma) at a final concentration of 2.5 pg/ml was used during

3o stimulation with ligands. Cell death of HT1080, HeLa and WM35 was quantified by staining with buffer KV (0.5% crystal violet, 20% methanol). After staining, the wells were washed with water and air-dried. The dye was eluted with methanol and optical density at 595 nm was measured with an

-392017201232 23 Feb 2017

ELISA reader. Viability of Colo205 cells was quantified by MTS assay (Promega).

1.5 Hepatocellular cytotoxicity assay

To determine the effect of TRAIL fusion proteins, primary human hepatocytes were prepared from healthy donors and cultured in Williams E medium using 25,000 cells per well in 96-well plates. At day two, medium was changed to DMEM-F12 supplemented with 10% FCS, human insulin, Pen/Strep, minimum essential medium (MEM), sodium pyruvate and 10 mM io Hepes and cultured for another day. Cells were stimulated at day three with varying concentrations of indicated proteins in presence or absence of crosslinking antibodies (StrepMablmmo, IBA GmbH). To evaluate the potential hepatotoxic effect of a cotreatment of ligands with chemotherapeutic agents, TRAIL-ASPD_F335D was coincubated at varying concentrations together is with 5 mM of doxorubicin or 5 mM gemcitabine. Cells were incubated for 5 or 24 hours at 37°C and 5% CO₂ and were then lysed for determination of caspase activity as described in section „Celi death assays.

1.6 Streptactin-ELISA

To determine the binding of receptors to constructed ligands, streptactincoated 96-well microplates were used. Therefore, supernatants from transiently transfected HEK293 cells, mouse sera or purified proteins were immobilized on streptactin-plates (IBA GmbH) for 1-3 hours in PBS. Samples were diluted in ELISA binding/blocking buffer (PBS, 0.1% Tween-20,

20% SuperBlock T20-PBS (Pierce)). Plates were washed with PBS +

0.1% Tween-20 and incubated with mouse-anti-TRAIL antibody (Pharmingen, clone RIK-2), TRAIL-Receptor 1-Fc (R&D Systems), TRAIL-Receptor 2-Fc (R&D Systems), TACI-Fc (R&D Systems) or HVEM-Fc (R&D Systems) for one hour at room temperature. Plates were again washed and Fc-proteins were detected with anti-human- or anti-mouse-Fcspecific peroxidase-conjugated antibodies (Sigma). Colour reaction was done by addition of 100 μΙ per well of TMB substrate (Kem-En-Tec Diagnostics) and the absorbance at 450 nm and 630 nm was determined

-402017201232 23 Feb 2017 with an ELISA reader after addition of 25 μΙ of 25% H₂SO₄ as stop-solution. Values were calculated as 450 nm - 630 nm with MS Excel.

1.7 Mannan-binding assay

ELISA plates (Nunc Maxisorp) were incubated over night at 4°C with 10 pg/well of yeast mannan (Sigma) in sterile coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, 0.025% NaN₃, pH 9.6). Plates were first incubated for one hour at room temperature with buffer BB (20 mM Tris, 140 mM NaCI, 5 mM CaCI₂, 0.1% BSA and 20% SuperBlock T20-PBS (Pierce)) and secondly for io additional 90 minutes with varying concentrations of indicated ligands in buffer BB. Plates were washed with buffer WB (20 mM Tris, 140 mM NaCI, 5 mM CaCI₂, 0.05% Tween-20) and detection was done by using streptactinHRP (IBA GmbH) in buffer BB. Plates were washed and developed with TMB substrate (Kem-En-Tec Diagnostics). The absorption at 450 nm and 630 nm is was determined with an ELISA reader after addition of 25 μΙ of 25% H₂SO₄ as stop-solution. Values were calculated as 450 nm - 630 nm with MS Excel.

1.8 Pharmacokinetics of TRAIL-SPD fusion proteins

Male CD1 mice (Charles River) were intravenously injected with 10 pg protein dissolved in 300 μΙ PBS (Invitrogen). Blood was collected after 0 min (predose), 5 min, 30 min, 2 hours, 6 hours and 24 hours. For each time point, two samples were collected. Blood samples were processed to obtain serum and were stored at -15°C. The concentration of TRAIL-fusion proteins was determined using an ELISA as described below (chapter 1.9) and half25 lives were calculated (GraphPad Prism v4.0).

1.9 ELISA for the quantitation of TRAIL-constructs in mouse sera

To quantitate the concentration of TRAIL proteins in mouse sera (originating from pharmacokinetic studies), an ELISA method employing 96-well microplates was used.

ELISA plates were coated for 1 h at 37°C with 2 pg/ml mouse-anti-TRAIL

-41 2017201232 23 Feb 2017 (clone RIK-2; Pharmingen). After washing with PBS + 0.1% Tween-20 and blocking the plate for 30 min at 37°C with StartingBlock™ (Pierce), serum samples at a concentration of 0.2 % and 5 %, calibration samples and control samples were added and incubated for 1 h at 37°C. Calibration and control samples were prepared from the respective TRAIL batch (TRAILASPD or TRAIL-ASPD-F335A or TRAIL-ASPD-F335D) and were supplemented with 0.2 % or 5 % non-treated pooled CD1-mouse serum to account for potential matrix effects. Control samples (high, medium and low concentration of the TRAIL-construct) were added as quality controls to io ensure precision and accuracy of the TRAIL-quantitation in the given assay window. Plates were again washed and the StrepTag-containing TRAILconstructs were detected with 1:1000 diluted StrepTactin-POD (IBA). All samples and proteins were diluted with ELISA buffer (PBS, 0.1% Tween-20, 5% StartingBlock (Pierce)). The colour reaction started after addition of 100 is pi per well TMB substrate (Kem-En-Tec Diagnostics), the absorbance at 450 nm and 630 nm was determined with an ELISA reader after addition of 25 μΙ of 25% H2SO4 as stop-solution. Values were calculated as 450 nm 630 nm with MS Excel.

2. Results

2.1 Characterization of CD95L fusion protein (CD95L-ASPD)

From the Streptactin-affinity purified CD95L-ASPD 0.5 ml (0.86 mg protein) were loaded with a flow rate of 0.5 ml/min onto a Superdex200 column using

PBS as running buffer. Fractions of 0.5 ml were collected (A1 to A11 are indicated). The retention volume of the major peak at 11.92 ml corresponded to 170 kDa as determined from size exclusion standard. This indicated that the protein is a trimer composed of glycosylated monomers. The calculated molecular weight of the monomeric polypeptide is 38 kDa. An aliquot of

3o fractions A1 to A11 was used for SDS-PAGE and caspase activity. Only the defined trimeric peak (fractions A7 to A10) was used for final analyses. The results are shown in Fig. 1.

-422017201232 23 Feb 2017

An aliquot from size exclusion chromatography of affinity purified CD95LASPD was used for reducing SDS-PAGE followed by silver staining. The band detected at approximately 40-45 kDa (indicated by an arrow) corresponded to CD95L-ASPD. The trimeric species was present in fractions

A7 to A10. The results are shown in Fig. 2.

Jurkat cells were incubated with aliquots at a final 8-fold dilution from fractions A1 to A15 from SEC with affinity purified CD95L-ASPD. Cells were lysed after 3h incubation and the caspase activity was determined with a io fluorogenic assay. The fractions corresponding to the trimeric peak (fractions

A7-A10) induced clear but weak caspase activity in Jurkat as these cells are known to require extensively cross-linked ligand. The aggregated and undefined species in fractions A1-A6 is therefore a potent inducer of caspase activation (not used further). Importantly, only the defined trimeric is species (A7 to A10) was collected and used for final analyses. The results are shown in Fig. 3.

The human cancer cell lines HT1080 (A), HeLa (B) or WM35 (C) were incubated with indicated concentrations of purified, trimeric CD95L-ASPD in the presence or absence of cross-linking antibody (2.5 microgram/ml of antiStrep-tag II). Cells were incubated for 18h and cytotoxicity was analyzed by crystal violet staining. As a result, CD95L-ASPD induced cell death in HeLa cervix cacinoma and HT1080 fibrosarcoma, but not in WM35 melanoma cells. The results are shown in Fig. 4.

The amino acid sequence of CD95L-ASPD is shown below.

SEQID 40 Sp-CD95L-ASPD

Total amino acid number: 346, MW=37682 30 ORIGIN

METDTLLLWV LLLWVPGSTG ELRKVAHLTG KSNSRSMPLE WEDTYGIVLL

SGVKYKKGGL

VINETGLYFV YSKVYFRGQS CNNLPLSHKV YMRNSKYPQD LVMMEGKMMS 35 YCTTGQMWAR

121 SSYVGAVFNL TSADHLYVNV SELSLVNFEE SQTFFGLYKL GSSGSSGSSG

SGLPDVASLR

-432017201232 23 Feb 2017

181 QQVEALQGQV QHLQAAFSQY KKVELFPNGQ SVGEKIFKTA GFVKPFTEAQ

LLCTQAGGQL

241 ASPRSAAENA ALQQLWAKN EAAFLSMTDS KTEGKFTYPT GESLVYSNWA

PGEPNDDGGS

301 EDCVEIFTNG KWNDRACGEK RLWCEFGGS PSSSSSSAWS HPQFEK

- 20: Secretion signal peptide (Sp; underlined)

- 160: CD95L-receptor binding domain

161 - 171: Flexible linker element (A-linker; italic)

172 - 209: Coiled coil “neck” region of human SP-D

210 - 327: C-type lectin domain of human SP-D 328 - 338: Linker element (GGSPSSSSSSA)

339 - 346: Strep-tag II (WSHPQFEK) is 2.2 Characterization of LIGHT Fusion Proteins (LIGHT-ASPD)

From affinity purified LIGHT-ASPD 0.5 ml (1.56 mg) were loaded onto a Superdex 200 column and resolved at 0.5 ml/min using PBS as running buffer. The major peak detected at 11.96 ml corresponded to a size of 170-180 kDa indicating that LIGHT-ASPD is a trimer composed of three glycosylated monomers. The trimeric peak (fractions A7 to A10) was collected and used for final analyses. The inset shows the silver stained SDS-PAGE of two independent purified and trimeric LIGHT-ASPD batches (designated 0917 and 0918). The results are shown in Fig. 5.

Varying concentrations (0-10 microgram/ml) of affinity and SEC purified, trimeric LIGHT-ASPD were used for immobilized via the Strep-tag II on Streptactin-coated microplates. LIGHT-ASPD was then detected in a ELISA set-up using 100 ng/ml of Fc-fusion proteins of the receptors HVEM and TRAIL-Receptor 1, respectively. Whereas the ELISA signal increased for

HVEM-Fc with increasing amounts of immobilized ligand, no signal was detected for TRAIL-Receptor 1-Fc over the whole range analyzed. This indicated that LIGHT-ASPD is a functional molecule that could bind to its receptor HVEM. The results are shown in Fig. 6.

The amino acid sequence of the LIGHT-ASPD fusion protein is shown below:

SEQID 41 Sp-LIGHT-ASPD

Total amino acid number: 356, MW=37931

-442017201232 23 Feb 2017

	ORIGIN 1 AFLRGLSYHD 61	METDTLLLWV GALWTKAGY	LLLWVPGSTG YYIYSKVQLG	EVNPAAHLTG GVGCPLGLAS	ANSSLTGSGG TITHGLYKRT	PLLWETQLGL PRYPEELELL
5	VSQQSPCGRA
	121 GSSGSSGSSG	TSSSRVWWDS	SFLGGWHLE	AGEEVWRVL	DERLVRLRDG	TRSYFGAFMV
	181 GFVKPFTEAQ	SGLPDVASLR	QQVEALQGQV	QHLQAAFSQY	KKVELFPNGQ	SVGEKIFKTA
10	241 GESLVYSNWA	LLCTQAGGQL	ASPRSAAENA	ALQQLWAKN	EAAFLSMTDS	KTEGKFTYPT
	301 HPQFEK	PGEPNDDGGS	EDCVEIFTNG	KWNDRACGEK	RLWCEFGGS	PSSSSSSAWS

1 - 20: Secretion signal peptide (Sp; underlined)

- 170: LIGHT-receptor binding domain

171 - 181: Flexible linker element (A-linker; italic) 182 - 219: Coiled coil “neck” region of human SP-D 220 - 337: C-type lectin domain of human SP-D

338 - 348: Linker element (GGSPSSSSSSA)

349 - 356: Strep-tag II (WSHPQFEK)

2.3 Characterization of TRAIL Fusion Proteins

HEK293 cells were transiently transfected with 24 different expression vectors encoding for TRAIL fusion proteins (Table 6).

No	Ligand	Linker	Trimerization motif
1	TRAIL	A/B/C/D	69
2	TRAIL	A/B/C/D	T4
3	TRAIL	A/B/C/D	SPD
4	TRAIL	A/B/C/D	CCSPD
5	TRAIL	A/B/C/D	Col11
6	TRAIL	A/B/C/D	CC11

Table 6: Overview fusion proteins produced by transient transfection of expression vecors. The ligand TRAIL was transfected as fusion proteins

3o comprising one of six stabilzing trimerization motifs and the linker element (A, B, C and D linker).

Supernatants were used for SDS-PAGE and TRAIL-constructs were detected by Western Blot analysis employing an antibody specific for Strep35 tag II.

Specific bands detected are indicated by an arrow. The expression strength depended on the type of the trimerization motif employed for construction,

-452017201232 23 Feb 2017 (SPD> 69/T4/Collectin11/CCSPD/CC11) as well as on the length of the linker element (A>B>C>D). The results are shown in Fig. 7.

Jurkat cells were incubated for three hours in the presence (filled bars, anti5 Strep-tag II) or absence (clear bars) of a cross-linking antibody (2.5 micrograms/ml anti-Strep-tag II) with supernatants from transiently transfected HEK cells. Supernatants contained TRAIL-fusion proteins with different trimerization motifs (T4, 69, SPD, CCSPD, Col11, CC11) fused through varying linker elements (A, B, C and D linker). As negative control, io cell supernatant from untransfected cells was used. Jurkat cells were lysed and analyzed for caspase activity with a fluorogenic assay.

As a result, the caspase activity decreased with the type of linker element employed (A>B>C>D) and on the Fold-On employed. Collectin-11 or coiled is coil of Collectin-11 (CCColH) containing TRAIL constructs are expressed (shown by Western Blot analyses), however were not functional, whereas

SPD-derived fold-on motifs yielded functional TRAIL-ligands. The results are shown in Fig. 8.

Affinity purified TRAIL-ASPD was subjected to SEC by loading 0.5 ml (0.4 mg protein) to a Superdex200 column at 0.5 ml/min with PBS as running buffer. Protein elution was monitored by absorption at 280 nm and 0.5 ml fractions were collected. The retention volume of 12.28 ml corresponds to 135-140 kDa as determined from size exclusion standard. This indicated that

TRAIL-ASPD is a homotrimer, as the calculated molecular weight of the monomeric polypeptide is 40 kDa. Importantly, for all fusion proteins analyzed by SEC consisting of the wild-type TRAIL-RBD sequence, an additional peak at around 8 ml corresponding to aggregated and non-active TRAIL-fusion protein was observed. From the collected fractions A1-A14 only the trimeric peak (A8 - A10) was used for further analyses. The results are shown in Fig. 9.

The human cancer cell lines HeLa, HT1080, Colo205 or WM35 were

-462017201232 23 Feb 2017 incubated for 18 hours with indicated concentrations of purified, trimeric TRAIL-ASPD in the presence or absence of cross-linking antibody (2.5 microgram/ml of anti-Strep-tag II). Cell death was quantified by crystal violet staining (HeLa, WM35 and HT1080) or by MTS assay (Colo205). The rise in the viability of Colo205 cells at high ligand concentration is likely due to limitation of cross-linking antibody. The results are shown in Fig. 10.

Varying (A) or a constant (B) concentration of affinity and SEC purified, trimeric TRAIL-ASPD was used for immobilization on Streptactin-coated 96io well plates. Plates were then incubated for 5h with 100,000 Jurkat cells per well at 37°C, 5% CO2 and the caspase activity was determined with a fluorogenic assay. To analyze specificity, plate (B) was incubated for 30 minutes with indicated varying concentrations of an antagonistic anti-TRAIL antibody (clone RIK-2, Pharmingen) prior addition of cells. The results are is shown in Fig. 11.

HT1080 cells were incubated on the same 96-well plate with purified and trimeric TRAIL-ASPD or TRAIL-DSPD at indicated concentrations. Cell death was quantified the following day by crystal violet staining. The use of the D2o linker reduced the bioactivity approximately 4.5-fold, as indicated by the EC50 values of 27 ng/ml and 6 ng/ml for TRAIL-DSPD and TRAIL-ASPD, respectively. The results are shown in Fig. 12.

The nucleic acid and amino sequences of TRAIL fusion polypeptides are 25 shown below.

SEQID 42: Expression cassette of Sp-TRAIL-ASPD

Endonuclease restriction sites are underlined (Hindlll, AAGCTT; BamHI, GGATCC; Notl, GCGGCCGC). The translational start codon is in boldface.

ORIGIN

GCTGTGGGTT

CAGGAGTAAC

121

CAACTCCTGG

AAGCTTGCCG

CCTGCAGGTA

ACCCTGAGTT

CCACCATGGA

ATGGTCAAAG

CTCCCAATTC

GACCGATACA

AGTCGCAGCT

CAAGAACGAG

CTGCTCTTGT

CACATCACTG

AAAGCCCTGG

GGGTGCTCTT

GGACTAGAGG

GTAGGAAGAT

-472017201232 23 Feb 2017

	181 TGGCGAACTT	GAAAGCTCCA	GAAGCGGCCA	TAGCTTTCTT	AGCAACCTCC	ACTTGAGGAA
	241 GTTCCAGGAG	GTGATCCATG	AGAAGGGCTT	CTACTACATC	TACAGCCAGA	CGTACTTCAG
5	301 CAAGTACACG	GAAATCAAGG	AGAACACCAA	GAACGACAAG	CAGATGGTGC	AATACATCTA
	361 GAGCAAAGAC	TCATACCCTG	ATCCTATACT	GCTGATGAAG	TCCGCCAGAA	ACAGTTGCTG
10	421 GGAGAACGAC	GCTGAATACG	GCCTGTATTC	CATCTATCAG	GGCGGTATCT	TTGAACTCAA
	481 AGCGTCTTTC	AGGATCTTCG	TGTCTGTGAC	AAACGAGCAT	CTGATCGACA	TGGACCATGA
	541 TGGATTGCCA	TTCGGTGCCT	TCTTGGTGGG	ATCCTCTGGT	TCGAGTGGTT	CGAGTGGTTC
15	601 GCACTTGCAG	GACGTTGCTT	CTTTGAGACA	ACAGGTTGAG	GCTTTGCAGG	GTCAAGTCCA
	661 TGTTGGCGAA	GCTGCTTTCT	CTCAATACAA	GAAGGTTGAG	TTGTTCCCAA	ACGGTCAATC
20	721 ATTGTGTACT	AAGATTTTCA	AGACTGCTGG	TTTCGTCAAA	CCATTCACGG	AGGCACAATT
	781 CTTGCAACAG	CAGGCTGGTG	GACAGTTGGC	CTCTCCACGT	TCTGCCGCTG	AGAACGCCGC
	841 GACAGAGGGC	TTGGTCGTAG	CTAAGAACGA	GGCTGCTTTC	TTGAGCATGA	CTGATTCCAA
25	901 TGGAGAGCCC	AAGTTCACCT	ACCCAACAGG	AGAATCCTTG	GTCTATTCTA	ATTGGGCACC
	961 GTGGAATGAC	AACGATGATG	GCGGCTCAGA	GGACTGTGTG	GAAATCTTCA	CCAATGGCAA
	1021	AGAGCTTGTG	GAGAGAAGCG	TTTGGTGGTC	TGTGAGTTCG	GAGGCAGTCC

TTCATCTTCA

1081 TCTAGCTCTG CCTGGTCGCA TCCACAATTC GAGAAATAAT AGCGGCCGC

SEQID 43 Sp-TRAIL-ASFD

	Total amino acid number: 367, MW=40404
35	ORIGIN 1	METDTLLLWV	LLLWVPAGNG	QRVAAHITGT	RGRSNTLSSP	NSKNEKALGR
	KINSWESSRS 61	GHSFLSNLHL	RNGELVIHEK	GFYYIYSQTY	FRFQEEIKEN	TKNDKQMVQY
40	IYKYTSYPDP 121	ILLMKSARNS	CWSKDAEYGL	YSIYQGGIFE	LKENDRIFVS	VTNEHLIDMD
	HEASFFGAFL 181	VGSSGSSGSS	GSGLPDVASL	RQQVEALQGQ	VQHLQAAFSQ	YKKVELFPNG
	QSVGEKIFKT 241	AGFVKPFTEA	QLLCTQAGGQ	LASPRSAAEN	AALQQLWAK	NEAAFLSMTD
45	SKTEGKFTYP 301	TGESLVYSNW	APGEPNDDGG	SEDCVEIFTN	GKWNDRACGE	KRLWCEFGG
	SPSSSSSSAW 361 SHPQFEK

50	1	20
	21 -	181
	182 -	192
	193 -	230
	231 -	348
55	349 -	359
	360 -	367

Secretion signal peptide (Sp; underlined) TRAIL-receptor binding domain Flexible linker element (A-linker; italic) Coiled coil “neck” region of human SP-D C-type lectin domain of human SP-D Linker element (GGSPSSSSSSA)

Strep-tag II (WSHPQFEK)

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SEQID 44 Sp-TRAIL-ACCSPD

Total amino acid number: 246, MW=27534

ORIGIN

1 METDTLLLWV LLLWVPAGNG QRVAAHITGT RGRSNTLSSP NSKNEKALGR

KINSWESSRS

GHSFLSNLHL RNGELVIHEK GFYYIYSQTY FRFQEEIKEN TKNDKQMVQY

IYKYTSYPDP

121 ILLMKSARNS CWSKDAEYGL YSIYQGGIFE LKENDRIFVS VTNEHLIDMD

HEASFFGAFL

181 VGSSGSSGSS GSGLPDVASL RQQVEALQGQ VQHLQAAFSQ YKKVELFPNG PSSSSSSAWS

241 HPQFEK

1 - 20: Secretion signal peptide (Sp; underlined)

- 181: TRAIL-receptor binding domain

182 - 192: Flexible linker element (A-linker; italic)

193 - 230: Coiled coil “neck” region of human SP-D 231 - 238: Linker element (PSSSSSSA)

20	239 - 246:	Strep-tag II	(WSHPQFEK)
	SEQID 45 Sp-TRAIL-AColll Total amino acid number: 365, MW=40806 ORIGIN
25	1 KINSWESSRS	METDTLLLWV	LLLWVPAGNG ORVAAHITGT RGRSNTLSSP NSKNEKALGR
	61 IYKYTSYPDP	GHSFLSNLHL	RNGELVIHEK GFYYIYSQTY FRFQEEIKEN TKNDKQMVQY
30	121 HEASFFGAFL	ILLMKSARNS	CWSKDAEYGL YSIYQGGIFE LKENDRIFVS VTNEHLIDMD
	181 LLVKEEKRYA	VGSSGSSGSS	GSQLRKAIGE MDNQVSQLTS ELKFIKNAVA GVRETESKIY
	241 VYSDHSPMRT	DAQLSCQGRG	GTLSMPKDEA ANGLMAAYLA QAGLARVFIG INDLEKEGAF
35	301 FNKWRSGEPN SSSSSSAWSH 361 PQFEK	NAYDEEDCVE MVASGGWNDV ACHTTMYFMC EFDKENMGSP

- 20: Secretion signal peptide (Sp; underlined)

21 - 181: TRAIL-receptor binding domain

182 - 192: Flexible linker element (A-linker; italic)

193 - 224: Coiled coil “neck” region of human Collectin-11 225 - 347: C-type lectin domain of human Collectin-11 348 - 357: Linker element (GSPSSSSSSA)

358 - 365: Strep-tag II (WSHPQFEK)

	SEQID 46 Sp-TRAIL-ACCll Total amino acid number: 246, MW=27431
50	ORIGIN 1	METDTLLLWV	LLLWVPAGNG	QRVAAHITGT	RGRSNTLSSP	NSKNEKALGR
	KINSWESSRS 61	GHSFLSNLHL	RNGELVIHEK	GFYYIYSQTY	FRFQEEIKEN	TKNDKQMVQY
55	IYKYTSYPDP 121	ILLMKSARNS	CWSKDAEYGL	YSIYQGGIFE	LKENDRIFVS	VTNEHLIDMD

-492017201232 23 Feb 2017

HEASFFGAFL

181 VGSSGSSGSS GSGSQLRKAI GEMDNQVSQL TSELKFIKNA VAGVRETESG PSSSSSSAWS

241 HPQFEK

- 20: Secretion signal peptide (underlined)

- 181: TRAIL-receptor binding domain

182 - 193: Flexible linker element (A-linker; GSS GSS GSS GSG italic)

194 - 229: Coiled coil “neck” region of human Collectin-11

230 - 238: Linker element (GPSSSSSSA)

239 - 246: Strep-tag II (WSHPQFEK)

2.4 Characterization of Receptor-selective TRAIL (’mutein') fusion is proteins

HEK293 cells were transiently transfected with expression plasmids encoding for different TRAIL receptor-selective SPD constructs:

No 1 2

5

9

10

12

Transfected Expression Vector

TRAILR1mut-A-SPD

TRAILR1mut-A-CCSPD

TRAILR1mut-D-SPD

TRAILR1mut-D-CCSPD

TRAILR2mut-A-SPD

TRAILR2mut-A-CCSPD

TRAILR2mut-D-SPD

TRAILR2mut-D-CCSPD

TRAIL-A-SPD

TRAIL-A-CCSPD

TRAIL-D-SPD

TRAIL-D-CCSPD

Supernatants were collected three days post-transfection and an aliquot was 35 used for SDS-PAGE and Western Blotting employing an antibody specifc for

Strep-tag II. Specific bands were detected at around 38 kDa (SPD-fusion proteins) and 28 kDa (coiled-coil-SPD fusion proteins). The amount of expressed protein depended on the ligand itself

-502017201232 23 Feb 2017 (TRAILR1mutein>TRAILR2mutein>TRAIL), secondly the linker length used (A>D) and third the trimerization motif used (SPD>CCSPD). Apparent molecular weights were as expected from the calculated sizes (40 kDa and 27 kDa for SPD and CCSPD fusion proteins, respectively). The results are shown in Fig. 13.

The selectivity of TRAIL-Receptor 1 or TRAIL-Receptor 2 towards fusion proteins of SPD/ccSPD and TRAIL, TRAILRImut and TRAILR2mut was shown by Streptactin-ELISA. Therefore, TRAIL-SPD-fusion proteins in w supernatants from transiently transfected HEK293 cells were immobilized on

Streptactin coated microplates. Cell supernatant from untransfected cells served as negative control. The results are shown in Fig. 14. Specifically bound proteins were detected with constant (A, B) or varying (C, D) concentrations of either TRAIL-Receptor 1-Fc or TRAIL-Receptor 2-Fc. As is shown in (A), the ligand TRAILRImut fused to SPD variants is deteced by

TRAIL-Receptor 1, whereas the ligand TRAILR2mut is not. As shown in (B), the ligand TRAILR2mut is preferentially detected by TRAIL-Receptor 2, whereas TRAILRImut- and TRAIL wild-type constructs are equally well detected. As shown in C, TRAIL-Receptor 1-Fc bound to

TRAIL-R1mut-ASPD and TRAIL-ASPD equally well over the whole receptor titration range, whereas TRAIL-R2mut-ASPD is not detected. As shown in D, TRAIL-Receptor 2-Fc bound to TRAIL-R2mut-ASPD and TRAIL-ASPD equally well over the receptor titration range analyzed, whereas the signal for TRAIL-R1mut-ASPD decreased rapidely with decreasing concentrations of receptor.

One microgram/ml of affinity purified, trimeric TRAIL-ASPD, TRAILRImutASPD or TRAILR2mut-ASPD in 100 microliter of PBS were used for immobilization via the Strep-tag II on Streptactin-coated microplates. Bound

3o ligands were detected in a ELISA set-up using Fc-fusion proteins of TRAILReceptor 1 (A) or TRAIL-Receptor 2 (B). As shown in (A), TRAIL-Receptor 1 bound preferentially to the receptor-selective TRAILR1mut-ASPD as compared to TRAILR2mut-ASPD. As shown in (B), TRAIL-Receptor 2

-51 2017201232 23 Feb 2017 preferentially bound to TRAILR2mut-ASPD as compared to TRAILR1mut-ASPD. In conclusion, the constructed TRAIL variants fused to SPD are receptor selective. The results are shown in Fig. 15.

Affinity purified TRAILR1mut-ASPD was subjected to SEC by loading 0.5 ml (0.95 mg protein) on a Superdex200 column. The results are shown in Fig. 16. Proteins were resolved at 0.5 ml/minute with PBS as running buffer and 0.5 ml fractions were collected (fractions A1 to A14 are indicated). The retention volume of 12.46 ml corresponded to 140 -145 kDa as determined io by size exclusion standard. A minor peak at 10.83 ml indicated some aggregated species, importantly however, no peak was detected at the running front (8ml) indicating that this molecule is much more soluble as compared to proteins containing parts of the wild-type TRAIL amino acid sequence.

An aliquot from size exclusion chromatography of affinity purified TRAILR1mut-ASPD was used for non-reducing (A) or reducing (B) SDSPAGE followed by silver staining as shown in Fig. 17. Under non-reducing conditions, two bands were detected at 35 and 70 kDa, whereas a single band of 40kDa (indicated by an arrow) was detected under reducing conditions. This indicated the formation of disulphide bridged molecules. The trimeric species was present in fractions A8 to A11 and was used for later analyses.

Jurkat cells were incubated in the absence (open bars) or presence (filled bars) of 2.5 microgram/ml of cross-linking antibody with aliquots at a final 80fold dilution from fractions A1 to A14 from SEC of affinity purified TRAILR1mut-ASPD. The results are shown in Fig. 18. As negative control, Jurkat cells were incubated with medium only. Jurkat cells were lysed after

3h incubation and the caspase activity was determined with a fluorogenic assay. As Jurkat cells have been shown to mainly express TRAIL-Receptor 2, no fraction induced significant caspase activity, even when TRAILRImitASPD was cross-linked by Strep-tag II specific antibody. This indicated that

-522017201232 23 Feb 2017

TRAILR1mut-ASPD does not bind to TRAIL-Receptor 2.

Affinity purified TRAILR2mut-ASPD was subjected to size exclusion chromatography by loading 0.5 ml (0.5 mg protein) to a Superdex 200 column as shown in Fig. 19. Proteins were resolved at 0.5 ml/minute with PBS as running buffer and 0.5 ml fractions were collected (fractions A1 to A14 are indicated). The retention volume of 12.60 ml corresponds to 130 135 kDa as determined from size exclusion standard. This indicated that TRAILR2mut-ASPD is a homotrimer as calulated from the expected io monomeric weight of 40 kDa. Importantly, more than 95% was present in the trimeric peak fraction and no aggregates were detected. The trimeric peak was used for later analyses.

An aliquot from size exclusion chromatography of affinity purified is TRAILR2mut-ASPD was used for non-reducing (A) or reducing (B) SDSPAGE followed by silver staining as shown in Fig. 20. Under non-reducing conditions, two bands were detected at 35 and 70 kDa, whereas a single band of approximately 40kDa (indicated by an arrow) was detected under reducing conditions. This indicated the formation of disulphide bridged molecules. The trimeric species was present in fractions A9 to A11 and was used for later analyses.

The results from a Jurkat cell kill assay with TRAILR2-mut-ASPD are shown in Fig. 21. Jurkat cells were incubated in th absence (clear bars) or presence (filled bars) of cross-linking antibodies (2.5 microgram/ml anti-Strep-tag II) with aliquots from fractions A1 to A14 from SEC of affinity purified TRAILR2mut-ASPD. Samples were used at at final 640-fold dilution. Cells were lysed after 3h of incubation and the caspase activity was determined with a fluorogenic assay. As Jurkat cells have been shown to mainly express

TRAIL-Receptor 2 that requires multimerized ligand forms for efficient signalling, TRAILR2mut-ASPD induced caspase activity when cross-linked. This indicated that TRAILR2mut-ASPD is a functional molecule.

-532017201232 23 Feb 2017

The cytotoxic activity of TRAIL-ASPD, TRAILR1mut-ASPD and TRAILR2mut-ASPD on different human cancer cells is shown in Fig. 22. The indicated cell lines HT1080 (A and B), Hela (C and D) or Colo205 (E and F) were treated with varying concentrations of purified and trimeric TRAIL5 ASPD, TRAILR1mut-ASPD or TRAILR2mut-ASPD in the absence (A, C and E) or presence (B, D and F) of cross-linking antibody (anti-Strep-tag II). Cells were incubated for 18 hours with indicated concentrations of ligands and cell death was quantified by crystal violet staining (HT1080 and HeLa) or MTS assay (Colo205). As a result, the ligand TRAIL-ASPD induced cell death on io the three cell lines tested and TRAILR2mut-ASPD showed superior cell killing activity. In contrast, TRAIL-Receptor 1 selective TRAILR1mut-ASPD was not active on any cell line tested.

Affinity purified TRAILR2mut-ASPD was concentrated 20-fold in PBS by is centrifugation through a 10 kDa membrane to give a solution of 2.5 mg/ml.

From the concentrate, 0.1 ml were subjected to size exclusion chromatography. As a result, only the trimeric peak and no aggregates were detected, indicating that this composition has improved production capabilities (Fig. 23). Similar results were achieved for TRAILR1mut-ASPD, where a concentrated solution of even 5.4 mg/ml showed no signs of aggregation (not shown). In contrast, all fusion proteins tested containing the receptor binding domain composed of the wild type TRAIL sequence showed aggregation with 40% aggregates at concentrations as low as 0.4 mg/ml.

The amino acid sequences of receptor-selective TRAIL mutein fusion polypeptides are shown in the following.

SEQID 47 Sp-TRAILRlmut-ASPD

Total amino acid number: 367, MW=40335

ORIGIN

METDTLLLWV LLLWVPAGNG QRVAAHITGT RGRSNTLSSP

KINSWESSRS

GHSFLSNLHL RNGELVIHEK GFYYIYSQTA FRFSEEIKEV

IYKWTDYPDP

121 ILLMKSARNS CWSKDAEYGL YSIYQGGIFE LKENDRIFVS

HEASFFGAFL

181 VGSSGSSGSS GSGLPDVASL RQQVEALQGQ VQHLQAAFSQ

NSKNEKALGR

TRNDKQMVQY

VTNEHLIDMD

YKKVELFPNG

-542017201232 23 Feb 2017

QSVGEKIFKT

241 AGFVKPFTEA QLLCTQAGGQ LASPRSAAEN AALQQLWAK NEAAFLSMTD

SKTEGKFTYP

301 TGESLVYSNW APGEPNDDGG SEDCVEIFTN GKWNDRACGE KRLWCEFGG

SPSSSSSSAW

361 SHPQFEK

- 20: Secretion signal peptide (Sp; underlined)

- 181: TRAILRlmut-receptor binding domain

182 - 192: Flexible linker element (A-linker; italic)

193 - 230: Coiled coil “neck” region of human SP-D 231 - 348: C-type lectin domain of human SP-D 349 - 359: Linker element (GGSPSSSSSSA)

360 - 367: Strep-tag II (WSHPQFEK)

SEQID 48 Sp-TRAILR2mut-ASPD

	Total amino acid number: 367, MW=40401 ORIGIN
20	1	METDTLLLWV	LLLWVPAGNG	QRVAAHITGT	RGRSNTLSSP	NSKNEKALGR
	KINSWESSRS 61	GHSFLSNLHL	RNGELVIHEK	GFYYIYSQTQ	FKFREEIKEN	TKNDKQMVQY
	IYKYTSYPDP 121	ILLMKSARNS	CWSKDAEYGL	YSIYQGGIFE	LKENDRIFVS	VTNERLLQMD
25	HEASFFGAFL 181	VGSSGSSGSS	GSGLPDVASL	RQQVEALQGQ	VQHLQAAFSQ	YKKVELFPNG
	QSVGEKIFKT 241	AGFVKPFTEA	QLLCTQAGGQ	LASPRSAAEN	AALQQLWAK	NEAAFLSMTD
30	SKTEGKFTYP 301	TGESLVYSNW	APGEPNDDGG	SEDCVEIFTN	GKWNDRACGE	KRLWCEFGG

SPSSSSSSAW

	361	SHPQFEK
	1	- 20: Secretion signal peptide (Sp; underlined)
35	21	- 181: TRAILR2mut-receptor binding domain
	182	- 192: Flexible linker element (A-linker; italic)
	193	- 230: Coiled coil “neck” region of human SP-D
	231	- 348: C-type lectin domain of human SP-D
	349	- 359: Linker element (GGSPSSSSSSA)
40	360	- 367: Strep-tag II (WSHPQFEK)

2.5 Characterization of SPD Carbohydrate-variants

Affinity purified TRAIL-ASPD_F335A was subjected to Size Exclusion 45 Chromatography by loading 0.5 ml PBS solution (0.4 mg protein) to a

Superdex 200 column as shown in Fig. 24. Proteins were resolved at 0.5 ml/ minute with PBS as running buffer and 0.5 ml fractions were collected (A1 to A13 are indicated). The retention volume of 12.27 ml corresponds to 135-145 kDa as determined from size exclusion standard. This indicated that so TRAIL-ASPD_F335A is a homotrimer as calulated from the expected

-552017201232 23 Feb 2017 monomeric weight of 40 kDa. Two additional peaks at 8.32 and 10.68 ml indicated the formation of TRAIL-ASPD_F335A aggregates. Only the trimeric peak was used for later analyses.

From Size exclusion chromatography an aliquot from collected fractions A1 to A13 was resolved by reducing SDS-PAGE and the gel was silver stained (Fig. 25). The band detected at approximately 40 kDa corresponded to the calculated molecular weight of 40 kDa for TRAIL-ASPD_F335A. Positive fractions corresponding the trimeric molecule (A8, A9, A10) of the SEC run io were pooled and used for further analyses.

The amino acid sequences of TRAIL-SPD carbohydrate variant fusion proteins is shown in the following.

15	SEQID 49: Sp Total amino ORIGIN 1 KINSWESSRS	—TRAIL-ASPD_F335A acid number: 367, MW=40328
METDTLLLWV	LLLWVPAGNG	QRVAAHITGT	RGRSNTLSSP	NSKNEKALGR
20	61 IYKYTSYPDP	GHSFLSNLHL	RNGELVIHEK	GFYYIYSQTY	FRFQEEIKEN	TKNDKQMVQY
	121 HEASFFGAFL	ILLMKSARNS	CWSKDAEYGL	YSIYQGGIFE	LKENDRIFVS	VTNEHLIDMD
25	181 QSVGEKIFKT	VGSSGSSGSS	GSGLPDVASL	RQQVEALQGQ	VQHLQAAFSQ	YKKVELFPNG
	241 SKTEGKFTYP	AGFVKPFTEA	QLLCTQAGGQ	LASPRSAAEN	AALQQLWAK	NEAAFLSMTD
30	301 TGESLVYSNW SPSSSSSSAW 361 SHPQFEK	APGEPNDDGG	SEDCVEIATN	GKWNDRACGE	KRLWCEFGG

- 20: Secretion signal peptide (Sp; underlined)

- 181: TRAIL-receptor binding domain

182 - 192: Flexible linker element (A-linker; italic)

193 - 230: Coiled coil “neck” region of human SP-D

231 - 348: C-type lectin domain of human SP-D (Phe mutation in boldface)

349 - 359: Linker element (GGSPSSSSSSA)

360 - 367: Strep-tag II (WSHPQFEK)

SEQID 50: Sp-TRAIL-ASPD_F335D

Total amino acid number: 367, MW=40372 ORIGIN

METDTLLLWV LLLWVPAGNG QRVAAHITGT RGRSNTLSSP NSKNEKALGR

KINSWESSRS

-562017201232 23 Feb 2017

	61	GHSFLSNLHL	RNGELVIHEK	GFYYIYSQTY	FRFQEEIKEN	TKNDKQMVQY
	IYKYTSYPDP 121	ILLMKSARNS	CWSKDAEYGL	YSIYQGGIFE	LKENDRIFVS	VTNEHLIDMD
5	HEASFFGAFL 181	VGSSGSSGSS	GSGLPDVASL	RQQVEALQGQ	VQHLQAAFSQ	YKKVELFPNG
	QSVGEKIFKT 241	AGFVKPFTEA	QLLCTQAGGQ	LASPRSAAEN	AALQQLWAK	NEAAFLSMTD
	SKTEGKFTYP 301	TGESLVYSNW	APGEPNDDGG	SEDCVEIDTN	GKWNDRACGE	KRLWCEFGG
10	SPSSSSSSAW 361 SHPQFEK

- 20: Secretion signal peptide (Sp; underlined)

- 181: TRAIL-receptor binding domain

182 - 192: Flexible linker element (A-linker; italic)

193 - 230: Coiled coil “neck” region of human SP-D

231 - 348: C-type lectin domain of human SP-D (Asp mutation in boldface)

349 - 359: Linker element (GGSPSSSSSSA)

360 - 367: Strep-tag II (WSHPQFEK)

The cytotoxic effect of TRAIL-ASPD_F335A on human cancer cells is shown in Fig. 26. Indicated human cancer cell lines were incubated over night with varying concentrations of affinity and SEC purified, trimeric TRAILASPD_F335A in the presence or absence of cross-linking antibody (2.5 microgram/ml of anti Strep-tag II). Cell viability was quantified by crystal violet staining (HT1080, HeLa and WM35) or MTS (Colo205). The rise of Colo205 cell viability at high ligand concentrations is likely due to limitation of cross-linking antibody.

Affinity purified TRAIL-ASPD_F335D was subjected to Size Exclusion Chromatography by loading 0.5 ml (0.2 mg protein) to a Superdex 200 column as shown in Fig. 27. Proteins were resolved at 0.5 ml/minute with

PBS as running buffer and 0.5 ml fractions were collected (A1 to A13 are indicated). The retention volume of 12.29 ml corresponds to 135-145 kDa as determined from size exclusion standard. This indicated that TRAILASPD_F335D is a homotrimer as calulated from the expected monomeric weight of 40 kDa. The peak at 8.35 corresponded to inactive

TRAIL-ASPD_F335D aggregates typically found for all fusion proteins containing parts of the wild type TRAIL amino acid sequence.

-572017201232 23 Feb 2017

From Size exclusion chromatography aliquots of affinity purified TRAILASPD_F335D from the collected fractions A1 to A13 were resolved by reducing SDS-PAGE and the gel was silver stained (Fig. 28). The bands detected at approximately 40 kDa (indicated by an arrow) corresponded to the calculated molecular weight of 40 kDa for TRAIL-ASPD_F335D. Fractions containing trimeric protein (fractions A8 to A10) were pooled and used for further analyses.

io The human cancer cell lines HT1080 (A), HeLa (B), WM35 (C) or Colo205 (D) were incubated over night with varying concentrations of affinity purified, trimeric TRAIL-ASPD_F335D in the presence or absence of cross-linking antibodies (anti-Strep-tag II). Ceil viability was quantified by crystal violet staining (HT1080, HeLa and WM35) or MTS (Colo205). The data show that is TRAIL-ASPD_F335D is capable of inducing cell death in exemplified cancer cell lines (Fig. 29). The rise of Colo205 cell viability at high concentrations of ligand is likely due to limitation of cross-linking antibody.

2.6 Analysis of Carbohydrate binding characteristics of the SPD trimerization motif variants

It has been shown that wild-type, full length and oligomeric SP-D protein from several species, as well as the trimeric neck+CRD of human SP-D bind to several different carbohydrates. In addition, the neck+CRD of human SPD also has been shown to excert immunomodulatory effects by serving as a chemotactic factor for immuno cells such as neutrophils (Cai et al., 1999, Am J Physiol Lung Cell Mol Physiol 276:131-136). Other cells may also be recruited by SP-D. The chemotactic effect of neck+CRD of human SP-D has been shown to depend on the glycobinding function, as the addition of maltose inhibited the chemotactic function. Thus, a ligand of the TNFSF with a SP-D-mediated chemotactic function may be of superior activity as compared to ligands or constructs thereof with natural amino acid sequences. For instance, in a scenario where cellular effects are desirable such as in cancer treatment such a described ligand may be desirable.

-582017201232 23 Feb 2017 ln addition, a ligand where SP-D has no carbohydrate function may be desirable in other settings. For human SP-D a mutant has been described in which amino acid phenylalanine 335 (corresponding to amino acid 355 of

SEQ ID NO:21) has been mutated to alanine (SPD_F335A, Crouch et al., JBC 281: 18008-18014). This mutant showed very weak carbohydrate binding. However, introducing a charged amino acid (e.g. an acidic amino acid) may be even better as compared to F335A if no carbohydrate binding is desired. Therefore the mutant SPD_F335D may be superior towards io F335A mutant.

To analyze the binding of TRAIL-fusion proteins to carbohydrates, mannan from yeast was immobilized on microplates and the binding of TRAIL-SPD, TRAIL-SPD_F335A or TRAIL-SPD_F335D was detected by ELISA. The is results are shown in Fig. 30. As expected, the ELISA signal increased with increasing concentrations of TRAIL-ASPD. In contrast, the carbohydratemutant form TRAIL-ASPD_F335A showed a very low ELISA signal. In addition, the new constructed variant TRAIL-ASPD_F335D displayed the lowest ELISA signal (see inset and arrow). This indicated that the mutant

F335D has a lower mannan-binding affinity as compared to the previously described SP-D mutant form F335A.

2.7 Pharmacokinetics of TRAIL-SPD Fusion Proteins

To determine the half-lifes of TRAIL-SPD fusion protein, ten micrograms of

TRAIL-ASPD (A) or TRAIL-ASPD_F335D (B) were injected intraveneously into male CD1 mice and serum samples were collected after several time points (predose, 5 min., 30 min., 2h, 6h and 24h). TRAIL proteins in sera of mice were quantified by an ELISA and the data was used to calculate halflifes. The results are shown in Fig. 31. For the two proteins analyzed, a so halflife of 7 to 14 hours for TRAIL-ASPD (A) and TRAIL-ASPD_F335D (B) were calculated. No animal died or showed signs of intolerance during the period observed. The data indicate an at least 80-fold improvement of the serum halftime as compared to wild type TRAIL that was reported to have a

-592017201232 23 Feb 2017 half time in the range of three to five minutes in rodents (Kelley et. al 2001).

2.8 Cytotoxicity of TRAIL-ASPD Fusion Proteins

To analyze potential hepatotoxic effects of TRAIL-ASPD, TRAIL5 ASPD_F335A or TRAIL-ASPD_F335D, primary human hepatocytes (PHH) were incubated with varying concentrations of indicated TRAIL-SPD-fusion proteins, with or without cross-linking antibodies (anti-Strep-tag II). As a control, a stabilized variant of CD95L, CD95L-T4 (described in W02008/025516) was used. The results are shown in Fig. 32.

In addition, the effect of a simultaneous incubation of PHH with 5 mM of chemotherapeutic drugs was analyzed for TRAIL-ASPD_F335D. After 5h (A,B and E) or 24h (C, D and F) of incubation, cells were lysed and caspase activity was assessed with a fluorogenic assay.

As a result, all analyzed TRAIL-SPD fusion proteins induced no hepatotoxic effects, even if ligands were secondarily cross-linked by antibodies. In contrast, CD95L-T4 is hepatotoxic as indicated by an increase of active caspase (A to D). Five hours of co-incubation of primary human hepatocytes with trimeric TRAIL-ASPD_F335D together with chemotherapeutic drugs induced no caspase activity (E). However, after 24h of co-incubation with doxorubicin, soluble TRAIL-ASPD_F335D induced a strong caspase activity signal (F).

This indicates that TRAIL fusion proteins of the present invention may not show undesired hepatotoxicity in medical use. Thus, TRAIL fusion proteins are preferably administered in combination with drugs, which are apoptosis sensitizers and/or apoptosis inducers, e.g. a chemotherapeutic drug such as oxaliplatin, cisplatin, 5-fluorouracil, etoposide, gemcitabine, irinotecan and

3o others, or Bcl2 binding molecules, e.g. small molecules or peptidic compounds, which bind to polypeptides of the Bcl2 family, particularly Bcl2 or Bclxl.

-602017201232 23 Feb 2017

2.9 Characterization of APRIL Fusion Proteins

HEK293 cells were transiently transfected with expression vectors encoding for APRIL-A69 (W02008025516), APRIL-ASPD, APRIL-ACCSPD or APRIL-ACol11. After three days supernatants were analyzed for secreted proteins by Western Blotting. The results are shown in Fig. 33. For the detection of APRIL-fusion proteins an antibody specifc for Strep-tag II was used. Arrows indicate specific bands that were detected around 40 kDa (APRIL-ASPD and APRIL-AColH, respectively), as well as at around 25 kDa (APRIL-A69 and APRIL-ACCSPD, respectively). Thus APRIL expression io cassettes are functional and the secretion of protein indicated that the proteins are properly folded. As for other TNFSF proteins analyzed, the highest secreted protein levels were found for APRIL fused to the trimerization motif composed of coiled coil “neck” + CRD of human SP-D (APRIL-ASPD, lane No. 2). APRIL-ASPD was used to analyze the binding to is the receptor TACI.

To show that the constructed APRIL-ASPD fusion protein is functional, the binding to a known receptor of APRIL, namely TACI, was assessed (Fig. 34). Therefore, APRIL-ASPD in supernatant from transiently transfected HEK293 cells was immobilized on Streptactin coated microplates. Cell supernatant from untransfected HEK293 cells served as negative control. Specifically bound proteins were detected with varying concentrations of TACI-Fc followed by incubation with an anti-human, Fc-specific antibody conjugated with peroxidase. As a result, the ELISA signal increased with increasing concentrations of TACI-Fc, indicating that APRIL-ASPD is a functional molecule.

The amino acid sequence of an APRIL fusion protein is shown below.

SEQID 51: Sp-APRIL-ASPD

Total amino acid number: 344, MW=37120 ORIGIN

METDTLLLWV LLLWVPAGNG KQHSVLHLVP INATSKDDSD VTEVMWQPAL

RRGRGLQAQG

61 YGVRIQDAGV YLLYSQVLFQ DVTFTMGQW SREGQGRQET LFRCIRSMPS

HPDRAYNSCY

-61 2017201232 23 Feb 2017

121

LPDVASLRQQ

181

CTQAGGQLAS

241

EPNDDGGSED 301 C'

SAGVFHLHQG

VEALQGQVQH

PRSAAENAAL

DILSVIIPRA

LQAAFSQYKK

QQLWAKNEA

RAKLNLSPHG

VELFPNGQSV

AFLSMTDSKT

TFLGFVKLGS SGSSGSSGSG

GEKIFKTAGF VKPFTEAQLL

EGKFTYPTGE SLVYSNWAPG

CVEIFTNGKW NDRACGEKRL WCEFGGSPS SSSSSAWSHP QFEK

- 20: Signal secretion peptide (underlined)

21 - 158: APRIL-RBD

159 - 169: Flexible linker element (A-linker; GSS GSS GSS GS italic) 170 - 207: Coiled coil “neck” region of human SP-D 208 - 325: C-type lectin domain of human SP-D 326 - 336: Linker element (GGSPSSSSSSA)

337 - 344: Strep-tag II (WSHPQFEK)

-622017201232 23 Feb 2017

References

1. Locksley RM, Killeen N and Lenardo MJ (2001) Cell 104: 487-501

2. Bodmer JL, Schneider P and Tschopp J (2002) Trends Biochem. Sci. 27:

s 19-26

3. Grell M, Douni E, Wajant H, Lohden M., Clauss M, Maxeiner B, Georgopoulos S, Lesslauer W, Kollias G, Pfizenmaier K and Scheurich P (1995) Cell 83: 793-802

4. Schneider P, Holler N, Bodmer JL, Hahne M, Frei K, Fontana A and io Tschopp J (1998) J. Exp. Med. 187: 1205-1213

5. Wajant H, Moosmayer D, Wuest T, Bartke T, Gerlach E, Schonherr U, Peters N, Scheurich P and Pfizenmaier K (2001) Oncogene 20: 4101-4106

6. Haswell LE, Glennie MJ and Al-Shamkhani A (2001) Eur. J. Immunol, is 31:3094-31008

7. Holler N, Tardivel A, Kovacsovics-Bankowski M, Hertig S, Galde 0, Martinon F, Tinel A, Deperthes D, Calderara S, Schulthess T, Engel J, Schneider P and Tschopp J (2003) Mol. Cell. Biol. 23: 1428-1440

8. Stone GW, Barzee S, Snarsky V, Kee K, Spina CA, Yu XF and Kornbluth

RS (2006) J. Virol. 80: 1762-177216

9. Mundle SD and Raza A (2002) Trends Immunol. 23: 187-194

10. Siegel RM, Muppidi JR, Sarker M, Lobito A, Jen M, Martin D, Straus SE and Lenardo MJ (2004) J. Cell Biol. 167: 735-744

11. Henkler F, Behrle E, Dennehy KM, Wicovsky A, Peters N, Warnke C,

Pfizenmaier K and Wajant H (2005) J. Cell Biol. 168: 1087-1098

2017201232 06 Feb 2018

Claims (34)

1. A fusion protein comprising:

(i) TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or a receptor binding domain thereof, and (ii) a collectin trimerization domain comprising a neck domain or a neck and carbohydrate binding domain of surfactant protein D, wherein (ii) is located C-terminally of (i).

2. The fusion protein of claim 1, additionally comprising a flexible linker between (i) and (ii), wherein the flexible linker is a glycine/serine linker and has a length of 3-20 amino acids.

3. The fusion protein of claim 2, wherein the flexible linker element has the amino acid sequence of SEQ ID NO: 53, which is (GSS)_a(SSG)_b(GSG)_c wherein a, b, c is each 0, 1, 2, 3, 4, 5 or 6.

4. The fusion protein of claim 2, wherein the flexible linker element has a length of 9-15 amino acids.

5. The fusion protein of any one of claims 1 to 4, wherein the TRAIL has the sequence of SEQ ID NO: 10.

6. The fusion protein of any one of claims 1 to 4, wherein (i) comprises amino acids 95-281, 116-281, 117-281, 118-281, 119-281 or 120-281 of human TRAIL (SEQ ID NO :10).

7. The fusion protein of any one of claims 1 to 4, wherein (i) comprises a mutant of SEQ ID NO: 10 or a receptor binding domain thereof, which binds (14173247_1):GGG

2017201232 06 Feb 2018 and/or activates TRAIL-receptor 1 and/or TRAIL-receptor 2, wherein the mutant comprises amino acid substitutions only in one or more of the following amino acid positions of SEQ ID NO:10: R130, G160, Y189, R191, Q193, E195, N199, K201, Y213, T214, 5215, H264, 1266, D267, and D269.

8. The fusion protein of claim 7, wherein the amino acid substitutions are one or more of the following: R130E, G160M, Y189A, Y189Q, R191K, Q193S, Q193R, E195R, N199V, N199R, K201R, Y213W, T214R, S215D, H264R, 1266L, D267Q, D269H, D269R, and D269K.

9. The fusion protein of any one of claims 1 to 8, wherein (ii) comprises amino acids 217-375, 218-375, 219-375, 220-375, 221-375, 222-375, 223-375, 224-375, or 225-375 of human surfactant protein-D of SEQ ID NO:21.

10. The fusion protein of any one of claims 1 to 8, wherein (ii) comprises amino acids 217-257, 218-257, 219-257, 220-257, 221-257, 222-257, 223-257, 224-257, or 225-257 of human surfactant protein-D of SEQ ID NO:21.

11. The fusion protein of any one of claims 1 to 8, wherein (ii) comprises a mutant of human surfactant protein-D of SEQ ID NO:21 having only one amino acid substitution.

12. The fusion protein of claim 11, wherein the amino acid substitution is one of the following: F355A, F355S, F355T, F355E, F355D, F355K, or F355R.

13. The fusion protein of claim 11, wherein (ii) comprises a mutant which does not bind to mannose.

14. The fusion protein of any one of the preceding claims, which additionally comprises an N-terminal signal peptide domain.

(14173247_1):GGG

2017201232 06 Feb 2018

15. The fusion protein of claim 1, which comprises the sequence of SEQ ID NO: 43 or 44.

16. The fusion protein of any one of the preceding claims, wherein the fusion protein further comprises a recognition/purification domain located at the N-terminus or at the C-terminus.

17. The fusion protein of claim 16, wherein the recognition/purification domain is a Strep-tag or a poly His-domain.

18. The fusion protein of any one of the preceding claims, which additionally comprises a terminal flexible element.

19. A trimeric complex comprising three fusion proteins of any one of the preceding claims.

20. The trimeric complex of claim 19, wherein the complex is formed by covalent linkage between the three fusion proteins.

21. The trimeric complex of claim 19, wherein the complex consists of three identical fusion proteins.

22. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 18.

23. The nucleic acid molecule of claim 22, which is operatively linked to an expression control sequence.

24. A vector comprising the nucleic acid molecule of claim 22 or 23.

(14173247_1):GGG

2017201232 06 Feb 2018

25. An isolated cell transformed or transfected with the nucleic acid molecule of claim 22 or 23, or the vector of claim 24.

Apogenix AG

Patent Attorneys for the Applicant/Nominated Person

SPRUSON & FERGUSON (14173247_1):GGG

1/34

2017201232 23 Feb 2017

Figure 1

SEC of affinity purified CD95L-ASPD

I

I*

A1 -A11

I *· I

2/34

2017201232 23 Feb 2017

Figure 2

Silver gel of SEC fractions A1-A11 from affinity purified CD95L-ASPD

CD95L-ASPD (SEC Fractions A1 - A11)

2017201232 23 Feb 2017

3/34

Figure 3

Caspase activity on Jurkat cells induced by SEC fractions A1-A15 from affinity purified CD95L-ASPD

4/34

2017201232 23 Feb 2017

Figure 4

Cytotoxicity of CD95L-ASPD on WM35, HT1080 and HeLa cells

WM35

I I-1-1-1-1-1—

0.01 0.1 1 10 100 1000

CD95L-ASPD [ng/ml]

5/34

2017201232 23 Feb 2017

Figure 5

SEC of affinity purified LIGHT-ASPD

6.0 8.0 10.0 | 12.0 , 14.0 16.0 18.0 I ιΑ7-Α1θ!

20.0

22.0 ml

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Figure 6

Binding of HVEM-Fc to immobilized LIGHT-ASPD

LIGHT-ASPD [ng/ml]

2017201232 23 Feb 2017

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Figure 7

Western blot from transiently transfected HEK cells transiently transfected with TRAIL-constructs

TRAIL-ATRAIL-B-

TRAIL-C- TRAIL-D-

1. T4

2. 69

3. SPD

4. CCSPD

5. Col11

6. CC11

1. T4

2. 69

3. SPD

4. CCSPD

5. Col11

6. CC11

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2017201232 23 Feb 2017

Figure 8

Caspase activity in Jurkat T-cells

Trimerization motifs

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Figure 9

Size exclusion chromatography of TRAIL-ASPD

A1 -A14

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Figure 10

Cytotoxic activity of TRAIL-ASPD against human cancer cells

HT1080

TRAIL-ASPD [ng/ml]

WM35

TRAIL-ASPD [ng/ml]

He La

Colo205

0.1 1 10 100 1000 10000 TRAIL-ASPD [ng/ml]

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Figure 11

TRAIL-ASPD induced caspase activity in Jurkat

TRAIL-ASPD [ng/ml] anti-TRAIL [ng/ml]

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Figure 12

Cytotoxicity assay with TRAIL-ASPD or TRAIL-DSPD on HT1080 cells

Concentration [ng/ml]

2017201232 23 Feb 2017

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Figure 13

Western blot from transiently transfected HEK cells transiently transfected with TRAIL-SPD-constructs or TRAIL-receptor selective SPD constructs.

TRAILRlmutein

TRAILR2mutein

TRAIL wild type

Q ◄<SPD-constructs ccSPD-constructs

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Figure 14 ο

(Μ

2017201232 23 Feb

TRAIL-Receptor selective ligands (TRAILRImut and TRAILR2mut) immobilized on Streptactin plates, are differentially detected by TRAIL-Receptor 1-Fc or TRAILReceptor 2-Fc

TRAIL-Receptor 1 binding TRAIL-Receptor 2 binding

TRAILRI-Fc binding

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Figure 15

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Binding of TRAIL-Receptors to Receptor-selective “mutein“ ligands

TRAILR2-FC [nM]

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Figure 16

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Size exclusion chromatography of affinity purified TRAILR1mut-ASPD

A1-A14 -►'

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Figure 17

2017201232 23 Feb 2017

Silver stained SDS-PAGE of SEC fractions A1-A14 from affinity purified TRAILR1 mut-ASPD

TRAIL-R1 mut-ASPD (SEC fractions A1-A14) g TRAIL-R1 mut-ASP D (SEC fractions A1 -A14)

M 1 2 3 4 S 6 7 8 9 10 11 12 13 14

100

85 —

ΦΟβΒ,β® •x

40 30 —

25 — 20 15

W18/34

Figure 18

2017201232 23 Feb 2017

Caspase activity of SEC fractions A1-A14 from affinity purified TRAILR1mut-ASPD on Jurkat cells

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Figure 19

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Size exclusion chromatography of affinity purified TRAILR2mut-ASPD

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Figure 20

2017201232 23 Feb 2017

Silver stained SDS-PAGE of SEC fractions A1-A14 from affinity purified TRAILR2mut-ASPD

TRAIL-R2mut-ASPD (SEC fractions A1-A14)

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

TRAIL-R2mut-ASPD (SEC fractions A1-A14)

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Figure 21

Jurkat Kill Assay Jurkat of SEC fractions A1-A14 from affinity purified TRAILR2mut ASPD

SEC Fractions

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Figure 22

2017201232 23 Feb 2017

Cytotoxic activity of TRAIL-ASPD, TRAILR1mut-ASPD and TRAILR2mut-ASPD on human cancer cells.

HT1080

Concentration [ng/ml] C HeLa

Concentration [ng/ml]

Colo205

Concentration [ng/ml]

Colo205

Ό-TRAIL-ASPD -t-cross-linWng

-ώ-TRAJLRI mut-ASPD +cross-finkfng

-*-TRAILR2mut-ASPD +cross-BnWng

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Figure 23

Receptor selective TRA1L-SPD proteins are highly souble

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Figure 24

SEC of affinity purified TRAIL-ASPD_F335A

A1 -A13

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Figure 25

Silver stained SDS-PAGE of SEC fractions A1-A13

TRAIL-ASPD_F335A (SEC Fractions A1-A13)

M 1 2 3 4 5 6 7 8 9 10 11 1213

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Figure 26

Cytotoxic effect of TRAIL-ASPD_F335A on human cancer cells

2017201232 23 Feb 2017

TRAIL-ASPD P335A

HeLa

WM35

I I ¹ I i-1 ——|

0.1 1 10 100 1000 10000

TRAlL-ASPD_F335A

Colo205

0.1 1 10 100 1000 10000 TRAIL-ASPD F335A

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Figure 27

2017201232 23 Feb 2017

SEC of affinity purified TRAIL-ASPD_F335D

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Figure 28

Silver stained SDS-PAGE of SEC from affinity purified TRAIL-ASPD_F335D

TRAIL-ASPD F335D (SEC Fractions A1 -A13)

M 1 2 3 4 5 6 7 8 9 10 11 12 13

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Figure 29

Cytotoxic effect TRAIL-SPD_F335D on human cancer cells

2017201232 23 Feb 2017

A

HT1080

TRA1L-ASPDF335D [ng/ml]

TRAIL-ASPDF335D [ng/ml]

WM35

Colo205

TRAIL-ASPD_F335D [ng/ml]

0.1 1 10 100 1000 10000 TRAIL-ASPD_F335D [ng/ml]

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Figure 30

2017201232 23 Feb 2017

Binding of TRAIL-ASPD fusion protein to carbohydrates

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Figure 31

2017201232 23 Feb 2017

Pharmacokinetics ofTRAIL-ASPD (A) or TRAIL-ASPD_F335 D (B) Fusion Proteins

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Figure 32

2017201232 23 Feb 2017

Caspase activity in primary human hepatocytes

A 5h Incubation, no cross-linking —ώ—CD95L-AT4 -H-TRAIL-ASPD -*-7RAIL-ASPD_F335A -Θ—7RAIL-ASPDF335D

24h Incubation, no cross-linking

CD95L-AT4 •TRA1L-ASPD

TRAIL-ASPD_F335A

TRAIL-ASPD_F335D

24h Incubation, + cross-linking

E TRAIL-ASPDF335D + Chemo

5 h Incubation

250η

-H-+ Daxoniilcin -O-+ Gemcltabine

O—O—<5—O—O-Ο—O

-,-,-_r

0.01 0.1 1 10

Ligand concentration [ gg/nd]

F TRAlL-ASPD_F335D + Chemo

24h incubation

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Figure 33

2017201232 23 Feb 2017

Western Blot of supernatants from HEK293 cells transiently transfected with trimerized APRIL constructs

APRIL

- 1 2 3 4 M

Lane Construct 1 APRIL-A69 2 APRIL-ASPD 3 APRIL-ACCSPD 4 APRIL-ACoM 1

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Figure 34

2017201232 23 Feb 2017

TACI-Fc binds to APRIL-ASPD