HK1246315B - Cytokine fusion proteins - Google Patents

Description

Cytokine fusion proteins

Technical Field

The present invention relates to cytokine fusion proteins and nucleic acid molecules encoding such cytokine fusion proteins. The present invention also relates to cells, non-human organisms, pharmaceutical compositions and kits comprising the cytokine fusion proteins or nucleic acid molecules encoding the cytokine fusion proteins, and their use as medicines.

Background Art

Ligands of the tumor necrosis factor (TNF) superfamily play important roles in normal developmental processes, including apoptosis, regulation of immune cell function, and other cell-type-specific responses. They also play a crucial role in a variety of acquired and inherited diseases, including cancer and autoimmune diseases.

The TNF ligand family is characterized by a conserved extracellular C-terminal domain called the TNF homology domain (THD) (Bodmer, J.L. et al. (2002), TRENDS in Biochemical Sciences, 27(1): 19-26). The THD shares an almost identical tertiary fold and exhibits approximately 20% to 30% sequence identity between family members, is responsible for receptor binding, and interacts non-covalently to form a (homo-)trimeric complex that is subsequently recognized by its specific receptor. Although most ligands are synthesized as membrane-bound proteins, more particularly type II (i.e., intracellular N-terminus and extracellular C-terminus) transmembrane proteins, proteolytic cleavage of the extracellular domain containing the THD can produce soluble cytokines (Bodmer, J.L. et al. (2002), TRENDS in Biochemical Sciences, 27(1): 19-26).

One object of the present invention is to provide a multifunctional, particularly bifunctional or dual-acting cytokine fusion protein comprising at least two different cytokines. Another object of the present invention is to provide a nucleic acid molecule, particularly an RNA molecule, encoding such a cytokine fusion protein.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a cytokine fusion protein comprising (i) three extracellular domains of a first ligand of the tumor necrosis factor (TNF) superfamily, or a fragment or variant thereof, forming a first homotrimer, wherein the first homotrimer is capable of binding to a receptor for the first ligand; and (ii) three extracellular domains of a second ligand of the TNF superfamily, or a fragment or variant thereof, forming a second homotrimer, wherein the second homotrimer is capable of binding to a receptor for the second ligand, wherein the first ligand and the second ligand are different, and wherein the first homotrimer and the second homotrimer are covalently linked, preferably via one or more peptide linkers.

In one embodiment, the three extracellular domains of the first ligand or a fragment or variant thereof and/or the three extracellular domains of the second ligand or a fragment or variant thereof are covalently linked.

In one embodiment, the cytokine fusion protein comprises a molecule/structure having the following general formula:

N'-AL _A -AL _A -ALBL _B -BL _B -B-C' (Formula I),

wherein A comprises the extracellular domain of a first ligand, or a fragment or variant thereof, and B comprises the extracellular domain of a second ligand, or a fragment or variant thereof, and

wherein L comprises a peptide linker, and

_LA and _LB at each occurrence are independently selected from a covalent bond and a peptide linker.

In one embodiment, L further comprises a multimerization domain, preferably a dimerization domain, that allows for multimerization, preferably dimerization, of the cytokine fusion protein.

In one embodiment, the dimerization domain is selected from IgE heavy chain domain 2 (EHD2), IgM heavy chain domain 2 (MHD2), IgG heavy chain domain 3 (GHD3), IgA heavy chain domain 3 (AHD2), IgD heavy chain domain 3 (DHD3), IgE heavy chain domain 4 (EHD4), IgM heavy chain domain 4 (MHD4), Fc domain, uteroglobin dimerization domain, and functional variants of any one of the foregoing.

In one embodiment, the cytokine fusion protein exists as a multimeric complex, preferably a dimeric complex.

In another embodiment, the cytokine fusion protein comprises at least one, preferably three, subunits having the following general formula:

N'-A-L-B-C' (Formula II),

wherein A comprises the extracellular domain of a first ligand, or a fragment or variant thereof, and B comprises the extracellular domain of a second ligand, or a fragment or variant thereof,

wherein L comprises a peptide linker, and

Among them, preferably, three subunits form the cytokine fusion protein.

In another aspect, the present invention relates to a cytokine fusion protein comprising a first segment comprising three covalently linked extracellular domains of a first ligand of the tumor necrosis factor (TNF) superfamily, or a fragment or variant thereof, and a second segment comprising three covalently linked extracellular domains of a second ligand of the TNF superfamily, or a fragment or variant thereof, wherein the first ligand and the second ligand are different, and wherein the first segment is covalently linked to the second segment.

In one embodiment, the three extracellular domains of the first ligand, or fragments or variants thereof, form a first homotrimer capable of binding to a receptor for the first ligand, and the three extracellular domains of the second ligand, or fragments or variants thereof, form a second homotrimer capable of binding to a receptor for the second ligand.

In one embodiment, the three extracellular domains of the first ligand and/or the three extracellular domains of the second ligand and/or the first segment and the second segment are covalently linked via a peptide linker.

In one embodiment, the cytokine fusion protein comprises a molecule/structure having the following general formula:

N'-AL _A -AL _A -ALBL _B -BL _B -B-C' (Formula I),

wherein A comprises the extracellular domain of a first ligand, or a fragment or variant thereof, and B comprises the extracellular domain of a second ligand, or a fragment or variant thereof, and

wherein L comprises a peptide linker, and

_LA and _LB at each occurrence are independently selected from a covalent bond and a peptide linker.

In one embodiment, L further comprises a multimerization domain, preferably a dimerization domain, that allows for multimerization, preferably dimerization, of the cytokine fusion protein.

In one embodiment, the dimerization domain is selected from IgE heavy chain domain 2 (EHD2), IgM heavy chain domain 2 (MHD2), IgG heavy chain domain 3 (GHD3), IgA heavy chain domain 3 (AHD2), IgD heavy chain domain 3 (DHD3), IgE heavy chain domain 4 (EHD4), IgM heavy chain domain 4 (MHD4), an Fc domain, a uteroglobin dimerization domain, and a functional variant of any one of the foregoing.

In one embodiment, the cytokine fusion protein exists as a multimeric complex, preferably a dimeric complex.

In another aspect, the present invention relates to a cytokine fusion protein comprising the extracellular domain of a first ligand of the tumor necrosis factor (TNF) superfamily, or a fragment or variant thereof, and the extracellular domain of a second ligand of the TNF superfamily, or a fragment or variant thereof, wherein the first ligand and the second ligand are different, and wherein the extracellular domains or fragments or variants thereof are covalently linked.

In one embodiment, the extracellular domains are covalently linked via a peptide linker.

In one embodiment, the cytokine fusion protein comprises a molecule/structure having the following general formula:

N'-A-L-B-C' (Formula II),

wherein A comprises the extracellular domain of a first ligand, or a fragment or variant thereof, and B comprises the extracellular domain of a second ligand, or a fragment or variant thereof, and

wherein L comprises a peptide linker.

In one embodiment, the cytokine fusion protein exists as a trimeric complex in which the three extracellular domains of a first ligand, or a fragment or variant thereof, form a first homotrimer capable of binding to a receptor for the first ligand, and the three extracellular domains of a second ligand, or a fragment or variant thereof, form a second homotrimer capable of binding to a receptor for the second ligand.

According to the present invention, the first ligand and the second ligand mentioned herein are preferably selected from CD40L, CD27L, 4-1BBL, OX40L, APRIL, CD30L, EDA-A1, EDA-A2, FasL, GITRL, LIGHT, LT-α, TL1A, TNF-α, TRAIL, RANKL and TWEAK, more preferably selected from CD40L, CD27L, 4-1BBL and OX40L.

In one embodiment,

- the first ligand is CD40L and the second ligand is CD27L;

- the first ligand is CD27L and the second ligand is CD40L;

- the first ligand is CD40L and the second ligand is 4-1BBL;

- the first ligand is 4-1BBL and the second ligand is CD40L;

- the first ligand is CD27L and the second ligand is 4-1BBL;

- the first ligand is 4-1BBL and the second ligand is CD27L;

- the first ligand is CD40L, and the second ligand is OX40L;

- the first ligand is OX40L, and the second ligand is CD40L;

- the first ligand is CD27L and the second ligand is OX40L;

- the first ligand is OX40L and the second ligand is CD27L;

- the first ligand is OX40L and the second ligand is 4-1BBL; or

- The first ligand is 4-1BBL and the second ligand is OX40L.

In one embodiment, the extracellular domain of CD40L comprises or consists of amino acid residues 51 to 261 or 116 to 261 of SEQ ID NO: 1, the extracellular domain of CD27L comprises or consists of amino acid residues 52 to 193 of SEQ ID NO: 2, the extracellular domain of 4-1BBL comprises or consists of amino acid residues 71 to 254 of SEQ ID NO: 3, and/or the extracellular domain of OX40L comprises or consists of amino acid residues 51 to 183 of SEQ ID NO: 4.

In one embodiment, the cytokine fusion protein further comprises at least one label or tag that allows for detection and/or isolation of the cytokine fusion protein.

In one embodiment, the cytokine fusion protein further comprises one or more modifications that increase the stability of the cytokine fusion protein.

In another aspect, the present invention relates to a nucleic acid molecule encoding a cytokine fusion protein as defined above or a subunit thereof.

In one embodiment, the nucleic acid molecule is operably linked to an expression control sequence.

In one embodiment, the nucleic acid molecule is contained in a vector.

In one embodiment, the nucleic acid molecule is an RNA molecule, preferably an in vitro transcribed (IVT) RNA molecule.

In another aspect, the invention relates to a cell transformed or transfected with a nucleic acid molecule as defined above.

In one embodiment, the cell is a prokaryotic cell.

In one embodiment, the cell is a eukaryotic cell, preferably a mammalian cell, more preferably a human cell.

In another aspect, the present invention relates to a non-human organism transformed or transfected with a nucleic acid molecule as defined above.

In another aspect, the present invention relates to a pharmaceutical composition comprising as active agent a cytokine fusion protein as defined above, a nucleic acid molecule as defined above or a cell as defined above.

In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In another aspect, the present invention relates to a kit comprising a cytokine fusion protein as defined above, a nucleic acid molecule as defined above, a cell as defined above or a pharmaceutical composition as defined above.

In another aspect, the present invention relates to a cytokine fusion protein as defined above, a nucleic acid molecule as defined above, a cell as defined above or a pharmaceutical composition as defined above for use as a medicament.

In another aspect, the present invention relates to a cytokine fusion protein as defined above, a nucleic acid molecule as defined above, a cell as defined above or a pharmaceutical composition as defined above for use in treating a disease selected from the group consisting of cancer, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases and transplant rejection.

In another aspect, the present invention relates to the use of the cytokine fusion protein as defined above, the nucleic acid molecule as defined above, the cell as defined above or the pharmaceutical composition in the preparation of a medicament for treating a disease selected from the group consisting of cancer, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases and transplant rejection.

In another aspect, the present invention relates to a method for treating a disease selected from the group consisting of cancer, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases and transplant rejection, the method comprising administering an effective amount of a cytokine fusion protein as defined above, a nucleic acid molecule as defined above, a cell as defined above or a pharmaceutical composition as defined above to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic combination of Duokine, scDuokine, and EHD2-scDuokine.

Figure 2. SDS-PAGE analysis of purified Duokine using 12% polyacrylamide gel under reducing and non-reducing conditions (1, CD40L-CD27L; 2, CD27L-CD40L; 3, CD40L-4-1BBL; 4, 4-1BBL-CD40L; 5, CD27L-4-1BBL; 6, 4-1BBL-CD27L; 7, CD40L-OX40L; 8, OX40L-CD40L; 9, CD27L-OX40L; 10, OX40L-CD27L; 11, 4-1BBL-OX40L; 12, OX40L-4-1BBL). Proteins were visualized by staining with Coomassie Brilliant Blue G250.

Figure 3 A and B. Duokine size exclusion chromatography (SEC) analysis shows the integrity of the fusion protein. High performance liquid chromatography (HPLC) was performed using a YarraSEC-2000 (Phenomenex) at a flow rate of 0.5 mL/min. Thyroglobulin, alcohol dehydrogenase, bovine serum albumin, carbonic anhydrase, and FLAG peptide were used as standard proteins.

Figure 4. Binding of Duokine (100 nM) to immobilized CD40-, CD27-, 4-1BB-, and OX40-Fc fusion proteins in ELISA. All Duokines bound to the corresponding receptor Fc fusion proteins, and no cross-reactivity was detected.

Figures 5A and B. Binding of Duokine to immobilized CD40-, CD27-, 4-1BB-, and OX40-Fc fusion proteins in ELISA (n=3±SD). Duokine was titrated in duplicate starting at a concentration of 316 nM. All Duokines bound to the corresponding receptor-Fc fusion proteins in a dose-dependent manner with _EC50 values in the low nanomolar range. Protein concentrations were based on the trimeric molecule.

Figure 6A and B. Binding of Duokine (100 nM) to HT1080 cells expressing CD40, CD27, 4-1BB, and OX40 analyzed by flow cytometry. Bound Duokine was detected using PE-labeled anti-FLAG antibody (grey, cells alone; thin line, cells incubated with PE-labeled anti-FLAG antibody; thick line, cells incubated with Duokine).

Figure 7A and B. Duokine's bispecificity was analyzed by flow cytometry. After Duokine (100 nM) was bound to HT1080 cells expressing CD40, CD27, 4-1BB, and OX40, Duokine was detected using the corresponding receptor Fc fusion protein (10 nM) and PE-labeled anti-human Fc antibodies. TNFR1-Fc was included as a negative control (gray, cells incubated with PE-labeled anti-human Fc antibodies; thin lines, cells incubated with Duokine and TNFR1-Fc; thick lines, cells incubated with CD40-, CD27-, 4-1BB-, or OX40-Fc).

Figure 8. SDS-PAGE analysis of purified single-chain Duokine using 10% polyacrylamide gel under reducing and non-reducing conditions (1, scCD40L-scCD27L; 2, scCD27L-scCD40L; 3, scCD40L-sc4-1BBL; 4, sc4-1BBL-scCD40L; 5, scCD27L-sc4-1BBL; 6, sc4-1BBL-scCD27L; 7, scCD40L-scOX40L; 8, scOX40L-scCD40L; 9, scCD27L-scOX40L; 10, scOX40L-scCD27L; 11, sc4-1BBL-scOX40L; 12, scOX40L-sc4-1BBL). Proteins were visualized by staining with Coomassie Brilliant Blue G250.

Figure 9 A and B. Size exclusion chromatography (SEC) analysis of single-chain Duokine demonstrates the integrity of the fusion protein. High performance liquid chromatography (HPLC) was performed using a Yarra SEC-2000 (Phenomenex) at a flow rate of 0.5 mL/min. Thyroglobulin, alcohol dehydrogenase, bovine serum albumin, carbonic anhydrase, and FLAG peptide were used as standard proteins.

Figure 10. Binding of single-chain Duokine (100 nM) to immobilized CD40-, CD27-, 4-1BB-, and OX40-Fc fusion proteins in ELISA (n=3±SD). All single-chain Duokines bound to the corresponding receptor Fc fusion proteins, and no cross-reactivity was detected.

Figures 11A and B. Binding of single-chain Duokines to immobilized CD40-, CD27-, 4-1BB-, and OX40-Fc fusion proteins in ELISA (n=3±SD). Duokines were titrated in duplicate starting at a concentration of 316 nM. All single-chain Duokines bound to the corresponding receptor Fc fusion proteins in a dose-dependent manner with _EC50 values in the low nanomolar range.

Figure 12A and B. Binding of single-chain Duokine (100 nM) to HT1080 cells expressing CD40, CD27, 4-1BB, and OX40 analyzed by flow cytometry. Bound single-chain Duokine was detected with PE-labeled anti-FLAG antibody (grey, cells only; thin line, cells incubated with PE-labeled anti-FLAG antibody; thick line, cells incubated with single-chain Duokine).

Figure 13A and B. Analysis of the bispecificity of single-chain Duokine by flow cytometry. After single-chain Duokine (100nM) was bound to HT1080 cells expressing CD40, CD27, 4-1BB, and OX40, single-chain Duokine was detected using the corresponding receptor Fc fusion protein (10nM) and PE-labeled anti-human Fc antibodies. TNFR1-Fc was included as a negative control (gray, cells incubated with PE-labeled anti-human Fc antibodies; thin lines, cells incubated with single-chain Duokine and TNFR1-Fc; thick lines, cells incubated with single-chain Duokine and CD40-, CD27-, 4-1BB-, or OX40-Fc).

Figure 14. SDS-PAGE analysis of purified single-chain Duokine using 4-15% polyacrylamide gel under reducing and non-reducing conditions (1, sc4-1BBL-EHD2-scCD40L; 2, sc4-1BBL-EHD2-scCD27L; 3, scCD40L-scCD27L). Proteins were visualized by staining with Coomassie Brilliant Blue G250.

Figure 15. Size exclusion chromatography (SEC) analysis of EHD2-scDuokine demonstrates the integrity of the fusion protein. High performance liquid chromatography (HPLC) was performed using a Yarra SEC-2000 (Pheuomenex) at a flow rate of 0.5 mL/min. Thyroglobulin, alcohol dehydrogenase, bovine serum albumin, carbonic anhydrase, and FLAG peptide were used as standard proteins.

Figure 16. Binding of EHD2-scDuokine (100 nM) to immobilized CD40-, CD27-, 4-1BB-, and OX40-Fc fusion proteins in ELISA. All EHD2-scDuokines bound to the corresponding receptor Fc fusion proteins, and no cross-reactivity was detected.

Figure 17. Binding of EHD2-linked single-chain Duokines to immobilized CD40-, CD27-, 4-1BB-, and OX40-Fc fusion proteins in ELISA (n=3±SD). EHD2-scDuokine was titrated in duplicate starting at a concentration of 316 nM. All EHD2-scDuokines bound to the corresponding receptor-Fc fusion proteins in a dose-dependent manner with _EC50 values in the low nanomolar range.

Figure 18. Flow cytometry analysis of EHD2-scDuokine (100 nM) binding to HT1080 cells expressing CD40, CD27, and 4-1BB. Bound EHD2-scDuokine was detected using a PE-labeled anti-FLAG antibody (grey, cells alone; thin line, cells incubated with a PE-labeled anti-FLAG antibody; thick line, cells incubated with EHD2-scDuokine).

Figure 19. Analysis of the bispecificity of EHD2-scDuokine by flow cytometry. Following binding of EHD2-scDuokine (100 nM) to HT1080 cells expressing CD40, CD27, 4-1BB, and OX40, EHD2-scDuokine was detected using the corresponding receptor-Fc fusion proteins (10 nM) and a PE-labeled anti-human Fc antibody. TNFR1-Fc was included as a negative control (grey, cells incubated with PE-labeled anti-human Fc antibody; thin line, cells incubated with EHD2-scDuokine and TNFR1-Fc; thick line, cells incubated with EHD2-scDuokine and CD40-, CD27-, 4-1BB-, or OX40-Fc).

Figure 20. Receptor activation of EHD2-scDuokine analyzed by IL-8 release from HT1080 cells expressing CD40 and 4-1BB (n=3±SD) or from HT1080 cells expressing CD27 (n=1±SD). 2×10 ⁴ HT1080 cells were incubated with serially diluted EHD2-scDuokine, and the amount of IL-8 in the supernatant was measured by ELISA after 18 hours of incubation. Monomeric ligands and their single-chain derivatives were included as controls.

Figure 21. Proliferation of T cells after stimulation with Duokine or single-chain Duokine. 1.5 x 10 ⁵ CFSE-stained human PBMCs (population) were incubated with serially diluted Duokine or single-chain Duokine in the presence of a cross-linked anti-human CD3 antibody as an initial suboptimal stimulator. After 6 days, T cell proliferation was assessed by flow cytometry.

Figure 22. Vector design for in vitro transcription of mRNA encoding the extracellular domain of TNFR ligands and their fusion proteins. The plasmid construct pST1-hAg-Kozak-sec(opt)-INSERT-2hBgUTR-A120 was used as a template for in vitro transcription of RNA encoding TNF receptor (TNFR) ligands and their fusion proteins. The extracellular domain of TNFR ligands encoded by the INSERT is a functionally active homotrimer. Two different coding sequences for those TNF receptor ligands were generated: (i) an insert containing a single extracellular domain of the TNF receptor, thereby encoding a non-covalently bound trimer; and (ii) a single-chain construct containing three copies of the extracellular domain separated by a short linker domain (encoded amino _acids : _G3SG3 ), thereby encoding a covalently bound trimer. The two TNFR ligands were linked via a linker to generate their fusion proteins. Nomenclature: Constructs encoding the human protein sequence are abbreviated as "h," and the murine protein sequence is abbreviated as "m." The numbers "1" and "3" indicate the number of copies of the encoded TNFR ligand extracellular domain.

Figure 23. Intracellular expression of fusion proteins after IVT-RNA electroporation. K562 cells were electroporated with IVT-RNA encoding the extracellular domain of a TNFR ligand or its fusion protein. Six hours after electroporation, protein export was blocked using GolgiPlug and GolgiSTOP, and after 12 hours of incubation, cells were intracellularly stained for CD27L, CD40L, OX40L, or 4-1BBL. As a negative control, K562 cells were electroporated without RNA (MOCK) and stained as appropriate. (A) Intracellular staining of TNFR ligands after electroporation of the h3_CD40L-h3_CD27L construct compared to the h3_CD27L and h3_CD40L constructs alone. (B) Intracellular staining of TNFR ligands after electroporation of the h3_CD40L-h3_OX40L construct compared to the h3_CD40L and h3_CD40L constructs alone. (C) Intracellular staining of TNFR ligands after electroporation of h3_4-1BBL-h3_CD27L construct compared to h3_CD27L and h3_4-1BBL single constructs. (D) Intracellular staining of TNFR ligands after electroporation of h1_CD40L-h1_CD27L, h1_4-1BBL-h1_CD40L and h1_4-1BBL-h1_CD27L constructs.

Figure 24. Cell surface expression of TNF receptors on stable HT1080 and K562 transfectants after transient transfection. (A) The TNF receptor transfectants of stable HT1080 were stained with anti-CD27-PE, anti-CD40-FITC, anti-4-1BB-PE and anti-OX40-PE. (B) K562 cells were electroporated with plasmids encoding the full-length coding sequences of human CD27, CD40, 4-1BB and OX40. One day after electroporation, cells were stained with anti-CD27-PE, anti-CD40-FITC, anti-4-1BB-PE and anti-OX40-PE.

Figure 25. Enhanced TNF receptor activation by TNFL(1)-TNFL(2) fusion constructs in a trans-presentation setting. K562 cells were electroporated in multiwell electroporation plates (96 wells) with varying amounts of IVT-RNA encoding the extracellular domain of a TNFR ligand or its fusion protein. RNA amounts are expressed as pmol RNA relative to the corresponding encoded protein. After overnight incubation, the supernatant was transferred to confluent cell layers of two corresponding stable TNF receptor transfectants of the HT1080 cell line (see Figure 24A). K562 cells electroporated the previous day with MOCK (as a control) or with the corresponding TNF receptor plasmid were added to the confluent cell layers and supernatant of the HT1080-TNFR transfectants to create a trans-presentation setting for the fusion protein (cell to cell transactivation). After 8 hours of co-incubation, cell-free supernatants were collected and the concentration of IL-8 released by HT1080 cells following TNF receptor-dependent NF-κB activation was measured. (A) IL-8 release from K562 cells electroporated with IVT-RNA encoding h3_CD27L-h3_CD40L upon activation of HT1080_CD40 following incubation with supernatants is shown. h3_CD40L single constructs and the fusion construct h3_CD27L-h3_CD40L (+K562_MOCK) without trans-presentation elicited IL-8 secretion following electroporation of at least 1 pmol of RNA relative to the encoded protein. Under conditions of trans-presentation mediated by K562_CD27, the fusion construct h3_CD27L-h3_CD40L induced CD40 activation to the same extent as an approximately 100-fold lower amount of IVT-RNA relative to the encoded protein. (B) CD27 activation was not detected by measuring IL-8 secretion after electroporation of IVT-RNA encoding h3_CD27L or h3_CD27L-h3_CD40L without trans-presentation. CD27 activation was detected after K562 electroporation of the h3_CD27L-h3_CD40L fusion construct in the case of trans-presentation by K562-CD40. (C) IL-8 release caused by activation of HT1080-OX40 after incubation with supernatant of IVT-RNA encoding h3_CD27L-h3_OX40L is shown. h3_OX40L alone and the fusion construct h3_CD27L-h3_OX40L (+K562_MOCK) without trans-presentation produced IL-8 secretion after electroporation of approximately 1 pmol RNA relative to the encoded protein and more. Under K562-CD27-mediated trans-presentation conditions, the fusion construct h3_CD27L-h3_OX40L induced CD40 activation to the same extent as the approximately 10-fold lower amount of IVT-RNA relative to the encoded protein. (D) CD27 activation was not detected by measuring IL-8 secretion after electroporation of IVT-RNA encoding h3_CD27L or h3_CD27L-h3_OX40L in the absence of trans-presentation conditions. In the case of trans-presentation via K562-OX40, CD27 activation was detected after K562 electroporation of the h3_CD27L-h3_CD40L RNA construct. (E) IL-8 concentrations produced by activation of HT1080-CD27 after incubation with supernatant of IVT-RNA encoding h3_CD27L-h3_4-1BBL are shown. H3_CD27L single construct and the fusion construct h3_CD27L-h3_4-1BBL (+K562_MOCK) without trans presentation do not induce IL-8 secretion.Under the trans presentation conditions mediated by K562_4-1BB, fusion construct h3_CD27L-h3_4-1BBL induces the activation of CD27. (F) h3_4-1BBL single construct and the fusion construct h3_CD27L-h3_4-1BBL (+K562_MOCK) without trans presentation do not induce IL-8 secretion.Under the trans presentation conditions mediated by K562_CD27, fusion construct h3_CD27L-h3_4-1BBL induces the activation of 4-1BB. (G) Shows IL-8 release from K562 cells electroporated with IVT-RNA encoding h3_41BBL-h3_CD40L and h1_41BBL-h1_CD40L upon activation of HT1080_CD40 following incubation with supernatant. h3_CD40L alone and two fusion constructs without trans-presentation (+K562_MOCK) elicited IL-8 secretion after electroporation of at least 1 pmol of RNA relative to the encoded protein. Under K562_41BB-mediated trans-presentation conditions, both fusion constructs, h3_41BBL-h3_CD40L and h1_41BBL-h1_CD40L, induced CD40 activation to the same extent as approximately 10-fold less IVT-RNA relative to the encoded protein. (H) h3_4-1BBL single construct and two fusion constructs h3_41BBL-h3_CD40L and h1_41BBL-h1_CD40L (+K562_MOCK) without trans presentation do not induce IL-8 secretion. Under trans presentation conditions mediated by K562_CD40, these two fusion constructs induce the activation of 4-1BB to the same extent.

Figure 26. Effect of h3_CD27L-h3_CD40L and h3_CD40L-h3_CD27L fusion constructs on CD8 ⁺ T cell proliferation. iDCs were electroporated with claudin-6 IVT-RNA + IVT-RNA encoding h3_CD27L-h3_CD40L, h3_CD40L-h3_CD27L, and the single construct h3_CD27L + h3_CD40L, or control RNA. CD8+ T cells (HLA-A2 ⁺ donors) were electroporated with IVT-RNA encoding either claudin-6-specific CD8 ⁺ T cell receptor or TPTE-specific CD8 ⁺ T cell receptor, followed by CFSE staining. Electroporated iDCs and CD8 ⁺ T cells were co-cultured at a 1:10 ratio for 5 days before FACS analysis of CD8 ⁺ T cell proliferation. Representative histograms of CFSE analysis of claudin-6-TCR ⁺ CD8 ⁺ T cells are shown in (A) and those of TPTE-TCR ⁺ CD8 ⁺ T cells are shown in (B). Detailed analysis of proliferation was performed by FlowJo software based on the peaks representing cell division. In this way, the percentage of T cells that entered division was calculated, expressed as "% dividing cells"; and the average number of divisions of cells that entered division, expressed as "proliferation index", were calculated, both of which are shown in (C). Administration of both h3_CD27L-h3_CD40L and h3_CD40L-h3_CD27L fusion constructs resulted in increased proliferation of CD8 ⁺ T cells in an antigen-specific manner, while administration of two RNAs encoding two corresponding TNFR ligands had no effect on proliferation.

Figure 27. Effect of h3_4-1BBL-h3_CD27L and h1_4-1BBL-h1_CD27L fusion constructs on CD8 ⁺ T cell proliferation. iDCs were electroporated with claudin-6 IVT-RNA + IVT-RNA encoding h3_4-1BBL-h3_CD27L, h1_4-1BBL-h1_CD27L, and single constructs h3_CD27L + h3_4-1BBL, or control RNA. CD8+ T cells (HLA-A2 ⁺ donor) were electroporated with IVT-RNA encoding claudin-6-specific CD8 ⁺ T cell receptor or TPTE-specific CD8 ⁺ T cell receptor, followed by CFSE staining. Electroporated iDCs and CD8 ⁺ T cells were co-cultured at a ratio of 1:10 for 5 days before FACS analysis of CD8 ⁺ T cell proliferation. Representative histograms of CFSE analysis of claudin-6-TCR ⁺ CD8 ⁺ T cells are shown in (A), and those of TPTE-TCR ⁺ CD8 ⁺ T cells are shown in (B). Detailed analysis of proliferation was performed by FlowJo software based on the peaks representing cell division. In this way, the percentage of T cells that entered division was calculated, expressed as "% dividing cells"; and the average number of divisions of cells that entered division, expressed as "proliferation index", are shown in (C). Administration of both h3_4-1BBL-h3_CD27L and h1_4-1BBL-h1_CD27L fusion constructs resulted in increased proliferation of CD8 ⁺ T cells in an antigen-specific manner, while administration of two RNAs encoding two corresponding TNFR ligands had no effect on proliferation.

Figure 28. Effect of recombinant Duookine on CD8 ⁺ T cell proliferation. iDC was electroporated with claudin-6 IVT-RNA. CD8 ⁺ T cells (HLA-A2 ⁺ donor) were electroporated with IVT-RNA encoding claudin-6 specific CD8 ⁺ T cell receptor and then stained with CFSE. One day after electroporation, iDC and CD8 ⁺ T cells were co-cultured for 4 days at a ratio of 1:10; 10 nM of each of the indicated recombinant proteins was added to the co-culture. CD8 ⁺ T cell proliferation was analyzed by FACS. Representative histograms of claudin-6-TCR ⁺ CD8 ⁺ T cell CFSE analysis are shown in (A). Detailed analysis of proliferation was performed by FlowJo software based on the peak indicating cell division. In this way, the percentage of T cells that entered division was calculated, expressed as "% dividing cells"; and the average number of divisions of cells that entered division was expressed as "proliferation index", both of which are shown in (B) and (C), respectively. Addition of all three Duoks resulted in enhanced proliferation of CD8 ⁺ T cells in an antigen-specific manner, whereas addition of the two corresponding individual TNFR ligands had no effect on proliferation.

Figure 29. Simultaneous binding of Duokine to immobilized receptors and PBMCs causes activation and proliferation of T cells. 200 ng/well receptor Fc was immobilized overnight at 4°C on microtiter plates. Residual binding sites were blocked with RPMI 1640 + 10% FCS for 1 hour. Serial dilutions of Duokine were incubated with immobilized receptors for 1 hour, followed by washing away unbound proteins. 1.5 x 10 ⁵ CFSE-stained human PBMCs (population) were added to microtiter plates in the presence (or absence) of a cross-linked anti-human CD3 antibody as an initial suboptimal stimulator. After 6 days, the proliferation of CD4 ⁺ and CD8 ⁺ T cells was assessed by flow cytometry using CFSE dilution.

Figure 30. Simultaneous binding of single-chain Duokine to immobilized receptors and PBMCs leads to activation and proliferation of T cells. 200 ng/well receptor Fc was immobilized overnight at 4°C on microtiter plates. Residual binding sites were blocked with RPMI 1640 + 10% FCS for 1 hour. Serial dilutions of scDuokine were incubated with immobilized receptors for 1 hour and then unbound proteins were washed away. 1.5 x 10 ⁵ CFSE-stained human PBMCs (population) were added to microtiter plates in the presence (or absence) of a cross-linked anti-human CD3 antibody as an initial suboptimal stimulator. After 6 days, the proliferation of CD4 and CD8 T cells was assessed by flow cytometry based on CFSE dilution.

Figure 31. Stability of selected Duokines and single-chain Duokines in human plasma. 200 nM (functional TNF ligand unit) purified Duokines and single-chain Duokines were prepared in 50% human plasma. Samples were frozen at -20°C immediately after preparation (day 0) or after incubation at 37°C for 1 day, 3 days, and 7 days. The level of intact protein was determined by ELISA through binding of the C-terminal homotrimeric ligand unit to immobilized receptor (150 ng/well) and detection of the N-terminal FLAG tag. The protein concentration in the diluted plasma samples was interpolated from the standard curve of the purified protein. The amount of fusion protein detected on day 0 was set to 100%.

Figure 32. Pharmacokinetic properties of selected murine duokines and single-chain duokines in CD1 mice. 25 μg of purified protein was injected into the tail vein of female CD1 mice (12 to 16 weeks, 30 to 35 g, 3 mice per construct) in a total volume of 150 μl. Blood samples were collected at 3 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 1 day, and 3 days after injection, incubated on ice for 30 minutes, and centrifuged at 13,000 g for 30 minutes at 4°C. Serum samples were stored at -20°C. Serum levels of the fusion proteins were determined by ELISA based on binding to immobilized receptors corresponding to the C-terminal ligand (150 ng/well) and detection via the N-terminal FLAG tag. Serum concentrations of all proteins were obtained by interpolation from a standard curve of purified proteins. For comparison, the concentration at 3 minutes was set to 100%. Initial and terminal half-lives (t _1/2 α _{3-60 minutes} , t _1/2 β _{1-24 hours} ) and AUC were calculated using Excel.

Figure 33. Receptor expression on human PBMC and the binding of single-chain Duokine to immune cell subsets. 2.5 × 10 ⁵ human PBMCs (populations) were incubated with 10 nM single-chain Duokine in the presence or absence of cross-linked anti-human CD3 antibodies as initial suboptimal stimulants. After 3 days at 37 ° C, different subpopulations were identified by CD marker staining (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD20, and anti-CD56) using flow cytometry, and the binding of single-chain Duokine to different subpopulations was assessed by detecting its FLAG tag. In addition, stimulated and unstimulated PBMCs were also incubated without single-chain Duokine, and subpopulations were identified after 3 days of cultivation and the surface expression of CD40, CD27, 4-1BB, and OX40 was determined by antibody staining.

Figure 34. Binding of cis-acting single-chain duokine to human immune cells and induction of T cell proliferation. 2.5×10 ⁵ human PBMCs (populations) were incubated with 10 nM sc4-1BBL-scCD27L in the presence or absence of cross-linked anti-human CD3 antibodies as initial suboptimal stimulants. After 3 days at 37°C, different subpopulations were identified by flow cytometry using CD marker staining (anti-CD3, anti-CD4, anti-CD8, anti-CD20, and anti-CD56), surface expression of CD27 and 4-1BB was determined by antibody staining, and the binding of single-chain duokine was assessed by detecting its FLAG tag. 1.5×10 ⁵ CFSE-labeled PBMCs (populations, different PBMC batches) were incubated with 30, 3, 0.3, or 0 nM sc4-1BBL-scCD27L in the presence or absence of cross-linked anti-human CD3 antibodies as initial suboptimal stimulants. After 6 days, the proliferation of CD4 and CD8 T cells was determined by flow cytometry using CFSE dilution.

Figure 35. Binding of trans-acting single-chain duokine to human immune cells and induction of T cell proliferation. 2.5×10 ⁵ human PBMCs (population) were incubated with 10 nM sc4-1BBL-scCD40L in the presence or absence of a cross-linked anti-human CD3 antibody as an initial suboptimal stimulus. After 3 days at 37°C, flow cytometry was used to identify different subpopulations by CD marker staining (anti-CD3, anti-CD4, anti-CD8, anti-CD20, and anti-CD56), surface expression of CD40 and 4-1BB was determined by antibody staining, and binding of single-chain duokine was assessed by detecting its FLAG tag. 1.5×10 ⁵ CFSE-labeled PBMCs (population, different PBMC batches) were incubated with 30, 3, 0.3, or 0 nM sc4-1BBL-scCD40L in the presence or absence of a cross-linked anti-human CD3 antibody as an initial suboptimal stimulus. After 6 days, the proliferation of CD4 and CD8 T cells was determined by flow cytometry using CFSE dilution.

Figure 36. Binding of trans-acting single-chain duokine to human immune cells and induction of T cell proliferation. 2.5×10 ⁵ human PBMCs (population) were incubated with 10 nM scCD40L-scCD27L in the presence or absence of a cross-linked anti-human CD3 antibody as an initial suboptimal stimulus. After 3 days at 37°C, flow cytometry was used to identify different subpopulations by CD marker staining (anti-CD3, anti-CD4, anti-CD8, anti-CD20, and anti-CD56), surface expression of CD40 and CD27 was determined by antibody staining, and the binding of single-chain duokine was assessed by detecting its FLAG tag. 1.5×10 ⁵ CFSE-labeled PBMCs (population, different PBMC batches) were incubated with 30, 3, 0.3, or 0 nM scCD40L-scCD27L in the presence or absence of a cross-linked anti-human CD3 antibody as an initial suboptimal stimulus. After 6 days, the proliferation of CD4 and CD8 T cells was determined by CFSE dilution using flow cytometry.

Detailed Description of the Invention

Although the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

Hereinafter, the key elements of the present invention will be described. These key elements and specific embodiments are listed together, but it should be understood that they can be combined in any way and in any quantity to produce other embodiments. The various embodiments and preferred embodiments described should not be interpreted as limiting the present invention to only the embodiments clearly described. This specification should be understood as supporting and including embodiments that will clearly describe the embodiment and any number of disclosures and/or preferred key element combinations. In addition, unless the context indicates otherwise, any arrangement and combination of all described key elements in this application should be considered as disclosed by the specification of the application.

Preferably, the terms herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel and H. eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques as explained in the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Unless the context requires otherwise, in the entire specification and claims below, the word "comprise/include" and its variations should be understood to mean including the group of the element, whole or step or element, whole or step, without excluding any other element, whole or step or element, whole or step group, although in some embodiments, such other element, whole or step or element, whole or step group may not be included, that is, the subject matter is to include the group of the element, whole or step or element, whole or step. Unless otherwise noted herein or clearly contradicted with the context, in the context of describing the present invention (especially in the context of the claims), nouns modified by quantifiers represent one/kind or more/kind. The recording of the range of values herein is only intended to be used as a shorthand method for individually referring to each individual value falling within the range. Unless otherwise noted herein, each individual value is incorporated into this specification as if it were individually recorded herein. Unless otherwise noted herein or clearly contradicted with the context, all methods described herein can be performed in any appropriate order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the invention and does not limit the scope of the invention otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate the disclosure of prior inventions.

The term "cytokine" generally refers to proteins that are important in cell signaling and act through receptors. In the context of the present invention, the term particularly refers to ligands of the TNF superfamily, more particularly the extracellular domains of these ligands that form soluble active homotrimers.

The term "ligand of the TNF superfamily" herein also includes variants of a given TNF superfamily ligand, provided that these variants are functional, more particularly have an extracellular domain capable of forming homotrimers capable of binding to the receptor of said ligand.

The term "variant of the ligand of the TNF superfamily" according to the present invention refers in particular to mutants, splice variants, conformational variants, isoforms, allelic variants, species variants and species homologs, particularly naturally occurring ones. Allelic variants relate to changes in the normal sequence of a gene, and their significance is often unclear. Complete gene sequencing typically identifies a variety of allelic variants of a given gene. Species homologs are nucleic acids or amino acid sequences with species origins that are different from a given nucleic acid or amino acid sequence. The term "variant of the ligand of the TNF superfamily" should include any variants and conformational variants modified after transfer.

According to the present invention, the first ligand and the second ligand of the TNF superfamily are preferably selected from CD40L, CD27L, 4-1BBL, OX40L, APRIL, CD30L, EDA-A1, EDA-A2, FasL, GITRL, LIGHT, LT-α, TL1A, TNF-α, TRAIL, RANKL and TWEAK, more preferably selected from CD40L, CD27L, 4-1BBL and OX40L.

CD40 ligand (CD40L), also known as CD154, TNFSF5, TRAP, or gp39, is a type II transmembrane glycoprotein belonging to the TNF superfamily. In one embodiment, the term CD40L herein refers to human CD40L. The UniProt accession number for human CD40L is P29965. In one embodiment, CD40L has the amino acid sequence of SEQ ID NO: 1.

CD27 ligand (CD27L), also known as CD70 or TNFSF7, is a type II transmembrane glycoprotein belonging to the TNF superfamily. In one embodiment, the term CD27L herein refers to human CD27L. The UniProt accession number for human CD27L is P32970. In one embodiment, CD27L has the amino acid sequence of SEQ ID NO: 2.

4-1BB ligand (4-1BBL) is a type II transmembrane glycoprotein belonging to the TNF superfamily, also known as TNFSF9. In one embodiment, the term 4-1BBL herein refers to human 4-1BBL. The UniProt accession number of human 4-1BBL is P41273. In one embodiment, 4-1BBL has the amino acid sequence of SEQ ID NO: 3.

OX40 ligand (OX40L), also known as gp34 or TNFSF4, is a type II transmembrane glycoprotein belonging to the TNF superfamily. In one embodiment, the term OX40L herein refers to human OX40L. The UniProt accession number for human OX40L is P23510. In one embodiment, OX40L has the amino acid sequence of SEQ ID NO: 4.

Proliferation-inducing ligand (APRIL), also known as TALL-2, TRDL-1 or TNFSF13, is a type II transmembrane protein that is a member of the TNF superfamily. In one embodiment, the term APRIL herein refers to human APRIL. The UniProt accession number of human APRIL is 075888.

CD30 ligand (CD30L), also known as TNFSF8, is a type II membrane protein belonging to the TNF superfamily. In one embodiment, the term CD30L herein refers to human CD30L. The UniProt accession number for human CD30L is P32971.

Ectodysplasin-A1 (EDA-A1) is a type II transmembrane protein belonging to the TNF superfamily. It is a splice variant of Ectodysplasin-A (EDA). In one embodiment, the term EDA-A1 herein refers to human EDA-A1. The UniProt accession number for human EDA-A1 is Q92838-1.

Exodiol-A2 (EDA-A2) is a type II transmembrane protein belonging to the TNF superfamily. It is a splice variant of exodiol-A (EDA). In one embodiment, the term EDA-A2 herein refers to human EDA-A2. The UniProt accession number for human EDA-A2 is Q92838-3.

Fas ligand (FasL), also known as CD95L or TNFSF6, is a type II transmembrane protein belonging to the TNF superfamily. In one embodiment, the term FasL herein refers to human FasL. The UniProt accession number for human FasL is P48023.

GITR ligand (GITRL) is a type II transmembrane protein belonging to the TNF superfamily and has been named TNFSF18. In one embodiment, the term GITRL herein refers to human GITRL. The UniProt accession number for human GITRL is Q9UNG2.

LIGHT, also known as HVEML or TNFSF14, is a type II transmembrane protein belonging to the TNF superfamily. In one embodiment, the term LIGHT herein refers to human LIGHT. The UniProt accession number of human LIGHT is O43557.

Lymphotoxin-α (LT-α), also known as TNF-β or TNFSF1, is a member of the TNF superfamily. In one embodiment, the term LT-α herein refers to human LT-α. The UniProt accession number for human LT-α is P01374.

TL1A is a type II transmembrane protein belonging to the TNF superfamily and has been named TNF superfamily member 15 (TNFSF15). In one embodiment, the term TL1A herein refers to human TL1A. The UniProt accession number of human TL1A is O95150-1.

Tumor necrosis factor alpha (TNF-α), also known as cachectin or TNFSF2, is a type II transmembrane protein belonging to the TNF superfamily. In one embodiment, the term TNF-α herein refers to human TNF-α. The UniProt accession number for human TNF-α is P01375.

The apoptosis-inducing ligand (TRAIL) associated with TNF, also known as Apo-2 ligand or TNFSF10, is a type II transmembrane protein belonging to the TNF superfamily. In one embodiment, the term TRAIL herein refers to human TRAIL. The UniProt accession number of human TRAIL is P50591.

Receptor activator of NF-kB (RANK) ligand (RANKL), also known as TRANCE, ODF, OPGL or TNFSF11, is a type II transmembrane protein belonging to the TNF superfamily. In one embodiment, the term RANKL herein refers to human RANKL. The UniProt accession number for human RANKL is O14788.

TWEAK is a type II transmembrane protein belonging to the TNF superfamily, also known as APO3 ligand or TNFSF12. In one embodiment, the term TWEAK herein refers to human TWEAK. The UniProt accession number of human TWEAK is O43508.

In one embodiment, the receptor for the first ligand and the receptor for the second ligand are located on the same cell ("cis"), wherein preferably the first ligand and the second ligand are selected from CD27L, 4-1BBL and OX40L. In one embodiment, the cell is a T cell, preferably a CD4 ⁺ and/or CD8 ⁺ T cell. In one embodiment, the T cell is an activated T cell.

In another embodiment, the receptor for the first ligand and the receptor for the second ligand are located on different cells ("trans"). The different cells can be of the same type or of different types. In one embodiment, the different cells are antigen presenting cells (APCs), such as dendritic cells and T cells. In another embodiment, the different cells are B cells and T cells. In one embodiment, the first ligand or the second ligand is CD40L, and the corresponding other ligand is selected from CD27L, 4-1BBL and OX40L. In one embodiment, the T cells are CD4 ⁺ and/or CD8 ⁺ T cells. In one embodiment, the T cells are activated T cells.

In one embodiment, the cytokine fusion protein activates the receptor of the first ligand and/or the receptor of the second ligand. In one embodiment, the cytokine fusion protein is a cis-activating cytokine fusion protein, which simultaneously activates the receptor of the first ligand and the receptor of the second ligand located on the same cell. In another embodiment, the cytokine fusion protein is a trans-activating cytokine fusion protein, which simultaneously activates the receptor of the first ligand and the receptor of the second ligand located on different cells. In one embodiment, the cytokine fusion protein activates the NF-κB pathway in cells expressing the receptor of the first ligand and/or the second ligand and/or induces the release of IL-8 therefrom.

In one embodiment, the cytokine fusion protein activates T cells and/or induces proliferation of T cells. In one embodiment, the cytokine fusion protein induces antigen-specific proliferation of T cells. In one embodiment, the T cells are CD4 ⁺ and/or CD8 ⁺ T cells. In one embodiment, the T cells are activated T cells.

The term "extracellular domain" herein refers to the extracellular C-terminal portion of a ligand of the TNF superfamily comprising a TNF homology domain (THD). The ectodomain is characterized by its ability to form (homo-) trimers that can bind to the receptor of the ligand and is also referred to as a "receptor binding domain."

" Fragment or variant " of the extracellular domain that can be used according to the present invention is a functional fragment or variant of a functional extracellular domain, which has the ability to form a (homo-) trimer that can bind to the receptor of the part. Therefore, suitable parts or variants at least include a functional TNF homology domain (THD).

The terms "portion" or "fragment" are used interchangeably herein to refer to a continuous element. For example, a portion of a structure (e.g., an amino acid sequence or protein) refers to a continuous element of that structure. A portion or fragment of a protein sequence preferably comprises a sequence of at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, at least 100, at least 150, or at least 200 consecutive amino acids of the protein sequence.

For the purposes of the present invention, "variants" of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. Amino acid deletion variants comprising deletions at the N-terminus and/or C-terminus of a protein are also referred to as N-terminal and/or C-terminal truncation variants.

Amino acid insertion variants comprise the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion under appropriate screening of the resulting product is also possible.

Amino acid addition variants comprise amino- and/or carboxyl-terminal fusions of one or more amino acids (eg, 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids).

Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be at any position in the protein.

Amino acid substitution variants are characterized in that at least one residue in the sequence is removed and another residue is inserted in its place. Preference is given to positional modifications in amino acid sequences that are not conserved between homologous proteins or peptides and/or replacement of amino acids with amino acids having similar properties. Preferably, the amino acid substitutions in protein variants are conservative amino acid substitutions. Conservative amino acid substitutions include replacements of amino acids with another amino acid from the same amino acid family, i.e., replacements of amino acids related to their side chains (e.g., in terms of charge and/or size). Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Preferably, the degree of similarity or identity given for an amino acid region is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% over the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids, preferably contiguous amino acids. In some preferred embodiments, the similarity or identity is given for the entire length of the reference amino acid sequence.

Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using optimal sequence alignment, e.g. using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

"Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between these sequences.

The term "percent identity" is intended to indicate the percentage of identical amino acid residues between the two sequences to be compared, obtained after optimal alignment, which percentage is purely statistical and the differences between the two sequences are randomly distributed over their entire lengths. Sequence comparisons between two amino acid sequences are conventionally performed by comparing these sequences after optimal alignment, either by segments or by a "comparison window" to identify and compare local regions of sequence similarity. In addition to manual work, optimal alignments of sequences for comparison can be generated by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the search similarity method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

Percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing that number by the number of positions being compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

The term "fusion protein" generally refers to a protein produced by linking two or more different (poly)peptides or proteins, preferably head to tail (i.e., N-terminus to C-terminus or vice versa), to produce a single protein with functional properties derived from each of the original proteins. According to the present invention, the term "cytokine fusion protein" also encompasses multimeric (e.g., dimer or trimer) complexes of different fusion proteins, which are also referred to herein as "subunits." Preferably, the subunits are non-covalently or covalently (e.g., via disulfide bonds) bound to form the cytokine fusion protein.

Preferred subunits according to the present invention have the following general formula as defined herein:

N'-A-L-B-C' (Formula II),

Preferably, three of these subunits are non-covalently associated via the extracellular domain of the first ligand or a fragment or variant thereof and the extracellular domain of the second ligand or a fragment or variant thereof to form a cytokine fusion protein.

Another preferred subunit according to the present invention has the following general formula as defined herein:

N'-AL _A -AL _A -ALBL _B -BL _B -B-C' (Formula I),

Wherein L further comprises a multimerization domain, preferably a dimerization domain, which allows the formation of a multimerized, preferably dimerized, cytokine fusion protein.

The term "segment" as used herein refers to a molecular unit/entity comprising three covalently linked extracellular domains of a ligand of the TNF superfamily, or a fragment or variant thereof. In one embodiment, the segment has the general formula AL _A -AL A- _A or BL _B -BL _B -B, wherein A, B, _LA , and _LB are as defined herein. In one embodiment, the segment comprises or consists of an amino acid sequence according to one of SEQ ID NOs: 9 to 12.

The term "covalently linked" as used herein refers to linkage via a covalent bond or via a covalent linker molecule (eg, a peptide linker).

The term "peptide linker" as used herein refers to a peptide suitable for binding/connecting a protein portion (e.g., the extracellular domain of a ligand of the TNF superfamily, or a segment thereof, or a homotrimer formed by these extracellular domains). The peptide linker according to the present invention may be of any length, i.e., comprise any number of amino acid residues. However, it is preferably long enough to provide a sufficient degree of flexibility to prevent the binding/connected portions from interfering with each other's activity - for example, the ability of the extracellular domain of a ligand of the TNF superfamily to form a homotrimer capable of binding to a receptor for the ligand, and/or the ability of two different homotrimers to bind to two different receptors on the same cell ("cis") or on different cells ("trans"), for example, via steric hindrance and allow proper protein folding; it is also preferably short enough to provide stability in the cell (e.g., proteolytic stability).

In some preferred embodiments, the length of the peptide linker is 1 to 30 amino acid.Therefore, according to the present invention, the peptide linker can be made up of single amino acid residues.Preferably, the extracellular domain of the first part or its fragment or variant and the extracellular domain of the second part or its fragment or variant (for example, the first homotrimer and the second homotrimer or the first section and the second section) of the long peptide linker are connected, and generally speaking, short peptide linkers are used for respectively connecting two extracellular domains of the first or second part or its fragment or variant (that is, two extracellular domains of the same part or its fragment or variant).In the case of part 4-1BBL, preferably long peptide linkers are used for connecting two of its extracellular domains or its fragment or variant.Short peptide linkers can be made up of 12 or less (for example 11,10,9,8,7,6,5,4,3,2 or 1) amino acid and preferably 1 to 7 amino acid.Long peptide linkers can be made up of 12 or more (for example 12 to 30 or 12 to 25 or 12 to 20) amino acid.

The amino acids of the peptide linker can be selected from all naturally or non-naturally occurring amino acids, wherein the amino acids glycine (Gly, G), serine (Ser, S) and threonine (Thr, T) are preferred. In one embodiment, the peptide linker is a glycine-serine-threonine-rich linker or a glycine-serine-rich linker, wherein at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90% of the amino acids are glycine or serine or threonine residues or glycine or serine residues, respectively. In another embodiment, the amino acids are selected from glycine, serine and threonine, i.e., the peptide linker consists only of glycine, serine and threonine residues (referred to as a glycine-serine-threonine linker). In another embodiment, the peptide linker consists only of glycine and serine residues (referred to as a glycine-serine linker).

Preferred peptide linkers according to the invention have the general formula (GGGGX) _n , wherein X is independently selected at each occurrence from S and T, and n is an integer selected from 1 to 6, preferably 1 to 5; or the general formula selected from GXG, GGXGG and GGGXGGG, wherein X is S or T.

Preferred short peptide linkers have a general formula selected from GXG, GGXGG, GGGXGGG and GGGGXGGGG, wherein X is S or T, preferably S. A particularly preferred short peptide linker is GGGXGGG, wherein X is S or T, preferably S.

Preferred long peptide linkers have the general formula (GGGGX) _n , wherein X at each occurrence is independently selected from S and T, and n is an integer selected from 3 to 6, preferably 3 to 5, more preferably 3 and 4. Particularly preferred long peptide linkers are selected from (GGGGS) ₃ (SEQ ID NO: 19), GGGGSGGGTGGGGS (SEQ ID NO: 20) and (GGGGS) ₄ (SEQ ID NO: 21).

Preferably, where the cytokine fusion protein comprises a molecule/structure having the general formula of Formula I as defined herein,

L comprises a long peptide linker as defined herein and/or

_LA and _LB are independently at each occurrence selected from a covalent bond (eg, a peptide bond), a short peptide linker as defined herein, and a long peptide linker as defined herein.

In particular, when A or B comprises the extracellular domain of 4-1BBL or a fragment or variant thereof, _LA or _LB are independently selected from the long peptide linker as defined herein at each occurrence. Preferably, when neither A nor B comprises the extracellular domain of 4-1BBL or a fragment or variant thereof, _LA and _LB are independently selected from the covalent bond (e.g., peptide bond) and the short peptide linker as defined herein at each occurrence.

According to the present invention, _LA and _LB may be the same or different.

According to the present invention, where the cytokine fusion protein comprises a molecule/structure having the general formula I as defined herein,

L may also comprise a multimerization domain that allows for multimerization of the cytokine fusion protein.

In some such cases, L may comprise a peptide linker as defined herein into which a multimerization domain has been inserted. In an alternative embodiment, L may comprise two peptide linkers as defined herein inserted into a multimerization domain, wherein the two peptide linkers may be identical or different. In one embodiment, the two peptide linkers are selected from short peptide linkers as defined herein. In another embodiment, the multimerization domain represents a peptide linker comprised by L.

Multimerization can occur via non-covalent and/or covalent interactions, in particular via one or more disulfide bonds between multiple (eg, 2, 3 or 4, preferably 2 or 3, more preferably 2) multimerization domains.

Suitable multimerization domains are known to those skilled in the art and include, for example, trimerization domains, such as the tenascin trimer motif, collectin trimerization domain, and streptavidin; and dimerization domains, such as IgE heavy chain domain 2 (EHD2), IgM heavy chain domain 2 (MHD2), IgG heavy chain domain 3 (GHD3), IgA heavy chain domain 3 (AHD2), IgD heavy chain domain 3 (DHD3), IgE heavy chain domain 4 (EHD4), IgM heavy chain domain 4 (MHD4), Fc domains, and uteroglobin dimerization domains. In one embodiment, the dimerization domain is an EHD2 domain or an MHD2 domain, for example, as described in WO 2013/156148 A1. In one embodiment, the dimerization domain is a human EHD2 domain, preferably comprising or consisting of the amino acid sequence of SEQ ID NO: 23. Also included are functional variants of any of the aforementioned domains, e.g., domains that have been modified to extend their half-life and/or improve their potency. Suitable modifications are known to those skilled in the art and include, but are not limited to, modifications of the Fc domain that improve its affinity for FcRn as described in Zalevsky, J. et al. (2010), Nature Biotechnology, 28(2): 157-9 (e.g., N434S, V259I/V308F, M252Y/S254T/T256E, M428L/N434S, and V259I/V308F/M428L).

Where the cytokine fusion protein comprises a molecule/structure (or at least one, preferably three subunits) having the general formula of Formula II as defined herein,

L preferably comprises a long peptide linker as defined herein.

The peptide linkers as described herein may be replaced with non-peptide molecules, for example, with non-peptide oligomers and polymers of appropriate length. Such equivalent embodiments are expressly encompassed by the present invention.

Preferably, in the cytokine fusion protein according to the present invention,

- the first ligand is CD40L and the second ligand is CD27L;

- the first ligand is CD27L and the second ligand is CD40L;

- the first ligand is CD40L and the second ligand is 4-1BBL;

- the first ligand is 4-1BBL and the second ligand is CD40L;

- the first ligand is CD27L and the second ligand is 4-1BBL;

- the first ligand is 4-1BBL and the second ligand is CD27L;

- the first ligand is CD40L, and the second ligand is OX40L;

- the first ligand is OX40L, and the second ligand is CD40L;

- the first ligand is CD27L and the second ligand is OX40L;

- the first ligand is OX40L and the second ligand is CD27L;

- the first ligand is OX40L and the second ligand is 4-1BBL; or

- The first ligand is 4-1BBL and the second ligand is OX40L.

In one embodiment, the cytokine fusion protein comprises a molecule/structure (or at least one, preferably three subunits) having the general formula of Formula II as defined herein, wherein the second ligand (i.e., the C-terminal ligand) is CD40L.

"A marker or tag that allows detection and/or isolation of a cytokine fusion protein" is intended to include any marker/tag known in the art for these purposes. Particularly preferred are affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), and poly (His) (e.g., His ₆ ); solubilization tags, such as thioredoxin (TRX) and poly (NANP); chromatography tags, such as FLAG tags; epitope tags, such as V5 tags, myc tags, and HA tags; and fluorescent or luminescent markers or tags, such as fluorescent proteins (e.g., GFP, YFP, RFP, etc.), fluorescent dyes, and luciferases. In one embodiment, the marker/tag is a FLAG tag.

The amino acid sequence of the (poly)peptide tag or label can be introduced at any position within the amino acid sequence of the cytokine fusion protein and can, for example, take the shape of a ring within the structure of the encoded protein (e.g., within any peptide linker described herein or even within the extracellular domain of the first and second ligands, as long as the tag/label does not interfere with its function), or it can be N-terminally or C-terminally fused. The tag or label may also comprise a cleavage site that allows the tag or label to be removed from the cytokine fusion protein. Similarly, non-peptide tags or labels (e.g., fluorescent dyes) can be conjugated to the cytokine fusion protein at any suitable site.

The cytokine fusion proteins according to the present invention may also comprise an amino acid sequence for promoting secretion of the molecule, such as an N-terminal secretion signal. Preferably, the secretion signal is a signal sequence that allows for adequate passage through the secretory pathway and/or secretion into the extracellular environment. Preferably, the secretion signal sequence is cleavable and removable from the mature cytokine fusion protein. The secretion signal sequence is preferably selected with respect to the cell or organism in which the cytokine fusion protein is produced. In one embodiment, the secretion signal sequence comprises or consists of the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 22.

The cytokine fusion proteins of the present invention may also comprise a binding domain for, for example, enhancing selectivity for a particular cell type. This can be achieved, for example, by providing a binding domain that binds to a specific antigen expressed on the surface of the cell type.

The cytokine fusion protein according to the present invention may also comprise one or more modifications that improve the stability of the cytokine fusion protein. The term "stability" of a cytokine fusion protein relates to the "half-life" (e.g., in vivo) of the cytokine fusion protein. "Half-life" relates to the time required to eliminate half the activity, amount, or number of a molecule.

Cytokine fusion proteins can, for example, be conjugated to half-life extending molecules. Such molecules are known to those skilled in the art and include, for example, albumin, albumin binding domains, immunoglobulin binding domains, FcRn binding motifs, and in particular polymers. Particularly preferred polymers include polyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid, and PEG mimetic peptide sequences.

The term "binding" according to the present invention preferably relates to specific binding. A binding agent (eg a cytokine fusion protein according to the present invention) is specific for a predetermined target if it is able to bind to said target but not to other targets.

According to the present invention, a "nucleic acid sequence" is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA (e.g., mRNA), and most preferably in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, the nucleic acid molecule can be single-stranded or double-stranded and can be linear or covalently closed to form a circular molecule.

In the context of the present invention, the term "DNA" refers to a molecule comprising deoxyribonucleotide residues and preferably consisting entirely or substantially of deoxyribonucleotide residues."Deoxyribonucleotides" refer to nucleotides that do not have a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. The term "DNA" includes isolated DNA, such as partially or completely purified DNA, substantially pure DNA, synthetic DNA, and recombinantly produced DNA, and includes modified DNA that is different from naturally occurring DNA by the addition, deletion, replacement, and/or change of one or more nucleotides. Such changes can include changes in non-nucleotide substances (such as at the end or inside of the DNA, such as one or more nucleotides of the DNA). The nucleotides in the DNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides. The DNA of these changes can refer to analogs or isotypes of naturally occurring DNA.

In the context of the present invention, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or substantially of ribonucleotide residues." Ribonucleotides" refer to nucleotides having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term "RNA" includes isolated RNA, such as partially or completely purified RNA, substantially pure RNA, synthetic RNA, and recombinantly produced RNA, and includes modified RNA that is different from naturally occurring RNA by the addition, deletion, replacement, and/or change of one or more nucleotides. Such changes may include changes in non-nucleotide substances (such as at the end or inside of the RNA, such as one or more nucleotides of the RNA). The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. The RNA of these changes may refer to analogs or isotypes of naturally occurring RNA. According to the present invention, "RNA" refers to single-stranded RNA or double-stranded RNA.

According to the present invention, the term "messenger RNA (mRNA)" refers to a "transcript" that can be produced using a DNA template and that can encode a peptide or protein. Generally speaking, mRNA comprises a 5'-untranslated region, a protein coding region, and a 3'-untranslated region. In the context of the present invention, mRNA can be produced by in vitro transcription from a DNA template. In vitro transcription methodology is known to the skilled person. For example, a variety of in vitro transcription kits are commercially available.

According to the present invention, RNA can be modified. For example, RNA can be stabilized by one or more modifications that have a stabilizing effect on RNA.

The term "modification" in the context of an RNA as used according to the invention includes any modification of the RNA that is not naturally occurring in said RNA.

In one embodiment of the invention, the RNA used according to the invention does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase.

The RNA according to the present invention may have modified naturally occurring or non-naturally occurring (synthetic) ribonucleotides to improve its stability and/or reduce cytotoxicity and/or modulate its immunostimulatory potential. For example, in one embodiment, in the RNA used according to the present invention, uridine is partially or completely substituted by pseudouridine, preferably completely substituted.

In one embodiment, the term "modification" relates to providing an RNA with a 5'-cap or a 5'-cap analog. The term "5'-cap" refers to a cap structure found at the 5'-end of an mRNA molecule and is generally composed of a guanosine nucleotide linked to the mRNA by a unique 5' to 5' triphosphate bond. In one embodiment, the guanosine is methylated at position 7. The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably a 7-methylguanosine cap (m ⁷ G). In the context of the present invention, the term "5'-cap" includes 5'-cap analogs that resemble RNA cap structures and are modified to have the ability to stabilize RNA (if attached thereto), preferably in vivo and/or within a cell. Providing an RNA with a 5'-cap or a 5'-cap analog can be achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analog, wherein the 5'-cap is co-transcriptionally incorporated into the resulting RNA chain, or the RNA can be produced, for example, by in vitro transcription, and the 5'-cap can be produced post-transcriptionally using a capping enzyme (e.g., a capping enzyme from vaccinia virus).

The RNA may comprise other modifications. For example, the modification of the mRNA used in the present invention may be an extension or truncation of the naturally occurring poly(A) tail.

The term "stability" of RNA relates to the "half-life" of the RNA. "Half-life" relates to the time required to eliminate half of the activity, amount or number of a molecule. In the context of the present invention, the half-life of an RNA indicates the stability of the RNA.

According to the present invention, if it is desired to reduce the stability of RNA, RNA can also be modified so as to interfere with the function of the elements that increase the stability of RNA as described above.

According to the present invention, RNA can be obtained by chemical synthesis or by in vitro transcription of an appropriate DNA template. In the context of the present invention, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. Thereafter, RNA can be translated into protein. According to the present invention, the term "transcription" includes "in vitro transcription," wherein the term "in vitro transcription" refers to a process in which RNA (particularly mRNA) is preferably synthesized in vitro in a cell-free system using a suitable cell extract. Preferably, a cloning vector is used for the production of transcripts. These cloning vectors are generally designated as transcription vectors and are encompassed by the term "vector" according to the present invention. The promoter used to control transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. The DNA template for in vitro transcription can be obtained by cloning nucleic acids (particularly cDNA) and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA. Preferably, a cloning vector is used to produce transcripts generally designated as transcription vectors.

The term "translation" according to the present invention relates to the process by which a strand of messenger RNA directs the assembly of an amino acid sequence in the ribosomes of a cell to produce a peptide or protein.

The term "peptide" according to the present invention includes oligopeptides or polypeptides and refers to a substance comprising two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids covalently linked by peptide bonds. The term "protein" refers to large peptides, preferably peptides having more than 100 amino acid residues, but in general, the terms "peptide" and "protein" are synonymous and are used interchangeably herein.

As used herein, the term "expression control sequence" is intended to refer to nucleic acid sequences that allow expression of an operably linked nucleic acid molecule in a desired host cell or in an in vitro environment. Suitable expression control sequences are known to those skilled in the art and include promoters, such as RNA promoters, for example, T7, T3 or SP6 promoters.

The nucleic acid molecules according to the present invention may be included/included in a vector. The term "vector" herein includes any vector known to the skilled person, including plasmid vectors; cosmid vectors; phage vectors, such as lambda phage; viral vectors, such as adenovirus or baculovirus vectors; or artificial chromosome vectors, such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). The vector includes expression vectors and cloning vectors. Expression vectors include plasmid vectors and viral vectors, and typically contain the desired coding sequence and appropriate DNA sequence necessary for the expression of the coding sequence operably connected to a specific host organism (e.g., bacteria, yeast, plants, insects or mammals) or in vitro expression system. Cloning vectors are typically used to design and amplify a desired DNA fragment, and may lack the functional sequences required for the expression of the desired DNA fragment.

The term "cell" or "host cell" preferably relates to an intact cell, i.e. a cell with an intact membrane that has not yet released its normal intracellular components (e.g. enzymes, organelles or genetic material). An intact cell is preferably a living cell, i.e. a living cell that is able to carry out its normal metabolic functions. Preferably, according to the present invention, the term relates to any cell that can be transfected or transformed with an exogenous nucleic acid. Preferably, when transfected or transformed with an exogenous nucleic acid and transferred to a recipient, the cell can express said nucleic acid in said recipient. The term "cell" includes prokaryotic cells, such as bacterial cells, and eukaryotic cells, such as yeast cells, fungal cells or mammalian cells. Suitable bacterial cells include cells from gram-negative bacterial strains, such as cells from strains of Escherichia coli, Proteus, and Pseudomonas, and cells from gram-positive bacterial strains, such as cells from Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include cells from Trichoderma, Neurospora, and Aspergillus species. Suitable yeast bacteria include cells from the genus Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula species. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK, and the like. However, amphibian cells, insect cells, plant cells, and any other cells known in the art for expression of heterologous proteins may also be used. Mammalian cells are particularly preferred for adoptive transfer, such as cells from humans, mice, hamsters, pigs, goats, and primates. These cells can be derived from a wide variety of tissue types and include primary cells and cell lines, such as cells of the immune system, particularly antigen-presenting cells (APCs), such as dendritic cells, B cells, and T cells; stem cells, such as hematopoietic stem cells and mesenchymal stem cells; and other cell types. Antigen-presenting cells are cells that display antigens in the context of a major histocompatibility complex on their surface. T cells can recognize this complex using their T cell receptors (TCRs).

As used herein, the term "non-human organism" is intended to include non-human primates or other animals, particularly mammals, such as cows, horses, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters, or rodents (e.g., mice and rats).

The pharmaceutical compositions of the present invention are preferably sterile and contain an effective amount of the cytokine fusion proteins, nucleic acid molecules or cells described herein to produce a desired response or desired effect.

The pharmaceutical compositions are generally provided in uniform dosage form and can be prepared in a manner known per se. The pharmaceutical compositions can be in the form of solutions or suspensions, for example.

The pharmaceutical composition may further comprise one or more carriers and/or excipients, all of which are preferably pharmaceutically acceptable.The term "pharmaceutically acceptable" as used herein refers to the non-toxicity of a substance which preferably does not interact with the action of the active agent of the pharmaceutical composition.

The term "carrier" refers to an organic or inorganic component of natural or synthetic nature, with which the active ingredient is combined to promote, enhance or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration to a subject.

Possible carrier materials for parenteral administration are, for example, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers.

The term "excipient" herein is intended to include all substances that may be present in pharmaceutical compositions and which are not active ingredients, such as salts, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffer substances, flavorings or colorings.

Non-pharmaceutically acceptable salts can be used to prepare pharmaceutically acceptable salts and are included in the present invention. Such pharmaceutically acceptable salts include, in a non-limiting manner, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts (e.g., sodium, calcium, or calcium salts).

Suitable preservatives for pharmaceutical compositions include benzalkonium chloride, chlorobutanol, parabens and thimerosal.

Suitable buffer substances for use in pharmaceutical compositions include acetate, citrate, borate and phosphate.

The agents and compositions described herein can be administered by any conventional route, for example by parenteral administration including by injection or infusion. Administration is preferably parenteral, for example intravenous, intraarterial, subcutaneous, intradermal or intramuscular.

Pharmaceutical compositions suitable for parenteral administration generally include a sterile aqueous or non-aqueous preparation of the active compound that is preferably isotonic with the blood of the recipient. Examples of compatible carriers/solvents/diluents are Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solution or suspension media.

The agents and compositions described herein are administered in an effective amount. An "effective amount" refers to an amount that achieves a desired reaction or desired effect, either alone or in combination with another dosage. In the case of treating a specific disease or a specific condition, the desired reaction preferably involves the inhibition of the course of the disease. This includes slowing down the progression of the disease, and particularly interrupting or reversing the progression of the disease. The desired reaction in the treatment of a disease or condition can also be a delayed onset or prevention of the onset of the disease or condition. The effective amount of the agents or compositions described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the subject (including age, physiological condition, body size and body weight), the duration of treatment, the type of concomitant therapy (if present), the specific route of administration, and similar factors. Therefore, the dosage of the agents described herein can depend on a variety of such parameters. In the case where the reaction in the subject is insufficient due to the initial dose, a higher dosage (or an effective higher dosage achieved by a different, more localized route of administration) can be used.

As used herein, the term "kit of parts" (abbreviated as: kit) refers to an article comprising one or more containers and optionally a data carrier. The one or more containers can be filled with one or more of the above-mentioned means or reagents. Additional containers can be included in the kit containing, for example, diluents, buffers and other reagents. The data carrier can be a non-electronic data carrier, for example a graphic data carrier such as an information brochure, an information sheet, a bar code or an access code; or an electronic data carrier, for example a floppy disk, a compact disk (CD), a digital versatile disk (DVD), a microchip or other semiconductor-based electronic data carrier. The access code can allow access to a database, such as an Internet database, a centralized database or a decentralized database. The data carrier can contain instructions for use of the cytokine fusion protein, nucleic acid molecule, cell and/or pharmaceutical composition of the present invention.

The agents and compositions described herein can be administered to a subject, eg, in vivo, to treat or prevent a variety of disorders, such as those described herein.

According to the present invention, the term "disease" refers to any pathological state, in particular cancer, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune diseases, degenerative diseases, apoptosis-related diseases and transplant rejection.

The term "cancer" as used herein includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion and/or migration. "Cancer cell" means an abnormal cell that grows with rapid, uncontrollable cell proliferation and continues to grow after the stimulus causing new growth has ceased. The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, stomach cancer, intestinal cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer and metastasis thereof. Examples thereof are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer or metastasis of the above-mentioned cancer types or tumors.

Term " cancer " according to the present invention also comprises cancer metastasis." metastasis " means that cancer cell spreads to another part of health from its original position.The formation of metastasis is very complicated process, and depends on that malignant cell breaks away from primary tumor, invades extracellular matrix, passes endothelial basement membrane to enter body cavity and blood vessel, then, after being transported by blood, infiltrates target organ.Finally, the growth of new tumor (i.e. secondary tumor or metastasis) depends on angiogenesis in target spot.Even after removing primary tumor, tumor metastasis also often occurs, because tumor cell or component can remain and develop transfer potential.In one embodiment, term " metastasis " according to the present invention relates to " distant metastasis ", it relates to the metastasis away from primary tumor and regional lymph node system.

The term "infectious disease" refers to any disease that can be transmitted from individual to individual or from organism to organism and is caused by a microbial agent (e.g., a cold). Examples of infectious diseases include viral infectious diseases, such as AIDS (HIV), hepatitis A, B, or C, herpes, herpes zoster (fowl pox), German measles (rubella virus), yellow fever, flaviviruses such as dengue fever, influenza virus, hemorrhagic infectious diseases (Marburg or Ebola virus), and severe acute respiratory syndrome (SARS); bacterial infectious diseases, such as Legionnaires' disease (Legionella spp.), sexually transmitted diseases (e.g., chlamydia or gonorrhea), gastric ulcers (Helicobacter spp.), cholera (Vibrio spp.), tuberculosis, diphtheria, E. coli (E. coli), and SARS. coli), Staphylococci, Salmonella or Streptococci (tetanus); infections with protozoan pathogens, such as malaria, sleeping sickness, leishmaniasis; toxoplasmosis, i.e., infections with Plasmodium, Trypanosoma, Leishmania and Toxoplasma; or fungal infections, such as those caused by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis or Candida albicans.

The term "inflammatory disease" refers to any disease characterized by or associated with high levels of inflammation in tissues, particularly connective tissue, or degeneration of these tissues. Chronic inflammatory diseases are medical conditions characterized by persistent inflammation. Examples of (chronic) inflammatory diseases include celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, chronic active hepatitis, dermatitis, and psoriasis.

The term "metabolic disease" refers to any disease or condition that disrupts normal metabolism. Examples include cystinosis, diabetes, dyslipidemia, hyperthyroidism, hypothyroidism, hyperlipidemia, hypolipidemia, galactosemia, Gaucher's disease, obesity, and phenylketonuria.

The term "autoimmune disease" refers to any disease/disorder to which the body produces immunogenicity (i.e., immune system) responses to a component of its own tissue. In other words, the immune system loses its ability to identify a certain tissue or system in its own body and targets and attacks it, as if it were foreign. Autoimmune diseases can be divided into those (e.g., hemolytic anemia and anti-immune thyroiditis) in which an organ is mainly affected and those (e.g., systemic lupus erythematosus) in which the autoimmune disease process is spread by many tissues. For example, multiple sclerosis is considered to be caused by T cell attacks on the myelin sheaths of the nerve fibers around the brain and spinal cord. This results in loss of coordination, weakness and blurred vision. Autoimmune diseases are known in the art and include, for example, Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, etc.

The term "degenerative disease" refers to any disease in which the function or structure of the affected tissue or organ gradually deteriorates over time. Examples include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, macular degeneration, multiple sclerosis, muscular dystrophy, Niemann-Pick disease, osteoporosis, and rheumatoid arthritis.

The term "apoptosis-related disease" refers to any disease in which changes in apoptosis are involved. Examples include cancer, neurological diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and stroke), cardiac diseases (e.g., ischemia-reperfusion and chronic heart failure), infectious diseases, and autoimmune diseases.

The term "transplant rejection" refers to the rejection of a transplanted tissue or organ by the recipient's immune system, which can ultimately destroy the transplanted tissue or organ.

As used herein, the term "drug" refers to a substance/composition used in therapy (ie, in the treatment of a disease).

"Treatment" means administering a compound or composition, or a combination of compounds or compositions, to a subject to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in a subject; arresting or slowing the development of a disease in a subject; inhibiting or slowing the development of new disease in a subject; reducing the frequency or severity of symptoms and/or recurrences in a subject currently suffering from or previously suffering from a disease; and/or prolonging (i.e., increasing) the lifespan of a subject.

In particular, the term "treating a disease" includes curing, shortening the duration, ameliorating, preventing, slowing or inhibiting the progression or worsening, or preventing or delaying the onset of the disease or its symptoms.

According to the present invention, the term "subject" refers to a therapeutic subject, in particular a diseased subject (also referred to as a "patient"), including humans, non-human primates or other animals, in particular mammals, such as cows, horses, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, hamsters or rodents such as mice and rats. In a particularly preferred embodiment, the subject/patient is a human.

Example

Example 1: Cloning, production and purification of Duokine

DNA encoding the extracellular portion of human CD40L (aa 116-261), human CD27L (aa 52-193), human 4-1BBL (aa 71-254), and human OX40L (aa 51-183) was codon-optimized for expression in human cells and synthesized by Geneart (Life Technologies, Carlsbad, USA) by adding appropriate cloning sites. Duokines were generated by fusion of single subunits of two of these different cytokines via a glycine-serine-rich linker at amino acid position 15 (in the case of Duokine without 4-1BBL) or amino acid position 20 (in the case of Duokine containing 4-1BBL) and cloned into the expression plasmid pIRESpuro3 (Clontech, Mountain View, USA). At the N-terminus, Duokines have a VH leader sequence for secretion and a FLAG tag for purification and detection. The following Duokines were produced: CD40L-CD27L, CD27L-CD40L, CD40L-4-1BBL, 4-1BBL-CD40L, CD27L-4-1BBL, 4-1BBL-CD27L, CD40L-OX40L, OX40L-CD40L, CD27L-OX40L, OX40L-CD27L, 4-1BBL-OX40L, and OX40L-4-1BBL (Table 1). All Duokines were produced by stably transfected HEK293 cells and purified from cell culture supernatants by one-step FLAG affinity chromatography (Sigma-Aldrich, St. Louis, USA), yielding supernatants ranging from 0.3 mg/L to 1.9 mg/L.

Duokine was analyzed using 12% polyacrylamide gels in SDS-PAGE under reducing and non-reducing conditions and visualized by staining with Coomassie Brilliant Blue G250. Considering the presence of possible N-glycosylation sites in CD40L (1 site; aa 240), CD27L (2 sites; aa 63 and 170), and OX40L (4 sites; aa 90, 114, 152, and 157), SDS-PAGE analysis showed the expected molecular weight of the monomeric polypeptide chain (approximately 33 kDa for Duokine without 4-1BBL and 37 kDa for Duokine containing 4-1BBL) (Fig. 2, Table 1). In addition, under non-reducing conditions, all proteins showed a second minor band corresponding to the molecular weight of the dimer. The homotrimeric assembly of Duokine was determined using size exclusion chromatography using high performance liquid chromatography at a flow rate of 0.5 mL/min on a Yarra SEC-2000 (Phenomenex). Duokines CD40L-4-1BBL, 4-1BBL-CD40L, CD27L-4-1BBL, and 4-1BBL-CD27L eluted as major peaks with apparent molecular weights of 80 kDa to 100 kDa, thus slightly smaller than the calculated molecular weight of 112 kDa, likely due to the more compact structure of the 4-1BBL-containing duokines. All other duokines eluted as major peaks with apparent molecular weights of 130 kDa to 160 kDa, 30% to 50% larger than the calculated molecular weight, likely due to N-glycosylation. In addition, duokines 4-1BBL-CD27L, 4-1BBL-CD40L, CD40L-OX40L, OX40L-CD40L, and OX40L-4-1BBL exhibited secondary peaks that most likely corresponded to higher molecular weight complexes (Figures 3A, B).

Table 1: Duokine and its biochemical properties

Example 2: Receptor Binding Properties of Duokine

The receptor binding of Duokine was analyzed by ELISA using fusion proteins (CD40-Fc, CD27-Fc, 4-1BB-Fc, OX40-Fc) of the extracellular regions of CD40, CD27, 4-1BB and OX40 and the human Fcγ1 region containing the hinge region for covalent assembly. Receptor-Fc fusion protein (200ng/well) was coated overnight at 4°C and the residual binding sites were blocked with PBS and 2% skim milk powder (2% MPBS). Duokine was titrated in duplicate starting at a concentration of 316nM, and the bound Duokine was detected with a mouse anti-FLAG antibody conjugated to HRP. All Duokines showed specific binding to their corresponding receptors, and no cross-reactivity with other receptor Fc fusion proteins was observed (Figure 4). The Duokine-receptor interaction was dose-dependent, with _EC50 values in the low nanomolar range (Figure 5A, B, Table 1).

In addition, the binding of Duokine to fibrosarcoma cell lines HT1080 (HT1080-CD40, HT1080-CD27, HT1080-4-1BB, HT1080-OX40) designed to express CD40, CD27, 4-1BB or OX40 receptors, respectively, was analyzed by flow cytometry. Among them, 1.5 × 10 ⁵ cells were incubated with 100nM Duokine, and binding was detected using PE-labeled mouse anti-FLAG antibody. Cells were analyzed using a MACSQuant 10 analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and data were analyzed using FlowJo (Tree Star, Ashland, USA). Flow cytometry showed that all Duokines bound to cell lines expressing their corresponding receptors (Figure 6A, B).

Example 3: Duokine's Bispecific Receptor Binding Properties

The bispecificity of Duokine was assessed by flow cytometry. 1.5 × ¹⁰ HT1080 cells (HT1080-CD40, HT1080-CD27, HT1080-4-1BB, HT1080-OX40) expressing CD40, CD27, 4-1BB or OX40 receptors were incubated with 100 nM Duokine, followed by incubation with the corresponding receptor Fc fusion protein (10 nM) to detect the second binding site. Cell-bound Duokine-receptor complexes were detected using PE-labeled mouse anti-human Fc antibodies, so only Duokine that recognizes two receptors (one on the cell and the other provided as a soluble Fc fusion protein) can generate fluorescent signals. Cells were analyzed using MACSQuant 10 analyzers (Miltenyi Biotec) and data were analyzed using FlowJo (Tree Star, Ashland, USA). Here, it was shown that all Duokines were able to bind to both receptors simultaneously, demonstrating the dual binding capacity of Duokines ( FIG. 7A , B ).

Example 4: Cloning, production and purification of single-chain Duokine (scDuokine)

DNA encoding single-chain derivatives of the extracellular portion of human CD40L (aa 116-261), human CD27L (aa 52-193), human 4-1BBL (aa 71-254), and human OX40L (aa 51-183) was codon-optimized for expression in human cells and synthesized by adding a suitable cloning site from Geneart (Life Technologies). In the case of scCD40L, scCD27L, and scOX40L, the three single subunits of the cytokine were connected by a GGGSGGG linker, while a (GGGGS) ₃ linker was used for sc4-1BBL. Two of these different single-chain cytokines were fused via a 15 amino acid glycine-serine linker to produce a single-chain Duokine (scDuokine) and cloned into the expression plasmid pIRESpuro3 (Clontech). At the N-terminus, scDuokine has a VH leader sequence for secretion and a FLAG tag for purification and detection. The following scDuokines were produced: scCD40L-scCD27L, scCD27L-scCD40L, scCD40L-sc4-1BBL, sc4-1BBL-scCD40L, scCD27L-sc4-1BBL, sc4-1BBL-scCD27L, scCD40L-scOX40L, scOX40L-scCD40L, scCD27L-scOX40L, scOX40L-scCD27L, sc4-1BBL-scOX40L, and scOX40L-sc4-1BBL (Table 2). All scDuokines were produced by stably transfected HEK293 cells and purified from cell culture supernatants by one-step FLAG affinity chromatography (Sigma-Aldrich), yielding harvests of 1.4 to 4.0 mg/L supernatant.

Single-chain Duokines were analyzed by SDS-PAGE using 10% polyacrylamide gels under reducing and non-reducing conditions and visualized by staining with Coomassie Brilliant Blue G250. Considering the presence of potential N-glycosylation sites in CD40L (1 site; aa 240), CD27L (2 sites; aa 63 and 170), and OX40L (4 sites; aa 90, 114, 152, and 157), SDS-PAGE analysis under reducing and non-reducing conditions showed the expected molecular weight of the trimeric polypeptide chain (approximately 97 kDa for Duokine without 4-1BBL and 111 kDa for Duokine containing 4-1BBL) ( FIG8 , Table 2). In addition, all proteins showed a second band corresponding to a higher molecular weight complex under reducing conditions. The appearance of the higher molecular weight complex was particularly prominent for scDuokine containing scOX40L. The preferential homotrimeric assembly of scDuokines was determined using size exclusion chromatography on a Yarra SEC-2000 (Phenomenex) at a flow rate of 0.5 mL/min. ScCD40L-scCD27L and scCD27L-scCD40L eluted as major peaks with an apparent molecular weight of 125 kDa, thus slightly exceeding the calculated molecular weight of 98 kDa. All scDuokines containing scOX40L showed major peaks corresponding to 140 kDa and 160 kDa and a longer retention time, which may be due to the structure of scOX40L. The scDuokine composed of scCD40L, sc4-1BBL, and scCD27L eluted as a major peak corresponding to a calculated molecular weight of 110 kDa. In addition, scDuokines scCD40L-sc4-1BBL, sc4-1BBL-scCD40L, scCD40L-scOX40L, scOX40L-scCD40L, sc4-1BBL-scOX40L, and scOX40L-sc4-1BBL showed secondary peaks that most likely corresponded to higher molecular weight complexes ( FIG. 9A , B ).

Table 2: Single-chain Duokine and its biochemical properties

Example 5: Receptor Binding Properties of Single-Chain Duokine

The receptor binding of single-chain Duokine was analyzed by ELISA using fusion proteins of the extracellular regions of CD40, CD27, 4-1BB and OX40 and the human Fcγ1 region containing the hinge region for covalent assembly (CD40-Fc, CD27-Fc, 4-1BB-Fc, OX40-Fc). The receptor Fc fusion protein (200 ng/well) was coated overnight at 4°C, and the remaining binding sites were blocked with PBS and 2% skim milk powder (2% MPBS). Single-chain Duokine was titrated in duplicate starting at a concentration of 316 nM, and the bound scDuokine was detected with a mouse anti-FLAG antibody conjugated to HRP. All scDuokines showed specific binding to their corresponding receptors, and no cross-reactivity with other receptor Fc fusion proteins was observed (Figure 10). The interaction between scDuokine and the receptor is dose-dependent, with an EC ₅₀ value in the low nanomolar range (Figure 11 A, B, Table 2)

In addition, the binding of single-chain Duokine to fibrosarcoma cell lines HT1080 (HT1080-CD40, HT1080-CD27, HT1080-4-1BB, HT1080-OX40) designed to express CD40, CD27, 4-1BB or OX40 receptors, respectively, was analyzed by flow cytometry. Among them, 1.5×10 ⁵ cells were incubated with 100nM scDuokine, and binding was detected using PE-labeled mouse anti-FLAG antibody. Cells were analyzed using a MACSQuant10 analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and data were analyzed using FlowJo (Tree Star, Ashland, USA). Flow cytometry showed that all scDuokines bound to cell lines expressing their corresponding receptors (Figures 12A, B).

Example 6: Bispecific receptor binding properties of single-chain Duokine

The bispecificity of single-chain Duokine was assessed by flow cytometry. 1.5 × ¹⁰ HT1080 cells (HT1080-CD40, HT1080-CD27, HT1080-4-1BB, HT1080-OX40) expressing CD40, CD27, 4-1BB or OX40 receptors were incubated with 100 nM scDuokine, followed by incubation with the corresponding receptor Fc fusion protein (10 nM) to detect the second binding site. scDuokine-receptor complexes were detected using PE-labeled mouse anti-human Fc antibodies, so only scDuokine that recognized these two receptors (one on the cell and the other provided as a soluble Fc fusion protein) was able to generate a fluorescent signal. Cells were analyzed using MACSQuant 10 analyzers (Miltenyi Biotec, Bergisch Gladbach, Germany) and data were analyzed using FlowJo (Tree Star, Ashland, USA). Among them, it was shown that all scDuokines were able to bind to both receptors simultaneously, demonstrating the dual binding ability of single-chain Duokines ( FIG. 13A , B ).

Example 7: Cloning, production and purification of single-chain Duokine linked to EHD2 (EHD2-scDuokine)

EHD2-scDuokines were generated by fusion of one single-chain cytokine N-terminus and a second single-chain cytokine C-terminus to the IgE heavy chain domain 2 (EHD2) via a GGSGG linker and cloned into the expression plasmid pSecTagA (Life Technologies, Carlsbad, USA). At the N-terminus, the EHD2-scDuokines contained an Igκ leader sequence for secretion and a FLAG tag for purification and detection. The following EHD2-scDuokines were produced: sc4-1BBL-EHD2-scCD40L, sc4-1BBL-EHD2-scCD27L, and scCD40L-EHD2-scCD27L (Table 3). All EHD2-scDuokines were produced from stably transfected HEK293 cells and purified from cell culture supernatants by one-step FLAG affinity chromatography (Sigma-Aldrich), yielding harvests ranging from 3.7 to 8.0 mg/L supernatant.

EHD2-scDuokine was analyzed by SDS-PAGE using 4% to 15% polyacrylamide gels under reducing and non-reducing conditions and visualized by staining with Coomassie Brilliant Blue G250. Given the presence of potential N-glycosylation sites in CD40L (one site; aa 240) and CD27L (two sites; aa 63 and 170), SDS-PAGE analysis under reducing conditions revealed the expected molecular weight of the hexavalent polypeptide chain (approximately 120 kDa) (Figure 14, Table 3). In addition, all proteins showed a second band under non-reducing conditions corresponding to dimers formed through disulfide bonds between the EHD2 domains. Approximately 50% of EHD2-scDuokine exhibited covalent linkage. Homodimeric assembly of EHD2-scDuokine was confirmed by size exclusion chromatography on a Yarra SEC-2000 (Phenomenex) high-performance liquid chromatography at a flow rate of 0.5 mL/min. Sc4-1BBL-EHD2-scCD27L eluted as a major peak with an apparent molecular weight of 120 kDa (calculated molecular weight 122 kDa), corresponding to the hexavalent monomer, and a minor peak with an apparent molecular weight of 200 kDa (calculated molecular weight 245 kDa), most likely corresponding to the disulfide-linked dimer. Similarly, scCD40L-EHD2-scCD27L eluted as a major peak with an apparent molecular weight of 150 kDa, corresponding to the monomer, and a minor peak with an apparent molecular weight of 270 kDa, corresponding to the dimer. However, the molecular weight determined by SEC was slightly higher than the calculated molecular weight (110 kDa and 220 kDa, respectively). For sc4-1BBL-EHD2-scCD40L, the distribution between monomer and smaller fragments was comparable, with two major peaks eluting at apparent molecular weights of 123 kDa and 74 kDa. In addition, all EHD2-scDuokines showed minor peaks that most likely corresponded to higher molecular weight complexes (Figure 15).

Table 3: EHD2-scDuokine and its biochemical properties

Example 8: Receptor Binding Properties of EHD2-Linked Single-Chain Duokine (EHD2-scDuokine)

Receptor binding of EHD2-scDuokine was analyzed by ELISA using fusion proteins of the extracellular regions of CD40, CD27, and 4-1BB, each containing a human Fcγ1 region containing a hinge region for covalent assembly (CD40-Fc, CD27-Fc, and 4-1BB-Fc). Receptor-Fc fusion proteins (200 ng/well) were coated overnight at 4°C, and remaining binding sites were blocked with PBS and 2% nonfat dry milk (2% MPBS). EHD2-scDuokine was titrated in duplicate starting at a concentration of 316 nM, and bound EHD2-scDuokine was detected using an HRP-conjugated mouse anti-FLAG antibody. All EHD2-scDuokines showed specific binding to their corresponding receptors, and no cross-reactivity with other receptor-Fc fusion proteins was observed (Figure 16). The interaction between EHD2-scDuokine and the receptors was dose-dependent, with _EC50 values in the low nanomolar range (Figure 17, Table 3).

In addition, the binding of EHD2-scDuokine to the fibrosarcoma cell line HT1080 (HT1080-CD40, HT1080-CD27, HT1080-4-1BB), which was designed to express CD40, CD27, or 4-1BB receptors, was analyzed by flow cytometry. 1.5×10 ⁵ cells were incubated with 100 nM EHD2-scDuokine, and binding was detected using a PE-labeled mouse anti-FLAG antibody. Cells were analyzed using a MACSQuant 10 analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and data were analyzed using FlowJo (Tree Star, Ashland, USA). Flow cytometry showed that all EHD2-scDuokines bound to cell lines expressing their corresponding receptors ( FIG. 18 ).

Example 9: Bispecific receptor binding properties of single-chain Duokine linked to EHD2 (EHD2-scDuokine)

The bispecificity of EHD2-scDuokine was analyzed by flow cytometry. 1.5×10 ⁵ HT1080 cells expressing CD40, CD27, or 4-1BB receptors (HT1080-CD40, HT1080-CD27, HT1080-4-1BB) were incubated with 100 nM EHD2-scDuokine, followed by incubation with the corresponding receptor Fc fusion protein (10 nM) to detect the second binding site. The EHD2-scDuokine-receptor complex was detected using a PE-labeled mouse anti-human Fc antibody, so only EHD2-scDuokine that recognized both receptors (one on the cell and the other provided as a soluble Fc fusion protein) would generate a fluorescent signal. Cells were analyzed using a MACSQuant 10 analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany) and data were analyzed using FlowJo (Tree Star, Ashland, USA). Here, it was shown that all EHD2-scDuokines were able to bind to both receptors simultaneously, demonstrating the dual binding capacity of EHD2-scDuokines ( FIG. 19 ).

Example 10: Receptor Activation by EHD2-Linked Single-Chain Duokine (EHD2-scDuokine)

To evaluate the biological activity of EHD2-scDuokine, receptor activation was analyzed by EHD2-scDuokine-mediated secretion of IL-8 from HT1080 cells (Figure 20, Table 3). Therefore, 2×10 ⁴ HT1080 cells expressing CD40, CD27, or 4-1BB were seeded overnight in 96-well microtiter plates, and the supernatant was replaced the next day to remove the continuously produced IL-8. The cells were then incubated in duplicate for 18 hours with serial dilutions of EHD2-scDuokine starting at a concentration of 316 nM. The amount of IL-8 in the supernatant was determined using an IL-8 ELISA kit (Immunotools, Freiburg, Germany) according to the manufacturer's instructions. For comparison, the corresponding monospecific trimeric soluble ligands (CD40L, CD27L) or their soluble single-chain derivatives (scCD40L, scCD27L, sc4-1BBL) were included. On HT1080-CD40 cells, the tested EHD2-scDuokine induced strong IL-8 secretion in a dose-dependent manner, producing IL-8 concentrations as high as 225 ng/mL. Both scCD40Ls containing EHD2-scDuokine (sc4-1BBL-EHD2-scCD40L and scCD40L-EHD2-scCD27L) caused stronger CD40 receptor activation than monospecific soluble scCD40L. Although IL-8 secretion induced by 4-1BB activation was observed for both 4-1BB-targeting EHD2-scDuokines, sc4-1BBL-EHD2-scCD40L showed significantly improved activity relative to sc4-1BBL-EHD2-scCD27L, which had the same activity as monospecific soluble sc4-1BBL. Although both sc4-1BBL-EHD2.scCD27L and scCD40L-EHD2-scCD27L were able to induce IL-8 secretion following CD27 activation, the combination of sc4-1BBL and scCD27L within EHD2-scDuokine showed a stronger induction of IL-8 release (Figure 20). These experiments established that EHD2-scDuokine is capable of inducing receptor activation that results in IL-8 release in this test system.

Example 11: Effects of Duokine and Single-chain Duokine on T Cell Proliferation

In order to evaluate the proliferation capacity of Duokine and scDuokine, the proliferation of T cells in a large number of PBMC colonies was analyzed. To this end, human PBMCs were isolated from healthy donors, stored frozen at -80 ° C, and thawed one day before the experiment. According to the manufacturer's instructions, PBMCs were stained with 625nM carboxyfluorescein diacetate succinimidyl ester (CFSE) at a concentration of 1 × 10 ⁶ cells/mL using the CellTrace CFSE cell proliferation kit (Life Technologies). For the primary stimulation of T cells, cross-linked anti-human CD3 monoclonal antibodies (UCHT-1, R&D systems, Minneapolis, USA) were used. The proliferation induced by the fusion protein in solution was assessed as follows: 1.5 × 10 ⁵ PBMCs/well were incubated with serially diluted different Duokine or scDuokine for 6 days, followed by flow cytometry analysis. Additional antibody-mediated staining was performed to identify T cells (CD3-PE, Immunotools, Friesoythe, Germany).

All PBMC batches tested required primary stimulation with a cross-linked anti-human CD3 mAb for proliferation. Suboptimal antibody concentrations (5-30 ng/ml) induced T cell proliferation at only 20% of T cell levels. All tested Duokine and scDuokine (combinations of CD40L and 4-1BBL, CD27L and 4-1BBL, or CD40L and OX40L) were inactive when administered without a primary stimulus, but induced T cell proliferation in a dose-dependent manner when administered in combination with a cross-linked anti-human CD3 mAb. Proliferation increased two to threefold at low nanomolar concentrations of the fusion protein. In all cases, no difference in proliferation effects could be detected between the different orientations of the cytokines within Duokine or single-chain Duokine. Fusion proteins containing CD40L and 4-1BBL or CD27L and 4-1BBL showed the same proliferation capacity regardless of the format used ( FIG. 21 ).

Example 12: Vector Design of In Vitro Transcribed RNA (IVT-RNA) (mRNA Encoding Duokine and scDuokine) Design, cloning and production

Plasmid constructs (pST1-hAg-Kozak-sec-2hBgUTR-A120) used as templates for in vitro transcription of RNA encoding tumor necrosis factor receptor (TNFR) ligands and their fusion proteins were obtained from pST1-2hBgUTR-A120 (Holtkamp S. et al. (2006), Blood, 108(13): 4009-17). The plasmid backbone was derived from pCMV-Script (Stratagene, LaJolla/CA, USA) by introducing a T7 promoter, a 5'-human α-globin UTR, a KozRk sequence, a 78-bp signal peptide (Sec; secretion signal) derived from MHC class I molecules, two copies of the human 3'β-globin UTR, a 120 bp poly (A) tail, and a kanamycin resistance gene. TNFR ligands or their fusion proteins (consistent with Duokine or scDuokine) were introduced by cold fusion reaction with PCR products (Cold fusion kit, Biocat). The DNA sequences encoding the relevant proteins are listed in Table 4 (Table 4).

The coding sequences of trimeric human TNFR ligands (CD40L, CD27L, OX40L, and 4-1BBL) were introduced by two different strategies: (i) one copy of the extracellular sequence of a given human TNFR ligand, designated as h1_TNFR-ligand (h1_CD27L, h1_CD40L, h1_OX40L, or h1_41BBL); and (ii) three copies of the extracellular sequence of a given human TNFR ligand, linearly linked and separated by a GS-linker. Such human inserts are designated as h3_TNFR-ligand (h3_CD40L, h3_CD27L, h3_OX40L, or h3_41BBL) and correspond to the single-chain proteins described above. To obtain the coding sequences for the fusion proteins, the two h1_TNFR-ligand or h3_TNFR-ligand sequences, respectively, were linked by a 15-amino acid linker ((G4S) ₃ -linker) ( FIG. 22 ). The RNA transcripts h1_TNFR-L(1)-h1_TNFR-L(2) correspond to Duokine, and the transcripts h3_TNFR-L(1)-h3_TNFR-L(2) correspond to scDuokine. The protein sequences of the single h1_TNFR-ligand and h3_TNFR-ligand are listed in Table 5 (Table 5).

To generate IVT-RNA, the plasmid was linearized downstream of the poly(A) tail using a class II restriction endonuclease. The linearized plasmid was purified using magnetic beads (MyOne™ Carboxylic Acid; Invitrogen) according to the manufacturer's instructions, quantified spectrophotometrically, and subjected to in vitro transcription using T7 RNA polymerase (ThermoScientific). Furthermore, the cap analog β-S-ARCA (D2) was incorporated, and finally, the RNA was purified using the MEGA kit (Ambion).

Table 4: DNA/amino acid sequences of specific parts of pST1-hAg-Kozak-sec-INSERT-2hBgUTR-A120 and other plasmids used according to the present invention

Table 5: Amino acid sequences of pST1-hAg-Kozak-sec-INSERT-2hBgUTR-A120 and the variable insert of the plasmid encoding the extracellular domain of TNFR ligands (linker sequences are underlined)

Example 13: Intracellular expression of TNF receptor ligands after IVT-RNA electroporation

K562, a human cell line derived from chronic myeloid leukemia (obtained from ATCC, Manassas, Va., USA), was cultured in RPMI 1640 GlutaMAX supplemented with 5% FCS (all Gibco), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco). For electroporation of K562 cells in a 96-well plate system, cells were washed once with X-Vivo 15 medium (Lonza) and resuspended again in X-Vivo 15 to 500,000 cells/150 μl. 150 μl of the cell suspension was pipetted into a 96-well plate (Biorad) containing the desired wells for multiwell electroporation. After mixing, electroporation was performed using a Gene Pulser MXcell electroporation system (250 V, 1×30 ms pulse) from Biorad, which served as a 96-well electroporation device. Immediately after electroporation, cells were transferred to a new culture plate by adding 100 μl of fresh culture medium and left in an incubator for approximately 6 hours. For intracellular staining of K562, cells were then incubated with GolgiPlug and GolgiStop (BD Biosciences, San Jose, CA) for 16 hours (overnight) according to the manufacturer's protocol. The next day, cells were washed with PBS and fixed with BD Cytofix Buffer (BD Biosciences) for 20 minutes at room temperature. Thereafter, cells were washed again in PBS and permeabilized by washing and transferring the cells to 1× Perm/Wash Buffer (BD Biosciences). After incubation for 10 minutes, cells were stained with anti-TNFR-ligand antibodies diluted in 1× Perm/Wash Buffer for 30 minutes at room temperature, followed by 3 wash steps with 1× Perm/Wash Buffer. Cells were then directly analyzed by flow cytometry using a FACSCanto II flow cytometer (BD Biosciences). Analysis was performed using FlowJo software (Tree Star, Ashland, Oregon, USA).

Electroporation of mRNA-encoded TNFR ligands, Duokine and scDuokine, resulted in intracellular protein expression, which was detected by intracellular antibody staining. Following electroporation of individual TNFR ligand constructs, CD27L, CD40L, OX40L, and 4-1BBL were detected using their corresponding antibodies ( Figures 23A-C ). ScDuokine, encoded by h3_CD40L-h3_CD27L, h3_CD40L-h3_OX40L, and h3_4-1BBL-h3_CD27L, was detected using two respective anti-TNFR-ligand antibodies ( Figures 23A-C ). Duokine, encoded by h1_CD40L-h1_CD27L, h1_41BBL-h1_CD40L, and h1_4-1BBL-h1_CD27L, was detected using two respective anti-TNFR-ligand antibodies ( Figures 23D ).

Example 14: Encoding TNFL(1)-TNFL(2) fusion constructs (corresponding to Duokine and scDuokine, respectively) Receptor Activation Properties of Proteins Expressed after IVT-RNA Electroporation

Preparation of cell lines/cells expressing TNF receptors (TNFRs): The HT1080 cell line and its stable TNFR transfectants were cultured in RPMI 1640 GlutaMAX supplemented with 5% FCS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Cell surface expression of TNF receptors in HT1080 transfectants was analyzed by FACS. To this end, cells were stained with antibodies against CD40-FITC (Biolegend), CD27-PE (BD), OX40-PE (BD), and 41BB-PE (BD) ( FIG24A ).

To produce K562 transiently expressing TNF receptors, K562 cells were washed once in X-Vivo 15 culture medium (Lonza) and resuspended in X-Vivo 15 to a final concentration of 8 × ¹⁰ cells/250 μl. 8 × 10 K562 cells were electroporated in 250 μl culture medium with 20 to 40 μg of plasmid DNA encoding the full-length TNF receptors of human CD40, CD27, OX40 or 4-1BB. Electroporation was performed in 250 μl X-VIVO 15 in a ⁴ mm electroporation cup (electroporation cuvette) using a BTX830 Electroporation System (BTX, Holliston, Mass., USA) device (200 V, 3 × 8 ms pulses). Immediately after electroporation, cells were transferred to a new culture plate containing fresh culture medium without antibiotics. The next day, cell surface expression of TNF receptors was examined by FACS analysis ( FIG. 24B ).

Generation of supernatant containing TNFR-ligand fusion protein: K562 cells were electroporated as described above. Immediately after electroporation, cells were transferred to a new culture plate by adding 100 μl of fresh culture medium and allowed to rest in an incubator for approximately 3 hours. The cells were then centrifuged and the cell pellet was resuspended in 250 μl of RPMI 1640 GlutaMAX with 0.5% FCS for an overnight incubation (approximately 16 hours). The following day, 100 μl of the supernatant containing the secreted fusion protein was transferred to a confluent layer of HT1080-TNF receptor transfectants.

NF-κB pathway activation after TNF-receptor activation measured by IL-8 release from HT1080 cells: TNF receptor transfectants of the HT1080 cell line were used for reporter assays to measure TNF- receptor activation. The following stable transfectants expressing human TNF receptors were used: HT1080_CD40, HT1080_CD27, HT1080_OX40, and HT1080_4-1BB; cell surface expression before reporter assays was examined by FACS analysis ( FIG. 24A ). Cells were seeded (2×10 ⁴ cells/well) in 96-well tissue culture plates with RPMI 1640 medium with 5% FCS and cultured overnight. The culture medium was aspirated and 100 μl of cell culture supernatant from electroporated K562 was added. If desired, K562 cells designated to express TNF receptors were additionally added to 100 μl of RPMI 1640 GlutaMAX medium with 0.5% FCS. After 6 to 8 hours of incubation, cell-free supernatants were collected and IL-8 concentrations were measured by IL-8 ELISA kit (Biolegend) according to the manufacturer's protocol.

Activation of the CD40 receptor by HT1080-CD40 was detected after electrophoresis of h3-CD40L and h3-CD27L-h3-CD40L. However, after administration of h3-CD27L-h3-CD40L, a strong increase in CD40 activation was detected in a trans-presentation setting, which correlated with the amount of RNA administered, and this was achieved by the addition of K562-CD27, thus enabling cell-cell interactions mediated by translated duokines ( FIG. 25A ). CD27 activation without trans-presentation was not detected after electroporation of IVT-RNA encoding h3-CD27L or h3-CD27L-h3-CD40L by measuring IL-8 secretion. In a trans-presentation setting, CD27 activation mediated by K562-CD40 was detected after K562 electroporation of the h3-CD27L-h3-CD40L fusion construct ( FIG. 25B ).

h3_OX40L and h3_CD27L-h3_OX40L constructs mediate activation of the OX40 receptor. A significant increase in OX40 activation related to the amount of RNA administered was again detected after administration of h3_CD27L-h3_OX40L in a trans-presentation setting mediated by K562_CD27 ( FIG. 25C ). CD27 activation by h3_CD27L-h3_OX40L was only detectable in a trans-presentation setting mediated by K562_OX40 ( FIG. 25D ).

41BB and CD27 activation after h3_CD27L-h3_4-1BBL administration were both clearly detectable only in the trans-presentation setting mediated by K562_CD27 and K562_4-1BB, respectively ( FIG. 25E , F).

Even if h3_4-1BBL-h3_CD40L and h1_4-1BBL-h1_CD40L constructs do not present in trans, they mediate the activation of CD40 receptors. However, under the trans presentation setting mediated by K562_4-1BB, a strong improvement (Figure 25 G) of CD40 activation related to the amount of RNA administered was detected. After h3_4-1BBL-h3_CD40L and h1_4-1BBL-h1_CD40L were applied, the activation of 41BB was only clearly detected under the trans presentation setting mediated by K562_CD40 (Figure 25 H).

Example 15: Effects of Duokine and mRNA-encoded scDuokine on the proliferation of antigen-specific CD8 ⁺ T cells

HLA-A2 ⁺ peripheral blood mononuclear cells (PBMCs) were obtained from blood donations from the Transfusionszentrale of the University Hospital in Mainz, Germany. Monocytes were isolated from PBMCs using anti-CD14 microbeads (Miltenyi) by magnetic-activated cell sorting (MACS) technology; peripheral blood lymphocytes (PBL, CD14 ^- fraction) were frozen for future T cell isolation. To differentiate into immature DCs (iDCs), monocytes were cultured for 4 to 5 days in RPMIGlutaMAX containing 5% human AB serum (Gibco), sodium pyruvate (Gibco), non-essential amino acids, 100 IU/mL penicillin and 100 μg/mL streptomycin, 1000 IU/mL granulocyte macrophage colony stimulating factor, and 1000 IU/mL IL-4 (all from Miltenyi). During these 4 to 5 days, half of the culture medium was replaced once with fresh culture medium. iDCs were harvested and washed once with X-Vivo 15 culture medium before electroporation and resuspended again with X-Vivo 15 to 300,000 to 500,000 cells/150 μl. 150 μl of the cell suspension was pipetted into a 96-well plate for multi-well electroporation, which already contained the required IVT-RNA, i.e., RNA encoding antigen sealant-6 and RNA encoding TNFR-ligand fusion protein as specified, or an unrelated RNA (encoding luciferase) as a control. After mixing the cell suspension with the RNA, electroporation was performed in a multi-well electroporation device (300 V, 1×12 ms pulse). Immediately after electroporation, the cells were transferred to a new culture plate by adding 100 μl of IMDM culture medium supplemented with 5% human AB serum and allowed to stand in an incubator for about 1 to 3 hours.

CD8 ⁺ T cells were isolated from the remaining HLA-A2 ⁺ peripheral blood lymphocytes frozen after CD14 ⁺ -MACS separation using anti-CD8 MicroBeads (Miltenyi) by MACS technology. The CD8 ⁺ T cells were washed once in X-vivo15 medium and resuspended again in X-Vivo15 to a final concentration of 10 × 10 ⁶ cells / 250 μl. 10 × 10 ⁶ CD8 ⁺ T cells were electroporated in 250 μl of culture medium with 10 μg of IVT-RNA encoding the α chain of the claudin-6-TCR (restricted to HLA-A2 ⁺ ) and 10 μg of IVT-RNA encoding the β chain of the claudin-6-TCR (restricted to HLA-A2 ⁺ ). As a control, RNA encoding TPTE-TCR (restricted to HLA-A2 ⁺ ) was used. Electroporation was performed in 250 μl X-Vivo15 using a BTX830 electroporation system device (500 V, 1 × 3 ms pulse) in a 4 mm electroporation cup. Immediately after electroporation, cells were transferred into fresh IMDM culture medium supplemented with 5% human AB serum and allowed to stand in an incubator for at least 1 hour. T cells were then stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) according to the manufacturer's protocol (Invitrogen).

To analyze the effects mediated by TNFR-ligand constructs (IVT-RNA), a CFSE proliferation assay was performed. To this end, a total of 5,000 electroporated DCs were added to wells of a 96-well plate containing 50,000 T cells electroporated with RNA encoding either the CLD6-TCR or the TPTE-TCR. The cells were incubated with RPMI 1640 supplemented with 5% human AB serum, 100 IU/mL penicillin, and 100 mg/mL GlutaMAX. After 5 days of incubation, PBMC proliferation was measured by flow cytometry and analyzed using FlowJo software.

Co-electroporation of DCs with claudin-6 antigen-RNA and h3_CD27L-h3_CD40L RNA or h3_CD40L-h3_CD27L RNA resulted in increased proliferation of CD8 ⁺ -T cells, more specifically, more T cells entered division (increased "% of dividing cells") and then also divided more frequently (increased "proliferation index") ( Figures 26A-C ). In contrast, co-electroporation of constructs encoding two separate proteins, h3_CD40L and h3_CD27L, with claudin-6 antigen-RNA did not result in increased T cell proliferation ( Figure 26A ). Furthermore, h3_CD27L-h3_CD40L did not induce significant proliferation of control T cells, TPTE-TCR ⁺ CD8 ⁺ T cells, indicating that the constructs did not activate T cell proliferation in an antigen-nonspecific manner ( Figure 26B ). h3_CD27L-h3_CD40L and h3_CD40L-h3_CD27L mediated comparable effects ( FIG. 26A ). h3_CD27L-h3_CD40L is an example of a fusion protein that can bind to and activate CD40 expressed on DCs and CD27 constitutively expressed on T cells, thereby trans-crosslinking these cells.

Likewise, DC co-electroporation with antigen-RNA and h3_4-1BBL-h3_CD27L RNA produced CD8 ⁺ -T cell proliferation that improved, while co-electroporation with antigen-RNA and a single construct encoding two separate proteins, h3_4-1BBL and h3_CD27L, did not produce improved T cell proliferation ( Figure 27 A ). In addition, h3_4-1BBL-h3_CD27L did not induce control T cell TPTE-TCR ⁺ CD8 ⁺ T cell proliferation, demonstrating that constructs do not activate T cell proliferation in an antigen-nonspecific manner ( Figure 27 B ).

H3_4-1BBL-h3_CD27L (corresponding to scDuokine) and h1_4-1BBL-h1_CD27L (corresponding to Duokine) mediate suitable effect (Figure 27 A).H3_4-1BBL-h3_CD27L is the example for fusion protein, which can be combined with the CD27 constitutively expressed on T cells and activated, and combined with the 4-1BB expressed on T cells after activation and activated, so as to be able to cis cross-linked receptors.

To analyze the effects of recombinant Duokine, iDC and T cell co-cultures were initiated one day after electroporation at the aforementioned cell counts and ratios. Simultaneously, recombinant proteins were added as indicated. PBMC proliferation was then measured after 4 days of incubation.

Adding the recombinant fusion proteins CD40L-CD27L, 41BBL-CD40L, and 4-1BBL-CD27L to T cell:DC co-cultures resulted in increased proliferation of CD8 ⁺ -T cells ( FIG. 28A ). More T cells entered division (increased "% of dividing cells") and then also divided more frequently (increased "proliferation index") ( FIG. 28B ). In contrast, adding either TNFR ligand protein alone did not result in increased T cell proliferation.

Example 16: Simultaneous binding of duokine to immobilized receptors and PBMCs leads to T cell activation and proliferation

It has been shown that duokines composed of 4-1BBL/CD40L, 4-1BBL/CD27L, and CD40L/CD27L in solution can activate T cells. In further experiments, the following situation was analyzed: whether those duokines also activate T cells when presented by binding to receptors immobilized on a plastic surface, limiting activation to the second receptor binding specificity. Therefore, 200ng/well receptor Fc (CD40-Fc, CD27-Fc, 4-1BB-Fc, OX40-Fc) were immobilized on microtiter plates overnight at 4°C. Residual binding sites were blocked with RPMI 1640 + 10% FCS for 1 hour. Serial dilutions of duokine (CD40L-CD27L, 4-1BBL-CD40L, 4-1BBL-CD27L, OX40L-CD40L, and OX40L-CD27L) were incubated with immobilized receptors for 1 hour, and unbound proteins were subsequently washed away. Simultaneously, human PBMCs at a concentration of 1×10 ⁶ cells/mL were stained with 625 nM carboxyfluorescein diacetate succinimidyl ester (CFSE) using the CellTrace CFSE Cell Proliferation Kit (Life Technologies) according to the manufacturer's instructions. For primary stimulation of T cells, anti-human CD3 monoclonal antibody (UCHT-1, R&D systems, Minneapolis, USA) was cross-linked with anti-mouse IgG at a molar ratio of 1:3. PBMCs (1.5×10 ⁵ cells per well) were added to the assay plate containing duokine bound to the immobilized receptors along with the primary stimulants. Six days later, T cell proliferation was measured by flow cytometry. T cells were identified by staining with antibodies to CD3-PE, CD4-VioBlue, and CD8-PE-Vio770. The observation of increased proliferation of CD4 ⁺ and CD8 ⁺ T cells with all duokines further demonstrates that both duokine binding sites are functional and are able to induce co-stimulation of T cells with the primary stimulus provided by CD3 ( FIG. 29 ). No proliferation was observed in the absence of CD3 stimulation, which supports the dependence of T cell activation on primary activation by duokine.

Example 17: Simultaneous binding of single-chain duokine to immobilized receptors and PBMCs leads to T cell activation and proliferation

Single-chain duokines composed of 4-1BBL/CD40L, 4-1BBL/CD27L, and CD40L/CD27L have been shown to activate T cells in solution. In further experiments, the following scenario was analyzed: whether those single-chain duokines also activate T cells when presented by binding to receptors immobilized on a plastic surface, restricting activation to the second receptor binding specificity. Therefore, 200 ng/well receptor-Fc (CD40-Fc, CD27-Fc, 4-1BB-Fc, OX40-Fc) were immobilized on microtiter plates overnight at 4°C. Residual binding sites were blocked with RPMI 1640 + 10% FCS for 1 hour. Serial dilutions of single-chain duokines (scCD40L-scCD27L, sc4-1BBL-scCD40L, sc4-1BBL-scCD27L, scOX40L-scCD40L, and scOX40L-scCD27L) were incubated with immobilized receptor for 1 hour, and unbound proteins were subsequently washed away. Simultaneously, human PBMCs at a concentration of 1×10 ⁶ cells/mL were stained with 625 nM carboxyfluorescein diacetate succinimidyl ester (CFSE) using the CellTrace CFSE Cell Proliferation Kit (Life Technologies) according to the manufacturer's instructions. For primary stimulation of T cells, anti-human CD3 monoclonal antibody (UCHT-1, R&D systems, Minneapolis, USA) was cross-linked to anti-mouse IgG at a 1:3 molar ratio. 1.5×10 ⁵ PBMC cells per well were added to the assay plate containing single-chain duokines bound to immobilized receptors along with the primary stimulant. After 6 days, T cell proliferation was measured by flow cytometry. T cells were identified by staining with CD3-PE, CD4-VioBlue, and CD8-PE-Vio770 antibodies. The observation of increased proliferation of CD4 ⁺ and CD8 ⁺ T cells with all duokines further demonstrates that both binding sites of single-chain duokines are functional and are able to induce co-stimulation of T cells with the primary stimulator provided by CD3 ( FIG. 30 ). No proliferation was observed in the absence of CD3 stimulators that support the dependence of T cell activation on primary activation via single-chain duokines.

Example 18: Stability of Selected Duokines and Single-Chain Duokines in Human Plasma

The stability of duokines and single-chain duokines was tested in vitro using human plasma. 200 nM purified duokines and single-chain duokines were prepared using 50% human plasma from healthy donors. Samples were frozen at -20°C immediately after preparation (day 0) or after incubation at 37°C for 1, 3, and 7 days. Intact protein levels were determined using ELISA by binding of the C-terminal homotrimeric ligand unit to immobilized receptors (150 ng/well) and detection of the N-terminal FLAG tag. Protein concentrations in diluted plasma samples were interpolated from a standard curve of purified protein. The amount of fusion protein detected on day 0 was set to 100%. Six duokines and six single-chain duokines were tested, covering all possible ligand combinations, and as shown by receptor binding assays, all constructs showed a time-dependent decrease in intact protein levels. After 7 days, 50% to 70% of intact duokine was detectable in plasma samples for most ligand combinations ( Figure 31 , left). Only in the case of 4-1BBL-CD27L did the amount of intact protein decrease to less than 10% after 7 days and by 40% on day 1. In general, single-chain duokines showed reduced stability, with 20% to 50% of intact protein remaining after 7 days ( Figure 31 , right).

Example 19: Pharmacokinetic Properties of Selected Murine Duokines and Murine Single-Chain Duokines in CD1 Mice

The in vivo bioavailability of selected mouse-specific duokines and their corresponding single-chain duokines was investigated by measuring serum concentrations after a single IV injection. Female CD1 mice (12 to 16 weeks, 30 to 35 g, 3 mice/duokine) each received a single intravenous injection of 25 μg of m4-1BBL-mCD40L and msc4-1BBL-mscCD40L (150 μl total volume each). Blood samples were collected from the tail vein at 3 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 1 day, and 3 days after injection, incubated on ice for 30 minutes, and centrifuged at 13,000 g for 30 minutes at 4°C. Serum was separated from cellular components and samples were stored at -20°C. Serum levels of the fusion proteins were determined in an ELISA by binding to immobilized receptors corresponding to the C-terminal ligands (150 ng/well) and by detection of the N-terminal FLAG tag. Serum concentrations of all proteins were obtained by interpolation from a standard curve of purified proteins. For comparison, the concentration at the 3rd minute was set to 100%. Initial and terminal half-lives (t _1/2 α _{3-60 minutes} , t _1/2 β _{1-24 hours} ) were calculated using Excel. Both m4-1BBL-mCD40L and msc4-1BBL-mscCD40L showed clearance from the bloodstream with terminal half-lives of 3.8 hours (single-chain duokine) and 5.6 hours (duokine) and short initial half-lives of 10 to 13 minutes in both cases (Figure 32). Relative to circulating IgG (cetuximab) mediated by FcRn, these two constructs were rapidly cleared. Comparison with the scFv-4-1BBL fusion protein with a terminal half-life of 6.6 hours (data not shown) shows that m4-1BBL-mCD40L and msc4-1BBL-mscCD40L are within the same range of those functionally related immunostimulatory fusion proteins. The overall rapid clearance of duokine-containing immunostimulatory fusion proteins is directed towards the specific depletion of target cells (ie, immune cells).

Example 20: Receptor Expression on Human PBMCs and Binding of Single-Chain Duokine to Immune Cell Subpopulations

To determine the potential target cell populations within the PBMC population and the actual target cells of single-chain duokine, human PBMCs were characterized in detail. PBMCs from healthy donors were thawed and incubated overnight at 4°C on cell culture plates. The next day, 2.5×10 ⁵ human PBMCs were incubated with 10 nM single-chain duokine in the presence or absence of a cross-linked anti-human CD3 antibody as the primary stimulator at a response-limiting suboptimal concentration. After 3 days at 37°C, flow cytometry was used to identify different subpopulations by staining with CD markers (anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD20, and anti-CD56), and the binding of single-chain duokine to different subpopulations was assessed by detecting their FLAG tags. In addition, stimulated and unstimulated PBMCs were incubated without single-chain duokine. After 3 days of culture, subpopulations were identified and surface expression of CD40, CD27, 4-1BB, and OX40 was determined by antibody staining. Unstimulated PBMC is composed of about 40% CD8 ⁺ T cells, 40% CD4 ⁺ T cells, 15% B cells and 5% NK cells (Figure 33, upper left). Due to the treatment of PBMC, all monocytes adhere to the plastic surface and are not present in the experiment. After 3 days of stimulation with anti-CD3mAb, the amount of CD8 ⁺ T cells increased to nearly 80%, accompanied by a slight decrease in the percentage of all other cell types (Figure 33, lower left). Receptors CD40 and CD27 are constitutively expressed on B cells (CD40) and these two types of T cells (CD27) that are not related to stimulation. While 4-1BB and OX40 are not present on any unstimulated PBMC, about 80% of stimulated CD4 ⁺ T cells and 40% of stimulated CD8 ⁺ T cells express these two receptors (Figure 33, middle figure). Consistent with receptor expression, the three tested trans-acting single-chain duokines (scCD40L-scCD27L, sc4-1BBL-scCD40L, and scOX40L-scCD40L) bound almost exclusively to stimulated and unstimulated B cells (Figure 33, upper right). Cis-acting single-chain duokines (sc4-1BBL-scCD27L and scOX40L-scCD27L) bound only to the fraction of CD4 ⁺ T cells in an unstimulated setting and to the majority of CD4 ⁺ and CD8 ⁺ T cells after stimulation (Figure 33, lower right). Taken together, these experiments show that trans-acting molecules target B cells independently of stimulation, while cis-acting constructs target activated CD8 ⁺ and CD4 ⁺ T cells.

Example 21: Binding of cis-acting single-chain duokine to human immune cells and induction of T cell proliferation

The link between binding to human immune cells and induction of T cell proliferation was verified for a selected cis-acting single-chain duokine. To this end, 2.5×10 ⁵ human PBMCs (population) were incubated with 10 nM sc4-1BBL-scCD27L in the presence or absence of a cross-linked anti-human CD3 antibody as a primary suboptimal stimulator. After 3 days at 37°C, different subpopulations were identified by flow cytometry using CD marker staining (anti-CD3, anti-CD4, anti-CD8, anti-CD20, and anti-CD56). Surface expression of CD27 and 4-1BB was determined by antibody staining, and binding of the single-chain duokine was assessed by detecting their FLAG tags. 1.5×10 ⁵ CFSE-labeled PBMCs (population, different PBMC batches) were incubated with 30, 3, 0.3, or 0 nM sc4-1BBL-scCD27L in the presence or absence of a cross-linked anti-human CD3 antibody as a primary suboptimal stimulator. After 6 days, the proliferation of CD4 ⁺ and CD8 ⁺ T cells was determined by CFSE dilution using flow cytometry. When administered in combination with a cross-linked anti-human CD3 antibody as the main stimulator, sc4-1BBL-scCD27L increased the initial CD3-mediated proliferation of CD4 ⁺ and CD8 ⁺ T cells by 30% as early as at a low concentration of 0.3 nM (Figure 34, bottom right). Generally speaking, more CD8 ⁺ T cells (80%) than CD4 ⁺ T cells (60%) begin to proliferate. This proliferation spectrum is consistent with the finding that sc4-1BBL-scCD27L combines with CD4 ⁺ and CD8 ⁺ T cells expressing both 4-1BB and CD27 in a stimulated environment (Figure 34, bottom left). Without anti-CD3 stimulation, no proliferative CD8 ⁺ T cells were observed at all, and only a slight proliferation of ~10% CD4 ⁺ T cells was found (Figure 34, top right). Although binding to CD27-expressing CD4 ⁺ T cells was noted, sc4-1BBL-scCD27L did not increase this basal proliferation rate ( FIG. 34 , upper left), indicating that cis-acting duokines do not act on resting T cells.

Example 22: Binding of cis-acting single-chain duokine to human immune cells and induction of T cell proliferation

The selected trans-acting single-chain duokine was validated for the connection between binding to human immune cells and induction of T cell proliferation. To this end, 2.5×10 ⁵ human PBMCs (populations) were incubated with 10 nM sc4-1BBL-scCD40L in the presence or absence of a cross-linked anti-human CD3 antibody as the primary suboptimal stimulator. After 3 days at 37°C, different subpopulations were identified by flow cytometry using CD marker staining (anti-CD3, anti-CD4, anti-CD8, anti-CD20, and anti-CD56). The surface expression of CD40 and 4-1BB was determined by antibody staining, and the binding of single-chain duokine was assessed by detecting its FLAG tag. 1.5×10 ⁵ CFSE-labeled PBMCs (populations, different PBMC batches) were incubated with 30, 3, 0.3, or 0 nM sc4-1BBL-scCD40L in the presence or absence of a cross-linked anti-human CD3 antibody as the primary suboptimal stimulator. After 6 days, the proliferation of CD4 ⁺ and CD8 ⁺ T cells was determined by CFSE dilution using flow cytometry. When administered in combination with a cross-linked anti-human CD3 antibody as the primary stimulator, sc4-1BBL-scCD40L strongly increased the initial CD3-mediated proliferation of CD4 ⁺ and CD8 ⁺ T cells by 50% to 60% as early as at a low concentration of 0.3 nM. Generally speaking, almost all CD8 ⁺ T cells (90%) and about 70% CD4 ⁺ T cells begin to proliferate ( Figure 35 , bottom right). sc4-1BBL-scCD40L initially binds to B cells constitutively expressing CD40B, allowing the single-chain duokine's 4-1BBL pattern to be trans-presented in the triggered 4-1BB-expressing T cells ( Figure 35 , bottom right). Without CD3 stimulation, only minimal (3%) CD8 ⁺ T cell proliferation was observed, and a slight increase to 9% of proliferative cells was observed after the addition of sc4-1BBL-scCD40L, indicating that a small fraction of CD8 ⁺ T cells can respond to transactivation without deliberate CD3 triggering (Figure 35, top panel), suggesting that these cells are in a pre-activated state.

Example 23: Binding of trans-acting single-chain duokine to human immune cells and induction of T cell proliferation

The second selected trans-acting single-chain duokine was tested for its association with human immune cell binding and T cell proliferation induction. To this end, 2.5×10 ⁵ human PBMCs (population) were incubated with 10 nM scCD40L-scCD27L in the presence or absence of a cross-linked anti-human CD3 antibody as a primary suboptimal stimulator. After 3 days at 37°C, different subpopulations were identified by flow cytometry using CD marker staining (anti-CD3, anti-CD4, anti-CD8, anti-CD20, and anti-CD56). Surface expression of CD40 and CD27 was determined by antibody staining, and binding of the single-chain duokine was assessed by detecting its FLAG tag. 1.5×10 ⁵ CFSE-labeled PBMCs (population, different PBMC batches) were incubated with 30, 3, 0.3, or 0 nM scCD40L-scCD27L in the presence or absence of a cross-linked anti-human CD3 antibody as a primary suboptimal stimulator. After 6 days, the proliferation of CD4 ⁺ and CD8 ⁺ T cells was determined by CFSE dilution using flow cytometry. When administered in combination with a cross-linked anti-human CD3 antibody as the primary stimulator, sc4-1BBL-scCD27L increased the initial CD3-mediated proliferation of CD4 ⁺ and CD8 ⁺ T cells by 20% to 35% as early as at a low concentration of 0.3 nM (Figure 36, lower right). Generally speaking, slightly more CD8 ⁺ T cells (60%) than CD4 ⁺ T cells (45%) began to proliferate. Antibody staining showed that CD27 was constitutively expressed on all CD8 ⁺ and CD4 ⁺ T cells, and the binding of scCD40L-scCD27L duokine was detected on 50% of all CD4 ⁺ T cells, but below the detection level on CD8 ⁺ T cells. It was also found that scCD40L-scCD27L bound to the entire population of B cells constitutively expressing CD40 (Figure 36, lower left). Thus, direct transactivation via scCD40L-scCD27L through CD27 ⁺ CD4 cell-CD40 ⁺ B cell interactions readily leads to CD4 ⁺ T cell proliferation, whereas in the additional case of CD8 ⁺ T cells, a hitherto undefined mechanism/cell appears to be necessary to enable robust transactivation by this single-chain duokine. In the absence of any CD3 stimulation, T cell proliferation in response to duokines scCD40L-scCD27L increased from 1% to 10% for CD8 ⁺ and from 5% to 25% for CD4 ⁺ T cells, respectively (Figure 36, top right), indicating that some T cells are able to respond to costimulatory signals without deliberate CD3 activation, again suggesting the presence of a subpopulation of pre-activated cells.

In summary, the considerable T cell activation mediated by scCD40L-scCD27L can be attributed to the constitutive expression of CD27 on all T cells and CD40 on all B cells ( Figure 36 , upper left), which leads to prolonged crosstalk and activation of different immune cells.

Sequence Listing

<110> Biotechnology RNA Pharmaceutical Co., Ltd.

University of Stuttgart

Johannes Gutenberg University Mainz Medical University Translational Oncology

<120> Cytokine fusion protein

<130> 674-132 PCT

<150> PCT/EP2015/050682

<151> January 15, 2015

<160> 23

<170> PatentIn version 3.5

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<212> PRT

<213> Artificial sequence

<220>

<223> h1_CD40L

<400> 5

Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val Phe Met

1 5 10 15

Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser Leu Leu

20 25 30

Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys Asp Ile

35 40 45

Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu Met Gln

50 55 60

Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala

65 70 75 80

Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr

85 90 95

Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr

100 105 110

Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys

115 120 125

Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys

130 135 140

Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn

145 150 155 160

Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly

165 170 175

Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr

180 185 190

Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu

195 200 205

Leu Lys Leu

210

<210> 6

<211> 142

<212> PRT

<213> Artificial sequence

<220>

<223> h1_CD27L

<400> 6

Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His Thr Gly Pro

1 5 10 15

Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg

20 25 30

Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His

35 40 45

Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys

50 55 60

Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly

65 70 75 80

Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe

85 90 95

His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg

100 105 110

Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg

115 120 125

Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro

130 135 140

<210> 7

<211> 184

<212> PRT

<213> Artificial sequence

<220>

<223> h1_4-1BBL

<400> 7

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Pro Ser Pro Arg Ser Glu

180

<210> 8

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> h1_OX40L

<400> 8

Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe

1 5 10 15

Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu

20 25 30

Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn Cys Asp

35 40 45

Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn

50 55 60

Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys

65 70 75 80

Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys

85 90 95

Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp

100 105 110

Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly

115 120 125

Glu Phe Cys Val Leu

130

<210> 9

<211> 452

<212> PRT

<213> Artificial sequence

<220>

<223> h3_CD40L

<400> 9

Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser

1 5 10 15

Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr

20 25 30

Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val

35 40 45

Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser

50 55 60

Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu

65 70 75 80

Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr

85 90 95

His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly

100 105 110

Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp

115 120 125

Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu

130 135 140

Lys Leu Gly Gly Gly Ser Gly Gly Gly Gly Asp Gln Asn Pro Gln Ile

145 150 155 160

Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu

165 170 175

Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr

180 185 190

Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr

195 200 205

Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln

210 215 220

Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu

225 230 235 240

Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys

245 250 255

Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly

260 265 270

Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly

275 280 285

Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly

290 295 300

Gly Gly Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu

305 310 315 320

Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr

325 330 335

Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu

340 345 350

Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe

355 360 365

Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu

370 375 380

Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala

385 390 395 400

Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu

405 410 415

Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val

420 425 430

Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly

435 440 445

Leu Leu Lys Leu

450

<210> 10

<211> 440

<212> PRT

<213> Artificial sequence

<220>

<223> h3_CD27L

<400> 10

Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His Thr Gly Pro

1 5 10 15

Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg

20 25 30

Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His

35 40 45

Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys

50 55 60

Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly

65 70 75 80

Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe

85 90 95

His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg

100 105 110

Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg

115 120 125

Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro Gly Gly

130 135 140

Gly Ser Gly Gly Gly Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu

145 150 155 160

Asn His Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly

165 170 175

Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly

180 185 190

Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val

195 200 205

Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr

210 215 220

Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu

225 230 235 240

Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu

245 250 255

Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr

260 265 270

Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp

275 280 285

Val Arg Pro Gly Gly Gly Ser Gly Gly Gly Ser Leu Gly Trp Asp Val

290 295 300

Ala Glu Leu Gln Leu Asn His Thr Gly Pro Gln Gln Asp Pro Arg Leu

305 310 315 320

Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg Ser Phe Leu His Gly Pro

325 330 335

Glu Leu Asp Lys Gly Gln Leu Arg Ile His Arg Asp Gly Ile Tyr Met

340 345 350

Val His Ile Gln Val Thr Leu Ala Ile Cys Ser Ser Thr Thr Ala Ser

355 360 365

Arg His His Pro Thr Thr Leu Ala Val Gly Ile Cys Ser Pro Ala Ser

370 375 380

Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe His Gln Gly Cys Thr Ile

385 390 395 400

Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg Gly Asp Thr Leu Cys Thr

405 410 415

Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg Asn Thr Asp Glu Thr Phe

420 425 430

Phe Gly Val Gln Trp Val Arg Pro

435 440

<210> 11

<211> 592

<212> PRT

<213> Artificial sequence

<220>

<223> h3_4-1BBL

<400> 11

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly

180 185 190

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro

195 200 205

Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly

210 215 220

Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro

225 230 235 240

Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly

245 250 255

Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala

260 265 270

Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala

275 280 285

Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu

290 295 300

Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro

305 310 315 320

Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg

325 330 335

Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr

340 345 350

Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val

355 360 365

Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser

370 375 380

Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

385 390 395 400

Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro

405 410 415

Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln

420 425 430

Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr

435 440 445

Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr

450 455 460

Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr

465 470 475 480

Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser

485 490 495

Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala

500 505 510

Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser

515 520 525

Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu

530 535 540

Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala

545 550 555 560

Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe

565 570 575

Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

580 585 590

<210> 12

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> h3_OX40L

<400> 12

Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe

1 5 10 15

Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu

20 25 30

Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn Cys Asp

35 40 45

Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn

50 55 60

Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys

65 70 75 80

Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys

85 90 95

Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp

100 105 110

Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly

115 120 125

Glu Phe Cys Val Leu Gly Gly Gly Ser Gly Gly Gly Gln Val Ser His

130 135 140

Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe Thr Glu Tyr Lys

145 150 155 160

Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu Asp Glu Ile Met

165 170 175

Lys Val Gln Asn Asn Ser Val Ile Ile Asn Cys Asp Gly Phe Tyr Leu

180 185 190

Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu His

195 200 205

Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys Lys Val Arg Ser

210 215 220

Val Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys Asp Lys Val Tyr

225 230 235 240

Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe His Val Asn

245 250 255

Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly Glu Phe Cys Val

260 265 270

Leu Gly Gly Gly Ser Gly Gly Gly Gln Val Ser His Arg Tyr Pro Arg

275 280 285

Ile Gln Ser Ile Lys Val Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly

290 295 300

Phe Ile Leu Thr Ser Gln Lys Glu Asp Glu Ile Met Lys Val Gln Asn

305 310 315 320

Asn Ser Val Ile Ile Asn Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys

325 330 335

Gly Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu His Tyr Gln Lys Asp

340 345 350

Glu Glu Pro Leu Phe Gln Leu Lys Lys Val Arg Ser Val Asn Ser Leu

355 360 365

Met Val Ala Ser Leu Thr Tyr Lys Asp Lys Val Tyr Leu Asn Val Thr

370 375 380

Thr Asp Asn Thr Ser Leu Asp Asp Phe His Val Asn Gly Gly Glu Leu

385 390 395 400

Ile Leu Ile His Gln Asn Pro Gly Glu Phe Cys Val Leu

405 410

<210> 13

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<223> hAg-Kozak

<400> 13

attcttctgg tccccacaga ctcagagaga acccgccacc 40

<210> 14

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> Sec

<400> 14

atgagagtga ccgcccccag aaccctgatc ctgctgctgt ctggcgccct ggccctgaca 60

gagacatggg ccggaagcgg atcc 84

<210> 15

<211> 28

<212> PRT

<213> Artificial sequence

<220>

<223> Sec

<400> 15

Met Arg Val Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala

1 5 10 15

Leu Ala Leu Thr Glu Thr Trp Ala Gly Ser Gly Ser

20 25

<210> 16

<211> 287

<212> DNA

<213> Artificial sequence

<220>

<223> 2hBgUTR

<400> 16

ctcgagagct cgctttcttg ctgtccaatt tctattaaag gttcctttgt tccctaagtc 60

caactactaa actgggggat attatgaagg gccttgagca tctggattct gcctaataaa 120

aaacatttat tttcattgct gcgtcgagag ctcgctttct tgctgtccaa tttctattaa 180

aggttccttt gttccctaag tccaactact aaactggggg atattatgaa gggccttgag 240

catctggatt ctgcctaata aaaaacattt attttcattg ctgcgtc 287

<210> 17

<211> 134

<212> DNA

<213> Artificial sequence

<220>

<223> A120

<400> 17

gagacctggt ccagagtcgc tagcaaaaaa aaaaaaaaaa aaaaaaaaaa aaaagcatat 60

gactaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaa 134

<210> 18

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<223> Connector

<400> 18

ggaggcggtg gtagtggagg tggcgggtcc ggtggaggtg gaagc 45

<210> 19

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 19

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 20

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 20

Gly Gly Gly Gly Ser Gly Gly Gly Thr Gly Gly Gly Gly Ser

1 5 10

<210> 21

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 21

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 22

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Sec

<400> 22

Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Ala Pro Gly

1 5 10 15

Ala His Ser

<210> 23

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> hEHD2

<400> 23

Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly

1 5 10 15

Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly

20 25 30

Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val

35 40 45

Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu

50 55 60

Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser

65 70 75 80

Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu

85 90 95

Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn

100 105

Claims (17)

1. Cytokine fusion proteins, selected from: A.) A cytokine fusion protein comprising an extracellular domain of a first ligand of the tumor necrosis factor (TNF) superfamily and an extracellular domain of a second ligand of the TNF superfamily, wherein the first ligand is different from the second ligand, and wherein the extracellular domain is covalently linked; and B.) A cytokine fusion protein comprising: (i) three extracellular domains of a first ligand of the tumor necrosis factor (TNF) superfamily forming a first homotrimer, the first homotrimer being capable of binding to a receptor of the first ligand; and (ii) three extracellular domains of a second ligand of the TNF superfamily forming a second homotrimer, the second homotrimer being capable of binding to a receptor of the second ligand, wherein the first ligand is different from the second ligand, and wherein the first homotrimer and the second homotrimer are covalently linked. Among the cytokine fusion proteins of A.) and B.) The first ligand is CD40L, and the second ligand is 4-1BBL, wherein the extracellular domain of CD40L consists of amino acid residues 51 to 261 or 116 to 261 of SEQ ID NO:1, and the extracellular domain of 4-1BBL consists of amino acid residues 71 to 254 of SEQ ID NO:3. The cytokine fusion protein of A.) contains three subunits of the following general formula: N’-A–L–B-C’ Formula II, Wherein A is composed of the extracellular domain of the first ligand, and B is composed of the extracellular domain of the second ligand. Where L contains a peptide linker, and The cytokine fusion protein of A.) exists as a trimer complex, wherein the three extracellular domains of the first ligand form a first homotrimer capable of binding to the receptor of the first ligand, and the three extracellular domains of the second ligand form a second homotrimer capable of binding to the receptor of the second ligand; and The cytokine fusion proteins of B.) contain molecules/structures with the following general formulas: N'-AL _A -AL _A -ALBL _B -BL _B -B-C' Formula I, Wherein A is composed of the extracellular domain of the first ligand, and B is composed of the extracellular domain of the second ligand, and Where L contains a peptide linker, and _LA and _LB are independently selected from covalent bonds and peptide linkers each time they appear. 2. The cytokine fusion protein according to claim 1, wherein the first isotrimester and the second isotrimester are covalently linked by one or more peptide linkers. 3. The cytokine fusion protein of claim 1, wherein L further comprises a multiplicative domain that allows the cytokine fusion protein to multiply. 4. The cytokine fusion protein according to claim 3, wherein the multimerization is dimerization and the dimerizing domain is selected from IgE heavy chain domain 2 (EHD2), IgM heavy chain domain 2 (MHD2), IgG heavy chain domain 3 (GHD3), IgA heavy chain domain 3 (AHD2), IgD heavy chain domain 3 (DHD3), IgE heavy chain domain 4 (EHD4), IgM heavy chain domain 4 (MHD4), Fc domain, uterine globin dimerizing domain, and functional variants of the foregoing. 5. The cytokine fusion protein according to claim 3 or 4, wherein it exists as a multimeric complex. 6. The cytokine fusion protein according to any one of claims 1 to 4, wherein the cytokine comprises an amino acid sequence for promoting the secretion of the molecule; and/or a marker or tag that allows detection and/or separation of the cytokine fusion protein, which is fused to the cytokine fusion protein via an N-terminus or a C-terminus. 7. The cytokine fusion protein of claim 6, wherein the amino acid sequence for promoting the secretion of the molecule is an N-terminal secretion signal. 8. A nucleic acid molecule encoding a cytokine fusion protein or a subunit thereof according to any one of claims 1 to 7. 9. The nucleic acid molecule according to claim 8, wherein it is effectively linked to an expression control sequence. 10. The nucleic acid molecule according to claim 8 or 9, wherein it is contained in a vector. 11. The nucleic acid molecule according to claim 8, wherein it is an RNA molecule. 12. The nucleic acid molecule according to claim 11, wherein it is an in vitro transcribed (IVT) RNA molecule. 13. A cell that has been transformed or transfected with a nucleic acid molecule according to any one of claims 8 to 12. 14. A pharmaceutical composition comprising, as an active agent, a cytokine fusion protein according to any one of claims 1 to 7, a nucleic acid molecule according to any one of claims 8 to 12, or a cell according to claim 13. 15. The pharmaceutical composition according to claim 14, further comprising a pharmaceutically acceptable carrier and/or excipients. 16. A kit comprising a cytokine fusion protein according to any one of claims 1 to 7, a nucleic acid molecule according to any one of claims 8 to 12, a cell according to claim 13, or a pharmaceutical composition according to claim 14 or 15. 17. Use of the cytokine fusion protein according to any one of claims 1 to 7, the nucleic acid molecule according to any one of claims 8 to 12, the cell according to claim 13, or the pharmaceutical composition according to claim 14 or 15 for the preparation of a medicament.