Multifunctional and polyvalent interleukin-TGF-beta receptor fusion polypeptide

Abstract

Multifunctional and multivalent fusion polypeptides are provided that include an interleukin polypeptide and two or more TGFβ ligand binding polypeptides. The compositions and methods provided herein are useful for preventing tumorigenesis and treating cancer.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 63 / 094,277, filed on October 20, 2020, and this application is incorporated herein by reference.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. The name of the ASCII copy, created on October 15, 2021, is 57949-701_601_SL.txt, and its size is 82,739 bytes. [Background technology]

[0003] Tumor formation, which drives cancer and leads to its development, can generally be characterized by abnormal cellular processes that result in, for example, persistent proliferative signaling, reduced response to growth inhibitors, increased resistance to cell death, replication immortality, and increased angiogenesis. These processes are thought to promote tumor invasion and metastasis. Therefore, the ability to target molecules involved in these processes may be important for the development of cancer therapeutics.

[0004] The transforming growth factor β (TGF-β) family or proteins are pleomorphic cytokines that promote angiogenesis and / or immunosuppression in tumorigenic environments. TGF-β proteins were originally named for their ability to transform normal fibroblasts into cells capable of independent growth. Produced primarily by hematopoietic and tumor cells, TGF-β proteins can regulate (e.g., stimulate or inhibit) cell growth and differentiation from a diversity of normal or neoplastic tissue origins. In particular, TGF-β proteins are known to be involved in many proliferative and non-proliferative cellular processes, such as cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses. Increased levels of TGF-β expression and activity are associated with numerous pathological conditions, including but not limited to tumorigenesis, and members of the TGF-β protein family are known to possess numerous biological activities related to tumorigenic processes (e.g., angiogenesis and immunosuppression) and metastasis.

[0005] Interleukin-2 (IL-2) is a potent cytokine that acts on the immune system primarily to generate cell-mediated immune responses. Generally, IL-2 is produced locally by high concentrations of immune cells near antigen sites, supplying the necessary costimulatory signals to generate an immune response to the antigen. Therefore, IL-2 is an immunoactivating molecule and may potentially promote immunotherapeutic responses in tumor treatment. For example, in addition to stimulating T cells, IL-2 has also been shown to stimulate lymphocytes (T cells, B cells, NK cells, and NKT cells). However, high-dose IL-2-based therapies have previously been associated with toxicity observed with systemic administration of IL-2. Such adverse effects hinder the successful utilization of IL-2 as an effective therapeutic agent. [Overview of the Initiative] [Means for solving the problem]

[0006] Provided herein are fusion proteins that function to activate the immune response and inhibit pro-tumorigenic immunosuppressive signaling. As described herein, immune activation and inhibition of immunosuppression are generally achieved by fusion proteins comprising interleukin polypeptides fused (e.g., linked) to two or more polypeptides that inhibit TGF-β activity (e.g., two or more polypeptides that bind to the TGF-β protein). The compositions provided herein are useful for or in inhibiting tumorigenesis (e.g., angiogenesis or metastasis). Therefore, methods for inhibiting and / or treating cancer using the fusion proteins disclosed herein are also disclosed herein.

[0007] This specification provides a fusion polypeptide comprising an interleukin (IL) polypeptide, a first TGFβ superfamily receptor polypeptide, and a second TGFβ superfamily receptor polypeptide. This specification also provides a fusion polypeptide comprising an IL polypeptide that binds to and activates the IL receptor, a first TGFβ superfamily receptor polypeptide that binds to and occludes soluble TGFβ, and a second TGFβ superfamily receptor polypeptide that binds to and occludes soluble TGFβ.

[0008] In some embodiments, the IL polypeptide comprises either the IL-2 polypeptide or IL-15. In some embodiments, the IL polypeptide comprises the IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises SEQ ID NO: 2. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 2. In some embodiments, the IL-2 polypeptide consists of SEQ ID NO: 2. In some embodiments, the IL polypeptide comprises either the IL-2 polypeptide or IL-15. In some embodiments, the IL polypeptide comprises the IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises SEQ ID NO: 3. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 3. In some embodiments, the IL-2 polypeptide consists of SEQ ID NO: 3. In some embodiments, the IL polypeptide comprises the IL-15 polypeptide. In some embodiments, the IL-15 polypeptide comprises SEQ ID NO: 5. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 5. In some embodiments, the IL-15 polypeptide consists of SEQ ID NO: 5.

[0009] In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide includes an activin receptor polypeptide or a fragment thereof, a bone morphogenetic protein (BMP) receptor polypeptide or a fragment thereof, a glial cell-derived neurotrophic factor (GDNF) receptor polypeptide or a fragment thereof, or a TGFβ receptor II polypeptide or a fragment thereof. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide includes a TGFβ receptor II (TBRII) polypeptide or a fragment thereof. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide includes a soluble TGFβ receptor II (TBRII) polypeptide or a fragment thereof. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide includes a soluble TGFβ receptor II (TBRII) polypeptide including SEQ ID NO: 8, SEQ ID NO: 9, or a combination thereof. In some embodiments, the first TGFβ superfamily receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the second TGFβ superfamily receptor polypeptide comprises a cleavage of the amino acid sequence of SEQ ID NO: 9. In some embodiments, the soluble TGFβ receptor II polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9.In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide is TGF-β1 polypeptide, TGF-β2 polypeptide, TGF-β3 polypeptide, activin βA polypeptide, activin βB polypeptide, activin βC polypeptide, activin βE polypeptide, bone morphogenetic protein (BMP) 2 polypeptide, BMP3 polypeptide, BMP4 polypeptide, BMP5 polypeptide, BMP6 polypeptide, BMP7 polypeptide, BMP8 polypeptide, BMP9 polypeptide, B The following polypeptides are conjugated: MP10 polypeptide, BMP11 polypeptide, BMP12 polypeptide, BMP13 polypeptide, BMP14 polypeptide, BMP15 polypeptide, growth differentiation factor (GDF) 1 polypeptide, GDF3 polypeptide, GDF8 polypeptide, GDF9 polypeptide, GDF15 polypeptide, Nodal polypeptide, inhibin α polypeptide, anti-Müllerian hormone polypeptide, Lefty 1 polypeptide, Lefty 2 polypeptide, arteman polypeptide, percephin polypeptide, or neuturin polypeptide. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide are conjugated to TGFβ1 polypeptide, TGFβ2 polypeptide, TGFβ3 polypeptide, or any combination thereof. In some embodiments, the first TGFβ superfamily receptor polypeptide and the second TGFβ superfamily receptor polypeptide are conjugated to TGFβ1 polypeptide.

[0010] In some embodiments, the cleaved TGFβ superfamily receptor polypeptide includes an N-terminal cleavage, a C-terminal cleavage, or a combination thereof. In some embodiments, the cleaved TGFβ superfamily receptor polypeptide is a cleaved soluble TGFβ receptor II (sTβRII) polypeptide. In some embodiments, the cleavage includes 2, 5, 7, 10, 15, 20, 25, or more than 30 amino acids. In some embodiments, the IL polypeptide includes a cleaved IL polypeptide. In some embodiments, the cleaved IL polypeptide includes an N-terminal cleavage, a C-terminal cleavage, or a combination thereof. In some embodiments, the cleaved IL polypeptide includes a cleaved IL-2 polypeptide or IL-15 polypeptide. In some embodiments, the fusion polypeptide includes a linker polypeptide or linker molecule that conjugates a first TGFβ superfamily receptor polypeptide and a second TGFβ superfamily receptor polypeptide. In some embodiments, the fusion polypeptide includes a linker polypeptide that fuses the IL polypeptide and the first TGFβ superfamily receptor polypeptide. In some embodiments, the fusion polypeptide includes a pharmacokinetic (PK) modifier.

[0011] In some embodiments, the pharmacokinetic modifier comprises an immunoglobulin constant-state (Fc) region polypeptide. In some embodiments, the immunoglobulin Fc region polypeptide is a human immunoglobulin Fc region polypeptide. In some embodiments, the immunoglobulin Fc region is an IgG Fc region. In some embodiments, the IgG Fc region is an IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the PK modifier comprises an albumin polypeptide. In some embodiments, the albumin polypeptide is a human albumin polypeptide.

[0012] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 10 to 25. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 12. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 13. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 14. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 15. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 16. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 17. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 18. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 19. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 20. In some embodiments, the fusion polypeptide contains the amino acid sequence of SEQ ID NO: 21. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the fusion polypeptide comprises any one of SEQ ID NOs: 10 to 25. In some embodiments, the fusion polypeptide activates target cells expressing IL-2 or IL-15 receptors. In some embodiments, the target cells are immune cells. In some embodiments, the immune cells are T cells, natural killer cells, NKT cells, B cells, or gamma delta T cells. Further pharmaceutical compositions are provided, comprising a fusion polypeptide comprising a pharmaceutically acceptable carrier or excipient.

[0013] Also provided herein are methods for inhibiting or reducing tumor growth and / or progression in an individual having cancer, comprising administering to the individual a fusion polypeptide comprising an interleukin (IL) polypeptide, a first TGFβ superfamily receptor polypeptide, and a second TGFβ superfamily receptor polypeptide, thereby inhibiting or reducing tumor growth and / or progression in the individual. In some embodiments, the tumor is a primary tumor. In some embodiments, the tumor is a metastatic tumor. In some embodiments, cancer includes solid tumors. In some embodiments, the fusion polypeptide activates immune cells within the tumor microenvironment. In some embodiments, the immune cells express IL-2.

[0014] In some embodiments, the immune cells comprise T cells, natural killer cells, NKT cells, B cells, or gamma-delta T cells. In some embodiments, the fusion polypeptide inhibits or reduces immunosuppression of immune cells in the tumor microenvironment, and / or reduces or inhibits activation of immunosuppressive cells in the tumor microenvironment. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a cancer comprising a solid tumor. Provided herein is a method of neutralizing or killing tumor cells in an individual having cancer, comprising administering to the individual a fusion polypeptide comprising an interleukin (IL) polypeptide, a first TGFβ superfamily receptor polypeptide, and a second TGFβ superfamily receptor polypeptide, thereby neutralizing or killing tumor cells in the individual. In some embodiments, the tumor is a primary tumor. In some embodiments, the tumor is a metastatic tumor. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the fusion polypeptide activates immune cells in the tumor microenvironment. In some embodiments, the immune cells express an IL-2 or IL-15 receptor. In some embodiments, the immune cells comprise T cells, natural killer cells, NKT cells, B cells, or gamma-delta T cells. In some embodiments, the fusion polypeptide inhibits or reduces immunosuppression of immune cells in the tumor microenvironment, and / or reduces or inhibits activation of immunosuppressive cells in the tumor microenvironment. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer comprises a solid tumor.

[0015] Furthermore, there is a method for treating or improving cancer in a subject having cancer, comprising administering a fusion polypeptide comprising an interleukin (IL) polypeptide, a first TGFβ superfamily receptor polypeptide, and a second TGFβ superfamily receptor polypeptide to the subject, thereby treating or improving cancer in the subject. In some embodiments, the tumor is a primary tumor. In some embodiments, the tumor is a metastatic tumor. In some embodiments, the cancer includes solid tumors. In some embodiments, the fusion polypeptide activates immune cells in the tumor microenvironment. In some embodiments, the immune cells express IL-2 or IL-15 receptors. In some embodiments, the immune cells include T cells, natural killer cells (NKT cells), B cells, or gamma delta T cells. In some embodiments, the fusion polypeptide inhibits or reduces immunosuppression of immune cells in the tumor microenvironment and / or reduces or inhibits the activation of immunosuppressive cells in the tumor microenvironment. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer includes solid tumors. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide conjugates a TGFβ1 polypeptide, a TGFβ2 polypeptide, a TGFβ3 polypeptide, or any combination thereof.

[0016] A method of activating a cell that expresses an interleukin (IL) receptor, the method further comprising contacting the cell with a fusion polypeptide comprising an interleukin (IL) polypeptide, a first TGFβ superfamily receptor polypeptide, and a second TGFβ superfamily receptor polypeptide. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell comprises a T cell, a natural killer cell, an NKT cell, a B cell, or a gamma delta T cell. In some embodiments, the immune cell is contacted in vivo. In some embodiments, the immune cell is contacted ex vivo. In some embodiments, a leukopack or leukapheresis product comprises the immune cell. In some embodiments, the immune cell is derived from an apheresis sample. In some embodiments, the immune cell is subsequently used for cell-based therapy. In some embodiments, the IL polypeptide is IL-2 or IL-15. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide comprises a soluble TGFβ receptor II (TBRII) polypeptide or a fragment thereof. A host cell comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a recombinant fusion polypeptide as described herein. In certain embodiments, for example, the following are provided: (Item 1) It is a fusion polypeptide, Interleukin (IL) polypeptides and The first TGFβ superfamily receptor polypeptide, A fusion polypeptide comprising a second TGFβ superfamily receptor polypeptide. (Item 2) It is a fusion polypeptide, A fusion polypeptide comprising an IL polypeptide that binds to and activates an IL receptor, a first TGFβ superfamily receptor polypeptide that binds to and occludes soluble TGFβ, and a second TGFβ superfamily receptor polypeptide that binds to and occludes soluble TGFβ. (Item 3) The fusion polypeptide according to item 1 or 2, wherein the IL polypeptide comprises an IL-2 polypeptide or an IL-15 polypeptide. (Item 4) The fusion polypeptide according to any one of items 1 to 3, wherein the IL polypeptide includes an IL-2 polypeptide. (Item 5) The fusion polypeptide according to item 4, wherein the IL-2 polypeptide includes SEQ ID NO: 2 or SEQ ID NO: 3. (Item 6) The fusion polypeptide according to item 4, wherein the IL-2 polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2, or an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 3. (Item 7) The fusion polypeptide according to item 4, wherein the IL-2 polypeptide consists of SEQ ID NO: 2 or SEQ ID NO: 3. (Item 8) The fusion polypeptide according to any one of items 1 to 3, wherein the IL polypeptide comprises an IL-15 polypeptide. (Item 9) The fusion polypeptide described in item 8, wherein the IL-15 polypeptide includes SEQ ID NO: 5. (Item 10) The fusion polypeptide according to item 8, wherein the IL-15 polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 5. (Item 11) The IL-15 polypeptide is the fusion polypeptide described in item 8, wherein the IL-15 polypeptide is sequence number 5. (Item 12) The fusion polypeptide according to any one of items 1 to 11, wherein the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide comprises an activin receptor polypeptide or a fragment thereof, a bone morphogenetic protein (BMP) receptor polypeptide or a fragment thereof, a glial cell-derived neurotrophic factor (GDNF) receptor polypeptide or a fragment thereof, or a TGFβ receptor II polypeptide or a fragment thereof. (Item 13) The fusion polypeptide according to any one of items 1 to 12, wherein the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide comprises a TGFβ receptor II (TBRII) polypeptide or a fragment thereof. (Item 14) The fusion polypeptide according to any one of items 1 to 13, wherein the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide comprises a soluble TGFβ receptor II (TBRII) polypeptide or a fragment thereof. (Item 15) The fusion polypeptide according to any one of items 1 to 14, wherein the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide comprises a soluble TGFβ receptor II (TBRII) polypeptide including SEQ ID NO: 8, SEQ ID NO: 9, or a combination thereof. (Item 16) The fusion polypeptide according to any one of items 1 to 15, wherein the first TGFβ superfamily receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the second TGFβ superfamily receptor polypeptide comprises a cleavage of the amino acid sequence of SEQ ID NO: 9. (Item 17) The fusion polypeptide according to any one of items 14 to 16, wherein the soluble TGFβ receptor II polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 9. (Item 18) The first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide is TGF-β1 polypeptide, TGF-β2 polypeptide, TGF-β3 polypeptide, activin βA polypeptide, activin βB polypeptide, activin βC polypeptide, activin βE polypeptide, bone morphogenetic protein (BMP) 2 polypeptide, BMP3 polypeptide, BMP4 polypeptide, BMP5 polypeptide, BMP6 polypeptide, BMP7 polypeptide, BMP8 polypeptide, BMP9 polypeptide, BMP10 polypeptide, BMP11 polypeptide A fusion polypeptide according to any one of items 1 to 17, conjugating a lipeptide, BMP12 polypeptide, BMP13 polypeptide, BMP14 polypeptide, BMP15 polypeptide, growth differentiation factor (GDF) 1 polypeptide, GDF3 polypeptide, GDF8 polypeptide, GDF9 polypeptide, GDF15 polypeptide, Nodal polypeptide, inhibin α polypeptide, anti-Müllerian hormone polypeptide, Lefty 1 polypeptide, Lefty 2 polypeptide, arteman polypeptide, percefin polypeptide, or neuturin polypeptide. (Item 19) The fusion polypeptide according to any one of items 1 to 18, wherein the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide conjugates a TGFβ1 polypeptide, a TGFβ2 polypeptide, a TGFβ3 polypeptide, or any combination thereof. (Item 20) A fusion polypeptide according to any one of items 1 to 19, wherein the first TGFβ superfamily receptor polypeptide and the second TGFβ superfamily receptor polypeptide bind to a TGFβ1 polypeptide. (Item 21) A fusion polypeptide according to any one of items 1 to 20, wherein the cleaved TGFβ superfamily receptor polypeptide comprises an N-terminal cleavage, a C-terminal cleavage, or a combination thereof. (Item 22) The fusion protein described in item 21, wherein the cleaved TGFβ superfamily receptor polypeptide is a cleaved soluble TGFβ receptor II (sTβRII) polypeptide. (Item 23) The fusion polypeptide according to item 22, wherein the cleavage comprises 2, 5, 7, 10, 15, 20, 25, or more than 30 amino acids. (Item 24) The fusion polypeptide according to any one of items 1 to 23, wherein the IL polypeptide includes a cleaved IL polypeptide. (Item 25) The fusion polypeptide according to item 24, wherein the cleavage-type IL polypeptide includes N-terminal cleavage, C-terminal cleavage, or a combination thereof. (Item 26) The fusion polypeptide according to item 24, wherein the cleaved IL polypeptide comprises a cleaved IL-2 polypeptide or an IL-15 polypeptide. (Item 27) A fusion polypeptide according to any one of items 1 to 26, further comprising a linker polypeptide or linker molecule that conjugates the first TGFβ superfamily receptor polypeptide and the second TGFβ superfamily receptor polypeptide. (Item 28) A fusion polypeptide according to any one of items 1 to 27, further comprising a linker polypeptide that fuses the IL polypeptide and the first TGFβ superfamily receptor polypeptide. (Item 29) A fusion polypeptide according to any one of items 1 to 28, further comprising a pharmacokinetic (PK) modifier. (Item 30) The fusion polypeptide according to item 29, wherein the pharmacokinetic modifier comprises an immunoglobulin constant-state (Fc) region polypeptide. (Item 31) The fusion polypeptide described in item 30, wherein the immunoglobulin Fc region polypeptide is a human immunoglobulin Fc region polypeptide. (Item 32) The fusion polypeptide described in item 30, wherein the immunoglobulin Fc region is an IgG Fc region. (Item 33) The fusion polypeptide according to item 30, wherein the IgG Fc region is an IgG1, IgG2, IgG3, or IgG4 Fc region. (Item 34) The fusion polypeptide described in item 29, wherein the PK modifier comprises an albumin polypeptide. (Item 35) The fusion polypeptide described in item 34, wherein the albumin polypeptide is a human albumin polypeptide. (Item 36) The fusion polypeptide according to any one of items 1 to 35, wherein the fusion polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of sequence numbers 10 to 25. (Item 37) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 10. (Item 38) The fusion polypeptide described in item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 11. (Item 39) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 12. (Item 40) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 13. (Item 41) The fusion polypeptide described in item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 14. (Item 42) The fusion polypeptide described in item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 15. (Item 43) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 16. (Item 44) The fusion polypeptide described in item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 17. (Item 45) The fusion polypeptide described in item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 18. (Item 46) The fusion polypeptide described in item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 19. (Item 47) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 20. (Item 48) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 21. (Item 49) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 22. (Item 50) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 23. (Item 51) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 24. (Item 52) The fusion polypeptide according to item 36, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 25. (Item 53) The fusion polypeptide described in any one of items 1 to 52, wherein the fusion polypeptide consists of one of sequence numbers 10 to 25. (Item 54) The fusion polypeptide according to any one of items 1 to 53, wherein the fusion polypeptide activates target cells expressing IL-2 or IL-15 receptors. (Item 55) The fusion polypeptide described in item 54, wherein the target cell is an immune cell. (Item 56) The fusion polypeptide according to item 55, wherein the immune cells are T cells, natural killer cells (NKT cells), B cells, or gamma delta T cells. (Item 57) A pharmaceutical composition comprising a fusion polypeptide as described in any one of items 1 to 56, comprising a pharmaceutically acceptable carrier or excipient. (Item 58) A method for inhibiting or reducing the growth and / or progression of a tumor in an individual with cancer, (a) Interleukin (IL) polypeptide and (b) The first TGFβ superfamily receptor polypeptide, (c) The second TGFβ superfamily receptor polypeptide, A method comprising administering a fusion polypeptide containing to the individual, thereby inhibiting or reducing the growth and / or progression of a tumor in the individual. (Item 59) The method described in item 58, wherein the tumor is a primary tumor. (Item 60) The method according to item 58 or 59, wherein the tumor is a metastatic tumor. (Item 61) The method according to any one of items 58 to 60, wherein the cancer includes solid tumors. (Item 62) The method according to any one of items 58 to 61, wherein the fusion polypeptide activates immune cells in the tumor microenvironment. (Item 63) The method according to item 62, wherein the immune cells express IL-2. (Item 64) The method according to item 63, wherein the immune cells include T cells, natural killer cells, NKT cells, B cells, or gamma delta T cells. (Item 65) The method according to any one of items 58 to 64, wherein the fusion polypeptide inhibits or reduces immunosuppression of immune cells in the tumor microenvironment and / or reduces or inhibits the activation of immunosuppressive cells in the tumor microenvironment. (Item 66) The method according to any one of items 58 to 65, wherein the cancer is a blood cancer. (Item 67) The method according to any one of items 58 to 66, wherein the cancer is a cancer including a solid tumor. (Item 68) A method for neutralizing or killing tumor cells in an individual with cancer, (a) Interleukin (IL) polypeptide and (b) The first TGFβ superfamily receptor polypeptide, (c) The second TGFβ superfamily receptor polypeptide, A method comprising administering a fusion polypeptide containing to an organism, thereby neutralizing or killing tumor cells in the organism. (Item 69) The method described in item 68, wherein the tumor is a primary tumor. (Item 70) The method according to item 68 or 69, wherein the tumor is a metastatic tumor. (Item 71) The method according to any one of items 68 to 70, wherein the cancer includes solid tumors. (Item 72) The method according to any one of items 68 to 71, wherein the fusion polypeptide activates immune cells in the tumor microenvironment. (Item 73) The method according to item 72, wherein the immune cells express IL-2 or IL-15 receptors. (Item 74) The method according to item 72, wherein the immune cells include T cells, natural killer cells (NKT cells), B cells, or gamma delta T cells. (Item 75) The method according to any one of items 68 to 74, wherein the fusion polypeptide inhibits or reduces immunosuppression of immune cells in the tumor microenvironment and / or reduces or inhibits the activation of immunosuppressive cells in the tumor microenvironment. (Item 76) The method according to any one of items 68 to 75, wherein the cancer is a blood cancer. (Item 77) The method according to any one of items 68 to 76, wherein the cancer includes solid tumors. (Item 78) A method for treating or improving cancer in a subject with cancer, (a) Interleukin (IL) polypeptide and (b) The first TGFβ superfamily receptor polypeptide, (c) The second TGFβ superfamily receptor polypeptide, A method comprising administering a fusion polypeptide containing to a subject, thereby treating or improving the cancer in the subject. (Item 79) The method described in item 78, wherein the tumor is a primary tumor. (Item 80) The method according to item 78 or 79, wherein the tumor is a metastatic tumor. (Item 81) The method according to any one of items 78 to 80, wherein the cancer includes solid tumors. (Item 82) The method according to any one of items 78 to 81, wherein the fusion polypeptide activates immune cells in the tumor microenvironment. (Item 83) The method according to item 82, wherein the immune cells express IL-2 or IL-15 receptors. (Item 84) The method according to item 82, wherein the immune cells include T cells, natural killer cells (NKT cells), B cells, or gamma delta T cells. (Item 85) The method according to any one of items 78 to 84, wherein the fusion polypeptide inhibits or reduces immunosuppression of immune cells in the tumor microenvironment and / or reduces or inhibits the activation of immunosuppressive cells in the tumor microenvironment. (Item 86) The method described in any one of items 78 to 85, wherein the cancer is a blood cancer. (Item 87) The method according to any one of items 78 to 86, wherein the cancer includes solid tumors. (Item 88) The method according to any one of items 78 to 87, wherein the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide conjugates a TGFβ1 polypeptide, a TGFβ2 polypeptide, a TGFβ3 polypeptide, or any combination thereof. (Item 89) A method for activating cells that express IL receptors, (a) Interleukin (IL) polypeptide and (b) The first TGFβ superfamily receptor polypeptide, (c) A method comprising contacting a cell with a fusion polypeptide comprising a second TGFβ superfamily receptor polypeptide. (Item 90) The method according to item 89, wherein the cells are immune cells. (Item 91) The method according to item 90, wherein the immune cells include T cells, natural killer cells (NKT cells), B cells, or gamma delta T cells. (Item 92) The method according to any one of items 89 to 91, wherein the immune cells are contacted in vivo. (Item 93) The method according to any one of items 89 to 91, wherein the immune cells are contacted ex vivo. (Item 94) The method according to item 93, wherein the leukopack or leukapheresis product contains the aforementioned immune cells. (Item 95) The method described in item 93, wherein the immune cells are derived from an apheresis sample. (Item 96) The method according to item 93, wherein the immune cells are subsequently used in cell-based therapy. (Item 97) The method according to any one of items 89 to 96, wherein the IL polypeptide is IL-2 or IL-15. (Item 98) The method according to any one of items 89 to 97, wherein the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide comprises a soluble TGFβ receptor II (TBRII) polypeptide or fragment. (Item 99) A host cell containing a nucleic acid molecule comprising a nucleic acid sequence encoding a recombinant fusion polypeptide as described in any one of items 1 to 56. (Item 100) Use of any one of items 1 to 56 to inhibit or reduce tumor growth and / or progression in an individual having cancer. (Item 101) Use of any one of items 1 to 56 for neutralizing or killing tumor cells in an individual with cancer. (Item 102) Use of any one of items 1 to 56 for the treatment or improvement of cancer in a subject with cancer. (Item 103) Use of any one of the fusion polypeptides described in item 1 to 56 to activate cells expressing the IL receptor. (Item 104) Use of any one of items 1 to 56 in the manufacture of a pharmaceutical product for inhibiting or reducing the growth and / or progression of a tumor in an individual with cancer. (Item 105) Use of any one of items 1 to 56 in the manufacture of a pharmaceutical product for neutralizing or killing tumor cells in an individual with cancer. (Item 106) Use of any one of items 1 to 56 in the manufacture of a drug for treating or improving cancer in a subject with cancer. (Item 107) Use of any one of items 1 to 56 in the manufacture of a drug for activating cells expressing the IL receptor. (Item 108) A fusion polypeptide as described in any one of items 1 to 56, for use in inhibiting or reducing the growth and / or progression of tumors in individuals with cancer. (Item 109) A fusion polypeptide as described in any one of items 1 to 56, for use in neutralizing or killing tumor cells in an individual with cancer. (Item 110) A fusion polypeptide as described in any one of items 1 to 56, for use in the treatment or improvement of cancer in subjects with cancer. (Item 111) A fusion polypeptide described in any one of items 1 to 56, for use in activating cells expressing the IL receptor.

[0017] Embedding by reference All publications, patents, and patent applications described herein are incorporated by reference in the same manner as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawing]

[0018] Novel features of this disclosure are specifically described in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of this disclosure are utilized, and to the following appended drawings.

[0019] [Figure 1-1] This report presents quantitative data on IFN-gamma production by human T cells previously stimulated with human monovalent FIST (a fusion protein containing interleukin-2 (IL-2) polypeptide and a single sTBRII receptor polypeptide) or bivalent FIST (also called bFIST, a fusion polypeptide containing interleukin-2 (IL-2) polypeptide, a first sTBRII receptor polypeptide, and a second sTBRII receptor polypeptide), and data on CXCL10 production by human NK cells stimulated with human FIST (monovalent FIST) or human bivalent FIST. [Figure 1-2] Same as above. [Figure 2] The data shows the quantification of active TGFβ1 detected in solution after a 30-minute incubation period with 5-20 nanomoles of human monovalent FIST (monovalent FIST), divalent FIST (bFIST), or sTBRII. [Figure 3] A schematic diagram of an exemplary bivalent FIST (bFIST) fusion polypeptide is shown. [Figure 4-1] This shows a comparison of bivalent FIST protein and monovalent FIST protein in blocking TGFβ isoforms. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5-1] This study demonstrates inhibition of TGFb1-mediated suppression of cytotoxic T cell proliferation. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above. [Figure 5-5] Same as above. [Figure 5-6] Same as above. [Figure 6-1] This study demonstrates the proliferation of effector memory T cells and terminally differentiated effector memory cells, as well as the quantitative analysis of IFNγ production by bivalent FIST, monovalent FIST, and control-stimulated T cells. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. [Figure 6-5] Same as above. [Figure 6-6] Same as above. [Figure 7-1] It exhibits bivalent FIST inhibition and / or reduction of TGFb1-mediated suppression of primary NK cell proliferation and IFNγ and CXC10 production. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 8-1] This study demonstrates that treating NK cells with bivalent FIST increases NK cell cytotoxicity and activation, as well as cytotoxicity against cancer cells. [Figure 8-2] Same as above. [Figure 9-1] This study demonstrates that treatment of B cells with bivalent FIST increases maturation, proliferation, and IFNγ production. [Figure 9-2] Same as above [Figure 9-3] Same as above [Figure 9-4] Same as above [Figure 10-1] This study demonstrates that treating lung cancer cells with bivalent FIST inhibits the upregulation of TGFb1-induced epithelial-to-mesenchymal transition (EMT) markers on lung cancer cells. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Modes for carrying out the invention]

[0020] Provided herein are multifunctional fusion proteins capable of, for example, activating immune responses, inhibiting and / or reducing immunosuppressive signaling, and / or suppressing or inhibiting angiogenesis or invasive signaling. In relation to cancer, immune activation is considered to be a physiological function that promotes the recognition and neutralization of transformed cells before they develop into tumors, or that kills (e.g., neutralizes) them after tumors have formed. Specific recognition of antigens based on heterogeneity, molecular weight, complexity, expression level, and degradability is thought to give rise to the immune system's ability to limit tumor growth. Conversely, tumor cells are thought to have the ability to limit their responsiveness through various mechanisms, for example, by producing immunosuppressive factors or molecules (e.g., immunosuppressive cytokines).

[0021] To activate the immune response and inhibit immunosuppressive signaling, the fusion proteins disclosed herein generally comprise an interleukin polypeptide fused (e.g., linked) to two or more polypeptides that inhibit TGFβ activity (e.g., two or more polypeptides that bind the TGFβ protein). The balance between activating and inhibitory signals is a crucial element in maintaining immune homeostasis. During tumorigenesis, immune stimulating (e.g., immune activation) signals (e.g., pro-inflammatory cytokines) are antagonized by tumor-derived immunosuppressive signaling molecules (e.g., immunosuppressive cytokines). Among the immunosuppressive cytokines considered important in driving tumorigenesis are members of the TGF superfamily of proteins (e.g., TGFβ).

[0022] The transforming growth factor beta (TGFβ) superfamily includes TGFβ protein, bone morphogenetic protein (BMP), growth differentiation factor (GDF), glial neurotrophic factor (GDNF), activin, inhibin, nodal, lefty, and Müllerian duct inhibitor (MIS). Generally, ligands of the TGFβ superfamily form dimers that bind to heterodimeric receptor complexes consisting of type I and type II receptor subunits having serine / threonine kinase domains.

[0023] The protein transformation growth factor β (TGFβ) superfamily are cytokines involved in essential cellular functions such as proliferation, differentiation, apoptosis, tissue remodeling, angiogenesis, immune responses, and cell adhesion (see, for example, Massague, J. TGFβ signaling in context. Nat Rev Mol Cell Biol 13, 616-630 (2012)). TGFβ superfamily proteins are also important factors in the pathophysiology of chronic inflammatory states and disease states such as cancer. Members of this family include the three isoforms of TGFβ: β1, β2, and β3, as well as bone morphogenetic proteins (BMPs) and activin. In addition to immunosuppression, TGFβ proteins can act as prometastatic and proangiogenic factors in late-stage cancer by constitutively inducing epithelial-to-mesenchymal transition (EMT) and tumor-associated angiogenesis.

[0024] Blocking tumor-derived active TGFβ has been widely explored as a therapeutic option. Many therapeutic approaches target the TGFβ pathway for the treatment of invasive cancers such as breast cancer and melanoma. For example, TGFβ antagonists that bind to its heteromeric receptor have shown reductions in tumor cell motility, intravascular invasion, and metastasis in three experimental models of breast cancer. However, these therapeutic strategies generally do not affect cell proliferation, which suggests that TGFβ blockers are not sufficient to promote the neutralization or killing of tumor cells.

[0025] The use of interleukin polypeptides (e.g., interleukin-2 or interleukin-15) in the fusion proteins disclosed herein is useful for activating the immune response, in addition to inhibiting TGFβ. The interleukin family of proteins generally promotes the activation, development, and differentiation of immune cells (e.g., T cells, NK cells, and other lymphocytes) and plays an essential role in both innate and adaptive immunity. Therefore, the activation, development, and differentiation of immune cells can be achieved by the fusion proteins disclosed herein.

[0026] Pro-inflammatory or immunostimulatory interleukins (e.g., interleukin-2 or interleukin-15) constitute useful adjuvants for activating immune responses. For example, interleukin-2 (IL-2) is a factor for lymphocyte activation and clonal proliferation, promoting the activation, development, and differentiation of cytotoxic T cells. IL-2 and IL-15 also stimulate the proliferation and cytotoxicity of NK cells. However, IL-2 can act as both an immunostimulator (e.g., an immunoactivator) and an immunosuppressant. As an immunosuppressant, IL-2 maintains peripheral tolerance by inducing the generation of regulatory cells. For these reasons, IL-2 is considered a double-edged sword. The versatility of interleukin function is influenced by interactions with the environment and signaling agents.

[0027] Accordingly, the fusion proteins disclosed herein are multifunctional in that they are useful for immunoactivation, inhibition of immunosuppression, and / or suppression or inhibition of angiogenesis or invasive signaling. In some embodiments, immunoactivation, inhibition of immunosuppression, and / or suppression or inhibition of angiogenesis or invasive signaling may be used to treat tumors. In some embodiments, activating immunity (e.g., activating cells expressing the IL-2 receptor), inhibiting immunosuppression by immune cells (e.g., blocking or inhibiting the interaction between cells and TGFβa), and / or suppression or inhibition of angiogenesis or invasive signaling in a subject may be used to treat tumors or cancer. As disclosed, activation of immune responses and inhibition of immunosuppressive responses are achieved at least partially by interleukin polypeptides. In turn, inhibition or reduction of immunosuppressive signaling or signaling molecules, and / or inhibition of angiogenesis or invasive signaling or signaling molecules are achieved at least partially by the use of one or more polypeptides that inhibit the activity of the TGFβ protein.

[0028] As used herein, the term “fusion protein” generally refers to a protein comprising polypeptide components derived from two or more parent proteins or polypeptides. Generally, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is added in a frame to a nucleotide sequence encoding a polypeptide sequence from another protein, and the nucleotide sequence is optionally separated from that nucleotide sequence by a linker. The fusion gene can then be expressed as a single protein by a recombinant host cell.

[0029] As used herein, a protein “domain” generally refers to any part of an entire protein, including but typically less than a complete protein. A domain can fold independently of the rest of the protein chain and / or does not need to correlate with any particular biological, biochemical, or structural function or location (e.g., a ligand-binding domain, or a cytoplasmic, transmembrane, or extracellular domain).

[0030] The term “recombinant” indicates that a material (e.g., nucleic acid or polypeptide) has been artificially or synthetically (e.g., unnaturally) modified by human intervention. The modification may be made to the material in its natural environment or state, or removed from its natural environment or state. For example, “recombinant nucleic acid” is made by recombining nucleic acids, for example, during cloning, DNA shuffling, or other well-known molecular biological procedures. A “recombinant DNA molecule” consists of DNA segments joined together by such molecular biological techniques. As used herein, the terms “recombinant protein” or “recombinant polypeptide” refer to protein molecules expressed using a recombinant DNA molecule. A “recombinant host cell” is a cell that contains and / or expresses recombinant nucleic acid.

[0031] Where used interchangeably herein, “polynucleotide sequence” or “nucleotide sequence” or “nucleic acid sequence” is, depending on the context, a polymer of nucleotides containing a string of characters representing oligonucleotides, DNA, and RNA, nucleic acids, or nucleotide polymers. From any particular polynucleotide sequence, either a given nucleic acid or a complementary polynucleotide sequence can be determined. It may be single-stranded or double-stranded and may contain genomic or synthetically derived DNA or RNA representing sense or antisense strands.

[0032] The polynucleotide and polypeptide sequences of this disclosure can be defined in terms of specific identity and / or similarity with specific polynucleotide and polypeptide sequences described herein. In some embodiments, sequence identity is typically greater than 60%, greater than 75%, greater than 80%, greater than 90%, and / or greater than 95%. Sequence identity and / or similarity may be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% compared to sequences disclosed herein. The optimal alignment can be determined by using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), Clustal W, Clustal X, BLAST, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, California)), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a BLAST search is performed with the BLAST program, score = 100, word length = 12, to obtain sequences with the desired percent sequence identity. In some embodiments, gapped BLAST is used to obtain gapped alignment for comparison purposes. When using BLAST and gapped BLAST programs, the default parameters for each program (NBLAST and XBLAST) can be used. In some embodiments, Clustal Omega is used.

[0033] As used herein, the terms “nucleic acid molecular code,” “DNA sequence code,” “nucleic acid sequence code,” or “DNA code” refer to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. These deoxyribonucleotide sequences determine the order of ribonucleotides along an mRNA chain and the order of amino acids along a polypeptide (protein) chain. Therefore, a DNA sequence encodes both an RNA sequence and an amino acid sequence.

[0034] "Genetic expression" or "nucleic acid expression" means, as indicated by the context, the transcription of DNA into RNA (optionally including modifications of RNA, e.g., splicing), the translation of RNA into polypeptides (possibly including subsequent post-translational modifications of polypeptides), or both transcription and translation.

[0035] The term “gene” is broadly used to refer to any nucleic acid associated with a biological function. A gene typically includes a coding sequence and / or regulatory sequence necessary for the expression of such a coding sequence. The term “gene” applies to a particular genome or recombinant sequence, as well as the cDNA or mRNA encoded by that sequence. A “fusion gene” includes a coding region that encodes a transgene. A gene also includes, for example, a non-expressed nucleic acid segment that forms a recognition sequence for another protein. A non-expressed regulatory sequence includes a transcriptional regulatory element to which regulatory proteins, such as transcription factors, bind, resulting in the transcription of adjacent or nearby sequences.

[0036] As used herein, the terms “coding region” or “coding sequence,” when used in reference to structural genes, refer to nucleotide sequences that encode amino acids found within a nascent polypeptide as a result of translation of an mRNA molecule. In eukaryotes, the coding region is bounded at the 5' end by the nucleotide triplet “ATG” encoding initiator methionine and at the 3' end by one of three triplets that identify a stop codon (e.g., TAA, TAG, TGA). Transcriptional regulatory signals in eukaryotes include “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that specifically interact with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including yeast, insects, and genes from mammalian cells and viruses (similar regulatory elements, e.g., promoters, are also found in prokaryotes). The selection of specific promoters and enhancers depends on the cell type used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range, while others function in a limited subset of cell types.

[0037] The term "expression vector" refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of an operably ligated coding sequence within a specific host cell. Nucleic acid sequences required for expression in prokaryotes include promoters, optionally operator sequences, ribosome binding sites, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. The expression vector operably ligated to the coding sequence of the toxin peptide analog of the present invention may also optionally encode a secretion signal peptide sequence, thereby enabling recombinant host cells to secrete the expressed toxin peptide analog, and, if necessary, allowing for easier isolation of the toxin peptide analog from the cell. Such techniques are well known to those skilled in the art.

[0038] As used herein, individual is synonymous with patient and / or subject, and includes and / or refers to a human being, which may be a human being diagnosed with a disease or condition as disclosed herein that requires treatment. However, examples are not limited to humans and include chimpanzees, marmosets, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. Individual is typically a human being, which may be a human being diagnosed with a disease or condition as disclosed herein that requires treatment.

[0039] As used herein, the terms “inhibit” or “inhibit” include and / or mean the reduction or suppression of a given condition, symptom, disorder, or disease, and / or a decrease in the baseline activity of a biological activity or process.

[0040] As used herein, the terms “to treat” or “treatment” include and / or include improving a disease or disorder or its symptoms (e.g., delaying, stopping, or reducing the onset of at least one of the disease or its clinical symptoms). In certain embodiments, “to treat” or “treatment” also includes and / or includes mitigating or improving at least one physical and / or biological parameter, including those not identifiable by the patient. In certain embodiments, “to treat” or “treatment” includes and / or modulating a disease, disorder, or biological process physically (e.g., stabilizing identifiable symptoms), physiologically (e.g., stabilizing physical and / or biological parameters), or both. In certain embodiments, “to treat” or “treatment” includes and / or includes preventing or delaying the onset or development or progression of a disease or disorder. In certain embodiments, “to treat” or “treatment” includes and / or means (i) preventing, delaying or inhibiting the deterioration of a healthy physiological state, or (ii) a baseline physiological state (e.g., the progression of a disease or disability).

[0041] As used herein, “a” or “an,” when used in conjunction with the term “including” in the claims and / or specification, means “one” and / or “one,” and is consistent with the meanings of “one or more,” “at least one,” and “one or more than one.” Similarly, the word “another” may mean at least one or more other things (a second or more).

[0042] As used herein, the terms “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “includes,” e.g., “includes” and “includes”), or “containing” (and any form of “contain,” e.g., “contain” and “contains”) are inclusive or open-ended and do not exclude any additional elements or process steps not described herein.

[0043] As used herein, the term “about” in the context of a given value or range includes and / or refers to values ​​or ranges within 20%, 10%, and / or 5% of the given value or range.

[0044] As used herein, the term “and / or” should be considered as a specific disclosure of each of two identified features or components, with or without the other. For example, “A and / or B” should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if they were described separately herein.

[0045] As used herein, “sample” includes and / or refers to any fluid or liquid sample being analyzed to detect and / or quantify an analyte. In some embodiments, the sample is a biological sample. Examples of samples include, but are not limited to, body fluids, extracts, solutions containing proteins and / or DNA, cell extracts, cell lysates, or tissue lysates. Non-limiting examples of body fluids include urine, saliva, blood, serum, plasma, cerebrospinal fluid, tears, semen, sweat, pleural fluid, liquefied fecal matter, and lacrimal gland secretions.

[0046] When used herein, “comprising” may be replaced with “consisting essentially of” and / or “consisting of” in any example or embodiment described herein. In any example or embodiment described herein, “comprises” may be replaced with “consists essentially of” and / or “consists of”

[0047] Interleukin The fusion proteins provided herein generally comprise an interleukin polypeptide fused (e.g., linked) to two or more polypeptides that inhibit TGFβ activity (e.g., two or more polypeptides that bind the TGFβ protein). Interleukins (ILs) are a class of cytokines that were initially thought to be expressed solely by leukocytes, but were later found to be produced by many other somatic cells. Interleukins play important roles, for example, in the activation and differentiation of immune cells, as well as in cell proliferation, maturation, migration, and adhesion. Therefore, the function of interleukins is to regulate growth, differentiation, and activation during inflammatory and immune responses. Thus, interleukin polypeptides in fusion proteins are useful for regulating (e.g., activation) immune responses.

[0048] Interleukin-2 (IL-2) is an interleukin useful for activating the immune response, in association with fusion proteins containing interleukin polypeptides fused (e.g., linked) to two or more polypeptides that inhibit TGFβ activity. Interleukin-2 (IL-2) is a pleiotropic cytokine induced by antigen stimulation and therefore plays an important role in regulating the immune response (see, for example, Spolski, R., Li, P. & Leonard, WJBiology and regulation of IL-2: from molecular mechanisms to human therapy. Nat Rev Immunol 18, 648-659 (2018)). For example, it can promote innate antitumor responses by inducing local tumor rejection, acting as an autocrine factor on T cells, supporting the development of cytotoxic T cells, and stimulating the proliferation and cytolytic activity of NK cells.

[0049] IL-2 is a small cytokine consisting of four α-helix bundles with a length of 15.5 kDa. It is primarily produced by antigen-simulated CD4+ T cells, but can also be produced by CD8+ cells, natural killer (NK) cells, and activated dendritic cells (DCs). IL-2 is a crucial factor in the maintenance of CD4+ regulatory T cells and plays a vital role in the differentiation of CD4+ T cells into various T cell subsets. It can promote the cytotoxic activity of CD8+ T cells and NK cells, modulate the T cell differentiation program in response to antigens, and promote naive CD4+ T cell differentiation into T helper 1 (Th1) and T helper 2 (Th2) cells while inhibiting T helper 17 (Th17) differentiation. Notably, IL-2 was one of the first FDA-approved immunotherapies for metastatic melanoma and renal cell carcinoma. However, IL-2 immunotherapy has not been widely applied due to its short in vivo half-life and severe toxicity at therapeutic doses.

[0050] The terms “interleukin-2” or “IL-2” refer to any natural IL-2 derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. IL-2 includes untreated IL-2, as well as any form of IL-2 resulting from intracellular processing. The term also encompasses naturally occurring variants of IL-2, such as splice variants or allele variants. An exemplary amino acid sequence of human IL-2 is shown in SEQ ID NO: 1. Untreated human IL-2 further includes an N-terminal 20-amino acid signal peptide that is not present in mature IL-2 molecules. IL-2 also includes “wild-type IL-2” or naturally occurring IL-2. The sequence of a natural human IL-2 molecule is shown in SEQ ID NO: 1. For the purposes of this disclosure, the term wild-type also encompasses forms of IL-2 that include one or more amino acid mutations. For example, the fusion proteins described herein may include IL-2 polypeptides having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 1.

[0051] In some embodiments, the fusion protein contains an IL-2 sequence having approximately 85% to 100% sequence identity with respect to SEQ ID NO: 1. In some embodiments, the fusion protein contains approximately 85% to 90%, 85% to 95%, 85% to 96%, 85% to 97%, 85% to 98%, 85% to 99%, 85% to 100%, 90% to 95%, 90% to 96%, 90% to 97%, 90% to 98%, 90% to 99%, 90% to 100%, and 95% to 9 The fusion protein contains an IL-2 sequence having approximately 6%, 95% to 97%, 95% to 98%, 95% to 99%, 95% to 100%, 96% to 97%, 96% to 98%, 96% to 99%, 96% to 100%, 97% to 98%, 97% to 99%, 97% to 100%, 98% to 99%, 98% to 100%, or approximately 99% to 100% sequence identity. In some embodiments, the fusion protein contains an IL-2 sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or approximately 100% sequence identity with respect to sequence number 1. In some embodiments, the fusion protein contains an IL-2 sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to SEQ ID NO: 1.

[0052] The IL-2 polypeptide may include the full-length native sequence or cleavage thereof. In some embodiments, the IL-2 polypeptide is a cleaved IL-2 polypeptide. The cleaved IL-2 polypeptide may include N-terminal cleavage, C-terminal cleavage, or a combination thereof. In some embodiments, the fusion protein contains IL-2 that has been cleaved by about 2 to about 20 amino acids (compared to, for example, the full-length IL-2 polypeptide). In some embodiments, the fusion protein contains IL-2 in which approximately 2 to 5 amino acids, approximately 2 to 7 amino acids, approximately 2 to 8 amino acids, approximately 2 to 10 amino acids, approximately 2 to 15 amino acids, approximately 2 to 20 amino acids, approximately 5 to 7 amino acids, approximately 5 to 8 amino acids, approximately 5 to 10 amino acids, approximately 5 to 15 amino acids, approximately 5 to 20 amino acids, approximately 7 to 8 amino acids, approximately 7 to 10 amino acids, approximately 7 to 15 amino acids, approximately 7 to 20 amino acids, approximately 8 to 10 amino acids, approximately 8 to 15 amino acids, approximately 8 to 20 amino acids, approximately 10 to 15 amino acids, approximately 10 to 20 amino acids, or approximately 15 to 20 amino acids have been cleaved (compared to, for example, full-length IL-2 polypeptide). In some embodiments, the fusion protein contains IL-2 cleaved by approximately 2 amino acids, approximately 5 amino acids, approximately 7 amino acids, approximately 8 amino acids, approximately 10 amino acids, approximately 15 amino acids, or approximately 20 amino acids (compared to, for example, the full-length IL-2 polypeptide). In some embodiments, the fusion protein contains IL-2 cleaved by at least approximately 2 amino acids, approximately 5 amino acids, approximately 7 amino acids, approximately 8 amino acids, approximately 10 amino acids, or approximately 15 amino acids (compared to, for example, the full-length IL-2 polypeptide). In some embodiments, the fusion protein contains IL-2 cleaved by at most approximately 5 amino acids, approximately 7 amino acids, approximately 8 amino acids, approximately 10 amino acids, approximately 15 amino acids, or approximately 20 amino acids (compared to, for example, the full-length IL-2 polypeptide). In some embodiments, the fusion protein contains an IL-2 polypeptide having the amino acid sequence of SEQ ID NO: 2.In some embodiments, the fusion protein comprises an IL-2 polypeptide having the amino acid sequence of SEQ ID NO: 3. A preferred IL-2 sequence or variant sequence (e.g., having one or more amino acid deletions, substitutions, and / or insertions) can be identified by any of the methods described herein (e.g., as described in any one of Examples 1 to 3).

[0053] In some embodiments, the fusion protein contains an IL-2 sequence having approximately 85% to 100% sequence identity with respect to SEQ ID NO: 2. In some embodiments, the fusion protein contains approximately 85% to 90%, 85% to 95%, 85% to 96%, 85% to 97%, 85% to 98%, 85% to 99%, 85% to 100%, 90% to 95%, 90% to 96%, 90% to 97%, 90% to 98%, 90% to 99%, 90% to 100%, and 95% to 9 The fusion protein contains an IL-2 sequence having approximately 6%, 95% to 97%, 95% to 98%, 95% to 99%, 95% to 100%, 96% to 97%, 96% to 98%, 96% to 99%, 96% to 100%, 97% to 98%, 97% to 99%, 97% to 100%, 98% to 99%, 98% to 100%, or approximately 99% to 100% sequence identity. In some embodiments, the fusion protein contains an IL-2 sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or approximately 100% sequence identity with respect to sequence number 2. In some embodiments, the fusion protein contains an IL-2 sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to SEQ ID NO: 2. A preferred IL-2 sequence or variant sequence (e.g., having one or more amino acid deletions, substitutions, and / or insertions) can be identified by any of the methods described herein (e.g., as described in any one of Examples 1 to 3).

[0054] In some embodiments, the fusion protein contains an IL-2 sequence having approximately 85% to 100% sequence identity with respect to SEQ ID NO: 3. In some embodiments, the fusion protein contains approximately 85% to 90%, 85% to 95%, 85% to 96%, 85% to 97%, 85% to 98%, 85% to 99%, 85% to 100%, 90% to 95%, 90% to 96%, 90% to 97%, 90% to 98%, 90% to 99%, 90% to 100%, and 95% to 9 The fusion protein contains an IL-2 sequence having approximately 6%, 95% to 97%, 95% to 98%, 95% to 99%, 95% to 100%, 96% to 97%, 96% to 98%, 96% to 99%, 96% to 100%, 97% to 98%, 97% to 99%, 97% to 100%, 98% to 99%, 98% to 100%, or approximately 99% to 100% sequence identity. In some embodiments, the fusion protein contains an IL-2 sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or approximately 100% sequence identity with respect to sequence number 3. In some embodiments, the fusion protein contains an IL-2 sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to SEQ ID NO: 3. A preferred IL-2 sequence or variant sequence (e.g., having one or more amino acid deletions, substitutions, and / or insertions) can be identified by any of the methods described herein (e.g., as described in any one of Examples 1 to 3).

[0055] Interleukin-15 (IL-15) is also an interleukin useful for activating the immune response, in association with fusion proteins containing interleukin polypeptides fused (e.g., linked) to two or more polypeptides that inhibit TGFβ activity. Interleukin-15 (IL-15) is a cytokine of approximately 12-14 kilodaltons containing four α-helix structures. IL-15 belongs to the cytokine family consisting of interleukins IL-2, IL-4, IL-7, IL-9, and IL-21. IL-15 signals via a receptor complex composed of the IL-2 / IL-15 receptor β (IL-15Rβ) (CD122) subunit, which is shared with IL-2, and the common gamma chain (γC) (CD132) receptor subunit, which is also utilized by all additional family members. In particular, IL-15 is a growth factor for T cells and NK cells, playing a crucial role in the development, proliferation, and activation of these immune cells. While IL-15 has potential therapeutic uses, natural IL-15 has problems with therapeutic development, namely low biological potency and a short half-life.

[0056] The terms “interleukin-15” or “IL-15” refer to any natural IL-15 derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. IL-15 includes untreated IL-15 as well as any form of IL-15 resulting from intracellular processing. The term also encompasses naturally occurring variants of IL-15, such as splice variants or allele variants. An exemplary amino acid sequence of human IL-15 is shown in SEQ ID NO: 4. IL-15 also includes “wild-type IL-15” or naturally occurring IL-15. The sequence of a natural human IL-15 molecule is shown in SEQ ID NO: 4. For the purposes of this disclosure, the term wild-type IL-15 also encompasses forms of IL-15 that include one or more amino acid mutations. For example, the fusion proteins described herein may contain IL-15 polypeptides having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4.

[0057] In some embodiments, the fusion protein contains an IL-15 sequence having approximately 85% to 100% sequence identity with respect to SEQ ID NO: 4. In some embodiments, the fusion protein contains approximately 85% to 90%, 85% to 95%, 85% to 96%, 85% to 97%, 85% to 98%, 85% to 99%, 85% to 100%, 90% to 95%, 90% to 96%, 90% to 97%, 90% to 98%, 90% to 99%, 90% to 100%, and 95% to 9 The fusion protein contains an IL-15 sequence having approximately 6%, 95% to 97%, 95% to 98%, 95% to 99%, 95% to 100%, 96% to 97%, 96% to 98%, 96% to 99%, 96% to 100%, 97% to 98%, 97% to 99%, 97% to 100%, 98% to 99%, 98% to 100%, or approximately 99% to 100% sequence identity. In some embodiments, the fusion protein contains an IL-15 sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or approximately 100% sequence identity with respect to sequence number 4. In some embodiments, the fusion protein contains an IL-15 sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to SEQ ID NO: 4. A suitable IL-15 sequence or variant sequence (e.g., an IL-15 polypeptide sequence having one or more amino acid deletions, substitutions, and / or insertions) can be identified by any of the methods described herein (e.g., as described in any one of Examples 1 to 3).

[0058] The IL-15 polypeptide may include the full-length native sequence or a cleavage thereof. In some embodiments, the IL-15 polypeptide is a cleaved IL-15 polypeptide. The cleaved IL-15 polypeptide may include N-terminal cleavage, C-terminal cleavage, or a combination thereof. In some embodiments, the fusion protein contains IL-15 that has been cleaved by about 2 to about 20 amino acids (compared to, for example, the full-length IL-15 polypeptide). In some embodiments, the fusion protein contains IL-15 cleaved at approximately 2 to 5 amino acids, approximately 2 to 7 amino acids, approximately 2 to 8 amino acids, approximately 2 to 10 amino acids, approximately 2 to 15 amino acids, approximately 2 to 20 amino acids, approximately 5 to 7 amino acids, approximately 5 to 8 amino acids, approximately 5 to 10 amino acids, approximately 5 to 15 amino acids, approximately 5 to 20 amino acids, approximately 7 to 8 amino acids, approximately 7 to 10 amino acids, approximately 7 to 15 amino acids, approximately 7 to 20 amino acids, approximately 8 to 10 amino acids, approximately 8 to 15 amino acids, approximately 8 to 20 amino acids, approximately 10 to 15 amino acids, approximately 10 to 20 amino acids, or approximately 15 to 20 amino acids (compared to, for example, full-length IL-15 polypeptide). In some embodiments, the fusion protein contains IL-15 cleaved by approximately 2 amino acids, approximately 5 amino acids, approximately 7 amino acids, approximately 8 amino acids, approximately 10 amino acids, approximately 15 amino acids, or approximately 20 amino acids (compared to, for example, the full-length IL-15 polypeptide). In some embodiments, the fusion protein contains IL-15 cleaved by at least approximately 2 amino acids, approximately 5 amino acids, approximately 7 amino acids, approximately 8 amino acids, approximately 10 amino acids, or approximately 15 amino acids (compared to, for example, the full-length IL-15 polypeptide). In some embodiments, the fusion protein contains IL-15 cleaved by at most approximately 5 amino acids, approximately 7 amino acids, approximately 8 amino acids, approximately 10 amino acids, approximately 15 amino acids, or approximately 20 amino acids (compared to, for example, the full-length IL-15 polypeptide). In some embodiments, the fusion protein contains an IL-15 polypeptide having the amino acid sequence of SEQ ID NO: 5.

[0059] In some embodiments, the fusion protein contains an IL-15 sequence having approximately 85% to 100% sequence identity with respect to SEQ ID NO: 5. In some embodiments, the fusion protein contains approximately 85% to 90%, 85% to 95%, 85% to 96%, 85% to 97%, 85% to 98%, 85% to 99%, 85% to 100%, 90% to 95%, 90% to 96%, 90% to 97%, 90% to 98%, 90% to 99%, 90% to 100%, and 95% to 9 The fusion protein contains an IL-15 sequence having approximately 6%, 95% to 97%, 95% to 98%, 95% to 99%, 95% to 100%, 96% to 97%, 96% to 98%, 96% to 99%, 96% to 100%, 97% to 98%, 97% to 99%, 97% to 100%, 98% to 99%, 98% to 100%, or approximately 99% to 100% sequence identity. In some embodiments, the fusion protein contains an IL-15 sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or approximately 100% sequence identity with respect to sequence number 5. In some embodiments, the fusion protein contains an IL-15 sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to SEQ ID NO: 5.

[0060] TGFβ inhibitory polypeptide As described herein, the fusion proteins provided herein generally comprise an interleukin polypeptide (e.g., IL-2 or IL-15) fused (e.g., linked) to two or more polypeptides that inhibit TGFβ activity (e.g., two or more polypeptides that bind the TGFβ protein). The transforming growth factor β (TGFβ) superfamily is a large group of soluble factors (e.g., proteins) that initiate and regulate the activation, proliferation, and differentiation of many cell types and therefore play a crucial role in embryonic development and homeostasis. The TGFβ superfamily includes several subfamilies: the activin / inhibin family, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), the TGFβ subfamily, and the glial cell line-derived neurotrophic factor (GDNF) family. There are three types of TGFβ superfamily: TGFβ1, TGFβ2, and TGFβ3. TGFβ superfamily proteins have been found in a variety of species, including invertebrates and vertebrates. TGFβ dysfunction can cause developmental disorders, severe organ dysfunction, and is associated with several diseases, including various types of cancer.

[0061] Inhibition and suppression of TGFβ superfamily activity can be achieved by using a fusion protein comprising two or more TGFβ superfamily receptor polypeptides in addition to an interleukin polypeptide. In association with the fusion protein described herein, two or more TGFβ superfamily receptor polypeptides produce a polyvalent single-chain polypeptide capable of binding two or more TGFβ proteins. Generally, TGFβ superfamily ligands bind to receptor complexes consisting of type I and type II receptor subunits. As disclosed herein, polypeptides derived from TGFβ superfamily receptors are useful for inhibiting or suppressing TGFβ superfamily ligands. For example, in some embodiments, the TGFβ superfamily receptor is selected from the group consisting of activin receptor polypeptide or fragment thereof, bone morphogenetic protein (BMP) receptor polypeptide or fragment thereof, glial cell-derived neurotrophic factor (GDNF) receptor polypeptide or fragment thereof, and TGFβ receptor polypeptide or fragment thereof.

[0062] Within the TGFβ superfamily of proteins, the TGFβ subfamily of proteins includes TGFβ1, TGFβ2, and TGFβ3. One of the biological effects of TGFβ proteins is the inhibition of the proliferation of most normal epithelial cells using an autocrine mechanism, which suggests a tumor suppressor role for TGFβ. Loss of autocrine TGFβ activity and / or responsiveness to exogenous TGFβ appears to provide some epithelial cells with a growth advantage that leads to malignant progression. This suggests a pro-oncogenic role for TGFβ in addition to its tumor suppressor role. Among TGFβ, β1, β2, β3, and TGFβ1 are the most potent immunosuppressive cytokines described to date, exerting detrimental effects (e.g., immunosuppression) on several components of the immune system response to cancer cells and are most frequently overexpressed by cancer. In particular, TGFβ proteins (e.g., TGFβ1) reduce or impair the effector function of macrophages, B cells, cytotoxic T cells, dendritic cells, and NK cells, where TGFβ acts as a negative regulator of IFNγ production via its mediators SMAD2, SMAD3, and SMAD4. In addition to immunosuppression, TGFβ proteins can act as prometastatic and pro-angiogenic factors in late-stage cancer by constitutively inducing epithelial-to-mesenchymal transition (EMT) and tumor-associated angiogenesis.

[0063] Most cell types express three sizes of TGFβ receptors, called type I (53 kDa), type II (70–85 kDa), and type III (250–350 kDa). The type I receptor is a membrane-bound serine / threonine kinase that clearly requires the presence of the type II receptor to bind TGFβ. The type II receptor is also a membrane-bound serine / threonine kinase that binds TGFβ1 and TGFβ3 with high affinity and TGFβ2 with much lower affinity. Both type I and type II receptors form a heterodimeric signaling complex essential for the transduction of the antiproliferative signal of TGFβ. The type III receptor is a transmembrane proteoglycan with a large extracellular domain and a 43-amino acid residue cytoplasmic domain. The cytoplasmic domain of the type III receptor lacks an obvious signaling motif, and the receptor may not be directly involved in signaling. In particular, the soluble extracellular domain of TβRII, which consists of the receptor's extracellular domain, binds TGF-β1 and TGF-β3 with high affinity.

[0064] Therefore, the TGFβ receptor TβRII polypeptide is useful as the TGFβ-binding polypeptide of the multifunctional fusion proteins described herein. In particular, the TGFβ receptor TβRII polypeptide is useful for inhibiting or reducing TGFβ1 activity or related signaling. As used herein, the term “soluble transforming growth factor (TGF)β receptor type IIB or sTβRII” refers to a soluble or non-membrane form of the ectodomain of a transforming growth factor βII receptor that has been spliced ​​alternatively, preferably from any species or source, including the full-length ectodomain and fragments or portions of the ectodomain. In some embodiments, sTβRIIB is human or mouse. Human TGFβ receptor II has the amino acid sequence of SEQ ID NO: 5 or 6 (short isoform and long isoform, respectively). As used herein, the term "sTβRII fragment" means at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the total length of the reference polypeptide. In one embodiment, IL-2 is cleaved at the N-terminus or C-terminus to enable cloning.

[0065] In some embodiments, the fusion protein contains an sTBRII sequence having approximately 85% to 100% sequence identity with respect to SEQ ID NO: 6. The fusion protein contains an sTBRII sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 6. In some embodiments, the fusion protein contains an sTBRII sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to SEQ ID NO: 6.

[0066] In some embodiments, the fusion protein contains an sTBRII sequence having approximately 85% to 100% sequence identity with respect to SEQ ID NO: 7. The fusion protein contains an sTBRII sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 7. In some embodiments, the fusion protein contains an sTBRII sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to SEQ ID NO: 7.

[0067] As described herein, two or more sTBRII receptor polypeptides utilize the ectodomain of the native sTBRII receptor. Therefore, the sTBRII receptor polypeptides used herein include SEQ ID NO: 8 (e.g., a short isoform of sTBRII), SEQ ID NO: 9 (e.g., a long isoform of sTBRII), or a combination thereof.

[0068] In some embodiments, the fusion protein contains an sTBRII sequence having approximately 85% to 100% sequence identity with respect to SEQ ID NO: 8. In some embodiments, the fusion protein contains an sTBRII sequence having approximately 85% to 90%, 85% to 95%, 85% to 96%, 85% to 97%, 85% to 98%, 85% to 99%, 85% to 100%, 90% to 95%, 90% to 96%, 90% to 97%, 90% to 98%, 90% to 99%, 90% to 100%, and 95% to 96% sequence identity with respect to SEQ ID NO: 8. The fusion protein contains an sTBRII sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 8. In some embodiments, the fusion protein contains a TBRII sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to SEQ ID NO: 8.

[0069] In some embodiments, the fusion protein contains an sTBRII sequence having approximately 85% to 100% sequence identity with respect to SEQ ID NO: 9. In some embodiments, the fusion protein contains approximately 85% to 90%, 85% to 95%, 85% to 96%, 85% to 97%, 85% to 98%, 85% to 99%, 85% to 100%, 90% to 95%, 90% to 96%, 90% to 97%, 90% to 98%, 90% to 99%, 90% to 100%, and 95% to 96% sequence identity with respect to SEQ ID NO: 9. The fusion protein contains an sTBRII sequence having approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to sequence number 9. In some embodiments, the fusion protein contains a TBRII sequence having at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with respect to SEQ ID NO: 9.

[0070] The sTBRII polypeptide of SEQ ID NO: 8 or SEQ ID NO: 9 may include additional cleavage. In some embodiments, the sTBRII polypeptide is a cleaved sTBRII polypeptide. A cleaved sTBRII polypeptide may include N-terminal cleavage, C-terminal cleavage, or a combination thereof. In some embodiments, the fusion protein contains sTBRII that has been cleaved by approximately 2 to 20 amino acids (compared to, for example, full-length sTBRII polypeptide). In some embodiments, the fusion protein contains sTBRII in which approximately 2 to 5 amino acids, approximately 2 to 7 amino acids, approximately 2 to 8 amino acids, approximately 2 to 10 amino acids, approximately 2 to 15 amino acids, approximately 2 to 20 amino acids, approximately 5 to 7 amino acids, approximately 5 to 8 amino acids, approximately 5 to 10 amino acids, approximately 5 to 15 amino acids, approximately 5 to 20 amino acids, approximately 7 to 8 amino acids, approximately 7 to 10 amino acids, approximately 7 to 15 amino acids, approximately 7 to 20 amino acids, approximately 8 to 10 amino acids, approximately 8 to 15 amino acids, approximately 8 to 20 amino acids, approximately 10 to 15 amino acids, approximately 10 to 20 amino acids, or approximately 15 to 20 amino acids have been cleaved (compared to, for example, full-length sTBRII polypeptide). In some embodiments, the fusion protein contains sTBRII cleaved with approximately 2 amino acids, approximately 5 amino acids, approximately 7 amino acids, approximately 8 amino acids, approximately 10 amino acids, approximately 15 amino acids, or approximately 20 amino acids. In some embodiments, the fusion protein contains sTBRII cleaved with at least approximately 2 amino acids, approximately 5 amino acids, approximately 7 amino acids, approximately 8 amino acids, approximately 10 amino acids, or approximately 15 amino acids. In some embodiments, the fusion protein contains sTBRII cleaved with at most approximately 5 amino acids, approximately 7 amino acids, approximately 8 amino acids, approximately 10 amino acids, approximately 15 amino acids, or approximately 20 amino acids (relative to, for example, full-length sTBRII polypeptide).

[0071] Suitable sTBRII sequences or variant sequences (e.g., sTBRII polypeptide sequences having one or more amino acid deletions, substitutions, and / or insertions) can be identified by any of the methods for blocking or binding TGFβ described herein (e.g., as described in any one of Examples 1 to 3).

[0072] Multifunctional and multivalent fusion protein compositions This specification provides a fusion polypeptide comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide. This specification also provides a fusion polypeptide comprising a first IL polypeptide that binds to and activates the IL receptor, a second polypeptide that binds to and occludes soluble TGFβ, and a third polypeptide that binds to and occludes soluble TGFβ.

[0073] In some embodiments, the IL polypeptide is IL-2. In some embodiments, the IL polypeptide is IL-15. In certain embodiments, the IL-2 polypeptide includes SEQ ID NO: 2. In certain embodiments, the IL-2 polypeptide includes an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 2. In some embodiments, the IL polypeptide is IL-2. In some embodiments, the IL polypeptide is IL-15. In certain embodiments, the IL-2 polypeptide includes SEQ ID NO: 3. In certain embodiments, the IL-2 polypeptide includes an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 3. In certain embodiments, the IL-15 polypeptide includes SEQ ID NO: 5. In certain embodiments, the IL-15 polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 5. In certain embodiments, the IL polypeptide is a cleaved polypeptide. In some embodiments, the IL-2 sequence contains alanine at position 62 (as in SEQ ID NO: 3, for example).

[0074] In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide comprises a soluble TGFβ receptor II polypeptide. In some embodiments, the first TGFβ superfamily receptor polypeptide and the second TGFβ superfamily receptor polypeptide comprise a soluble TGFβ receptor II polypeptide comprising SEQ ID NO: 8, SEQ ID NO: 9, or a combination thereof. In some embodiments, the first TGFβ superfamily receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the second TGFβ superfamily receptor polypeptide comprises a cleavage of the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the soluble TGFβ receptor II polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 8 or SEQ ID NO: 9.

[0075] In another embodiment, the second polypeptide that binds to and occludes soluble TGFβ, and / or the third polypeptide that binds to and occludes soluble TGFβ, comprises a soluble TGFβ receptor II polypeptide. In some embodiments, the second polypeptide that binds to and occludes soluble TGFβ, and / or the third polypeptide that binds to and occludes soluble TGFβ, comprises a soluble TGFβ receptor II polypeptide comprising SEQ ID NO: 8, SEQ ID NO: 9, or a combination thereof. In certain embodiments, the soluble TGFβ receptor II polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 8 or SEQ ID NO: 9.

[0076] In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide targets TGF-β1 polypeptide, TGF-β2 polypeptide, TGF-β3 polypeptide, activin βA polypeptide, activin βB polypeptide, activin βC polypeptide, activin βE polypeptide, bone morphogenetic protein (BMP) 2 polypeptide, BMP3 polypeptide, BMP4 polypeptide, BMP5 polypeptide, BMP6 polypeptide, BMP7 polypeptide, BMP8 polypeptide, BMP9 polypeptide, BMP10 polypeptide, BMP11 polypeptide, BMP12 polypeptide, BMP13 polypeptide, BMP14 polypeptide, BMP15 polypeptide, growth differentiation factor (GDF) 1 polypeptide, GDF3 polypeptide, GDF8 polypeptide, GDF9 polypeptide, GDF15 polypeptide, nodal polypeptide, inhibin α polypeptide, anti-Müllerian hormone polypeptide, Lefty 1 polypeptide, Lefty 2 polypeptide, arteman polypeptide, percephin polypeptide, or neuturin polypeptide. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide conjugates a TGFβ1 polypeptide, a TGFβ2 polypeptide, a TGFβ3 polypeptide, or any combination thereof. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide conjugates a TGFβ1 polypeptide.

[0077] In another embodiment, the second polypeptide and / or the third polypeptide bind to and occlude the TGFβ1 polypeptide, the TGFβ2 polypeptide, the TGFβ3 polypeptide, or any combination thereof. In a particular embodiment, the second polypeptide and / or the third polypeptide bind to and occlude the TGFβ1 polypeptide.

[0078] The polypeptides of the fusion polypeptide can be “fused” or “linked” via a linker amino acid sequence. The linker may be a polypeptide linker or another suitable flexible linker, so as not to inhibit the binding of either targeted polypeptide (e.g., IL2 polypeptide or TBRII receptor polypeptide). The linker polypeptide may be unstructured (e.g., lacking secondary structure), structured, or a combination thereof. In some embodiments, the linker sequence is a native amino acid sequence of the IL polypeptide or soluble TBRII polypeptide. In some embodiments, the linker is not native to the IL polypeptide or TGF receptor polypeptide. For example, non-natural linkers may include polyglycine, polyalanine, polyserine amino acids, or combinations thereof (e.g., GSSG (SEQ ID NO: 26), GGSS (SEQ ID NO: 27), GSAGG (SEQ ID NO: 28), etc.).

[0079] In some embodiments, the linker is a non-natural or synthetic linker. As used herein, the term “synthetic linker” includes or includes a chemical moiety comprising a group of atoms covalently bonded to the targeting agent and also covalently bonded to the cytotoxic moiety, or a group of atoms derived therefrom. Examples of linkers include divalent radicals, such as alkylenes, arylenes, heteroarylenes, and -(CR2)nO(CR2)n- (wherein R2 is independently a repeating unit of alkyloxy (e.g., polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g., polyethyleneamino, polyetheramines such as Jeffamine®), and n is independently greater than 1, specifically n may be between 1 and 15); and the linker described in Example 1. Examples include compounds comprising N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) and N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB); as well as diacid esters and amides comprising succinates, succinamides, diglycolates, malonates, and caproamides, and peptides (e.g., repeating units of G, A, and C (e.g., up to 10) having one or more lys residues or other suitable chemical groups for linking to targeting agents and cytotoxic moieties). Optionally, the linker is a C1-30 alkylene that is unsubstituted or substituted with one or more substituents and / or optionally interrupted by one or more heteromolets independently selected from O, S, and NR1, and / or optionally interrupted by one or more C(O) and C(S) (wherein R1 is independently selected from H and C1-6 alkyl). The linker may contain uncleavable (stable linker) or cleavable (unstable linker) units such as peptide bonds or disulfide bonds. The linker may be conjugated to the targeting agent and / or cytotoxic moiety via a reactive functional group.

[0080] Both cleavable and incleavable linkers can be used in the synthesis of fusion proteins (ADCs). Examples of cleavable linkers include motifs that are either sensitive to lysosomal proteases or sensitive to acidic pH (e.g., hydrazones that hydrolyze and cleave the linker in gemtuzumab ozogamicin and inotuzumab ozogamicin), or they may contain disulfide crosslinks that can be reduced by glutathione. The steric hindrance of the disulfide crosslinks can be optimized to limit early cleavage within the cell. Generally, disulfide linkers are cleaved first to release thiol compounds. Acid-cleavable linkers, such as hydrazones, are designed to remain stable at neutral pH in blood circulation, but undergo hydrolysis in acidic cell compartments to release cytotoxic drugs.

[0081] In some embodiments, the linker (e.g., a peptide linker) contains about 10 to about 100 amino acids. In some embodiments, the linker has approximately 10 to 15 amino acids, approximately 10 to 20 amino acids, approximately 10 to 25 amino acids, approximately 10 to 30 amino acids, approximately 10 to 40 amino acids, approximately 10 to 50 amino acids, approximately 10 to 75 amino acids, approximately 10 to 100 amino acids, approximately 15 to 20 amino acids, approximately 15 to 25 amino acids, approximately 15 to 30 amino acids, approximately 15 to 40 amino acids, approximately 15 to 50 amino acids, approximately 15 to 75 amino acids, approximately 15 to 100 amino acids, approximately 20 to 25 amino acids, approximately 20 to 30 amino acids, and approximately 20 to 40 amino acids. Contains amino acids, approximately 20 to 50 amino acids, approximately 20 to 75 amino acids, approximately 20 to 100 amino acids, approximately 25 to 30 amino acids, approximately 25 to 40 amino acids, approximately 25 to 50 amino acids, approximately 25 to 75 amino acids, approximately 25 to 100 amino acids, approximately 30 to 40 amino acids, approximately 30 to 50 amino acids, approximately 30 to 75 amino acids, approximately 30 to 100 amino acids, approximately 40 to 50 amino acids, approximately 40 to 75 amino acids, approximately 40 to 100 amino acids, approximately 50 to 75 amino acids, or approximately 75 to 100 amino acids. In some embodiments, the linker contains about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, or about 100 amino acids.In some embodiments, the linker contains at least about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, or about 75 amino acids. In some embodiments, the linker contains at most about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, or about 100 amino acids.

[0082] In general, the different polypeptide elements of a fusion polypeptide can be arranged or ordered in any number of different combinations from the N-terminus to the C-terminus. For example, described herein is a fusion polypeptide comprising, from the N-terminus to the C-terminus, an IL polypeptide, a first TGFβ superfamily receptor polypeptide, and a second TGFβ superfamily receptor polypeptide. As a further example, the fusion polypeptide described herein may also include, from the N-terminus to the C-terminus, a first TGFβ superfamily receptor polypeptide, an IL polypeptide, and a second TGFβ superfamily receptor polypeptide. In some embodiments, the fusion polypeptide further comprises a pharmacokinetic extender polypeptide (e.g., an Fc polypeptide or an HSA polypeptide), and the fusion polypeptide further comprising the pharmacokinetic extender can be arranged or ordered in any number of different combinations from the N-terminus to the C-terminus. Figure 3 shows a non-limiting schematic diagram of an exemplary embodiment of a fusion polypeptide (e.g., SEQ ID NOs: 10-25) comprising an IL polypeptide, a first TGFβ superfamily receptor polypeptide, and a second TGFβ superfamily receptor polypeptide.

[0083] In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 12. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 13. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 14. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 15. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 16. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 20. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 21. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 23. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 25. In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 10 to 20.

[0084] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 10. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 10. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 10. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 10. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 10. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 10. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 10. In some embodiments, the fusion product contains SEQ ID NO: 10.

[0085] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 11. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 11. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 11. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 11. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 11. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 10. In some embodiments, the fusion product contains SEQ ID NO: 11.

[0086] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 12. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 12. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 12. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 12. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 12. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 12. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 10. In some embodiments, the fusion product contains SEQ ID NO: 12.

[0087] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 13. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 13. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 13. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 13. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 13. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 13. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 13. In some embodiments, the fusion product contains SEQ ID NO: 13.

[0088] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 14. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 14. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 14. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 14. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 14. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 14. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 14. In some embodiments, the fusion product contains SEQ ID NO: 14.

[0089] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 15. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 15. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 15. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 15. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 15. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 15. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 15. In some embodiments, the fusion product contains SEQ ID NO: 15.

[0090] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 16. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 16. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 16. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 16. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 16. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 16. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 16. In some embodiments, the fusion product contains SEQ ID NO: 16.

[0091] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 17. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 17. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 17. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 17. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 17. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 17. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 17. In some embodiments, the fusion product contains SEQ ID NO: 17.

[0092] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 18. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 18. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 18. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 18. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 18. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 18. In some embodiments, the fusion product contains SEQ ID NO: 18.

[0093] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 19. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 19. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 19. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 19. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 19. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 19. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 19. In some embodiments, the fusion product contains SEQ ID NO: 19.

[0094] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 20. In some embodiments, the fusion product contains SEQ ID NO: 20.

[0095] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 21. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 21. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 21. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 21. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 21. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 21. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 21. In some embodiments, the fusion product contains SEQ ID NO: 21.

[0096] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 22. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 22. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 22. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 22. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 22. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 22. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 22. In some embodiments, the fusion product contains SEQ ID NO: 22.

[0097] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 23. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 23. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 23. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 23. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 23. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 23. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 23. In some embodiments, the fusion product contains SEQ ID NO: 23.

[0098] In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to that of SEQ ID NO: 24. In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 24. In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 24. In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 24. In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 24. In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 24. In some embodiments, the fusion comprises SEQ ID NO: 24.

[0099] In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 25. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 25. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 85% sequence identity with respect to SEQ ID NO: 25. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO: 25. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 25. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 98% sequence identity with respect to SEQ ID NO: 25. In some embodiments, the fusion polypeptide contains an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 25. In some embodiments, the fusion product contains SEQ ID NO: 25.

[0100] In some embodiments, the bivalent FIST fusion polypeptide contains one or more amino acid modifications (e.g., substitutions, deletions, cleavages, etc.). In some embodiments, the fusion polypeptide sequence contains approximately more than 5 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to approximately 25 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.). In some embodiments, the fusion polypeptide sequence has more than 5 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to about 10 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.), about 5 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to about 15 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.), about 5 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to about 20 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.), about 5 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to about 25 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.), about 10 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to about 15 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.). It includes approximately 10 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to approximately 20 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.), approximately 10 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to approximately 25 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.), approximately 15 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to approximately 20 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.), approximately 15 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to approximately 25 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.), or approximately 20 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.) to approximately 25 amino acid modification mutations (e.g., substitutions, deletions, cleavages, etc.). In some embodiments, the fusion polypeptide sequence contains approximately 5 or more amino acid modification mutations (e.g., substitutions, deletions, cleavages), approximately 10 amino acid modification mutations (e.g., substitutions, deletions, cleavages), approximately 15 amino acid modification mutations (e.g., substitutions, deletions, cleavages), approximately 20 amino acid modification mutations (e.g., substitutions, deletions, cleavages), or approximately 25 amino acid modification mutations (e.g., substitutions, deletions, cleavages). In some embodiments, the modifications increase affinity for the IL-2 receptor.In certain embodiments, the IL-2 receptor comprises IL-2Rα (referred to as CD25). In certain embodiments, the IL-2 receptor comprises IL-2Rβ. In certain embodiments, the IL-2 receptor comprises IL-2Rγ. In certain embodiments, the modification increases affinity for IL-2Rβ and decreases affinity for IL-2Rα. Examples of such mutations affecting IL-2 affinity for the IL-2 receptor include those known in the art. Preferred bivalent FIST sequences or variant sequences (e.g., fusion polypeptide sequences having one or more amino acid deletions, substitutions, and / or insertions) can be identified by either a method of activating immune cells (e.g., increasing IFN-gamma secretion, increasing proliferation, etc.) or a method of blocking or binding TGFβ (e.g., TGFβ binding, etc.) as described herein (e.g., as described in any one of Examples 1 to 5).

[0101] In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 20. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion polypeptide consists of the amino acid sequence of SEQ ID NO: 25.

[0102] The fusion polypeptides disclosed herein may further comprise pharmacokinetic modifier polypeptides (e.g., polypeptides that improve the pharmacokinetic profile of a therapeutic agent). For example, the fusion protein may further comprise an immunoglobulin Fc polypeptide or a human serum albumin (HAS) polypeptide fused to the N-terminus of an IL polypeptide. In some embodiments, the immunoglobulin Fc region polypeptide is a human immunoglobulin Fc region polypeptide. In some embodiments, the immunoglobulin Fc region is an IgG Fc region. In some embodiments, the IgG Fc region is an IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the PK modifier comprises an albumin polypeptide. In some embodiments, the albumin polypeptide is a human albumin polypeptide.

[0103] In certain embodiments, the fusion polypeptide of the Disclosure is contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the fusion protein of the Disclosure is administered suspended in a sterile solution. In certain embodiments, the solution contains about 0.9% NaCl. In certain embodiments, the solution contains about 5.0% dextrose. In certain embodiments, the solution further comprises a buffer, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, e.g., polysorbate 80, polysorbate 20, and poloxamer 188; a polyol / disaccharide / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, e.g., glycine or arginine; an antioxidant, e.g., ascorbic acid, methionine, or a chelating agent, e.g., ethylenediaminetetraacetic acid or ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid.

[0104] In certain embodiments, the fusion protein of this disclosure is shipped / stored, lyophilized, reconstituted, and then administered. In certain embodiments, the lyophilized fusion polypeptide formulation contains a volume extender such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation may be contained in a vial made of glass or other suitable non-reactive material. Once formulated, whether reconstituted or not, the fusion protein may be buffered at a specific pH, generally below 7.0. In certain embodiments, the pH may be 4.5–6.5, 4.5–6.0, 4.5–5.5, 4.5–5.0, or 5.0–6.0.

[0105] In certain embodiments, a method for preparing a cancer treatment is described herein, comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with the fusion polypeptides of the Disclosure. In certain embodiments, a method for preparing a cancer treatment for storage or shipment is described herein, comprising lyophilizing one or more fusion polypeptides of the Disclosure.

[0106] The fusion polypeptides described herein (e.g., SEQ ID NOs: 10-25) may be encoded by nucleic acids. A nucleic acid is a type of polynucleotide containing two or more nucleotide bases. In certain embodiments, a nucleic acid is a component of a vector that can be used to transfer a polypeptide encoding a polynucleotide into a cell. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is an integrated vector, or “integrated vector,” which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an “episome” vector, e.g., a nucleic acid capable of extrachromosomal replication. Vectors capable of inducing the expression of a gene to which they are operably ligated are referred herein to as “expression vectors.” Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. Expression vector regulatory elements, such as promoters, enhancers, and polyadenylation signals for use in transcriptional control, may be derived from mammalian, microbial, viral, or insect genes. Selective genes that facilitate the ability to replicate in a host and the recognition of transformants, usually conferred by the origin of replication, may be additionally incorporated. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses may be used. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors may contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) into defined locations or a limited set of sites within the genome. Furthermore, vectors may contain sequences derived from transposable elements.

[0107] How to use The fusion polypeptides disclosed herein are useful as effective angiogenic inhibitors and anticancer compounds for cancer therapy. For example, the disclosed fusion polypeptides are effective immunotherapies that induce unique gene expression profiles downstream of the IL-2 receptor that cannot be achieved by their single component or combination, resulting in novel pharmacological properties. These novel multifunctional proteins can simultaneously activate several immune system mechanisms and signaling pathways that act synergistically to effectively eliminate cancer cells. At the cellular level, the immunotherapy platform activates a whole (panoply) of lymphoid cells (e.g., T, B, NK, and NK-T cells) and indirectly prime antigen-presenting cells (e.g., dendritic cells and macrophages), partly due to GM-CSF induction triggering a cascade of specific immune responses against malignant cells. At the molecular level, it activates key transcription factors and signaling molecules essential for enhancing innate and adaptive immune responses. The fusion polypeptides described herein target and synchronize various arms of the immune system against cancer.

[0108] Disclosed herein is a method for inhibiting or reducing tumor growth, angiogenesis, and / or progression in an individual having cancer, comprising administering a fusion polypeptide to the individual comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide, thereby inhibiting or reducing tumor growth and / or progression in the individual. Furthermore, provided is a fusion polypeptide for use in a method for inhibiting or reducing tumor growth, angiogenesis, and / or progression in an individual having cancer, wherein the method comprises administering a fusion polypeptide to an individual comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide, thereby inhibiting or reducing tumor growth and / or progression in the individual.

[0109] Disclosed herein is a method for inhibiting or reducing tumor growth, angiogenesis, and / or progression in an individual having cancer, comprising administering to the individual a fusion polypeptide comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide, wherein the fusion polypeptide activates immune cells. Furthermore, provided is a fusion polypeptide for use in a method for inhibiting or reducing tumor growth, angiogenesis, and / or progression in an individual having cancer, wherein the method comprises administering to the individual a fusion polypeptide comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide, wherein the fusion polypeptide activates immune cells. In some embodiments, the immune cells are selected from the group consisting of T cells, NK cells, B cells, or combinations thereof. In certain embodiments, activation includes increased cell proliferation, increased cytokine signaling (e.g., IFNγ), and / or reduced TGFβ1-mediated repression.

[0110] Furthermore, disclosed is a method for neutralizing or killing tumor cells in an individual having cancer, comprising administering a fusion polypeptide to the individual comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide, thereby neutralizing or killing tumor cells in the individual. Furthermore, provided is a fusion polypeptide for use in a method for neutralizing or killing tumor cells in an individual having cancer, wherein the method comprises administering a fusion polypeptide to an individual comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide, thereby neutralizing or killing tumor cells in the individual.

[0111] Also disclosed is a method for treating or improving cancer in an individual having cancer, comprising administering a fusion polypeptide comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide to a target, thereby treating or improving cancer in the individual. Furthermore, provided is a fusion polypeptide for a method of use for treating or improving cancer in an individual having cancer, wherein the method comprises administering a fusion polypeptide comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide to a target, thereby treating or improving cancer in the individual.

[0112] Also provided is a method for activating cells expressing an IL receptor, comprising contacting a cell with a fusion polypeptide comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide. In some embodiments, the cells are immune cells. The immune cells can be contacted in vivo or ex vivo and subsequently activated. In vivo, the immune cells may be present within the tumor microenvironment. Furthermore, provided is a fusion polypeptide for use in a method for activating cells expressing an IL receptor, wherein the method comprises contacting a cell with a fusion polypeptide comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide.

[0113] Methods utilizing the ex vivo activation of immune cells using the fusion polypeptides disclosed herein are also practiced. For example, a method for ex vivo activation of immune cells is disclosed, comprising contacting immune cells with a composition comprising (a) an interleukin (IL) polypeptide, (b) a first TGFβ superfamily receptor polypeptide, and (c) a second TGFβ superfamily receptor polypeptide. Such a method can be applied to the treatment of cancer or tumors by administering the activated immune cells to an individual having cancer or a tumor. For example, the fusion polypeptides described herein are useful in methods for ex vivo activation of immune cells. Ex vivo treatment of immune cells is also useful for the generation and proliferation of immune cells (e.g., CAR-T or CAR-NK cells and B cells) for the creation of chimeric antigen receptor cell therapies. For example, a leukocyte pack containing immune cells (e.g., a leukocyte apheresis product) can be contacted with the fusion polypeptides disclosed herein to proliferate T cells or NK cells used in chimeric antigen receptor cell therapy. In some embodiments, the immune cells express IL-2 or IL-15 receptors. In some embodiments, the immune cells include T cells, natural killer (NKT) cells, B cells, or gamma delta T cells. In some embodiments, the fusion polypeptide inhibits or reduces immunosuppression of immune cells in the tumor microenvironment and / or reduces or inhibits the activation of immunosuppressive cells in the tumor microenvironment. In some embodiments, the cancer is a hematological cancer.

[0114] In some embodiments, the IL polypeptide is IL-2. In some embodiments, the IL polypeptide is IL-15. In certain embodiments, the IL-2 polypeptide includes SEQ ID NO: 2. In certain embodiments, the IL-2 polypeptide includes an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 2. In some embodiments, the IL polypeptide is IL-2. In some embodiments, the IL polypeptide is IL-15. In certain embodiments, the IL-2 polypeptide includes SEQ ID NO: 3. In certain embodiments, the IL-2 polypeptide includes an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 3. In certain embodiments, the IL-15 polypeptide includes SEQ ID NO: 5. In certain embodiments, the IL-15 polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 5. In certain embodiments, the IL polypeptide is a cleaved polypeptide.

[0115] In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide comprises a soluble TGFβ receptor II polypeptide. In some embodiments, the first TGFβ superfamily receptor polypeptide and the second TGFβ superfamily receptor polypeptide comprise a soluble TGFβ receptor II polypeptide comprising SEQ ID NO: 8, SEQ ID NO: 9, or a combination thereof. In some embodiments, the first TGFβ superfamily receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 8, and the second TGFβ superfamily receptor polypeptide comprises a cleavage of the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the soluble TGFβ receptor II polypeptide comprises an amino acid sequence having more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 8 or SEQ ID NO: 9.

[0116] In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide targets TGF-β1 polypeptide, TGF-β2 polypeptide, TGF-β3 polypeptide, activin βA polypeptide, activin βB polypeptide, activin βC polypeptide, activin βE polypeptide, bone morphogenetic protein (BMP) 2 polypeptide, BMP3 polypeptide, BMP4 polypeptide, BMP5 polypeptide, BMP6 polypeptide, BMP7 polypeptide, BMP8 polypeptide, BMP9 polypeptide, BMP10 polypeptide, BMP11 polypeptide, BMP12 polypeptide, BMP13 polypeptide, BMP14 polypeptide, BMP15 polypeptide, growth differentiation factor (GDF) 1 polypeptide, GDF3 polypeptide, GDF8 polypeptide, GDF9 polypeptide, GDF15 polypeptide, nodal polypeptide, inhibin α polypeptide, anti-Müllerian hormone polypeptide, Lefty 1 polypeptide, Lefty 2 polypeptide, arteman polypeptide, percephin polypeptide, or neuturin polypeptide. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide conjugates a TGFβ1 polypeptide, a TGFβ2 polypeptide, a TGFβ3 polypeptide, or any combination thereof. In some embodiments, the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide conjugates a TGFβ1 polypeptide.

[0117] In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 10. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 12. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 13. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 14. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 15. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 16. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 20. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 21. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 22. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 23. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 24. In some embodiments, the fusion polypeptide includes the amino acid sequence of SEQ ID NO: 25.

[0118] In some embodiments, the fusion polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 10 to 20.

[0119] The term "cancer" includes primary malignancies (for example, those in which the cells have not spread to other parts of the body other than the site of the original tumor) and secondary malignancies (for example, those resulting from metastasis, the migration of tumor cells to a secondary site different from the site of the original tumor).

[0120] The term "tumor", regardless of whether it is malignant or benign, refers to the growth and proliferation of neoplastic cells, as well as all precancerous and cancerous cells and tissues.

[0121] The methods disclosed herein are useful for treating cancer including solid tumors or treating solid tumors. The term "solid tumor" refers to, for example, breast cancer, ovarian cancer, colon cancer, and generally cancers of the GI (gastrointestinal) tract, cervical cancer, lung cancer, particularly small cell lung cancer and non-small cell lung cancer, head and neck cancer, bladder cancer, prostate cancer, or Kaposi's sarcoma. The fusion polypeptide neutralizes and / or inhibits the growth of solid tumors. Furthermore, depending on the type of tumor and the particular combination used, a reduction in tumor volume can be achieved. The combinations disclosed herein are also suitable for preventing metastatic spread of tumors and the growth or development of micrometastases. The combinations disclosed herein are particularly suitable for the treatment of patients with poor prognosis, especially such patients with poor prognosis having metastatic melanoma or pancreatic cancer.

[0122] Furthermore, the methods disclosed herein are useful for treating hematological cancer. The term "hematological cancer" refers to cancer of the blood, and includes, inter alia, leukemia and malignant lymphoproliferative disorders. "Leukemia" refers to a cancer of the blood in which too many leukocytes or erythrocytes are produced, so that other components constituting blood, such as platelets and normal erythrocytes, are compressed. It is understood that cases of leukemia are classified as acute or chronic. Cancer cells in acute leukemia are blocked at an immature stage but continue to proliferate. Chronic leukemia progresses more slowly, and cancer cells develop until fully mature. Furthermore, leukocytes may be myeloid or lymphoid. Accordingly, certain forms of leukemia may be, by way of example, acute lymphocytic (or lymphoblastic) leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myeloid leukemia (CML), and myelodysplastic syndrome. "Malignant hematological disorder" may refer to, inter alia, Hodgkin lymphoma, non-Hodgkin lymphoma, or lymphomas such as multiple myeloma.

[0123] In certain embodiments, disclosed herein are fusion proteins useful for the treatment of cancer or tumors. Treatment refers to a method that seeks to improve or ameliorate a condition during treatment. With respect to cancer, treatment includes, but is not limited to, a reduction in tumor volume, a reduction in tumor volume increase, or an increase in progression-free survival or overall life expectancy. In certain embodiments, treatment may influence remission of cancer during treatment. In certain embodiments, treatment includes use as a prophylactic or maintenance dose intended to prevent recurrence or progression of a previously treated cancer or tumor. Those skilled in the art will understand that not all individuals will respond equally to or not to the treatment administered, and nevertheless, these individuals are considered to be treated.

[0124] The fusion polypeptides provided herein are useful for activating immune responses (e.g., anti-tumor immune responses). In some embodiments, the immune cells express IL-2 or IL-15 receptors. In some embodiments, the immune cells include T cells, natural killer (NKT) cells, B cells, or gamma delta T cells. In some embodiments, the fusion polypeptide inhibits or reduces immunosuppression of immune cells in the tumor microenvironment and / or reduces or inhibits the activation of immunosuppressive cells in the tumor microenvironment. Therefore, in some embodiments, treatment or therapy involves activating immune effector cells that target tumor cells and / or reducing the activation of immunosuppressive cells (e.g., Treg cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), tumor-associated neutrophils (TANs), and cancer-associated fibroblasts (CAFs)).

[0125] The term "T cell" refers to a subset of lymphocyte cells present in PBMCs that express the surface marker "CD3" (T cell receptor). Unless otherwise indicated, T cells are intended to include CD4+ (e.g., T helper cells) and CD8+ (e.g., cytotoxic killer cells).

[0126] The methods provided herein are also useful for suppressing or reducing immunosuppressive immune cells, such as T regulatory cells. The terms “Treg” or “regulatory T cell” refer to CD4+CD25+FOxP3+ T cells that suppress the proliferation and / or effector function of CD4+CD25- and CD8+ T cells and / or otherwise downregulate the immune response. In particular, Tregs can downregulate immune responses mediated by CD8 T cells, natural killer cells, natural killer T cells, and other immune cells. Some embodiments further encompass blocking or preventing the differentiation or activation of immunosuppressive cells.

[0127] In certain embodiments, cancer or tumor is a solid cancer or tumor. In certain embodiments, cancer or tumor is a blood cancer or tumor. In certain embodiments, cancer or tumor includes tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovaries, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, and liver. In certain embodiments, tumors that can be treated with the fusion proteins described herein include adenomas, adenocarcinomas, angiosarcomas, astrocytomas, epithelial carcinomas, germ cell tumors, glioblastomas, gliomas, hemangioendotheliomas, angiosarcomas, hematomas, hepatoblastomas, leukemias, lymphomas, medulloblastomas, melanomas, neuroblastomas, osteosarcomas, retinoblastomas, rhabdomyosarcomas, sarcomas, and / or teratomas.In certain embodiments, tumors / cancers include acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, Bartholin's gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoma, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, atypical endometrial hyperplasia, endometrial stromal sarcoma, endometrial adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia, gastronoma, germline tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hepatocellular adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinitis, intraepithelial squamous cell neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, nerve sheath tumor, medulloblastoma, medullary epithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate cancer, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma The group consists of carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar carcinoma, VIPpoma, and Wilms' tumor.In certain embodiments, tumors / cancers treated with one or more fusion proteins described herein include brain cancer, head and neck cancer, colorectal cancer, acute myeloid leukemia, pre-B cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III, or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell carcinoma and non-small cell carcinoma, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate cancer, and breast cancer, preferably ductal carcinoma, and / or breast carcinoma. In certain embodiments, cancers treated with the fusion proteins of this disclosure include glioblastoma. In certain embodiments, cancers treated with one or more fusion proteins of this disclosure include pancreatic cancer. In certain embodiments, cancers treated with one or more fusion proteins of this disclosure include ovarian cancer. In certain embodiments, cancers treated with one or more fusion proteins of this disclosure include lung cancer. In certain embodiments, cancers treated with one or more fusion proteins of the Disclosure include prostate cancer. In certain embodiments, cancers treated with one or more fusion proteins of the Disclosure include colon cancer. In certain embodiments, cancers treated include glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In certain embodiments, the cancer is refractory to other treatments. In certain embodiments, the treated cancer recurs. In certain embodiments, the cancer is recurrent / refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer.

[0128] The term "tumor microenvironment" refers to the microenvironment within and around a solid tumor (e.g., the region / area where malignant cells or clusters of malignant cells are located) that supports the growth and metastasis of tumor cells. The tumor microenvironment includes surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that can promote oncogenic transformation, support tumor growth and invasion, protect the tumor from host immunity, promote treatment resistance, and provide a niche for successful dormant metastases. The tumor and its surrounding microenvironment are closely related and constantly interacting. The tumor can influence its microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance, while immune cells in the microenvironment can influence the growth and evolution of cancer cells.

[0129] In certain embodiments, the fusion protein may be administered to a subject requiring it by any route suitable for the administration of the fusion protein-containing pharmaceutical composition, such as subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally. In certain embodiments, the fusion protein is administered intravenously. In certain embodiments, the fusion protein is administered subcutaneously. In certain embodiments, the fusion protein is administered intratumorally. In certain embodiments, the fusion protein is administered according to a suitable administration schedule, such as weekly, twice weekly, monthly, twice monthly, every two weeks, every three weeks, or once a month. In certain embodiments, the fusion protein is administered every three weeks. The fusion protein may be administered in any therapeutically effective dose. In certain embodiments, the therapeutically acceptable dose is about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, the therapeutically acceptable dose is about 1 mg / kg to about 40 mg / kg. In certain embodiments, the therapeutically acceptable dose is about 5 mg / kg to about 30 mg / kg. A therapeutically effective dose includes an amount sufficient to improve one or more symptoms associated with the disease or distress being treated.

[0130] Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art in which the claimed subject matter relates. In some cases, terms that have a commonly understood meaning are defined herein for clarity and / or for easy reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from the commonly understood meaning in the art.

[0131] As used herein, the terms “treatment” or “treating” are used in relation to a pharmaceutical or other intervention regimen for obtaining a beneficial or desired outcome in a recipient. Beneficial or desired outcomes include, but are not limited to, therapeutic benefits and / or preventive benefits. Therapeutic benefits may refer to the elimination or improvement of symptoms, or the elimination or improvement of an underlying disorder during treatment. Therapeutic benefits may also be achieved with the elimination or improvement of one or more physiological symptoms associated with an underlying disorder, such that improvement is observed in the subject, even though the subject may still have the underlying disorder. Preventive effects include delaying, preventing, or eliminating the onset of a disease or condition; delaying or eliminating the onset of symptoms of a disease or condition; slowing, stopping, or reversing the progression of a disease or condition; or any combination thereof. With respect to preventive benefits, subjects at risk of developing a particular disease, or those reporting one or more physiological symptoms of a disease, may be treated even if they have not been diagnosed with the disease.

[0132] The term “therapeutic dose” of a compound in this application refers to the amount of the compound that induces a biological or medical response in a subject, such as a reduction or inhibition of tumor cell proliferation, or improvement of symptoms, alleviation of a condition, slowing or delaying disease progression, or prevention of disease. In one non-limiting embodiment, the term “therapeutic dose” refers to the amount of the compound that, when administered to a subject, is effective in at least partially alleviating, inhibiting, preventing and / or improving a condition, disorder or disease, or in at least partially inhibiting the activity of a targeted enzyme or receptor.

[0133] The term “target cells” refers to a cell type, cell population, or composition of cells that are the desired cells to be collected, isolated, or separated by this disclosure. Target cells represent cells that require, or are designed to be purified, collected, manipulated, etc., by the various procedures described herein. What constitutes a particular cell depends on the context in which the term is used. For example, if the objective of a procedure is to isolate a particular type of stem cell, then that cell would be the target cell of the procedure. The terms “target cells” and “desired cells” are interchangeable and have the same meaning in this disclosure. Target cells may exist in a genus-species relationship. For example, if target cells include leukocytes, then target cells would include T cells.

[0134] The methods disclosed herein are particularly useful or involve the activation of immune cells. The term “immune cells” generally refers to cells of the immune system. Immune cells are derived from bone marrow cells or lymphocyte cell lineages. Generally, the methods disclosed herein target, but are not limited to, the activation of immune effector cells.

[0135] The term "immune effector cells" refers to cells involved in promoting immune responses, such as immune effector responses. Examples of immune effector cells include T cells, such as alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells. In certain embodiments, T cells, NK cells, B cells, NKT cells, and gamma / delta T cells, or combinations thereof, are activated by fusion polypeptides.

[0136] The terms “immune effector function” or “immune effector response,” as used herein, refer to the function or response of, for example, immune effector cells that enhance or promote an immune attack on target cells. For example, immune effector function or response refers to the properties of T cells or NK cells that promote the killing of target cells or the inhibition of the growth or proliferation of target cells. Immune effector function includes direct cytotoxicity, cytokine release, chemokine release, phagocytosis, or other immune functions that prime or perpetuate an immune response.

[0137] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including cytokine secretion.

[0138] The term "myelocytes" refers to terminally differentiated cells of the myeloid lineage. These cells include neutrophils, eosinophils and monocytes / macrophages, and myeloid dendritic cells. In one embodiment of any aspect of this disclosure, myelocytes are neutrophils, eosinophils, or monocytes / macrophages / dendritic cells.

[0139] The terms “macrophage” and / or “macrophage-like cell” generally refer to macrophages, monocytes, and cells of the macrophage / monocyte lineage, as well as any other similar cells that perform functions generally associated with macrophages, such as phagocytosis or antigen presentation to other classes of immune cells, such as T cells and B cells, in order to sensitize these cells to specific targets, including but not limited to these.

[0140] As used herein, the term “lymphocyte” includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic lymphocyte that represents a major component of the intrinsic immune system. NK cells kill virus-infected tumors and cells.

[0141] The term "natural killer (NK) cells" refers to cells of the immune system that kill target cells without restriction according to MHC class, in the absence of specific antigenic stimulation. Target cells may be tumor cells or virus-bearing cells. NK cells are characterized by the presence of CD56 and the absence of the CD3 surface marker.

[0142] The term "T cell" refers to a subset of lymphocyte cells that express the surface marker "CD3" (T cell receptor). Unless otherwise specified, T cells express CD4 + (For example, T helper cells) and CD8 + It is intended to include (for example, cytotoxic killer cells).

[0143] The term "endogenous cells" is used to refer to donor (or patient)-derived cells, distinct from cells from cell lines. Endogenous cells are generally a heterogeneous population of cells from which a particular cell type can be isolated or enriched. Endogenous cells may be intended for the patient's own or allogeneic treatment.

[0144] Throughout this application, various embodiments may be presented in range form. It should be understood that range form descriptions are merely for convenience and brevity and should not be interpreted as inflexible limitations on the scope of this disclosure. Therefore, range descriptions should be considered to specifically disclose all possible subranges and individual numbers within that range. For example, a range description such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0145] As used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context explicitly indicates otherwise. For example, the term "a sample" includes multiple samples (including mixtures thereof).

[0146] The terms “determining,” “measuring,” “evaluating,” “assessing,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present or not (e.g., detection). These terms may include quantitative, qualitative, or a combination of quantitative and qualitative determinations. Evaluation may be relative or absolute. “Detecting the presence of” may, depending on the context, include determining the quantity of something that is present, in addition to determining whether it is present or not.

[0147] The terms “subject,” “individual,” and “patient” are often used interchangeably herein. “Subject” may be a biological entity containing expressed genetic material. A biological entity may be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject may be a tissue, cell, or offspring of a biological entity obtained in vivo or cultured in vitro. A subject may be a mammal. A mammal may be a human. In some embodiments, a subject may be diagnosed or suspected of being at high risk of cancer. In some embodiments, a subject may be diagnosed or suspected of being at high risk of having a tumor. In some embodiments, a subject may not necessarily be diagnosed or suspected of being at high risk of cancer.

[0148] The term "in vivo" is used to describe events that occur within the body of a subject.

[0149] The term "ex vivo" is used to describe events that occur outside the body of a subject. Ex vivo assays are not performed on the subject; rather, they are performed on a sample isolated from the subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay.

[0150] The term "in vitro" is used to describe the event of a material being contained within a container for holding laboratory reagents, so that it is separated from the biological source from which the material is obtained. In vitro assays can include cell-based assays in which living or dead cells are used. In vitro assays can also include cell-free assays in which intact cells are not used.

[0151] As used herein, the term “approximately” refers to a number within plus or minus 10% of that number. The term “approximately” range refers to a range of minus 10% of its minimum value and a range of plus 10% of its maximum value.

[0152] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. [Examples]

[0153] In addition, the following embodiments are included for illustrative purposes only and are not intended to limit the scope of this disclosure.

[0154] Example 1 - Improved IL-2 activation of immune cells Exemplifying the fusion polypeptide compositions and methods disclosed herein, the following example demonstrates that a fusion polypeptide comprising interleukin-2 (IL-2) polypeptide, a first sTBRII receptor polypeptide, and a second sTBRII receptor polypeptide (bFIST) activates higher levels of IFN-gamma and CXCL10 in human NK cells compared to a fusion protein comprising IL-2 polypeptide and a single sTBRII receptor polypeptide (monovalent FIST).

[0155] Human T cells and NK cells were isolated from PBMCs by negative selection and stimulated for 48 hours with FIST (IL2-sTBRII, monovalent) or a FIST variant with a bivalent sTBRII trap (IL2-sTBRII-sTBRII). After 48 hours of incubation, the incubation medium was collected, and the levels of IFN-gamma from T cells and CXCL10 from NK cells were quantified by ELISA. As shown in the data in Figure 1, bFIST showed significantly higher IL-2 receptor activation than FIST (monovalent FIST).

[0156] Example 2 - Improved TGFβ1 binding Exemplifying the fusion polypeptide compositions and methods disclosed herein, the following examples demonstrate that a fusion polypeptide comprising an interleukin-2 (IL-2) polypeptide, a first sTBRII receptor polypeptide, and a second sTBRII receptor polypeptide (bFIST) binds active TGFβ1 more effectively than a fusion protein comprising an IL-2 polypeptide and a single sTBRII receptor polypeptide (monovalent FIST).

[0157] Human active TGFβ1 (2 ng / mL), pre-activated with 4 mM HCl containing 0.1% human or bovine serum albumin (BSA), was incubated with sTBRII isoform 2 (long), FIST (monovalent FIST), or bFIST (5-20 nmol) for 30 minutes at room temperature, and the free active TGFβ in the solution was quantified by ELISA (ELISA for human TGFβ1 immunoassay).

[0158] bFIST effectively blocks active TGFβ1 in solution at significantly lower concentrations than sTBRII, as shown in Figure 2. These results indicate that IL-2, as part of the fusion protein, also modifies its binding properties to the sTBRII moiety and active TGFβ. Furthermore, the divalent sTBRII trap variant exhibits more effective TGFβ blocking activity.

[0159] Example 3 - Blocking of TGFβ isoforms in solution Exemplifying the fusion polypeptide compositions and methods disclosed herein, the following examples demonstrate that a fusion polypeptide comprising an interleukin-2 (IL-2) polypeptide, a first sTBRII receptor polypeptide, and a second sTBRII receptor polypeptide more effectively binds to TGFβ isoforms.

[0160] Equimolar concentrations of bFISTv3 and FIST (monovalent FIST) were compared for their ability to inhibit TGFβ1-mediated suppression of CTLL-2 proliferation (IL-2-dependent T lymphocyte cell line). The blocking activity of TGFβ isoforms in solution by bFISTv3, FIST (monovalent FIST), or control (TbRII isoform 2) was determined by incubating bFISTv3, FIST (monovalent FIST), or control with 1 ng / mL of active TGFβ isoform. Equimolar concentrations (5-5 × 10⁻⁶) -6 bFISTv3, FIST (monovalent FIST), or control (TβRII isoform 2) in nmoL ranges were compared for their ability to block active TGFβ isoforms in solution. bFISTv3, FIST (monovalent FIST), or control were incubated with 1 ng / mL of active TGFβ isoform diluted in phosphate-buffered saline (PBS) for 30 minutes, and free TGFβ (unblocked) was quantified by ELISA specific to each TGFβ isoform.

[0161] bFISTv3 showed significantly increased affinity for picomolar titers of TGFb1 (9 picomoles) and TGFβ3 (70 picomoles) compared to FIST and control TβRII isoform 2 (Figures 4A-4C). Figure 4A shows blockade of TGFb1 binding. Figure 4B shows blockade of TGFb2 binding. Figure 4C shows blockade of TGFb3 binding. Table 1 shows the binding values ​​of TGFβ blocking activity in solution. TGFβ1 and TGFβ3 isoforms are the most oncogenic isoforms and are potent inducers of tumor immune resistance, angiogenesis, metastasis, and immunosuppression. TGFβ1 and TGFβ3 isoforms are also known inducers of fibrotic diseases. In contrast, TGFβ2 was not blocked by the fusion protein or control. TGFβ2 is an important regulator of hematopoiesis, cardiovascular function, glucose, and fatty acid metabolism. Table 1: IC50 values ​​of active TGFβ blocking activity in solution [Table 1]

[0162] Example 4 - Inhibition of TGFb1-mediated inhibition of CTLL-2 proliferation To exemplify the fusion polypeptide compositions and methods disclosed herein, the following example demonstrates that a fusion polypeptide comprising an interleukin-2 (IL-2) polypeptide, an sTβRII receptor polypeptide, and a second sTβRII receptor polypeptide more effectively induces cytotoxic T cell proliferation and prevents TGFβ1-mediated suppression.

[0163] Equimolar concentrations (50 to 5×10 -5 nmol range) of bFISTv3, FIST (monovalent FIST) or a control (IL-2 and TβRII isoform 2) were compared for their ability to inhibit active TGFβ1-mediated inhibition of CTLL-2 proliferation. CTLL-2 cells (5×10 3 cells / well) were stimulated with equimolar concentrations of bFISTv3, FIST (monovalent FIST) or control, with / without active TGFβ1 (1 ng / mL), at 37° C. for 72 hours. After the incubation period, cell proliferation was determined by a luminescent cell viability assay. Absolute IC50 was determined using dose-response curves using a non-linear regression model with sigmoidal dose-response. Viability (%)=(Lum 試験品 -Lum 培地対照 ) / (Lum ビヒクル対照 -Lum 培地(Control) × 100%. Cell count was quantified by luminescence signal versus cell dose curve. bFISTv3 induced CTLL-2 cell proliferation but more effectively prevented TGFβ1-mediated suppression of CTLL-2 proliferation than FIST (monovalent FIST). Figure 5A: Proliferation of CTLL-2 stimulated with bFISTv3. Figure 5B: Proliferation of CTLL-2 stimulated with bFISTv3 in the presence of active TGFβ1 (1 ng / mL). Figure 5C: Proliferation of CTLL-2 stimulated with FIST (monovalent FIST). Figure 5D: Proliferation of CTLL-2 stimulated with FIST (monovalent FIST) in the presence of active TGFβ1 (1 ng / mL). Figure 5E: Proliferation of CTLL-2 stimulated with IL-2. Figure 5F: CTLL-2 proliferation stimulated by IL-2 in the presence of active TGFβ1 (1 ng / mL). Data are shown as mean + SD, **p<0.005. TGFβ1 is known to inhibit IL-2 signaling and T cell proliferation via multiple pathways. TGFβ1 inhibits the phosphorylation and activation of components of the JAK / STAT cascade downstream of IL-2R, and exerts inhibitory activity at the nuclear level on a subset of IL-2 target genes, including c-Myc, cyclin D2, and cyclins. Table 2: IC50 values ​​for CTLL-2 cell proliferation in response to fusion protein or control stimulation [Table 2] Table 3: IC50 values ​​for inhibiting TGFb1-dependent suppression of CTLL-2 cell proliferation [Table 3]

[0164] Example 5 - bFIST proliferation of effector memory T cells and terminally differentiated effector memory cells Exemplifying the fusion polypeptide compositions and methods disclosed herein, a flowing example shows a fusion polypeptide comprising an interleukin-2 (IL-2) polypeptide, an sTβRII receptor polypeptide, and a second sTβRII receptor polypeptide, which is used to produce effector memory (T EM) and terminal differentiation effector memory (T EMRA This demonstrates that it more effectively induces cytokine production and proliferation in both types of CD8 T cells.

[0165] Purified human T cells derived from PBMCs stimulated with a CEF peptide pool were labeled with CellTrace® Violet, and T cell proliferation (CellTrace® mean fluorescence (MFI) decreases as cell proliferation increases) was tracked. CD8 T cells stimulated with equimolar concentrations of bFISTv3, FIST (monovalent FIST), or TbRII isoform 2 were also tracked. + T cells, effector memory T cells (CCR7-CD45RA-CD62L-CD8 + T cells), and terminal differentiation effector memory CD8 T cells (CCR7-CD45RA + CD62L-CD8 + T cell proliferation. Human PBMCs from healthy donors were stimulated overnight with a CEF peptide pool to induce the generation of antigen-specific T cells. T cells (CD3 + ) were purified from PBMCs derived from healthy donors by immunomagnetically negative selection using a human T cell isolation kit. The purified T cells were labeled with CellTrace® Violet and then stimulated with 5 nmoL of bFISTv3, FIST (monovalent FIST), and IL-2 for 48 hours. The stimulated T cells were labeled with a fluorescent conjugate antibody specific to the memory T cell population. After the incubation period, the cell supernatant was collected, and the concentration of IFNγ was quantified by ELISA. Cells were collected, and the cell proliferation of each T cell subpopulation was determined by flow cytometry. bFISTv3 induced IFNγ production from T cells, and effector memory (T EM ) and terminal differentiation effector memory (T EMRA It induces significantly higher proliferation of both CD8 T cells (e.g., compared to FIST). Figure 6A shows effector memory T cells (T EM ) and terminally differentiated effector memory cells (T EMRA Figure 6B shows bFISTv3-induced proliferation of effector memory T cells (T). EM) and terminally differentiated effector memory cells (T EMRA Figure 6C shows FIST-induced proliferation of effector memory T cells (T). EM ) and terminally differentiated effector memory cells (T EMRA Figure 6D shows IL-2-induced proliferation of stimulated T cells derived from two donors. EM Figure 6E shows the average CellTrace(trademark) MFI value (a decrease in MFI indicates increased proliferation). Stimulated T cells derived from two donors. EMRA The mean CellTrace™ MFI values ​​are shown (a decrease in MFI indicates increased proliferation). Figure 6F shows the 48-hour quantitative analysis of IFNγ production by T cells stimulated with bFISTv3, FIST (monovalent FIST), or IL-2. Data are shown as mean + SD, *p<0.05. Effector memory T lymphocytes are antigen-primed lymphocytes that can confer rapid immunoprotection against a second challenge by tumor-associated antigens. The memory T cell subpopulation CCR7- can migrate to inflammatory tissues (e.g., tumor sites) and exert immediate effector functions such as cytotoxicity against cancer cells. CCR7-memory CD8 + T cells are characterized by the production of IFNγ and perforin-containing granules, which are essential for antitumor cell lytic activity. In particular, perforin expression is associated with terminal differentiated effector memory T cells (CD45RA). + This is particularly noticeable in CCR7-CD62L-.

[0166] Example 6-bFIST inhibits TGFb1-mediated suppression of primary NK cell proliferation and increases IFNγ production. Exemplifying the fusion polypeptide compositions and methods disclosed herein, the following examples demonstrate that a fusion polypeptide comprising an interleukin-2 (IL-2) polypeptide, an sTβRII receptor polypeptide, and a second sTβRII receptor polypeptide more effectively inhibits and / or reduces TGFb1-mediated primary NK cell proliferation and IFNγ production.

[0167] Human NK cells were purified from healthy donor-derived PBMCs by immunomagnetically negative selection using a human NK cell isolation kit. Purified NK cells were labeled with equimolar concentrations (25 nM, 12.5 nM, and 6.25 nM) of bFISTv3, FIST (monovalent FIST), and IL-2 for 5 days. After incubation, the cell culture supernatant was collected, and the IFNγ concentration was quantified by ELISA. The number of viable cells was quantified using a fluorescence-based assay with a cell count / emission curve, and the percentage of cell proliferation was calculated as the maximum stimulation of NK cell proliferation for these experimental conditions, relative to the positive control (IL-2 + TbRII).

[0168] Purified human NK cells derived from peripheral mononuclear cells (PBMCs) were stimulated for 5 days with or without active TGFb1 (1 ng / mL) using equimolar concentrations of bFISTv3, FIST (monovalent FIST), or control (IL-2 + TbRII). The culture supernatant was collected, and the number of viable cells in culture was determined by quantifying the amount of IFNγ and the amount of ATP present by ELISA. This indicates metabolically active cells. Figure 7A shows the percentage of NK cell proliferation in the absence of active TGFb1. Figure 7B shows the percentage of NK cell proliferation in the presence of active TGFb1. Figure 7C shows the quantification of IFNγ produced by stimulated NK cells cultured in the absence of active TGFb1. Figure 7D shows the quantification of IFNγ produced by stimulated NK cells cultured with active TGFb1 (1 ng / mL). Figure 7E shows the quantification of CXCL10 produced by stimulated NK cells cultured with active TGFb1 (1 ng / mL). The data represent two independent experiments performed in overlapping manner. A statistical comparison between bFISTv3 and FIST is shown (data are shown as mean + SD, *p<0.05, **p<0.005, ***p<0.0005).

[0169] Example 7 - bFIST increases NK cell-mediated cytotoxicity and activation. Exemplifying the fusion polypeptide compositions and methods disclosed herein, the following examples demonstrate that a fusion polypeptide comprising an interleukin-2 (IL-2) polypeptide, an sTβRII receptor polypeptide, and a second sTβRII receptor polypeptide more effectively increases and / or stimulates natural killer (NK) cell-mediated cytotoxicity and activation.

[0170] NK92 cells were subjected to serum starvation for 9 hours, then stimulated for 48 hours with / without active TGFβ1 (1 ng / mL) equimolar concentrations of bFISTv3, FIST (monovalent FIST), and TβRII isoform 2 (control). After incubation, the amount of IFNγ was quantified by ELISA. NK92 cells were collected and co-cultured with target cells (K-562 cells, chronic myeloid leukemia) at different effector:target ratios (1:1, 3:1, 6:1). The NK92 cell-lytic activity against cancer cells was determined by measuring the percentage of specific tumor target death by fixable viability staining after NK:K-562 co-culture (3 hours in a 37°C, 5% CO2 incubator). The percentage of dead cells was calculated based on the following formula:

number

[0171] TGF-β is a potent immunosuppressive cytokine that significantly affects immune cell function, including NK cell activation, IFN-γ production, and the expression of activating receptors NKG2D and NKp30, thereby reducing the cytotoxicity of NK cells and impairing their antitumor function. bFIST induces both robust immune cell activation and effective blockade (with picomolar potency) of active TGFβ1. These two effects promote higher NK cell proliferation, activation, and cytotoxicity against cancer cells. Figure 8A shows the quantification of IFNγ production by NK92 cells as an indicator of NK92 cell activation (as described above, human NK92 cells were serum-starved for 9 hours, then stimulated for 48 hours with / without active TGFβ1 (1 ng / mL) with equimolar concentrations of bFISTv3, FIST (monovalent FIST), or control (IL-2 + TβRII), and the cell supernatant was collected). Figure 8B shows the induced cytotoxicity of bFISTv3 or FIST-stimulated NK92 cells against K-562 cells in a 6:1 ratio (effector:target). A statistical comparison of bFISTv3 and FIST is shown. Data are presented as mean + SD, *p<0.05.

[0172] Example 8 - bFIST stimulates and increases B cell maturation, proliferation, and IFNγ production. Exemplifying the fusion polypeptide compositions and methods disclosed herein, the following examples demonstrate that a fusion polypeptide comprising an interleukin-2 (IL-2) polypeptide, an sTβRII receptor polypeptide, and a second sTβRII receptor polypeptide (1) more effectively stimulates and / or increases B cell maturation, proliferation, and IFNγ production, and (2) reduces and / or inhibits TGFb1 suppression of B cell maturation, proliferation, and IFNγ production.

[0173] Human B cells were purified from healthy donor-derived PBMCs by immunomagnetically negative selection using a human B cell isolation kit. The purified B cells were pre-labeled with CFSE, and cell proliferation (marker intensity decreased as cell proliferation increased) was tracked. They were then stimulated for 5 days with equimolar concentrations (20 nM, 10 nM, and 5 nM) of bFISTv3, FIST, and IL-2. After the incubation period, the B cells were labeled with CD86 and HLA-DR, and double-positive (CD86) was detected. + HLA-DR + The percentage of B cells and proliferation was quantified by flow cytometry. Cell culture supernatant was collected, and the concentration of IFNγ was quantified by ELISA. A statistical comparison between bFISTv3 and FIST is shown. Data are shown as mean + SD, *p<0.05, **p<0.005, ***p<0.0005.

[0174] Bivalent FIST effectively increases and / or stimulates B cell activation, proliferation, and IFNγ production (e.g., compared to FIST or IL-2). Bivalent FIST also induces upregulation of the expression of costimulatory molecules (CD86) and HLA-DR (MHC class II) molecules that exhibit B cell responsiveness to bivalent FIST stimulation. Furthermore, bivalent FIST also activates B cells into effector cells with high expression of antigen-presenting cell (APC) maturation markers and IFNγ production.

[0175] Figures 9A to 9D show data from purified human B cells (CD19-CD3-) derived from peripheral mononuclear cells (PBMCs). Cell proliferation was tracked after labeling with carboxyfluorescein succinimimidyl ester (CFSE), and then the cells were stimulated for 5 days with or without active TGFb1 (1 ng / mL) using equimolar concentrations of bFISTv3, FIST (monovalent FIST), or control (IL-2+TbRII). Stimulated B cells were labeled with HLA-DR and CD86-specific conjugate antibodies, and the percentage of mature B cells was quantified by flow cytometry. Figure 9A shows double-positive CD86 cells cultured with and without active TGFb1. + HLA-DR +Figure 9B shows a comparison of bFISTv3 (20 nmol) and FIST (20 nmol) in B cell induction. CD86 cells stimulated with 20, 10, and 5 nmol of bFISTv3, FIST (monovalent FIST), or control (IL-2 + TbRII) with and without active TGFb1. + HLA-DR + Figure 9C shows the percentage of B cells with and without active TGFb1. + HLA-DR + This shows a comparison of bFISTv3 (20 nmoL) and FIST (20 nmoL) in inducing B cell proliferation. The mean fluorescence intensity (MFI) values ​​of CFSE (cell tracer) are shown. Figure 9D shows the quantification of IFNγ production in the supernatant of stimulated B cells cultured with and without active TGFb1.

[0176] Example 9 -- bFIST inhibits the upregulation of epithelial-to-mesenchymal transition (EMT) markers on lung cancer cells. Exemplifying the fusion polypeptide compositions and methods disclosed herein, the following examples demonstrate that a fusion polypeptide comprising an interleukin-2 (IL-2) polypeptide, an sTβRII receptor polypeptide, and a second sTβRII receptor polypeptide more effectively inhibits and / or reduces the upmodulation of epithelial-to-mesenchymal transition (EMT) markers on lung cancer cells.

[0177] A-549 lung cancer cells were treated for 72 hours with or without active TGFb1 in equimolar concentrations of bFISTv3, FIST (monovalent FIST), or control (IL2 and IL2+TbRII). After the incubation period, cells were collected and labeled with conjugated antibodies specific to E-cadherin, N-cadherin, and PD-L1. The percentage of cells expressing these EMT markers was quantified by flow cytometry. Epithelial-to-mesenchymal transition (EMT) allows cancer cells to suppress epithelial features as they transform into mesenchymal cells. As a result of the EMT process, cancer cells acquire mobility and the ability to migrate from the primary tumor site to distant organs and form metastases. Loss of E-cadherin expression is a critical step in EMT and a feature of the mesenchymal phenotype that characterizes the transformation from non-metastatic to metastatic cancer cells. Treatment with bFIST reduces the upregulation of TGFb1-mediated EMT markers (decreased E-cadherin, increased N-cadherin, and increased PD-L1 expression). Figure 10A shows the percentage of A-549 cells (lung cancer cells) expressing E-cadherin after 72 hours of treatment with / without active TGFb1 with equimolar concentrations of bFISTv3, FIST (monovalent FIST), or control (IL-2 and IL-2+TbRII). Figure 10B shows the percentage of A-549 cells expressing N-cadherin, and Figure 10C shows the percentage of A-549 cells expressing PD-L1. The data represent two independent experiments performed in overlapping manner. As shown, treatment of lung cancer cells with bFIST increases E-cadherin expression, decreases N-cadherin, and decreases PD-L1 expression.

[0178] While preferred embodiments of the Disclosure are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Many variations, alterations, and substitutions will arise herein without departing from the Disclosure. It should be understood that various alternatives to the embodiments of the Disclosure described herein may be used when carrying out the Disclosure. The following claims define the scope of the Disclosure, and methods and structures within the scope of these claims and their equivalents are intended to be covered thereby. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8]

Claims

1. An interleukin-2 (IL-2) polypeptide containing an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3, The first TGFβ superfamily receptor polypeptide, A second TGFβ superfamily receptor polypeptide, It is a fusion polypeptide, The first TGFβ superfamily receptor polypeptide comprises a first soluble TGFβ receptor II (sTβRII) polypeptide having an amino acid sequence having more than 95% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 9, A fusion polypeptide comprising a second TGFβ superfamily receptor polypeptide having an amino acid sequence having more than 95% sequence identity with SEQ ID NO: 8 or SEQ ID NO:

9.

2. The fusion polypeptide according to claim 1, wherein the first TGFβ superfamily receptor polypeptide binds to and occludes soluble TGFβ, and the second TGFβ superfamily receptor polypeptide binds to and occludes soluble TGFβ.

3. The fusion polypeptide according to claim 1, wherein the first TGFβ superfamily receptor polypeptide and / or the second TGFβ superfamily receptor polypeptide is conjugated to a TGFβ1 polypeptide, a TGFβ2 polypeptide, a TGFβ3 polypeptide, or any combination thereof.

4. The fusion polypeptide according to claim 1, wherein the first TGFβ superfamily receptor polypeptide and the second TGFβ superfamily receptor polypeptide bind to a TGFβ1 polypeptide.

5. The fusion polypeptide according to claim 1, wherein the first sTβRII polypeptide, the second sTβRII polypeptide, or both thereof, comprises an N-terminal cleavage, a C-terminal cleavage, or both of the above with respect to SEQ ID NO: 8 or SEQ ID NO: 9, wherein the cleavage is by only 2 to 18 amino acids.

6. The fusion polypeptide according to claim 1, comprising a linker polypeptide or linker molecule that binds the first TGFβ superfamily receptor polypeptide and the second TGFβ superfamily receptor polypeptide.

7. The fusion polypeptide according to claim 1, comprising a linker polypeptide that fuses the IL-2 polypeptide and the first TGFβ superfamily receptor polypeptide.

8. A fusion polypeptide according to claim 1, comprising a pharmacokinetic (PK) modifier, wherein the pharmacokinetic modifier comprises an immunoglobulin constant-state (Fc) region polypeptide or an albumin polypeptide.

9. The fusion polypeptide according to claim 8, wherein the pharmacokinetic modifier comprises an albumin polypeptide.

10. The fusion polypeptide according to claim 1, wherein the fusion polypeptide comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 10 to 22.

11. The fusion polypeptide according to claim 1, wherein the fusion polypeptide activates immune cells that express the IL-2 receptor.

12. The fusion polypeptide according to claim 11, wherein the immune cells are T cells, natural killer cells, NKT cells, B cells, or gamma delta T cells.

13. A pharmaceutical composition comprising a fusion polypeptide according to any one of claims 1 to 12, comprising a pharmaceutically acceptable carrier or excipient.

14. Use of a fusion polypeptide according to any one of claims 1 to 12 in the manufacture of a pharmaceutical product for inhibiting or reducing immunosuppression of immune cells in the tumor microenvironment and / or reducing or inhibiting the activation of immunosuppressive cells in the tumor microenvironment.

15. (i) Inhibition or reduction of tumor growth and / or progression in individuals with cancer; (ii) Neutralization or killing of tumor cells in individuals with cancer; (iii) Treatment or improvement of cancer in subjects with cancer; and / or (iv) Activation of cells expressing the IL receptor Use of a fusion polypeptide according to any one of claims 1 to 12 in the manufacture of a pharmaceutical product.

16. A composition for inhibiting or reducing immunosuppression of immune cells in the tumor microenvironment and / or reducing or inhibiting the activation of immunosuppressive cells in the tumor microenvironment, comprising the fusion polypeptide described in any one of claims 1 to 12.

17. (i) Inhibition or reduction of tumor growth and / or progression in individuals with cancer; (ii) Neutralization or killing of tumor cells in individuals with cancer; (iii) Treatment or improvement of cancer in subjects with cancer; and / or (iv) Activation of cells expressing the IL receptor A composition for which a fusion polypeptide according to any one of claims 1 to 12 is provided.

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