Designed interleukin-2 cytokine composition and method of use

Abstract

This disclosure provides a polypeptide comprising (a) an alpha-helix sequence comprising H1, H2, H3, and H4, wherein a first loop (LI) connects H1 and H4 from the amino terminus to the carboxy terminus, a second loop (L2) connects H4 and H2, and a third loop (L3) connects H2 and H3, and the polypeptide binds to the IL-2 receptor beta-gamma (IL-2Rβγ); and (b) a targeting moiety that binds to the sequence of the differentiation antigen group 8 (CD8) glycoprotein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 513,540, filed Jul. 13, 2023, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to the fields of immunology, gene therapy, protein design, cell signaling, biologics, and cell therapy.

Background Art

[0003] IL - 2 is promising as an anti - cancer immunotherapy, but its efficacy and safety are reduced by dose - limiting toxicities due to preferential stimulation of Treg cells and IL - 2Rα binding. This disclosure provides a non - naturally occurring designed cytokine as a solution to this unmet need in the art.

Summary of the Invention

[0004] This disclosure provides a non - naturally occurring designed cytokine that has enhanced function and improved stability compared to wild - type IL - 2. The non - naturally occurring designed cytokine of this disclosure, also referred to herein as an IL - 2 / 15 cytokine, is named for its dual functionality of signaling through both the IL - 2 receptor and the IL - 15 receptor, reduces preferential Treg stimulation, enhances T - cell subtype targeting, stabilizes protein folding, and reduces immunogenicity. In some embodiments, the designed cytokine eliminates preferential Treg stimulation. In some embodiments, the designed cytokine eliminates immunogenicity. In some embodiments, the designed cytokine provides enhanced function and improved stability with reduced post - translational modifications compared to post - translational modifications made to wild - type IL - 2. In some embodiments, the designed cytokine provides enhanced function and improved stability without post - translational modifications.

[0005] This disclosure provides a designed cytokine comprising alpha-helix H1, H2, H3, and H4, wherein a first loop (L1) connects H1 and H4 from the amino terminus to the carboxy terminus, a second loop (L2) connects H4 and H2, and a third loop (L3) connects H2 and H3, and the polypeptide binds to the IL-2 receptor βγ (IL-2Rβγ), also referred to as IL-2 / 15Rβγ. In some embodiments, the designed cytokine does not bind to the IL-2 receptor alpha (IL-2Rα).

[0006] In some embodiments of the designed cytokines of this disclosure, the designed cytokine comprises one or more sequences from sequence numbers 1 to 350.

[0007] In some embodiments of the cytokines designed in this disclosure, the polypeptide comprises one or more of the following: (a) sequences of sequence numbers 1 to 350, and (b) sequences having at least 70% identity with sequence (a).

[0008] In some embodiments of the cytokines designed in this disclosure, the polypeptide comprises sequences SEQ ID NOs: 1-38 or 150-350.

[0009] In some embodiments of the cytokines designed in this disclosure, the L3 loop includes the sequence of QSKNFHLR (SEQ ID NO: 132).

[0010] In some embodiments of the cytokines designed in this disclosure, the H1 helix comprises sequences of sequence numbers 39-51.

[0011] In some embodiments of the cytokines designed in this disclosure, the H2 helix comprises sequences SEQ ID NOs. 52–77.

[0012] In some embodiments of the cytokines designed in this disclosure, the H3 helix comprises sequences SEQ ID NOs. 78-97.

[0013] In some embodiments of the cytokines designed in this disclosure, the H4 helix comprises sequences 98-101.

[0014] In some embodiments of the cytokines designed in this disclosure, the L1 loop includes sequences of sequence numbers 102-111.

[0015] In some embodiments of the cytokines designed in this disclosure, the L2 loop includes sequences SEQ ID NOs. 112–131.

[0016] In some embodiments of the cytokines designed in this disclosure, the polypeptide comprises sequences SEQ ID NOs: 1-38 or 150-350.

[0017] In some embodiments of the cytokines designed in this disclosure, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK.

[0018] In some embodiments of the cytokines designed in this disclosure, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0019] In some embodiments of the cytokines designed in this disclosure, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0020] In some embodiments of the cytokines designed in this disclosure, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTLTAGGSLSGDLKHLQNLSEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0021] In some embodiments of the cytokines designed in this disclosure, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSVDPEELAKELQKLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0022] In some embodiments of the cytokines designed in this disclosure, the polypeptide is operably linked to a targeting moiety. In some embodiments, the polypeptide comprises a targeting moiety. In some embodiments, the fusion protein comprises a polypeptide and a targeting moiety. In some embodiments, the targeting moiety binds to a component of the tumor microenvironment (TME). In some embodiments, the targeting moiety binds to one or more of the following: T cell surface glycoprotein CD8 (also known as differentiation antigen group 8), programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1; also known as differentiation antigen group 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immune receptor (TIGIT) having Ig and ITIM domains, cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), and T cell immunoglobulin mucin receptor 3 (TIM3). In some embodiments, the targeting moiety binds to CD8. In some embodiments, the targeting moiety binds to PD-1. In some embodiments, the targeting moiety binds to PD-L1. In some embodiments, the targeting moiety includes an antibody, an antibody mimetic, or a functional fragment thereof. In some embodiments, the targeting moiety includes one or more of a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more heavy chain (VH) and light chain (VL) domains of an immunoglobulin (Ig) polypeptide or a gene encoding them, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and one or more nanobodies. In some embodiments, the targeting moiety includes scFv. In some embodiments, the targeting moiety includes VHH.

[0023] In some embodiments of the cytokines designed in this disclosure, the polypeptide is operably linked to a tether. In some embodiments, the polypeptide comprises a tether. In some embodiments, the fusion protein comprises a polypeptide and a tether. In some embodiments, the tether comprises one or more of a nucleic acid sequence, an amino acid sequence, or a small molecule. In some embodiments, the hinge region comprises a sequence isolated from or derived from a transmembrane sequence. In some embodiments, the tether comprises the sequence PLFIPVAVMVTAFSGLAFIIWLARRLKKGKK.

[0024] In some embodiments of the designed cytokines of this disclosure, the polypeptide is operably linked to a second cytokine or a second designed cytokine. In some embodiments, the polypeptide comprises a second cytokine or a second designed cytokine. In some embodiments, the fusion protein comprises a polypeptide and a second cytokine or a second designed cytokine.

[0025] In some embodiments of the cytokines designed in this disclosure, the second cytokine comprises a sequence isolated from or derived from one or more of the following: IL-2 polypeptide, IL-12 polypeptide, IL-15 polypeptide, IL-18 polypeptide, IL-21 polypeptide, IL-23 polypeptide, interferon alpha polypeptide, interferon beta polypeptide, interferon gamma polypeptide, and interferon omega polypeptide. In some embodiments, the polypeptide comprises a first targeting moiety, and the second cytokine comprises a second targeting moiety. In some embodiments, the first and second targeting moieties are identical. In some embodiments, the first and second targeting moieties are not identical.

[0026] In some embodiments of the engineered cytokines of the present disclosure, the second engineered cytokine comprises one or more sequences of SEQ ID NOs: 1-38 or 150-350. In some embodiments, the polypeptide comprises a first targeting moiety and the second engineered cytokine comprises a second targeting moiety. In some embodiments, the first targeting moiety and the second targeting moiety are the same. In some embodiments, the first targeting moiety and the second targeting moiety are not the same.

[0027] In some embodiments of the engineered cytokines of the present disclosure, the polypeptide comprises a first tether and the second cytokine comprises a second tether. In some embodiments, the first tether and the second tether are the same. In some embodiments, the first tether and the second tether are not the same.

[0028] In some embodiments of the engineered cytokines of the present disclosure, the polypeptide comprises a first tether and the second engineered cytokine comprises a second tether. In some embodiments, the first tether and the second tether are the same. In some embodiments, the first tether and the second tether are not the same.

[0029] The present disclosure provides a nucleic acid encoding an engineered cytokine of the present disclosure or a fusion protein comprising an engineered cytokine of the present disclosure. In some embodiments, the nucleic acid further comprises regulatory elements capable of driving the expression of the engineered cytokine. In some embodiments, the regulatory element comprises a promoter. In some embodiments, the promoter comprises a minimal promoter. In some embodiments, the minimal promoter comprises a sequence isolated from or derived from one or more of minimal promoter-1 (“minP”), YB-TATA, and human beta globin. In some embodiments, minP comprises the sequence AGAGGGTATATAAAAGCTCGACTTCCAG. In some embodiments, the minimal promoter is It contains the sequence of TAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC. In some embodiments, the minimal promoter contains the sequence of CTAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC. In some embodiments, the promoter is inducible. In some embodiments, the regulatory element contains a response element. In some embodiments, the regulatory element contains a non-coding sequence or untranslated sequence isolated from or derived from one or more of NFAT, NFkB, REL, RELA, IRF2, GATA3, and ATF3. In some embodiments, the regulatory element contains a non-coding sequence or untranslated sequence isolated from or derived from the GATA3 gene. In some embodiments, the regulatory element contains a non-coding sequence or untranslated sequence isolated from or derived from RELA. In some embodiments, the response element contains a repetitive sequence.

[0030] The present disclosure provides a vector containing the nucleic acid of the present disclosure. In some embodiments, the vector contains an expression vector. In some embodiments, the vector contains a delivery vector. In some embodiments, the vector further contains a sequence encoding an exogenous receptor. In some embodiments, the exogenous receptor contains an antigen-binding portion. In some embodiments, the exogenous receptor contains a T cell receptor (TCR). In some embodiments, the exogenous receptor contains a chimeric antigen receptor (CAR). In some embodiments, the antigen is expressed or secreted within one or more of tumor cells, cancer cells, components of the TME, and the TME.

[0031] In some embodiments of the present disclosure, the vector is a non-viral vector. In some embodiments, the non-viral vector contains one or more of plasmids, nucleic acids, polymers, micelles, polymerosomes, exosomes, lysosomes, nanoparticles, and any combination thereof.

[0032] In some embodiments of this disclosure, the vector is a viral vector. In some embodiments, the viral vector includes a sequence isolated from or derived from a virus, a lentivirus, or a lentiviral vector sequence.

[0033] This disclosure provides cells containing cytokines designed in this disclosure. This disclosure provides cells containing nucleic acids in this disclosure. This disclosure provides cells containing vectors in this disclosure. In some embodiments of this disclosure, the cells are mammalian cells. In some embodiments of this disclosure, the cells are human cells. In some embodiments of this disclosure, the cells are primary cells. In some embodiments of this disclosure, the cells are cultured cells. In some embodiments, the cultured cells are immortalized cells. In some embodiments, the cells exist ex vivo or in vitro. In some embodiments, the cells exist in vivo. In some embodiments, the cells are immune cells. In some embodiments, the cells are stem cells or progenitor cells capable of producing immune cells. In some embodiments, the stem cells are hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), or dedifferentiated immune cells. In some embodiments, the immune cells are T lymphocytes (T cells), B lymphocytes (B cells), macrophages, or natural killer (NK) cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are alpha-beta T cells. In some embodiments, the T cells are gamma-delta T cells. In some embodiments, the immune cells are NK cells.

[0034] This disclosure provides compositions comprising cytokines designed in this disclosure. This disclosure provides compositions comprising nucleic acids in this disclosure. This disclosure provides compositions comprising vectors in this disclosure. This disclosure provides compositions comprising cells in this disclosure.

[0035] This disclosure provides a pharmaceutical composition comprising (1) a cytokine designed by this disclosure, a nucleic acid by this disclosure, a vector by this disclosure, and a cell by this disclosure, and (2) a pharmaceutically acceptable carrier.

[0036] This disclosure provides the use of the engineered cytokines, nucleic acids, vectors, cells, or pharmaceutical compositions of the Disclosure in the manufacture of agents for the treatment of a disease or condition. In some embodiments, the disease or disorder includes cancer or its subtypes. In some embodiments, cancer or its subtypes includes liquid cancer. In some embodiments, cancer or its subtypes includes hematological cancer. In some embodiments, cancer or its subtypes includes solid cancer.

[0037] This disclosure provides cytokines designed by this disclosure, nucleic acids of this disclosure, vectors of this disclosure, cells of this disclosure, or pharmaceutical compositions of this disclosure for use in the treatment of a disease or condition. In some embodiments, the disease or disorder includes cancer or a subtype thereof. In some embodiments, cancer or a subtype thereof includes liquid cancer. In some embodiments, cancer or a subtype thereof includes hematological cancer. In some embodiments, cancer or a subtype thereof includes solid cancer.

[0038] This disclosure provides a method for treating a disease or disorder, comprising administering to a subject an effective amount of a cytokine designed by this disclosure, a nucleic acid of this disclosure, a vector of this disclosure, a cell of this disclosure, or a pharmaceutical composition of this disclosure, the severity of the signs or symptoms of the disease or disorder being reduced, thereby treating the disease or disorder. In some embodiments, the disease or disorder includes cancer or a subtype thereof. In some embodiments, cancer or a subtype thereof includes liquid cancer. In some embodiments, cancer or a subtype thereof includes hematological cancer. In some embodiments, cancer or a subtype thereof includes solid cancer.

[0039] This disclosure provides a method for preventing a disease or disorder, comprising administering to a subject an effective amount of a cytokine designed by this disclosure, a nucleic acid of this disclosure, a vector of this disclosure, a cell of this disclosure, or a pharmaceutical composition of this disclosure, such that the onset or recurrence of signs or symptoms of the disease or disorder is delayed or suppressed, thereby preventing the disease or disorder. In some embodiments, the disease or disorder includes cancer or a subtype thereof. In some embodiments, cancer or a subtype thereof includes liquid cancer. In some embodiments, cancer or a subtype thereof includes hematological cancer. In some embodiments, cancer or a subtype thereof includes solid cancer.

[0040] This disclosure provides a polypeptide comprising (a) an alpha-helix sequence comprising H1, H2, H3, and H4, wherein a first loop (L1) connects H1 and H4 from the amino terminus to the carboxy terminus, a second loop (L2) connects H4 and H2, and a third loop (L3) connects H2 and H3, and the polypeptide binds to the IL-2 receptor beta-gamma (IL-2Rβγ); and (b) a targeting moiety that binds to differentiation antigen group 8 (CD8). In some embodiments, the polypeptide does not bind to the IL-2 receptor alpha (IL-2Rα).

[0041] In some embodiments, the sequence of (a) includes one or more of the sequences of (i) sequence numbers 1 to 350 and (ii) sequences that have at least 70% identity with the sequence of (i).

[0042] In some embodiments, the sequence of (a) includes the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK.

[0043] In some embodiments, the sequence of (a) includes the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0044] In some embodiments, the sequence of (a) includes the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0045] In some embodiments, the sequence of (a) includes the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTLTAGGSLSGDLKHLQNLSEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0046] In some embodiments, the sequence of (a) includes the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSVDPEELAKELQKLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0047] In some embodiments, the sequence of (a) includes APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK.

[0048] In some embodiments, the sequence of (a) is operably linked to the targeting portion of (b). In some embodiments, the polypeptide further includes a linker that operably links the sequence of (a) to the targeting portion of (b). In some embodiments, the linker includes deoxyribonucleic acid, ribonucleic acid, amino acids, or any combination thereof. In some embodiments, the linker includes one or more of glycine (G) and serine (S). In some embodiments, the linker includes the GGGGS sequence or any number of repeats thereof. In some embodiments, the targeting portion includes an antibody, antibody mimetic, protein scaffold, or a functional fragment thereof. In some embodiments, the targeting portion includes a humanized form of an antibody, antibody mimetic, protein scaffold, or a functional fragment thereof. In some embodiments, the targeting portion includes one or more of the following: a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more heavy chain (VH) and light chain (VL) domains of an immunoglobulin (Ig) polypeptide or encoding genes, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and one or more nanobodies. In some embodiments, the targeting portion includes one or more humanized forms of the following: a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more heavy chain (VH) and light chain (VL) domains of an immunoglobulin (Ig) polypeptide or encoding genes, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and one or more nanobodies. In some embodiments, the targeting portion includes scFv. In some embodiments, the targeting portion includes VHH. In some embodiments, the targeting portion includes humanized VHH.

[0049] In some embodiments, the targeting portion includes one of the sequences from sequence numbers 9791 to 9800, or a sequence having at least 90% identity thereto. In some embodiments, the targeting portion includes the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYYDMDYWGQGTQVTVSS, or a sequence having at least 90% identity thereto. In some embodiments, the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYYDMDYWGQGTQVTVSS. In some embodiments, the polypeptide further comprises a signal sequence. In some embodiments, the signal sequence comprises MVLQTQVFISLLLWISGAYG. In some embodiments, the polypeptide is operably linked to a second polypeptide. In some embodiments, the polypeptide further comprises a second polypeptide. In some embodiments, the second polypeptide comprises the polypeptide described herein or any component thereof.

[0050] In some embodiments, the second polypeptide comprises a cytokine or a sequence isolated from or derived from any nucleic acid sequence encoding a cytokine. In some implementations, cytokines include interleukin-1 polypeptide (IL-1), interleukin-2 polypeptide (IL-2), interleukin-3 polypeptide (IL-3), interleukin-4 polypeptide (IL-4), interleukin-5 polypeptide (IL-5), interleukin-6 polypeptide (IL-6), interleukin-7 polypeptide (IL-7), interleukin-8 polypeptide (IL-8), interleukin-9 polypeptide (IL-9), interleukin-10 polypeptide (IL-10), interleukin-11 polypeptide (IL-11), interleukin-12 polypeptide (IL-12), interleukin-13 polypeptide (IL-13), interleukin-14 polypeptide (IL-14), interleukin-15 polypeptide (IL-15), interleukin-16 polypeptide (IL-16), interleukin-17 polypeptide (IL-17), interleukin-18 polypeptide (IL-18), and interleukin-17 polypeptide. It contains one or more of the following: interleukin 19 polypeptide (IL-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), and interleukin 36 polypeptide (IL-36).

[0051] In some embodiments, the second polypeptide comprises a sequence isolated from or derived from an interferon or any nucleic acid sequence encoding an interferon. In some embodiments, the interferon comprises one or more of interferon type I, interferon type II, and interferon type III. In some embodiments, the interferon comprises one or more of interferon alpha (IFN-α), interferon beta (IFN-β), and interferon epsilon (IFN-ε), interferon kappa (IFN-κ), interferon omega (IFN-ω), and interferon gamma (IFN-γ).

[0052] In some embodiments, the targeting portion of (b) is the first targeting portion, and the second polypeptide includes the second targeting portion. In some embodiments, the first targeting portion and the second targeting portion include the same sequence. In some embodiments, the first targeting portion and the second targeting portion do not include the same sequence.

[0053] This disclosure provides nucleic acids encoding polypeptides or any functional components thereof as disclosed herein. In some embodiments, when introduced into mammalian cells, the regulatory element drives the expression of the nucleic acid. In some embodiments, the regulatory element includes a promoter. In some embodiments, the regulatory element includes a minimal promoter. In some embodiments, the promoter includes the sequence AGAGGGTATATAATGGAAGCTCGAATTCCAG. In some embodiments, the regulatory element includes an inducible promoter. In some embodiments, the regulatory element includes a response element. In some embodiments, the regulatory element includes one or more repeats of the response element. In some embodiments, the regulatory element includes the sequence GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTCCAG. In some embodiments, the regulatory element further includes a sequence encoding one or more of the following: a 5-prime untranslated region, a 3-prime untranslated region, an intron, an exon, and an enhancer. In some embodiments, the regulatory element further includes a sequence encoding a 5-prime untranslated region (5'UTR). In some embodiments, the 5'UTR includes the sequence ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC. In some embodiments, the regulatory element includes a sequence isolated from or derived from a mammalian sequence. In some embodiments, the regulatory element includes a sequence isolated from or derived from a human sequence. In some embodiments, the regulatory element includes a recombinant sequence.

[0054] This disclosure provides vectors comprising nucleic acids as described herein. In some embodiments, the vector comprises an expression vector. In some embodiments, the vector comprises a delivery vector. In some embodiments, the delivery vector is a nonviral vector. In some embodiments, the delivery vector is a viral vector. In some embodiments, the expression vector is a lentiviral vector. In some embodiments, the vector further comprises a sequence encoding an exogenous receptor. In some embodiments, the exogenous receptor comprises an antigen-binding moiety. In some embodiments, the exogenous receptor comprises a T cell receptor (TCR) or a functional component thereof. In some embodiments, the exogenous receptor comprises a chimeric antigen receptor (CAR) or a functional component thereof. In some embodiments, the antigen-binding moiety binds to tumor cells, cancer cells, components of the tumor microenvironment (TME), or antigens expressed in or secreted within one or more of the TME. In some embodiments, the antigen-binding moiety binds to antigens expressed in or secreted within one or more of the cancer cells. In some embodiments, a liquid tumor comprises cancer cells. In some embodiments, a solid tumor comprises cancer cells. In some embodiments, the antigen-binding moiety binds to a mesothelin (MSLN) polypeptide or its sequence.

[0055] This disclosure provides cells comprising polypeptides as described herein. In some embodiments, the cells comprise nucleic acids as described herein. In some embodiments, the cells comprise vectors as described herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the cells are primary cells. In some embodiments, the cells are cultured cells. In some embodiments, the cells are immortalized cells. In some embodiments, the cells exist ex vivo or in vitro. In some embodiments, the cells exist in vivo. In some embodiments, the cells are immune cells. In some embodiments, the cells are stem cells or progenitor cells capable of producing immune cells. In some embodiments, the stem cells are hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), or dedifferentiated immune cells. In some embodiments, the immune cells are T lymphocytes (T cells), B lymphocytes (B cells), macrophages, or natural killer (NK) cells. In some embodiments, the immune cells are T cells. In some embodiments, the T cells are alpha-beta T cells. In some embodiments, the T cells are gamma delta T cells. In some embodiments, the immune cells are NK cells.

[0056] This disclosure provides compositions comprising polypeptides as described herein. This disclosure provides compositions comprising nucleic acids as described herein. This disclosure provides compositions comprising vectors as described herein. This disclosure provides compositions comprising cells as described herein.

[0057] This disclosure provides pharmaceutical compositions comprising (1) one or more polypeptides, nucleic acids, vectors, cells, and (2) pharmaceutically acceptable carriers.

[0058] This disclosure provides the use of the polypeptides, nucleic acids, vectors, cells, or pharmaceutical compositions described herein in the manufacture of agents for the treatment of a disease or condition. This disclosure provides the polypeptides, nucleic acids, vectors, cells, or pharmaceutical compositions described herein for use in the treatment of a disease or condition. In some embodiments, the disease or disorder includes cancer or a subtype thereof. In some embodiments, cancer or a subtype thereof includes liquid cancer. In some embodiments, cancer or a subtype thereof includes hematological cancer. In some embodiments, cancer or a subtype thereof includes solid cancer.

[0059] This disclosure provides a method for treating a disease or disorder, comprising administering to a subject an effective amount of a polypeptide, nucleic acid, vector, cell, or pharmaceutical composition described herein, wherein the severity of the signs or symptoms of the disease or disorder is reduced, thereby treating the disease or disorder. This disclosure also provides a method for preventing a disease or disorder, comprising administering to a subject an effective amount of a polypeptide, nucleic acid, vector, cell, or pharmaceutical composition described herein, wherein the onset or recurrence of the signs or symptoms of the disease or disorder is delayed or suppressed, thereby preventing the disease or disorder. In some embodiments, the disease or disorder includes cancer or a subtype thereof. In some embodiments, cancer or a subtype thereof includes liquid cancer. In some embodiments, cancer or a subtype thereof includes hematological cancer. In some embodiments, cancer or a subtype thereof includes solid cancer. [Brief explanation of the drawing]

[0060] [Figure 1A]This is a schematic diagram representing a naturally occurring form of IL-2 (left) and an exemplary naturally occurring, non-naturally occurring, engineered cytokine of the present disclosure, in which one or more loops are redesigned to alter the binding of the engineered cytokine to the naturally occurring or endogenous IL-2 receptor beta (IL-2Rβ) or IL-2 receptor gamma (IL-2Rγ, also known as the common gamma chain receptor), but not to alter IL-2 receptor alpha (IL-2Rα). As shown in this diagram, in some embodiments, the exemplary naturally occurring, non-naturally occurring, engineered cytokines of the present disclosure contain sequences isolated from or derived from an IL-2 cytokine helix having connectivity to either an engineered cytokine following the 1-4-2-3 plan represented herein or a structure of four helices connected by loops that lack the ability to bind to and / or activate IL-2 receptor alpha due to the absence of an interface for binding to IL-2 receptor alpha. [Figure 1B] The left half of the diagram shows a modern diagram illustrating the interaction with each subunit of the IL-2 receptor that enables IL-15 function, while the right half shows a correspondence between the abstract schematic and the ribbon schematic, following the diagram in Figure 1A. As shown in this diagram, the designed cytokine not only lacks an interface for binding to and / or activating the alpha subunit of the IL-2 receptor, but the "helix 4" of the designed cytokine also binds to the gamma subunit of the IL-2 receptor, which is shared with the IL-15 receptor, and the "helix 1" and "helix 3" of the designed cytokine also bind to the IL-2 receptor beta, which is shared with the IL-15 receptor. As a result, the designed cytokine of this disclosure functions via the IL-2 receptor and / or the IL-15 receptor. [Figure 2] This is a schematic diagram representing T cell subtype-specific IL-2WT signaling. As shown in Figures 1A and 1B, the cytokines designed in this disclosure signal via one or more of the beta and gamma subunits of the IL-2 receptor. [Figure 3A]This schematic diagram illustrates how the cytokines designed in this disclosure can target specific cell subtypes as beta / gamma binding agents. Furthermore, the cytokines designed in this disclosure may be linked to or contain a targeting portion. The targeting portion may bind to any target, including, but not limited to, antigens present or expressed within cells, or other components of the tumor microenvironment (TME), such as CD8 or PDL1. [Figure 3B]A schematic diagram illustrating how the engineered cytokines of this disclosure can target specific cell subtypes as a result of (1) being engineered as beta / gamma binding agents, (2) being targeted to cell subtypes, and (3) being anchored to T cells or their subtypes (e.g., alpha-beta, gamma-delta, CD8+, CD4+, natural killer T cells (NKT cells)), or any combination thereof. The engineered cytokines to be targeted can be tethered to or include a targeting portion that can bind to any target, including, but not limited to, antigens present or expressed intracellularly, or other components of the tumor microenvironment (TME), such as CD8 or PDL1. The anchored engineered cytokines can be tethered to or include a tether, which may include nucleic acids, amino acids, small molecules, or any combination thereof, and the tether can tether the engineered cytokine to a cell expressing the engineered cytokine (e.g., a T cell, NK cell, or other immune cell) or any component of that cell. In some embodiments, the use of a targeting portion may be advantageous for localizing a designed cytokine or the cells expressing it to a solid tumor or TME for localized signaling from the designed cytokine (as opposed to systemic signaling from the designed cytokine). In some embodiments, the use of a tether may be advantageous for minimizing bystander activity of cells proximal to either the target cell or the cells expressing the designed cytokine. In some embodiments, the use of a tether may be advantageous for treating liquid tumors or hematological malignancies. In some embodiments, the use of a tether may be advantageous for use with T cells, where the bystander cells may be natural killer (NK) cells. [Figure 4]This schematic diagram illustrates that the cytokine designed in this disclosure lacks an interface for binding to and / or activating the alpha subunit of the IL-2 receptor. Furthermore, the removal of this interface retains the natural interface for binding the IL-2 receptor beta and IL-2 receptor gamma subunits, which are shared between the IL-2 receptor and the IL-15 receptor. The design of short, structured loops between helices increases protein stability compared to the loop length, loop structure, and / or protein stability of wild-type (WT) IL-2. [Figure 5A] A pair of graphs demonstrating that a common structural configuration of the 38 (38) designed cytokines tested individually alongside WT IL-2 and a negative control removes the IL-2 receptor alpha binding in all of the designed cytokines (above), while preserving signaling via the beta and gamma subunits of the IL-2 receptor (the gamma subunit is shared with the IL-15 receptor). [Figure 5B] This is a series of graphs demonstrating that the designed cytokines of this disclosure bind to IL-2 / 15Rβγ with very low nanomolar affinity. For each plot shown, the affinity (measured in nanomolar (nm) concentration) of either WT IL-2 (top) or the designed cytokine (bottom) in contact with either IL-1Rα (left) or IL-2 / 15Rβγ (right) is shown as a function of time (measured in seconds (s)). These plots were prepared by testing compositions of either WT IL-2 or the designed cytokine at concentrations from 0.9 nM to 3000 nM. [Figure 6]This is a series of graphs demonstrating that the activity of the designed cytokines of this disclosure can be tuned over a wide range. As used throughout this disclosure, the designed cytokines can be “tuned” to perform optimally within any of the following: a desired cell type, a desired TME, a desired target cell, and / or a desired cytokine receptor (e.g., the ratio of activity between IL-2 and IL-15). The initial design of the designed cytokine may include the sequence of this disclosure or a sequence having at least 70% identity thereto. The optimized design of the designed cytokine may include the sequence of this disclosure or a sequence having at least 70% identity thereto. The optimized design of the designed cytokine may include a sequence derived from the sequence of this disclosure, generated according to the teachings provided by this disclosure. Plot above: Cell proliferation was assessed by measuring the phosphorylation signaling and percentage of activator 5 (STAT5) in CD8+ T cells after antigen stimulation as a function of the concentration of either WT IL-2 or the provided designed cytokine (concentration measured as nanograms per milliliter (ng / ml)). The designed cytokines of this disclosure may be “optimized” to have a desired activity profile, as shown in the plot in the upper right. With respect to IL-2 / 15Rβγ binding activity in contrast to IL-2Rα, the two lower plots show that the designed cytokines each bind to IL-2 / 15Rβγ with different activities, while the designed cytokines do not bind to IL-2Rα. In this figure, design 1 = designed cytokine No. 169, design 2 = designed cytokine No. 175, and design 3 = designed cytokine No. 153. [Figure 7]This series of graphs demonstrates that the engineered cytokines do not have a preference for regulatory T cells (Tregs) compared to WT IL-2, but the engineered cytokines retain NK and CD8+ T cell activity. CD8+ T cells, NK cells, or Tregs were exposed to either WT IL-2 (upper plot) or the engineered cytokines (lower plot), and cell proliferation was assessed by measuring the phosphorylation signaling and activator 5 (STAT5) percentage of CD8+ T cells after antigen stimulation as a function of the concentration of either WT IL-2 or the engineered cytokines (shown as nanomolar (nM) concentrations). Two cell donors are shown (left and right plots). WT IL-2 preferentially activates Tregs, while the engineered cytokines demonstrate reduced Treg potency, eliminating the WT IL-2 Treg preference. [Figure 8] This graph demonstrates in vitro cell proliferation in the presence of 1 nanomolar (nM) of WT IL-2 or a designed cytokine. The measurements on the Y axis are relative scales in which the measured cell proliferation is normalized to WT activity (held at a value of 1.0), as shown in Figure 7. Within a population of mixed peripheral blood mononuclear cells (PBMCs), or within either isolated populations of NK cells or activated CD8+ T cells, respectively, the designed cytokine eliminates the Treg preference shown by WT IL-2 while retaining the ability to proliferate NK and CD8+ T cells. [Figure 9] This series of graphs demonstrates that the engineered cytokine does not have preference for regulatory T cells (Tregs) compared to WT IL-2, but the engineered cytokine retains NK and CD8+ T cell activity. For each plot, either WT IL-2 (top) or the engineered cytokine (bottom) was provided in vitro at nanomolar (nM) concentrations to mixed PBMCs (left), isolated NK cells (middle), and CD3 / 28 activated T cells, and the proliferation ratio was measured. Notably, the bottom left plot shows a significant rightward shift in activity toward Treg cells, as the engineered cytokine has reduced potency toward Treg cells compared to WT IL-2. [Figure 10] These schematic diagrams and graphs demonstrate the enhanced activity of T cells expressing both the CARs and the designed cytokines of this disclosure. The schematic diagrams represent the experimental design used to produce the data on which the graphs are displayed. Cells transfected with sham antigens, cells transfected with only the chimeric antigen receptor (CAR), cells transfected with CAR in combination with a WT IL-2 construct, or cells transfected with constructs providing both the CARs and the designed cytokines of this disclosure were repeatedly stimulated with plate-bound antigens, and proliferation multipliers were assessed after each round. [Figure 11] Schematic diagrams and graphs demonstrate that T cell activation induced the secretion of a designed cytokine using an inducible promoter capable of conditionally driving the expression of the designed cytokine in response to antigen receptor signaling (e.g., CAR or TCR) or cellular conditions (stimulated T cell). The regulation of the expression and / or secretion of the designed cytokines in this disclosure reduces systemic exposure in vivo and provides a safety profile favorable to the designed cytokines. [Figure 12] These schematic diagrams and graphs demonstrate that incorporating a short, structured loop within the designed cytokine increases its protein stability compared to WT IL-2. The schematic diagram on the left shows the structural difference of the loop in WT IL-2 compared to the designed cytokine of this disclosure. The graph shows protein unfolding / instability (measured by autofluorescence) as a function of increasing molar concentration of guanidine hydrochloride ([GdnHCl(M)]), a protein denaturing / unfolding agent. The higher stability of the designed cytokine (circular data points) is indicated by lower fluorescence (right-shifted) at higher concentrations compared to WT IL-2. When used throughout this disclosure, the label “WT IL-2 internal” is intended to describe WT IL-2 having the same sequence as the commercially available WT IL-2 protein synthesized by the applicant. [Figure 13]This graph demonstrates that two exemplary engineered cytokines in this disclosure exhibit IL-15-like effects on NK cells in vitro. In this experiment, the activity of commercially available WT IL-2 was compared with that of internally produced WT IL-2. WT IL-15 polypeptide was used as a positive control and as a basis for comparing the effects against IL-15, in contrast to the IL-2-like effects. [Figure 14A] In some embodiments, the cytokines designed in this disclosure may be fused to the targeting moiety, or, to put it another way, a series of schematic diagrams demonstrating that the fusion protein may comprise the designed cytokines and the targeting moiety. The rightmost diagram represents signal transduction in trans. [Figure 14B] This graph demonstrates that the use of a targeting moiety to localize the physical presence and / or signaling activity of a designed cytokine, for example to a TME, does not impair any activity, receptor binding, or signaling from the designed cytokine. In this graph, the percentage of total cells binding to the designed cytokine is shown as a function of the nanomolar (nM) protein concentration of the targeting moiety that binds to the designed cytokine. [Figure 14C] This is a series of graphs demonstrating the use of targeting moieties by four exemplary engineered cytokines of this disclosure. In each plot, the cell proliferation activity of each engineered targeted cytokine is measured as a percentage of phosphorylated PSTAT5+ cells, as a function of the nanomolar concentration of the engineered targeted cytokine provided. The label "VHH-1" means that two VHH targeting moieties with different sequences are represented, in contrast to the label "VHH-2". [Figure 15]A series of schematic diagrams of two exemplary engineered cytokines of this disclosure demonstrate the structural differences between IL-2 and each engineered cytokine, in contrast to WT IL-2. Furthermore, the diagrams reflect the structural modifications to each engineered cytokine that result in functional changes to the protein. Each engineered cytokine contains a helix initiator (e.g., lysine(L)) on the front of the H4 helix, while engineered cytokines of “conjugation,” which more readily conjugate with the targeting moiety, have an elongated H2 helix and a shorter loop between the H4 and H2 helices. Both engineered cytokines have a substitution at a position of 3 amino acids from the H2 interhelix loop, but the substitution itself differs between these two engineered cytokines. [Figure 16] This series of graphs demonstrates that while the designed cytokine No. 201 does not show detectable binding to IL2Rα, WT IL-2 binds to IL2Rα with nanomolar affinity. [Figure 17A] This graph, which shows two replicated experiments (previous experiments shown in Figure 6) side by side, demonstrates that the activity of the exemplary designed cytokine can be tuned over a wide range. [Figure 17B] These graphs represent the subsequent replications of the experiment shown in Figures 6 and 17A. [Figure 18] This is a series of graphs showing the binding affinity (by Octet) to IL-2Rβγ for either WT IL-2 (left plot) or one of two exemplary designed cytokines (center and right plots). [Figure 19] This is a series of graphs demonstrating the cell proliferation capacity of three exemplary engineered cytokines compared to WT IL-2 in three cell types (NK cells, Treg cells, and CD8+ T cells). [Figure 20]This is a pair of schematic diagrams and graph coordinate pairs demonstrating sister-gating versus trans-targeting of the cytokines designed in this disclosure by altering the binding priority of the targeting portion. As shown in the upper row of the upper schematic diagram and graph, PD-1 and CD8 mediate sister-gating of activated T cells by the cytokines designed in this disclosure. As shown in the lower row of the lower schematic diagram and graph, PD-L1 mediates sister-gating and trans-presentation of the cytokines designed in this disclosure to activated cells. [Figure 21A] This is a schematic diagram showing the experimental design used to produce the data provided in Figures 21B and 21C. Briefly, CAR-T cells were sequentially loaded with H1975 tumor cells in the presence of recombinant engineered cytokines, engineered cytokines + target-binding domains (added separately in equimolar ratios), or engineered targeted cytokines: engineered targeted cytokine No. 169 was fused to a VHH-binding domain that binds to either PD-L1, PD-1, or CD8. [Figure 21B] This is a pair of graphs, corresponding to two different T-cell donors, demonstrating the cytokine-killing activity of the designed cytokine targeted by PDL1 in this disclosure. [Figure 21C] This is a pair of graphs, corresponding to two different T-cell donors, demonstrating the cytokine-killing activity of the designed cytokine targeted by PDL1 in this disclosure. [Figure 21D] This disclosure presents a pair of graphs, corresponding to two different T-cell donors, demonstrating the cytokine-killing activity of the designed cytokine targeted by CD8. [Figure 22A] This graph demonstrates the cell proliferation activity of a designed cytokine derived from engineered cytokine No. 153 in natural killer cells. [Figure 22B] This graph demonstrates the cell proliferation activity of a designed cytokine derived from designed cytokine No. 153 in CD8+ T cells. [Figure 23A]These are a series of schematic diagrams representing two vectors (2V), a single vector (1V), and an autoregulatory circuit incorporating an inducible engineered cytokine. In these schematics, the term "DC" refers to the engineered cytokine. In some embodiments, according to these diagrams, the expression of the inducible engineered cytokine is under the control of one or more response elements (RE arrays) and promoter sequences (which may be minimal promoter sequences (pMin)). In each of the 2V and 1V diagrams, the marker and CAR elements are under the control of the MND promoter. In the autoregulatory diagram, the expression of the CAR and engineered cytokine is under the control of an inducible promoter that is active during the period of cell stimulation. [Figure 23B] Figure 23C is a schematic diagram of the experimental design used to produce the data shown. In this experiment, CAR-T cells containing the vector or circuit shown in 23A were stimulated with an antigen bound to a plate. WT IL-2 was measured in the supernatant with control or secreted inducible engineered cytokines. [Figure 23C] This graph demonstrates that inducible engineered cytokines (DCs) show increased expression compared to control, CARs alone (without engineered cytokines), or no antigen stimulation. [Figure 24] This is a series of graphs demonstrating that exemplary inducible engineered cytokines retain better target cell-killing activity than CAR-only controls after three consecutive rounds of stimulation. [Figure 25]This graph demonstrates that the exemplary engineered cytokine (IL-2 / 15-CD8 targeting moiety) of this disclosure enhances TCR-T activity, both by itself and in combination with an inducible promoter (10×NFκB-minP). Tumor growth (measured as fluorescence) was measured as a function of culture time under multiple conditions. Constructs containing TCR alone under either a constitutive MND promoter (upward-pointing pointed triangle) or an inducible promoter (square data points) showed comparable results. After the addition of the engineered cytokine (MND, indicated by diamond-shaped data points, and the inducible promoter, indicated by downward-pointing pointed triangles), the same constructs each demonstrated significant improvement. The engineered cytokine shown in this figure contains the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK. Preferred anti-CD8 of the present disclosure, including the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYYDMDYWGQGTQVTVSS When combined with VHH, the resulting polypeptide may contain a linker and may also contain the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYYDMDYWGQGTQVTVSS. As shown in this figure, the designed cytokine may be under the control of an inducible promoter.The inducible promoter used to create this data includes the sequence GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTCCAG. In a preferred embodiment of the present disclosure, the inducible promoter shown in this figure may be used in combination with a 5' UTR sequence including ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC. When used in combination, the resulting inducible promoter + 5'UTR contains the sequence GGGGACTTTCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTCCAG ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC. [Figure 26] This graph demonstrates that TCR T cell activation is not impaired by the supraphysiological dose of the designed cytokine of this disclosure. Binding of anti-CD8α VHH does not impair the coreceptor activity necessary for TCR T cell activation. In this study, the percentage of interferon-gamma (IFN-γ+) T cells among EGFR+CD4+CD19-T cells was measured as a function of peptide concentration (measured in nanomoles). T2 antigen-presenting cells (APCs) were loaded with the indicated peptide concentrations. T cell activation (measured as IFNγ+%) is dependent on pMHCI interaction and CD8 coreceptor function. The designed cytokine shown in this figure is the humanized CD8 VHH of this disclosure. When combined with the preferred anti-CD8 VHH of this disclosure, the resulting polypeptide may contain a linker. [Figure 27]The following graphs show a series of schematic diagrams representing the constructs used to create the data presented. In summary, the data demonstrate that a combination of techniques, including CAR-expressing T cells capable of secreting the cytokines designed in this disclosure, progressively improves antitumor efficacy and proliferation. In the first “CAR only” construct, anti-MSLN chimeric antigen receptor expression is under the control of a constitutive MND promoter (and is also conjugated by a 2A sequence with an EGFR sequence that functions as both a cellular marker and a safety switch (see WO2021 / 189008)). In the second construct, anti-MSLN chimeric antigen receptor expression is under the control of an inductive promoter (and is also conjugated by a 2A sequence with an EGFR sequence that functions as both a cellular marker and a safety switch (see WO2021 / 189008)). The inducible promoter contains the sequence GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATATAATGGAAGCTCGAATTCCAG. In a preferred embodiment of the present disclosure, the inducible promoter shown in this figure may be used in combination with a 5' UTR sequence containing ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC. When used in combination, the resulting inducible promoter + 5'UTR contains the sequence GGGGACTTTCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTCCAG ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC.In the third construct, the expression of the anti-MSLN chimeric antigen receptor is under the control of an inducible promoter (and is combined with a 2A sequence containing an EGFR sequence that functions as both a cell marker and a safety switch (see WO2021 / 189008)), and is further combined with the designed cytokine of this disclosure by the 2A sequence. The designed cytokine shown in this figure contains the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK. The preferred anti-CD8 of this disclosure contains the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYDMDYWGQGTQVTVSS. When combined with VHH, the resulting polypeptide may contain a linker and may also contain the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYYDMDYWGQGTQVTVSS. Using these constructs, antitumor efficacy was evaluated by measuring tumor volume (mm3) as a function of days after tumor engraftment (see Figure 28 for a description of the xenograft mouse model used). As shown in the series of graphs in the lower left, the addition of inducible promoters, as well as the engineered cytokines, significantly improved the performance of CARs compared to constitutive expression alone. Using these constructs, CAR T cell proliferation was evaluated by measuring the hCD45+ cell count (cells / milliliter) as a function of days after T cell injection.As shown in the graph in the lower right, including the designed cytokines significantly increased T cell proliferation compared to the results obtained with inducible promoters or CAR expression alone. [Figure 28]The following is a series of schematic diagrams and graphs illustrating the elimination of intraperitoneal (IP) transplanted SKVO3 ovarian cancer cells after intravenous (IV) treatment with a CAR-T composition containing the cytokines designed in this disclosure. The schematic diagrams demonstrate the generation of the mouse xenograft model used in this study. Briefly, fluorescently labeled (FFluc) SKVO3 ovarian cancer cells from one of two donors were introduced into immunodeficient mice to generate intraperitoneal (IP) engraftment. Twenty days after injection of SKOV3 cells, the CAR-T composition was introduced intravenously via the tail vein. The T cell composition was provided in various doses of 50,000 cells per animal, 200,000 cells per animal, and 500,000 cells per animal. Each CAR-T provided for treatment contained constructs of the anti-MSLN chimeric antigen receptor expression, EGFRopt sequence (see WO2021 / 189008), and the cytokines designed in this disclosure under the control of an inducible promoter. The designed cytokine shown in this figure contains the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK. The preferred anti-CD8 of this disclosure contains the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYYDMDYWGQGTQVTVSS. When combined with VHH, the resulting polypeptide may contain a linker and may also contain the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYYDMDYWGQGTQVTVSS.The inducible promoter contains the sequence GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATATAATGGAAGCTCGAATTCCAG. In a preferred embodiment of the present disclosure, the inducible promoter shown in this figure may be used in combination with a 5' UTR sequence containing ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC. When used in combination, the resulting inducible promoter + 5'UTR contains the sequence GGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCCCGCTGGGGACTTTCCGCTAACGAGAGGGTATATAATGGAAGCTCGAATTCCAG ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCTAGCGCCGCCACC. The elimination of SKOV3 tumor cells was determined by measuring the radiation dose as a function of the number of days after engraftment. The data show that in both donors, and at doses of at least 200K and 500K cells per animal, fluorescent cells are eliminated as the brightness decreases rapidly. [Figure 29] These are a series of pseudo-colored photographs, providing a qualitative analysis of the reduction of fluorescent labeling based on the research conducted in Figure 28. [Figure 30] These schematic diagrams and graphs extend the study performed in Figure 28 using two additional T cell donors and a wider range of doses of the T cell composition (20,000 cells per animal, 50,000 cells per animal, 200,000 cells per animal, and 500,000 cells per animal). The results of this study support the results of the study shown in Figure 28. Elimination of SKOV3 tumor cells occurs in a dose-dependent manner after treatment with the T cell composition. [Modes for carrying out the invention]

[0061] This disclosure provides “engineered cytokines,” which are naturally occurring polypeptides, as illustrated in embodiments provided herein by the drawings and sequences, demonstrating the functional benefits of IL-2 and IL-15. Accordingly, “engineered cytokines” may be referred to as “IL-2 / 15 polypeptides” or “IL-2 / 15.” These terms are interchangeable with “engineered cytokines.”

[0062] This disclosure provides a designed cytokine that preferably exhibits one or more of the following attributes: (1) optionally reduces or eliminates the binding of the designed cytokine to the alpha subunit of the IL-2 receptor (IL-2Rα) without post-translational modification of the designed IL-2 polypeptide; (2) binds or retains the binding of the designed IL-2 polypeptide to the beta and / or gamma subunits of the IL-2 receptor (IL-2Rβ / γ); (3) preferentially stimulates or more potently stimulates T cells (CD8 T cells) that express more CD8 than regulatory T cells (Treg); (4) CD8 (5) to preferentially stimulate or more potently stimulate natural killer (NK) cells than T cells; (6) to enhance the activity of T cells expressing or secreting the designed IL-2 polypeptide of this disclosure; (7) to demonstrate stable folding of the designed IL-2 polypeptide into a protein in vitro or in vivo; and (8) to demonstrate reduced, minimal, or undetectable levels of immunogenicity when administered to a subject (e.g., a mouse, another animal species, or a human patient) compared to the level of immunogenicity demonstrated by wild-type IL-2 protein under the same circumstances. Alternatively or additionally, the designed cytokines of this disclosure may (1) bind to the IL-15 receptor (IL-15R), beta subunit, or gamma subunit, and / or (2) compete with IL-15 cytokines, including WT IL-15, for binding to the IL-15R, beta subunit, or gamma subunit.

[0063] In some embodiments, the designed cytokines of the Disclosure (1) localize the expression, translation, production, and / or secretion of the designed IL-2 polypeptide of the Disclosure to tumors, target cells, and / or tumor microenvironment (TME), (2) target the TME, and / or (3) maintain localization in the TME. In some embodiments of the designed cytokines of the Disclosure, including those in which the polypeptide targets to the TME, localizes to the TME, and / or maintains localization within the TME, the designed cytokine includes a targeting moiety. In some embodiments, the targeting moiety includes nucleic acids, amino acids, or combinations thereof that specifically bind to a target on or within the cells of a lymph node, tumor, tumor microenvironment, malignant tumor site, or metastatic site, or in each case, a target. In some embodiments, the fusion protein or targeting moiety includes an antibody, antibody mimetic, or a functional fragment thereof. In some embodiments, the targeting moiety includes scFv, VH, or VHH. In some embodiments, the targeting moiety includes scFv, VH, or VHH that specifically or selectively bind to T cell surface glycoprotein CD8 (also known as differentiation antigen group 8), programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1; also known as differentiation antigen group 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immune receptor (TIGIT) having Ig and ITIM domains, cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), or T cell immunoglobulin mucin receptor 3 (TIM3).

[0064] In some embodiments of the designed cytokines of this disclosure, the designed cytokine includes a “tether” that activatably links the designed cytokine to (1) cells expressing the designed cytokine, or (2) cells delivering the designed cytokine to target cells, immune synapses, and / or TMEs. In some embodiments, the tether includes DNA, RNA, amino acids, or any combination thereof. In some embodiments, the tether includes a defined secondary structure. In some embodiments, the tether has a rigid structure. In some embodiments, the tether has a flexible conformation. In some embodiments, the tether has an elastic conformation. In some embodiments, the tether includes a cleavable sequence. In some embodiments, the tether includes a transmembrane sequence or a membrane anchor sequence. In some embodiments, the tether is optionally linked to the designed cytokine by a linker sequence. In some embodiments, the tether is optionally linked to the designed cytokine by a linker sequence including a “GS” linker. In some embodiments, the tether includes the sequence “PLFIPVAVMVTAFSGLAFIIWLARRLKKGKK”.

[0065] In some embodiments of the designed cytokines of this disclosure, the construct includes an inducible promoter capable of expressing the designed cytokine and a sequence encoding the designed cytokine. In some embodiments, the construct may further include a sequence encoding a targeting moiety. Alternatively or in addition, in some embodiments, the construct may further include a sequence encoding a tether. In some embodiments, the construct may further include a linker located between the sequence encoding the designed cytokine and one or more of the sequences encoding the targeting moiety and the sequences encoding the tether.

[0066] In some embodiments of the designed cytokines of this disclosure, the designed cytokine comprises the designed cytokine and either a targeting moiety or a tether. In some embodiments of the fusion protein, a linker is located between the designed cytokine and either the targeting moiety or the tether.

[0067] In some embodiments of the designed cytokines of this disclosure, immune cells express the designed cytokines of this disclosure. In some embodiments, immune cells secrete the designed cytokines. In some embodiments, the designed cytokines include a tether that contacts the immune cells. In some embodiments, the tethered designed cytokines contact the plasma membrane or components of the immune cells. In some embodiments, the tethered designed cytokines contact the outer surface of the plasma membrane of the immune cells.

[0068] In some embodiments, the cytokines designed in this disclosure can be expressed alone or in combination with one or more antigen receptors (e.g., T cell receptors (TCRs), chimeric antigen receptors (CARs), or any combination thereof). In some embodiments, the vector includes a viral vector. In some embodiments, the vector includes a non-viral vector. In some embodiments, the vector consists of a single vector. In some embodiments, the viral vector includes a sequence that encodes the designed cytokine and a sequence that encodes an antigen receptor (e.g., T cell receptors (TCRs), chimeric antigen receptors (CARs), or any combination thereof). In some embodiments, the single viral vector includes a sequence that encodes the designed cytokine and a sequence that encodes an antigen receptor (e.g., T cell receptors (TCRs), chimeric antigen receptors (CARs), or any combination thereof). In some embodiments, the viral vector includes a sequence isolated from or derived from a lentiviral vector. In some embodiments, the viral vector includes a lentiviral vector.

[0069] In some embodiments, the engineered cytokines provided herein have high affinity for the target. In some embodiments, the engineered cytokines of this disclosure are also referred to herein as high-affinity engineered cytokines. In some embodiments, the engineered cytokines of this disclosure fused to VHH with CD8 have high affinity for the target. In some embodiments, the engineered cytokines provided herein have high activity. In some embodiments, the engineered cytokines of this disclosure are also referred to herein as high-activity engineered cytokines. In some embodiments, the engineered cytokines of this disclosure fused to VHH with CD8 have high activity.

[0070] Function of designed cytokines This disclosure provides engineered cytokines that demonstrate the functional benefits of IL-2 and IL-15. This disclosure provides engineered cytokines that preferably exhibit one or more of the following attributes: (1) optionally reduce or eliminate the binding of the engineered cytokine to the alpha subunit of the IL-2 receptor (IL-2Rα) without post-translational modification of the engineered IL-2 polypeptide; (2) bind or retain the binding of the engineered IL-2 polypeptide to the beta and / or gamma subunits of the IL-2 receptor (IL-2Rβ / γ); (3) preferentially stimulate or more potently stimulate T cells (CD8 T cells) that express more CD8 than regulatory T cells (Treg); (4) CD8 (5) to preferentially stimulate or more potently stimulate natural killer (NK) cells than T cells; (6) to enhance the activity of T cells expressing or secreting the designed IL-2 polypeptide of this disclosure; (7) to demonstrate stable folding of the designed IL-2 polypeptide into a protein in vitro or in vivo; and (8) to demonstrate reduced, minimal, or undetectable levels of immunogenicity when administered to a subject (e.g., a mouse, another animal species, or a human patient) compared to the level of immunogenicity demonstrated by wild-type IL-2 protein under the same circumstances. Alternatively or additionally, the designed cytokines of this disclosure may (1) bind to the IL-15 receptor (IL-15R), beta subunit, or gamma subunit, and / or (2) compete with IL-15 cytokines, including WT IL-15, for binding to the IL-15R, beta subunit, or gamma subunit.

[0071] Both wild-type IL-2 and IL-15 stimulate signaling via IL-2 / 15Rβ (also referred to as IL-2Rβ) and a common IL-2 / 15Rγ chain (also referred to as IL-2Rγ). Both IL-2 and IL-15 bind to the heterodimeric βγ receptor complex IL-2 / 15Rβγ (also referred to as IL-2Rβγ) to activate signaling, and their unique biology is primarily driven by their respective interactions via IL-2Rα (cis-presentation) and IL-15Rα (trans-presentation). The designed IL-2 polypeptide of this disclosure (also known as the IL-2 / 15 polypeptide of this disclosure) agonizes the βγ receptor pair shared by IL-2 and IL-15 while evading IL-2Rα and IL-15Rα. Therefore, the IL-2 / 15 polypeptides designed in this disclosure may demonstrate the activity of both IL-2 and IL-15 when expressed by different cell types or when in contact with different cell types.

[0072] IL-2 and IL-15 stimulate various types of lymphocytes and natural killer cells. Among the distinct functions of these two cytokines, IL-2 mediates regulatory T cell homeostasis and regulates T helper (TH) differentiation. Furthermore, IL-15 mediates the proliferation of CD8 memory T cells, NK cells, and NKT cells. The IL-2 / 15 polypeptides designed in this disclosure may demonstrate one or more activities of IL-2 and IL-15 in specific cell types.

[0073] Modified or optimized engineered cytokines The engineered cytokines of this disclosure may be “tuned” or “optimized” with respect to cytokine activity. The engineered cytokines of this disclosure may be “tuned” or “optimized” to demonstrate a preferred ratio of IL-2 activity to IL-15 activity. In some embodiments, the engineered cytokines may be tuned to achieve specific activity thresholds for wild-type IL-2 polypeptides and wild-type IL-15 polypeptides. In some embodiments, the tuning of the IL-2 / 15 polypeptides of this disclosure does not involve a change in the structure of the engineered cytokine (e.g., a 1-4-2-3 configuration of its helix). In some embodiments, the modification of the designed cytokines of this disclosure includes one or more of the following: (1) modifying the sequence of the designed cytokine or a part thereof (e.g., an alpha helix, a loop, or a combination thereof); (2) modifying the physical length of the folded designed cytokine or a part thereof (e.g., an alpha helix, a loop, or a combination thereof); (3) inserting a new sequence into an existing sequence of the designed cytokine or a part thereof (e.g., an alpha helix, a loop, or a combination thereof); and / or (4) removing a sequence of the designed cytokine or a part thereof (e.g., an alpha helix, a loop, or a combination thereof). In some embodiments, the modified designed cytokines of this disclosure include sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of identity between them and the designed cytokine of this disclosure that has not been subjected to the modification process. In some embodiments, the modified designed cytokines of this disclosure include sequences isolated from or derived from IL-15 sequences.

[0074] This disclosure provides engineered cytokines (formerly known as "IL-2 / 15 polypeptides") that can be “tuned” or “optimized” with respect to any activity of the cytokine to demonstrate an activity threshold in one or more cell types. In some embodiments, the tuning or optimization of the engineered cytokines of this disclosure for use in cell types does not involve a structural change of the engineered cytokine (e.g., a 1-4-2-3 arrangement of its helix). In some embodiments, the optimization of the engineered cytokines of this disclosure for use in cell types includes one or more of the following: (1) modifying the sequence of the engineered cytokine or part thereof (e.g., an alpha helix, a loop, or a combination thereof); (2) modifying the physical length of the folded engineered cytokine or part thereof (e.g., an alpha helix, a loop, or a combination thereof); (3) inserting a new sequence into an existing sequence of the engineered cytokine or part thereof (e.g., an alpha helix, a loop, or a combination thereof); and / or (4) removing a sequence of the engineered cytokine or part thereof (e.g., an alpha helix, a loop, or a combination thereof). In some embodiments, the optimized engineered cytokines of the Disclosure include sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage of identity with the engineered cytokines of the Disclosure that have not been subjected to the optimization process. In some embodiments, the optimized engineered cytokines of the Disclosure include sequences isolated from or derived from IL-15 sequences.

[0075] Designed combination of cytokines In some embodiments of this disclosure, the designed cytokine may be used in combination with a second cytokine or a second designed cytokine, or may be operably linked to a second cytokine. In some embodiments, the designed cytokine and the second cytokine or second designed cytokine may be independently modified, regulated, and / or targeted. In some embodiments, the designed cytokine and the second cytokine or second designed cytokine may be harmonized in one or more of the modifications, regulations, and / or targeting.

[0076] In some embodiments, the cytokines designed in this disclosure may be used in combination with any cytokine, whether naturally occurring or modified. In some embodiments, the cytokines designed in this disclosure may be used in combination with one or more of the following: IL-2 polypeptide, IL-12 polypeptide, IL-15 polypeptide, IL-18 polypeptide, IL-21 polypeptide, IL-23 polypeptide, and interferon polypeptides (including, but not limited to, interferon alpha, beta, gamma, and / or omega polypeptides). In some embodiments, the cytokines designed in this disclosure may be used in combination with IL-2 polypeptide. In some embodiments, the cytokines designed in this disclosure may be used in combination with IL-12 polypeptide. In some embodiments, the cytokines designed in this disclosure may be used in combination with IL-15 polypeptide. In some embodiments, the cytokines designed in this disclosure may be used in combination with IL-18 polypeptide. In some embodiments, the cytokines designed in this disclosure may be used in combination with IL-21 polypeptide. In some embodiments, the cytokines designed in this disclosure may be used in combination with IL-23 polypeptide. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptide. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon beta polypeptides. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon gamma polypeptides. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon omega polypeptides. In some embodiments, the second cytokine includes a wild-type sequence. In some embodiments, the second cytokine does not include a wild-type sequence. In some embodiments, the second cytokine includes one or more modifications to alter the cytokine's activity to one or more cytokine receptors.

[0077] In some embodiments, the cytokines designed in this disclosure may be used in combination with a second cytokine designed in this disclosure to generate a combination of a first cytokine and a second cytokine.

[0078] In some embodiments of this disclosure, the designed cytokine may be expressed together with a second cytokine or a second designed cytokine of this disclosure. In some embodiments, the expression may be simultaneous. In some embodiments, the expression may be sequential. In some embodiments, the expression may be modulated or inducible by the use of an inducible promoter of this disclosure.

[0079] In some embodiments of this disclosure, the designed cytokine may be secreted together with a second cytokine or a second designed cytokine of this disclosure. In some embodiments, the secretion may be simultaneous. In some embodiments, the secretion may be sequential. In some embodiments, the secretion may be modulated or inducible by the use of an inducible promoter of this disclosure.

[0080] In some embodiments of this disclosure, a designed cytokine may be operably linked to a second cytokine or a second designed cytokine of this disclosure. In some embodiments, the designed cytokine and the second cytokine or second designed cytokine may be operably linked via a linker sequence. In some embodiments, the linker sequence includes nucleic acids, amino acids, small molecules, or any combination thereof. In some embodiments, the linker is rigid. In some embodiments, the linker is flexible. For example, the linker sequence may include a “GS” sequence of any length.

[0081] In some embodiments, the designed cytokine and the second cytokine or second designed cytokine may be operably coupled via the targeting portion of the Disclosure. In some embodiments, the designed cytokine and the second cytokine or second designed cytokine may be operably coupled to the same targeting portion. In some embodiments, a composition comprising the designed cytokine and the second cytokine or second designed cytokine may include the same targeting portion. In some embodiments, the designed cytokine may include a first targeting portion and a second cytokine, or the second designed cytokine may include a second targeting portion. In some embodiments, the first targeting portion and the second targeting portion bind to the same target. In some embodiments, the first targeting portion and the second targeting portion do not bind to the same target. In some embodiments, the first targeting portion and the second targeting portion are identical. In some embodiments, the first targeting portion and the second targeting portion are not identical.

[0082] In some embodiments, the designed cytokine and the second cytokine or second designed cytokine may be operably coupled via the tether of this disclosure. In some embodiments, the designed cytokine and the second cytokine or second designed cytokine may be operably coupled to the same tether. In some embodiments, a composition comprising the designed cytokine and the second cytokine or second designed cytokine may comprise the same tether. In some embodiments, the designed cytokine may comprise the first tether and the second cytokine, or the second designed cytokine may comprise the second tether. In some embodiments, the first tether and the second tether bind to the same target. In some embodiments, the first tether and the second tether do not bind to the same target. In some embodiments, the first tether and the second tether are identical. In some embodiments, the first tether and the second tether are not identical.

[0083] In some embodiments, the designed cytokine is operably linked to one or more of the following: IL-2 polypeptide, IL-12 polypeptide, IL-15 polypeptide, IL-18 polypeptide, IL-21 polypeptide, IL-23 polypeptide, and interferon polypeptides (including, but not limited to, interferon alpha, beta, gamma, and / or omega polypeptides). In some embodiments, the designed cytokine is operably linked to IL-21 polypeptide.

[0084] Targeted, engineered cytokines In some embodiments of this disclosure, the designed cytokine targets, localizes to, or remains active in, one or more of the following: target cells, target cell types, organs, lymph nodes, tumors (including, but not limited to, liquid tumors, hematological malignancies, and solid tumors), the biological microenvironment, tumor microenvironment (TME), malignant tumor sites, metastatic sites, tumor angiogenesis sites, and any combination thereof. In some embodiments, the fusion protein includes the designed cytokine that targets, localizes to, or remains active in, one or more of the following: target cells, target cell types, organs, lymph nodes, tumors (including, but not limited to, liquid tumors, hematological malignancies, and solid tumors), the biological microenvironment, tumor microenvironment (TME), malignant tumor sites, metastatic sites, tumor angiogenesis sites, and any combination thereof. In some embodiments, the designed cytokine includes a targeting portion. In some embodiments, the designed cytokine is operably linked to the targeting portion. In some embodiments, the designed cytokine is operably linked to the targeting moiety by one or more of the following: covalent bonding, non-covalent bonding, hybridization, dimerization, complex formation, linkers, and tethers. In some embodiments, the designed cytokine is operably linked to the targeting moiety by a linker comprising one or more of the following: nucleic acid sequences, amino acid sequences, small molecules (organic or inorganic), and any combination thereof. In some embodiments, the designed cytokine is operably linked to the targeting moiety by a tether comprising one or more of the following: nucleic acid sequences, amino acid sequences, small molecules (organic or inorganic), and any combination thereof. In some embodiments, the fusion protein is operably linked to the targeting moiety by a tether bound to one or more of the following: target cells, target cell types, organs, lymph nodes, tumors (including, but not limited to, liquid tumors, hematological malignancies, and solid tumors), biological microenvironment, tumor microenvironment (TME), malignant tumor sites, metastatic sites, tumor angiogenesis sites, and any combination thereof.In some embodiments, the designed cytokine is operably linked to the targeting portion by a tether that is bound to a component of the immune cell expressing or secreting the designed cytokine.

[0085] In some embodiments of this disclosure, the targeting moiety includes nucleic acids, amino acids, or combinations thereof that specifically bind to one or more components of target cells, target cell types, organs, lymph nodes, tumors (including, but not limited to, liquid tumors, hematological malignancies, and solid tumors), biological microenvironments, tumor microenvironments (TMEs), malignant tumor sites, metastatic sites, tumor angiogenesis sites, and any combination thereof. In some embodiments, the targeting moiety includes binding domains, protein scaffolds, antibodies, antibody mimes, and / or functional fragments thereof. In some embodiments, the targeting moiety includes sequences isolated from or derived from any species, including, but not limited to, humans, non-human primates, rodents (including, but not limited to, mice), and camelid species. In some embodiments, the targeting moiety includes sequences that may be humanized, chimeric, recombinant, not naturally occurring, modified (e.g., to include synthetic nucleic acids or synthetic amino acids), and optimized (e.g., to reduce immunogenicity and / or aggregation during manufacturing).

[0086] In some embodiments of this disclosure, the targeting portion includes, but is not limited to, a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more heavy chain (VH) and light chain (VL) domains of an immunoglobulin (Ig) polypeptide or a gene encoding such domain, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and a nanobody. In some embodiments, the targeting portion includes scFv, VH, or VHH. In some embodiments, the targeting moiety includes scFv, VH, or VHH that specifically or selectively bind to targets including, but not limited to, T cell surface glycoprotein CD8 (also known as differentiation antigen group 8), programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1; also known as differentiation antigen group 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immune receptor (TIGIT) having Ig and ITIM domains, cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), and T cell immunoglobulin mucin receptor 3 (TIM3).

[0087] In some embodiments of this disclosure, the targeting portion includes an antibody mimetic comprising, but not limited to, one or more of the following: a manipulated protein scaffold, monobody, afibody molecule, adonectin molecule, affimer molecule, afitin molecule, afirin molecule, alphabody molecule, antikalin molecule, aptamer molecule, atrimer molecule, avimer molecule, DARPin molecule, finomer, armadillo repeat protein molecule, Knitz domain inhibitor molecule, Notchin molecule, designed ankyrin repeat protein molecule, nanophytin molecule, and centinrin molecule. In some embodiments, the targeting moiety includes antibodies that specifically or selectively bind to targets including, but not limited to, T cell surface glycoprotein CD8 (also known as differentiation antigen group 8), programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1; also known as differentiation antigen group 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immune receptor (TIGIT) having Ig and ITIM domains, cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), and T cell immunoglobulin mucin receptor 3 (TIM3).

[0088] In some embodiments, the cytokines designed herein include a targeting moiety comprising one of the binders listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0089] In some embodiments, the cytokines and polypeptides designed in this disclosure include a targeting moiety comprising one of the sequences in Table 2.

[0090] In some embodiments, the targeting portion of the Disclosure includes a humanized VHH that specifically binds to CD8. In preferred embodiments, the targeting portion of the Disclosure includes a humanized VHH provided in Table 2. [Table 2]

[0091] In some embodiments, the cytokines designed herein include a targeting moiety comprising one of the sequences listed in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15]

[0092] Inducible engineered cytokines This disclosure provides nucleic acids encoding polypeptides that do not exist in nature. In some embodiments, a promoter or inducible promoter capable of driving expression in mammalian cells controls the expression of nucleic acids encoding cytokines designed by this disclosure.

[0093] In some embodiments, the inducible promoter of the Disclosure comprises a minimal promoter. In some embodiments, the minimal promoter of the Disclosure comprises a sequence isolated from or derived from one or more of the following: minimal promoter-1 ("minP1"), YB-TATA, and human betaglobin. In some embodiments, the minimal promoter comprises "minP1" having the sequence AGAGGGTATATAAAAGCTCGACTTCCAG, "minP2" having the sequence TAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC, and It contains one or more "minP3" sequences that are CTAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC.

[0094] In some embodiments, the inducible promoters of this disclosure include sequences isolated from or derived from coding or non-coding sequences of genes associated with one or more of the following: BACH2, BARX1, BATF, ELF1, ELF2, Elf4, Elk1, ERF, ETV1, Fli1, FOXP1, GABPA, GATA3, IRF1, IRF2, IRF5, IRF7, IRF9, MAF, MAFF, Maz, Mef2d, MLX, MYB, NFAT, NFATC3, NFkB, NR4A1, Nur77, PATZ1, REL, RELA, RORa, RORg, RORgt, STAT2, Tbox, TFEB, TOX, USF1, ZBTB2, ZKSCAN3, ZNF12, ZNF140, ZNF263, ZNF282, ZNF304, ZNF398, ZNF708, and ZNF75D. In some embodiments, the inducing promoter of this disclosure includes sequences isolated from or derived from coding or non-coding sequences of genes related to NFAT, NFkB, REL, RELA, IRF2, GATA3, and ATF3. In some embodiments, the inducing promoter of this disclosure includes sequences isolated from or derived from coding or non-coding sequences of genes related to NFAT. In some embodiments, the inducing promoter of this disclosure includes sequences isolated from or derived from coding or non-coding sequences of genes related to NFkB. In some embodiments, the inducing promoter of this disclosure includes sequences isolated from or derived from coding or non-coding sequences of genes related to REL. In some embodiments, the inducing promoter of this disclosure includes sequences isolated from or derived from coding or non-coding sequences of genes related to RELA. In some embodiments, the inducing promoter of this disclosure includes sequences isolated from or derived from coding or non-coding sequences of genes related to IRF2. In some embodiments, the inducing promoter of this disclosure includes sequences isolated from or derived from coding or non-coding sequences of genes related to GATA3. In some embodiments, the inducible promoter of this disclosure includes a sequence isolated from or derived from a coding or non-coding sequence of a gene related to ATF3.

[0095] In some embodiments, the inducible promoter of this disclosure includes a response element and / or an enhancer sequence. In some embodiments, the response element and / or enhancer sequence includes a sequence isolated from or derived from a coding or non-coding sequence of a gene associated with one or more of the following genes: BACH2, BARX1, BATF, ELF1, ELF2, Elf4, Elk1, ERF, ETV1, Fli1, FOXP1, GABPA, GATA3, IRF1, IRF2, IRF5, IRF7, IRF9, MAF, MAFF, Maz, Mef2d, MLX, MYB, NFAT, NFATC3, NFkB, NR4A1, Nur77, PATZ1, REL, RELA, RORa, RORg, RORgt, STAT2, Tbox, TFEB, TOX, USF1, ZBTB2, ZKSCAN3, ZNF12, ZNF140, ZNF263, ZNF282, ZNF304, ZNF398, ZNF708, and ZNF75D. In some embodiments, the response element and / or enhancer sequence includes a sequence isolated from or derived from a coding or non-coding sequence of a gene associated with one or more of NFAT, NFkB, REL, RELA, IRF2, GATA3, and ATF3. In some embodiments, the response element and / or enhancer sequence includes a sequence isolated from or derived from NFAT. In some embodiments, the response element and / or enhancer sequence includes a sequence isolated from or derived from NFkB. In some embodiments, the response element and / or enhancer sequence includes a sequence isolated from or derived from REL. In some embodiments, the response element and / or enhancer sequence includes a sequence isolated from or derived from RELA. In some embodiments, the response element and / or enhancer sequence includes a sequence isolated from or derived from IRF2. In some embodiments, the response element and / or enhancer sequence includes a sequence isolated from or derived from GATA3. In some embodiments, the response element and / or enhancer sequence includes sequences isolated from or derived from ATF3.

[0096] In some embodiments, the inducible promoter of the Disclosure comprises two or more response elements and / or enhancer sequences. In some embodiments, the inducible promoter of the Disclosure comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 response elements and / or enhancer sequences. In some embodiments, the repeating response elements and / or enhancer sequences are identical. In some embodiments, the repeating response elements and / or enhancer sequences are not identical.

[0097] In some embodiments, the inducible promoter of the Disclosure does not include a combination of (1) an activated T cell nuclear factor (NFAT) sequence, an interferon regulator 4 (IRF4) sequence, an activated protein 1 (AP-1)-IRF complex element (AICE) sequence, or an interferon-stimulated response element (ISRE) sequence, and (2) a human betaglobin sequence. In some embodiments, the inducible promoter of the Disclosure is The sequence does not include GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGAATTCAGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC. As used throughout this disclosure, the AICE sequence includes or consists of a sequence isolated from or derived from IRF4 or IRF8, each together with BATF. In some embodiments, the AICE sequence may be derived from the untranslated regions of IRF4, IRF8, and / or BATF. As used throughout this disclosure, the ISRE sequence includes the consensus sequence "YAGTTTC(A / T)YTTTYCC" where "Y" is either C or T.

[0098] In some embodiments, the inducible promoter of the Disclosure does not include a combination of (1) an NFAT sequence and (2) a YB-TATA sequence. In some embodiments, the inducible promoter of the Disclosure does not include the sequence GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGAATTCCTAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC.

[0099] In some embodiments, the inducing promoter of the Disclosure comprises one or more transcription factor-binding motifs. In some embodiments, the inducing promoter of the Disclosure comprises a concatemer of two or more repeat sequences, each repeat sequence may comprise one or more transcription factor-binding motifs. In some embodiments, the two or more repeat sequences are identical. In some embodiments, the two or more repeat sequences are not identical. In some embodiments, the concatemer comprises a linking sequence located between the repeat sequences. In some embodiments, the linking sequence may comprise one or more of TACGCT, TGATCT, TGCTTT, and TGCCCGT.

[0100] In some embodiments, the inducible promoter of the Disclosure, comprising a concatemer, binds to more than one specific transcription factor. Alternatively or in addition, in some embodiments, the inducible promoter of the Disclosure, comprising a concatemer, binds to the same transcription factor at more than one site.

[0101] In some embodiments, the inducible promoter of this disclosure, which includes one or more transcription factor-binding motifs, is optionally organized as a concatemer and includes a sequence with a consensus sequence provided in Table 19.

[0102] In some embodiments, the inducing promoter of this disclosure, which optionally comprises one or more transcription factor-binding motifs organized as an concentrate, comprises one or more sequences selected from any of the sequences in Table 21.

[0103] In some embodiments, the inducement promoter of this disclosure, which includes a concatemer, includes a sequence comprising one or more of the sequences in Table 22.

[0104] In some embodiments, the inducible promoter of the Disclosure includes an enhancer sequence pairing (EP pairing) of an enhancer sequence and a promoter sequence. In some embodiments, the inducible promoter of the Disclosure, which includes one or more EP pairs, includes the sequences shown in Table 21.

[0105] In some embodiments, the cytokines designed in this disclosure are operably coupled to any of the nucleic acid constructs disclosed in U.S. Provisional Applications No. 63 / 479,176, No. 63 / 479,177, and No. 63 / 479,178, which are incorporated herein in their entirety by reference.

[0106] Designed cytokine structure This disclosure provides a designed cytokine comprising alpha-helix H1, H2, H3, and H4, wherein a first loop (L1) connects H1 and H4 from the amino terminus to the carboxy terminus, a second loop (L2) connects H4 and H2, and a third loop (L3) connects H2 and H3, the designed cytokine (formerly referred to as “interleukin-2 / 15 (IL-2 / 15) polypeptide”), which does not exist in nature. In some embodiments, the designed cytokine binds to the beta (β) and / or gamma (γ) subunits of the IL-2 receptor. In some embodiments, the designed cytokine binds to the IL-2 receptor β / γ heterodimer (IL-2Rβ / γ) as a heterodimeric receptor.

[0107] In some embodiments of the designed cytokines of this disclosure, the designed cytokine comprises one or more sequences from sequence numbers 1 to 350.

[0108] In some embodiments of the designed cytokines of this disclosure, the designed cytokine comprises one or more sequences of sequence numbers 1-38 or 150-350.

[0109] In some embodiments of the designed cytokines of this disclosure, the designed cytokines include a sequence isolated from or derived from an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide is a wild-type polypeptide. In some embodiments, the IL-2 polypeptide includes the sequence of SEQ ID NO: 500. In some embodiments, the designed cytokines of this disclosure may be used in combination with an IL-2 polypeptide containing the sequence of SEQ ID NO: 500.

[0110] Natural human (hIL-2) contains the sequence (sequence number 500): 1 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE 61 EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 121 WITFCQSIIS TLT.

[0111] hIL-2 has four helices connected by long, irregular loops. The N-terminal helix (H1) interacts with both the beta and gamma subunits, the third helix (H3) interacts with the beta subunit, and the C-terminal helix (H4) interacts with the gamma subunit. The alpha subunit interaction plane is formed by an irregular second helix (H2) and two long loops, one connecting H1 to H2 and the other connecting H3 and H4.

[0112] In some embodiments of the designed cytokines of this disclosure, the designed cytokines include a sequence isolated from or derived from an IL-15 polypeptide. In some embodiments, the IL-15 polypeptide is a wild-type polypeptide. In some embodiments, the IL-15 polypeptide includes the sequence of SEQ ID NO: 501. In some embodiments, the designed cytokines of this disclosure may be used in combination with an IL-15 polypeptide containing the IL-15 polypeptide having the sequence of SEQ ID NO: 501.

[0113] Natural human IL-15 (hIL-15) is (UniProtKB accession number P40933 and sequence number 501; signal sequence ;IL-15 chain; Glycosylated site Includes a sequence of disulfide bonds: [ka]

[0114] hIL-15 has a similar structure to hIL-2, but hIL-15 has less than 20% sequence identity with hIL-2.

[0115] In some embodiments, the cytokines designed in this disclosure may be used in combination with an IL-12 polypeptide comprising an IL-12 polypeptide which is subunit A having the following sequence: MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS.In some embodiments, the cytokines designed in this disclosure may be used in combination with an IL-12 polypeptide comprising an IL-12 polypeptide which is subunit B having the following sequence: MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSD PQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS.

[0116] In some embodiments, the cytokines designed in this disclosure may be used in combination with IL-18 polypeptides, including an IL-18 polypeptide having the sequence MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED.

[0117] In some embodiments, the cytokines designed in this disclosure may be used in combination with IL-21 polypeptides, including an IL-21 polypeptide having the sequence MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS.

[0118] In some embodiments, the cytokines designed in this disclosure may be used in combination with an IL-23 polypeptide comprising an IL-23 polypeptide which is subunit A having the following sequence: MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP.In some embodiments, the cytokines designed in this disclosure may be used in combination with an IL-23 polypeptide comprising an IL-23 polypeptide which is subunit B having the following sequence: MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSD PQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS.

[0119] In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 1 / 13, which is a polypeptide having the sequence MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 2, which is a polypeptide having the sequence MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 4, which is a polypeptide having the sequence MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRISHFSCLKDRHDFGFPEEEFDGHQFQKAQAISVLHEMIQQTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD.In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 5, which is a polypeptide having the sequence MALPFVLLMALVVLNCKSICSLGCDLPQTHSLSNRRTLMIMAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWDETLLDKFYTELYQQLNDLEACMMQEVGVEDTPLMNVDSILTVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSANLQERLRRKE. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 6, which is a polypeptide having the sequence MALPFALLMALVVLSCKSSCSLDCDLPQTHSLGHRRTMMLLAQMRRISLFSCLKDRHDFRFPQEEFDGNQFQKAEAISVLHEVIQQTFNLFSTKDSSVAWDERLLDKLYTELYQQLNDLEACVMQEVWVGGTPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSSSRNLQERLRRKE. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 7, which is a polypeptide having the sequence MARSFSLLMVVLVLSYKSICSLGCDLPQTHSLRNRRALILLAQMGRISPFSCLKDRHEFRFPEEEFDGHQFQKTQAISVLHEMIQQTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDFILAVRKYFQRITLYLMEKKYSPCAWEVVRAEIMRSFSFSTNLKKGLRRKD.In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 8, which is a polypeptide having the sequence MALTFYLLVALVVLSYKSFSSLGCDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFEFPQEEFDDKQFQKAQAISVLHEMIQQTFNLFSTKDSSAALDETLLDEFYIELDQQLNDLESCVMQEVGVIESPLMYEDSILAVRKYFQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSINLQKRLKSKE. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 10, which is a polypeptide having the sequence MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLGQMGRISPFSCLKDRHDFRIPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVRKYFQRITLYLIERKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 14, which is a polypeptide having the sequence MALPFALMMALVVLSCKSSCSLGCNLSQTHSLNNRRTLMLMAQMRRISPFSCLKDRHDFEFPQEEFDGNQFQKAQAISVLHEMMQQTFNLFSTKNSSAAWDETLLEKFYIELFQQMNDLEACVIQEVGVEETPLMNEDSILAVKKYFQRITLYLMEKKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD.In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 17, which is a polypeptide having the sequence MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGLPQEEFDGNQFQKTQAISVLHEMIQQTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNNLEACVIQEVGMEETPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSLSFSTNLQKILRRKD. In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon alpha polypeptides, including interferon alpha 21, which is a polypeptide having the sequence MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSATWEQSLLEKFSTELNQQLNDLEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE.

[0120] In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon beta polypeptides, including interferon beta polypeptides having the sequence MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN.

[0121] In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon-gamma polypeptides, including interferon-gamma polypeptides having the sequence MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ.

[0122] In some embodiments, the cytokines designed in this disclosure may be used in combination with interferon omega polypeptides, including an interferon omega polypeptide having the sequence MALLFPLLAALVMTSYSPVGSLGCDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGSS.

[0123] Exemplary designed cytokine sequences The exemplary designed cytokines of this disclosure include, but are not limited to, one or more polypeptides provided in any one of Tables 4-18. In some embodiments, the designed cytokines of this disclosure include polypeptides having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or any percentage identity with the designed cytokines of this disclosure. In some embodiments, the designed cytokines of this disclosure optionally include polypeptides having one or more sequences of helix H1, helix H4, helix H2, and helix H3 of the IL-2 / 15 polypeptides of this disclosure, where the helix order is H1, H4, H2, and then H3 from its amino terminus to its carboxyl terminus. In some embodiments, the IL-2 / 15 polypeptides of this disclosure include polypeptides having one or more sequences of helix H1, helix H4, helix H2, and helix H3 of the designed cytokines of this disclosure, where the helix order is H1, H4, H2, and then H3 from its amino terminus to its carboxyl terminus. In some embodiments, the cytokine designed by the Disclosure comprises a polypeptide having helix H1, helix H4, helix H2, and helix H3 of the cytokine designed by the Disclosure, from its amino terminus to its carboxyl terminus. In some embodiments, the cytokine designed by the Disclosure comprises a polypeptide having helix H1, helix H4, helix H2, and helix H3 of the cytokine designed by the Disclosure, in this order from its amino terminus to its carboxyl terminus, and optionally includes a loop connecting each helix having substantially the same topology and / or secondary structure as the loop in the cytokine designed by the Disclosure. [Table 4-1] [Table 4-2] [Table 4-3] [Table 5-1] Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 6-1 Table 6-2 Table 7-1 Table 7-2 Table 7-3 Table 7-4 Table 8-1 Table 8-2 Table 8-3 Table 8-4 Table 8-5 Table 8-6 Table 8-7 Table 8-8 Table 8-9 Table 9 Table 10-1 Table 10-2 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16-1 Table 16-2 Table 16-3 Table 16-4 Table 17-1 Table 17-2 [Table 17-3] [Table 18]

[0124] nucleic acid In some embodiments of this disclosure, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide,” “nucleotide sequence,” “polynucleotide,” and their grammatical variations are used interchangeably and refer to phosphate ester polymer forms of ribonucleosides (adenosine, guanosine, uridine, or cytidine; “RNA molecule”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecule”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters of either single-stranded or double-stranded helices. Single-stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double-stranded DNA-DNA helices, DNA-RNA helices, and RNA-RNA helices are possible.

[0125] In some embodiments of this disclosure, “nucleic acid,” particularly DNA or RNA molecules, refers only to the primary and secondary structures of the molecule and is not limited to any particular tertiary form. In some embodiments of this disclosure, “nucleic acid” includes, among other things, linear or round DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and double-stranded DNA found in chromosomes. When considering the structure of a particular double-stranded DNA molecule, sequences are provided in accordance with the usual convention of describing the sequence from left to right in a 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to messenger RNA or mRNA). Unless otherwise indicated, all nucleic acid sequences and nucleotide sequences are described from left to right in a 5' to 3' orientation.

[0126] Nucleotides are referred to by their commonly known single-letter symbols, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, "A" represents adenine, "C" represents cytosine, "G" represents guanine, "T" represents thymine, and "U" represents uracil.

[0127] In certain embodiments of this disclosure, the term “polynucleotide” refers to a polymer of nucleotides of any length or type, including ribonucleotides, deoxyribonucleotides, their analogues, or mixtures thereof. The term refers to the primary structure of the molecule. Therefore, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term also includes modified forms of polynucleotides, for example, by alkylation and / or capping, as well as unmodified forms. More specifically, “polynucleotides” include polydeoxyribonucleotides (containing 2-deoxy-D-ribose) and polyribonucleotides (containing D-ribose), including mRNA whether spliced ​​or unspliced, any other type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine base, as well as other polymers containing a nucleotide backbone, such as polyamides (e.g., peptide nucleic acids “PNA”) and polymorpholinopolymers, and other synthetic sequence-specific nucleic acid polymers (provided that the polymer contains nucleic acid bases in a configuration that allows for base pairing and base stacking as seen in DNA and RNA).

[0128] In some embodiments of this disclosure, the polynucleotide includes a DNA sequence. In some embodiments of this disclosure, the polynucleotide includes a vector or a DNA sequence inserted into a vector containing a DNA sequence.

[0129] In some embodiments of this disclosure, the polynucleotide comprises mRNA. In some embodiments, the mRNA is synthetic mRNA, or the mRNA comprises synthetic nucleotides.

[0130] In some embodiments of this disclosure, a polynucleotide comprises at least one non-natural, naturally occurring, or modified nucleic acid. In some embodiments, a polynucleotide comprises multiple non-natural, naturally occurring, or modified nucleic acids. In some embodiments, all nucleic acid bases of a particular class are non-natural, naturally occurring, or modified nucleic acids (for example, all uridines in a polynucleotide can be replaced with non-natural nucleic acid bases, such as 5-methoxyuridine).

[0131] In some embodiments of this disclosure, “expression” refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0132] In some embodiments of this disclosure, “expression vector” refers to a plasmid, virus, or other nucleic acid designed for polypeptide expression within a cell. The vector or construct is used to introduce a gene into a host cell, thereby causing the vector to interact with intracellular polymerases to express the protein encoded within the vector / construct. The expression vector may be extrachromosomal or intrachromosomal. The expression vector may include additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., a shuttle vector). The polynucleotides of this disclosure may be provided as components of the expression vector.

[0133] In some embodiments of this disclosure, “cloning vector” refers to a plasmid, virus, or other nucleic acid designed to produce copies of a polynucleotide. A cloning vector may include a transcription and translation initiation sequence, a transcription and translation termination sequence, and a polyadenylation signal. Such constructs typically include a 5'LTR, a tRNA binding site, a packaging signal, a second-strand DNA synthesis origin, and a 3'LTR, or a portion thereof. The polynucleotides of this disclosure may be provided as components of a cloning vector, which can be used to produce the polynucleotides of this disclosure.

[0134] In some embodiments of this disclosure, “encode” or such terms refer to the ability of a particular nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) to function as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Thus, a gene, cDNA, or RNA codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein within a cell or other biological system. Both the coding strand, in which the nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, used as a template for the transcription of the gene or cDNA, can be considered to code for a protein or other product of that gene or cDNA.

[0135] Unless otherwise specified, a nucleotide sequence that "encodes an amino acid sequence," for example, a polynucleotide that "encodes" a chimeric polypeptide as defined below in this disclosure, includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0136] Polypeptide Amino acids are referred to by either their commonly known three-letter or one-letter symbols, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acid residues are abbreviated as follows, with the abbreviations shown in parentheses: alanine (Ala;A), asparagine (Asn;N), aspartic acid (Asp;D), arginine (Arg;R), cysteine ​​(Cys;C), glutamic acid (Glu;E), glutamine (Gln;Q), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V).

[0137] Amino acid sequences are described from left to right, with the orientation from amino to carboxyl.

[0138] In some embodiments of this disclosure, “polypeptide” refers to a sequence of amino acid subunits. In some embodiments, “peptide” may be 50 amino acid length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid length or less. “Polypeptide” refers to proteins, polypeptides, and peptides of any length, size, structure, or function. “Polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length.

[0139] Polypeptides of this disclosure may include naturally occurring or synthetically produced or modified amino acids, e.g., any other operations or modifications such as disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or conjugation with labeling components. Also included in the definition are polypeptides in which, for example, one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids (e.g., including synthetic amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. Polypeptides also include gene products, homologs, orthologues, paralogs, fragments, and other equivalents, variants, and analogs as described above. Polypeptides may include a single polypeptide or multimolecular complexes such as dimers, trimers, or tetramers. Polypeptides of this disclosure may include single-chain polypeptides or polychain polypeptides. Most commonly, disulfide bonds are found in polychain polypeptides.

[0140] The polypeptides of this disclosure may include L-amino acid + glycine, D-amino acid + glycine (which exhibits resistance to L-amino acid-specific proteases in vivo), or combinations of D-amino acid + glycine and L-amino acid + glycine. The polypeptides described may be chemically synthesized or expressed recombinantly.

[0141] The polypeptides of this disclosure may include additional residues at the N-terminus, C-terminus, within the polypeptide, or in combination thereof, and these additional residues are not included in determining the identity percentage of the polypeptides of this disclosure to the reference polypeptide. Such residues may include, but are not limited to, tags, and may be any residue suitable for the intended use.

[0142] In some embodiments of this disclosure, “chimeric polypeptide” may refer to any polypeptide comprising a first amino acid sequence derived from a first source that is bound, covalently, or non-covalently to a second amino acid sequence derived from a second source, wherein the first and second sources are not the same. In some embodiments, the non-identical first and second sources may include two different biological entities, or two different proteins derived from the same biological entity, or a biological entity and a non-biological entity. A chimeric protein may include, for example, a protein derived from at least two different biological sources. In some embodiments, a chimeric polypeptide may include sequences from similar proteins derived from two different species. In some embodiments, a chimeric polypeptide may include sequences from different proteins derived from the same species. A biological source may include any non-synthetically produced nucleic acid or amino acid sequence (e.g., a genome or cDNA sequence, a plasmid or viral vector, a natural virion, or a variant or analog of any of the above). A synthetic source may include a protein or nucleic acid sequence produced chemically rather than by a biological system (e.g., solid-phase synthesis of amino acid sequences). A chimeric protein may also comprise a protein derived from at least two different synthetic sources, or a protein derived from at least one biological source and at least one synthetic source. A chimeric protein may also comprise a first amino acid sequence derived from a first source, covalently or noncovalently linked to a nucleic acid, or a small organic or inorganic molecule derived from any source. A chimeric protein may comprise a linker molecule between the first amino acid sequence and the second amino acid sequence, or between the first amino acid sequence and the nucleic acid, or between the first amino acid sequence and the small organic or inorganic molecule.

[0143] In some embodiments of this disclosure, a polypeptide “fragment” or “cleaved polypeptide” may refer to an amino acid sequence of a polypeptide that is shorter than the sequence of a reference polypeptide (which may be a naturally occurring sequence). Compared to the reference polypeptide, the fragment may include N-terminal and / or C-terminal deletions. Compared to the reference polypeptide, the fragment may include deletions in any part of the sequence, whether the deletions are contiguous or not. Polypeptides that have internal amino acid deletions compared to a naturally occurring sequence are also considered fragments. Various polypeptide components of this disclosure may be provided as fragments or cleaved forms of a reference protein.

[0144] In some embodiments of this disclosure, “functional fragment” may refer to a polypeptide fragment that retains the functionality of the polypeptide. In some embodiments, a functional fragment of a biologically active peptide (e.g., an enzyme) contains the catalytic domain of the enzyme, and therefore the functional fragment retains the ability to catalyze a biological action. Polypeptides of this disclosure may be provided as functional fragments or cleaved versions.

[0145] In some embodiments of this disclosure, “amino acid substitution” may mean replacing an amino acid residue present in a parent or reference sequence with another amino acid residue. In some embodiments, the parent or reference sequence includes a wild-type sequence. Amino acids may be substituted, for example, via chemical peptide synthesis or via recombination methods known in the art. For example, substitution of an amino acid residue with an alternative amino acid residue is performed by replacing a codon encoding a first amino acid with a codon encoding a second amino acid. Polypeptides of this disclosure having one or more amino acid substitutions may be provided.

[0146] In some embodiments of this disclosure, a “conservative amino acid substitution” is an amino acid substitution in which one amino acid residue is replaced by an amino acid residue having a chemically similar side chain. Families of amino acid residues having similar side chains are defined in the art, including acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the substitution is considered conservative. In some embodiments, amino acid chains can be conservatively replaced with chemically similar chains that differ in the order and / or composition of their side-chain family members. Various polypeptide components of this disclosure may be provided with conservative amino acid substitutions.

[0147] In some embodiments of the present disclosure, non-conservative amino acid substitutions include (i) substitutions in which a residue having a positively charged side chain (e.g., Arg, His, or Lys) is substituted for or by a negatively charged residue (e.g., Glu or Asp); (ii) substitutions in which a hydrophilic residue (e.g., Ser or Thr) is substituted for or by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) substitutions in which cysteine ​​or proline is substituted for or by any other residue; or (iv) substitutions in which a residue having a large hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) is substituted for or by a residue having a smaller side chain (e.g., Ala or Ser) or a residue without a side chain (e.g., Gly). Non-conservative amino acid substitutions may be provided for various polypeptide components of the present disclosure. The possibility that one of the aforementioned non-conservative substitutions can alter the functional properties of a protein correlates with the location of the substitution in a functionally important region of the protein; therefore, some non-conservative substitutions may have little or no effect on the biological properties. Various polypeptide components of this disclosure may be provided with non-conservative amino acid substitutions that do not significantly alter the functionality of the altered component.

[0148] Sequence analysis In some embodiments of this disclosure, “identity” refers to the conservation of total monomers between polymer molecules, for example, between polypeptide molecules or polynucleotide molecules. Without any additional qualifiers, “identity” means that the sequences are 100% identical (100% sequence identity), such as, for example, protein A being identical to protein B. To say that two sequences are, for example, “70% identical” is equivalent to saying that they have, for example, “70% sequence identity.”

[0149] If a position in a first sequence is occupied by the same amino acid as a corresponding position in a second sequence, then those molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences. Sequence comparison and determination of the percentage of identity between two sequences can be achieved using mathematical algorithms.

[0150] In a particular embodiment, the identity percentage (ID%) of a first amino acid (or nucleic acid) sequence to a second amino acid (or nucleic acid) sequence is calculated as ID% = 100(Y / Z), where Y is the number of amino acid (or nucleic acid base) residues that were scored as identical when the first and second sequences were aligned (by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the identity percentage of the first sequence to the second sequence is higher than the identity percentage of the second sequence to the first sequence.

[0151] The percentage of identity between two polypeptide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes. For example, gaps can be introduced into one or both of the first and second polypeptide sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes. In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. Then, the amino acids at the corresponding amino acid positions are compared.

[0152] The generation of sequence alignments for calculating sequence identity percentages is not limited to binary sequence comparisons driven solely by primary sequence data. It will also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystalline protein structure), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available at www.tcoffee.org, or, for example, on the European Bioinformatics Institute (EBI) website at ebi.ac.uk / Tools / psa. It will also be understood that the final alignments used to calculate sequence identity percentages can be automatically or manually selected.

[0153] Suitable software programs are available from various sources for both protein and nucleotide sequence alignment. One suitable program for determining sequence identity percentage is bl2seq, which is part of the BLAST program suite available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). B12seq performs the comparison between two sequences using either the BLASTN algorithm or the BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs include Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS bioinformatics program suite and are also available from EBI. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, ClustalX, or Clustal Omega), and MUSCLE. Different regions within a single polynucleotide or polypeptide target sequence aligned with a polynucleotide or polypeptide reference sequence may each have their own sequence identity percentage. Please note that array identity percentage values ​​are rounded to two decimal places. For example, values ​​from 80.11 to 80.14 are rounded down to 80.1, and values ​​from 80.15 to 80.19 are rounded up to 80.2. Please also note that length values ​​are always integers.

[0154] In some embodiments of this disclosure, “not naturally occurring” means a polypeptide or polynucleotide sequence that does not exist in nature. In some embodiments, a non-natural sequence does not exist in nature because the non-natural sequence is modified compared to a naturally occurring sequence. In some embodiments, a non-natural sequence does not exist in nature because the non-natural sequence is a combination of two known naturally occurring sequences that do not occur together in nature (e.g., a chimeric polypeptide). In some embodiments, a non-natural polypeptide is a chimeric polypeptide. In some embodiments, a polypeptide or polynucleotide does not exist in nature because the sequence contains a portion (e.g., a fragment) that cannot be found in nature, i.e., it is a novel sequence. Any of the polynucleotides described herein may be provided, for example, as a non-natural sequence having a modified sequence compared to a natural sequence, or as a polynucleotide linked to another polynucleotide in a manner not found in nature. Any of the polypeptides described herein may be provided, for example, as a non-natural sequence having a modified sequence compared to a natural sequence, or as a polypeptide linked to another polypeptide in a manner not found in nature.

[0155] treatment method In some embodiments of the present disclosure, the term “therapeutically effective” may mean conferring a beneficial effect to a recipient, for example, providing some reduction, alleviation, or decrease of at least one clinical symptom in the subject. The therapeutic effect of the present disclosure does not need to be complete or curative, as long as some benefit is provided to the subject. For example, a therapeutic regimen incorporating the cells of the present disclosure along with the polynucleotides, gene therapy vectors, or small molecules of the present disclosure may be structured so that the regimen as a whole is therapeutically effective.

[0156] In some embodiments of the present disclosure, the term “therapeutically effective amount” means a dose or quantity of the nucleic acids, vectors, polypeptides, compositions, pharmaceutical compositions, or cells of the present disclosure sufficient to impart a therapeutically effective benefit to a recipient. For example, the polynucleotides, gene therapy vectors, or cells of the present disclosure may be administered in a therapeutically effective amount. A subject who has been administered the polynucleotides, gene therapy vectors, or cells of the present disclosure may then be administered a therapeutically effective amount of small molecules of the present disclosure, i.e., sufficient to impart a beneficial effect to a recipient who has previously received a dose of the polynucleotides, gene therapy vectors, or cells.

[0157] cell therapy In some embodiments of this disclosure, the term “stem cell” may refer to an undifferentiated or partially differentiated cell that can differentiate into various cell types and proliferate indefinitely to produce more of the same stem cells.

[0158] In some embodiments of this disclosure, the term “pluripotent stem cell” (PSC) may refer to cells that can remain undifferentiated indefinitely and can differentiate into most, if not all, cells of the body.

[0159] In some embodiments of this disclosure, the term “induced pluripotent stem cell” (iPS or iPSC) may refer to pluripotent stem cells that can be generated directly from somatic cells. This includes, but is not limited to, specialized cells such as skin cells or blood cells derived from an adult.

[0160] In some embodiments of this disclosure, the term “multipotency” may refer to cells that can develop into two or more cell types, but are more limited than pluripotent cells. For example, adult stem cells and umbilical cord blood stem cells may be considered multipotent.

[0161] In some embodiments of this disclosure, the term “hematopoietic cells” may refer to cells arising from hematopoietic stem cells (hSCs). Hematopoietic stem cells as provided for in this disclosure include, but are not limited to, myeloid progenitor cells, lymphoid progenitor cells, megakaryocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, macrophages, platelets, monocytes, natural killer cells, T lymphocytes, B lymphocytes, and plasma cells.

[0162] In some embodiments of this disclosure, the terms “T lymphocyte” or “T cell” may refer to hematopoietic cells that typically develop within the thymus. T lymphocytes or T cells include, but are not limited to, natural killer T cells, regulatory T cells, helper T cells, cytotoxic T cells, memory T cells, gamma delta T cells, and mucosal invariant T cells.

[0163] In some embodiments of this disclosure, the term “mesenchyma” may refer to an animal tissue type comprising a protein and fluid mesh, i.e., loose cells embedded in an extracellular matrix. Mesenchyma give rise to the majority of the body’s connective tissue, including bone, cartilage, the lymphatic system, and the circulatory system.

[0164] In some embodiments of this disclosure, the term “mesenchymal cells” may refer to cells derived from mesenchymal tissue. In some embodiments, the cells of this disclosure may be mesenchymal cells.

[0165] In some embodiments of this disclosure, the term “mesenchymal stromal cells” (MSCs) may refer to spindle-shaped plastic-adherent cells isolated from bone marrow, adipose tissue, and other tissue sources that possess pluripotent differentiation potential in vitro. For example, mesenchymal stromal cells can differentiate into osteoblasts (osteocytes), chondrocytes (cartilage cells), myocytes (muscle cells), and adipocytes (fat cells that give rise to bone marrow adipose tissue). The term mesenchymal stromal cells has been suggested to replace the term “mesenchymal stem cells” in the scientific literature. In some cases, the cells of this disclosure may be mesenchymal stromal cells.

[0166] In some embodiments of this disclosure, “autologous cells” refers to cells obtained from the same individual that may be administered as therapy (these cells are self to the subject). Autologous cells in this disclosure include, but are not limited to, hematopoietic cells and stem cells such as hematopoietic stem cells.

[0167] In some embodiments of this disclosure, allogeneic cells are cells obtained from an individual that is not the intended recipient of the cells as therapy (these cells are allogeneic to the target). The allogeneic cells of this disclosure may be selected from immunologically compatible donors with respect to the target of the method of this disclosure. The allogeneic cells of this disclosure may be modified to produce “universal” allogeneic cells suitable for administration to any target without unintentional immunogenicity. The allogeneic cells of this disclosure include, but are not limited to, hematopoietic cells and stem cells such as hematopoietic cells and hematopoietic stem cells.

[0168] In some embodiments of this disclosure, the terms “transfect,” “transform,” or “transform” may refer to the process by which an exogenous nucleic acid is transferred to or introduced into a host cell. In some embodiments, a “transfected,” “transformed,” or “transformed” cell is a cell in which the exogenous nucleic acid or offspring of that cell has been transfected, transformed, or transformed.

[0169] In some embodiments of this disclosure, the term “cell therapy” may refer to providing or delivering cells to a recipient for therapeutic purposes.

[0170] formulation In some embodiments of this disclosure, the term “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form suitable for use in contact with human and animal tissues within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. For example, the small molecules, polynucleotides, polypeptides, gene therapy vectors, or cells of this disclosure may be administered as part of a composition together with other pharmaceutically acceptable components, including a pharmaceutically acceptable carrier.

[0171] In some embodiments of this disclosure, the term “pharmaceutically acceptable salt” refers to a derivative of a small molecule of the disclosure in which a given compound is converted to an acid or base salt thereof. Such pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Examples of pharmaceutically acceptable salts include conventional non-toxic or quaternary ammonium salts of parent compounds formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and isethionic acid. For example, the small molecules of this disclosure may be provided as pharmaceutically acceptable salts.

[0172] In some embodiments of the present disclosure, the term "excipient" refers to a pharmacologically inactive component that is not active in the body. For example, see Hancock, B.C., Moss, G.P., & Goldfarb, D.J. (2020). Handbook of pharmaceutical excipients. London: Pharmaceutical Press, the entire disclosure of which is incorporated herein by reference. The small molecules of the present disclosure can be mixed with pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preservatives, fillers, polymers, disintegrants, lubricants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, lubricants, acidifying agents, and dispensing agents, depending on the method of administration and the nature of the dosage form. Such components include pharmaceutically acceptable carriers and excipients that can be used to formulate oral dosage forms. Pharmaceutically acceptable carriers include water, ethanol, polyols, vegetable oils, fats, wax polymers including gelling and non-gelling polymers, and suitable mixtures thereof. Examples of excipients include starch, pregelatinized starch, Avicel, lactose, lactitol, sodium citrate, calcium carbonate, dicalcium phosphate, and lake blends. Examples of disintegrants include starch, arginine, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. For example, the small molecules, polynucleotides, gene therapy vectors, or cells of the present disclosure can be provided and administered in a composition containing a pharmaceutically acceptable excipient. Sequence [Table 19-1] [Table 19-2] [Table 19-3] [Table 19-4] [Table 19-5] [Table 19-6] [Table 19-7] [Table 19-8] [Table 19-9] [Table 19-10]

[0173] The identified consensus motifs in Table 19 contain some degree of variability within the identified sequences. In some embodiments, the identified consensus motifs in Table 19 may allow one or more nucleotide substitutions within the identified consensus motif. For example, within the consensus motifs in Table 19, "N" may allow any nucleotide including A, G, C, and T to be substituted at its position; "S" may allow either G or C to be substituted at its position; "R" may allow either A or G to be substituted at its position; and "W" may allow either A or T to be substituted at its position.

[0174] Table 20 below lists specific motifs that have been identified to reduce the variability of consensus motifs. Table 20 lists specific exemplary motifs present within the identified consensus motifs in Table 19. The transcription initiators and regulators of this disclosure may include one or more of the specific exemplary motifs in Table 19. [Table 20-1] [Table 20-2] [Table 20-3] Table 20-4 Table 20-5 Table 20-6 Table 20-7 Table 20-8 Table 20-9 Table 20-10 Table 20-11 Table 20-12 Table 20-13 Table 20-14 Table 20-15 Table 20-16 Table 20-17 Table 20-18 Table 21-1 Table 21-2 Table 21-3 Table 21-4 Table 21-5 Table 21-6 Table 21-7 Table 21-8 Table 21-9 Table 21-10 Table 21-11 Table 21-12 Table 21-13 Table 21-14 Table 21-15 Table 21-16 Table 21-17 Table 21-18 Table 21-19 Table 21-20 Table 21-21 Table 21-22 Table 21-23 Table 21-24 Table 21-25 Table 21-26 Table 21-27 Table 21-28 Table 21-29 Table 21-30 Table 21-31 Table 21-32 Table 21-33 Table 21-34 Table 21-35 Table 21-36 Table 21-37 Table 21-38 Table 21-39 Table 21-40 Table 21-41 Table 21-42 Table 21-43 Table 21-44 Table 22-1 Table 22-2 Table 22-3 Table 22-4 Table 22-5 Table 22-6 Table 22-7 Table 22-8 Table 22-9 Table 22-10 Table 22-11 Table 22-12 Table 22-13 Table 22-14 Table 22-15 Table 22-16 Table 22-17 Table 22-18 Table 22-19 Table 22-20 Table 22-21 Table 22-22 Table 22-23 Table 22-24 Table 22-25 Table 22-26 Table 22-27 Table 22-28 Table 22-29 Table 22-30 Table 22-31 Table 22-32 Table 22-33 Table 22-34 Table 22-35 Table 22-36 Table 22-37 Table 22-38 Table 22-39 Table 22-40 Table 22-41 Table 22-42 Table 22-43 Table 22-44 Table 22-45 Table 22-46 Table 22-47 Table 22-48 Table 22-49 Table 22-50 Table 22-51 Table 22-52 Table 22-53 Table 22-54 Table 22-55 Table 22-56 Table 22-57 Table 22-58 Table 22-59 Table 22-60 Table 22-61 Table 22-62 Table 22-63 Table 22-64 Table 22-65 Table 22-66 Table 22-67 Table 22-68 Table 22-69 Table 22-70 Table 22-71 Table 22-72 Table 22-73 Table 22-74 Table 22-75 Table 22-76 Table 22-77 Table 22-78 Table 22-79 Table 22-80 Table 22-81 Table 22-82 Table 22-83 Table 22-84 Table 22-85 Table 22-86 Table 22-87 Table 22-88 Table 22-89 Table 22-90 Table 22-91 Table 22-92 Table 22-93 Table 22-94 Table 22-95 Table 22-96 Table 22-97 Table 22-98 Table 22-99 Table 22-100 Table 22-101 Table 22-102 Table 22-103 Table 22-104 Table 22-105 Table 22-106 Table 22-107 Table 22-108 Table 22-109 Table 22-110 Table 22-111 Table 22-112 Table 22-113 Table 22-114 Table 22-115 Table 22-116 Table 22-117 Table 22-118 Table 22-119 Table 22-120 Table 22-121 Table 22-122 Table 22-123 Table 22-124 Table 22-125 Table 22-126 Table 22-127 Table 22-128 Table 22-129 Table 22-130 Table 22-131 Table 22-132 Table 22-133 Table 22-134 Table 22-135 Table 22-136 Table 22-137 Table 22-138 Table 22-139 Table 22-140 Table 22-141 Table 22-142 Table 22-143 Table 22-144 Table 22-145 Table 22-146 Table 22-147 Table 22-148 Table 22-149 Table 22-150 Table 22-151 Table 22-152 Table 22-153 Table 22-154 Table 22-155 Table 22-156 Table 22-157 Table 22-158 Table 22-159 Table 22-160 Table 22-161 Table 22-162 Table 22-163 Table 22-164 Table 22-165 Table 22-166 Table 22-167 Table 22-168 Table 22-169 Table 22-170 Table 22-171 Table 22-172 Table 22-173 Table 22-174 Table 22-175 Table 22-176 Table 22-177 Table 22-178 Table 22-179 Table 22-180 Table 22-181 Table 22-182 Table 22-183 Table 22-184 Table 22-185 Table 22-186 Table 22-187 Table 22-188 Table 22-189 Table 22-190 Table 22-191 Table 22-192 Table 22-193 Table 22-194 Table 22-195 Table 22-196 Table 22-197 Table 22-198 Table 22-199 Table 22-200 Table 22-201 Table 22-202 Table 22-203 Table 22-204 Table 22-205 Table 22-206 Table 22-207 Table 22-208 Table 22-209 Table 22-210 Table 22-211 Table 22-212 Table 22-213 Table 22-214 Table 22-215 Table 22-216 Table 22-217 Table 22-218 Table 22-219 Table 22-220 Table 22-221 Table 22-222 Table 22-223 Table 22-224 Table 22-225 Table 22-226 Table 22-227 Table 22-228 Table 22-229 Table 22-230 Table 22-231 Table 22-232 Table 22-233 Table 22-234 Table 22-235 Table 22-236 Table 22-237 Table 22-238 Table 22-239 Table 22-240 Table 22-241 Table 22-242 Table 22-243 Table 22-244 Table 22-245 Table 22-246 Table 22-247 Table 22-248 Table 22-249 Table 22-250 Table 22-251 Table 22-252 Table 22-253 Table 22-254 Table 22-255 Table 22-256 Table 22-257 Table 22-258 Table 22-259 Table 22-260 Table 22-261 Table 22-262 Table 22-263 Table 22-264 Table 22-265 Table 22-266 Table 22-267 Table 22-268 Table 22-269 Table 22-270 Table 22-271 Table 22-272

Table 22-273

Table 22-330

Table 22-332

Table 22-334

Table 22-339

Table 22-340

Table 22-343

Table 22-347

Table 22-356

Table 22-359

Table 22-360

Table 22-363

Table 22-367

Table 22-370

Table 22-378

Table 22-379

Table 22-380

[0175] definition In some embodiments of this disclosure, the term “Subject” refers to any mammal, including but not limited to humans.

[0176] Unless otherwise explicitly indicated by the context, the terms "a," "an," and "the" include their plural forms.

[0177] The term "and" is used interchangeably with "or" unless otherwise explicitly stated.

[0178] The term "and / or" should be interpreted as a specific disclosure of each of two specified features or components, whether or not they are accompanied by the other. Therefore, when "and / or" is used in phrases such as "A and / or B," it includes "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when "and / or" is used in phrases such as "A, B, and / or C," it is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0179] A numerical range includes the numbers that define the range. Where a range of values ​​is described, each integer value and each fraction thereof that lies between the enumerated upper and lower bounds of that range are also specifically disclosed as subranges between such values. The upper and lower bounds of any range may be independently included in or excluded from that range, and each range that includes either limit, neither limit, or both limits is also included in this disclosure. Thus, a range is understood to be an abbreviated representation of all values ​​within the range, including the enumerated endpoint. For example, the range 1 to 10 is understood to include any number, combination of numbers, or subrange selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0180] Where a value is explicitly stated, it should be understood that values ​​that are approximately the same quantity or amount as the stated value are also within the scope of this disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are disclosed individually, their combinations are also disclosed. Where any element of the disclosure is disclosed to have multiple substitutes, examples of that disclosure in which each substitute is excluded, either alone or in any combination with other substitutes are also disclosed herein, and two or more elements of the disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0181] Unless otherwise explicitly required by the context, words such as “including” and “including” should be interpreted in a comprehensive sense, not exclusive or exhaustive, throughout the modes for carrying out the invention and the claims, meaning “including, but not limited to.”

[0182] Singular and plural words also include plural and singular forms, respectively. Therefore, for example, when describing a gene of interest as described herein, this disclosure includes polynucleotides having a single gene of interest or polynucleotides having multiple genes of interest.

[0183] Units, prefixes, and symbols are shown in the forms recognized by their Systeme International de Unites (SI).

[0184] Headings are included herein for reference and to help locate various sections. These headings are not intended to limit the scope of the concepts described within them. Such concepts may be applicable throughout this specification.

[0185] Embodiment Embodiment 1. A designed cytokine comprising alpha-helix H1, H2, H3, and H4, From the amino terminus to the carboxyl terminus, The first loop (L1) connects H1 and H4, The second loop (L2) connects H4 and H2, The third loop (L3) connects H2 and H3, The polypeptide is the designed cytokine that binds to the IL-2 receptor βγ (IL-2Rβγ).

[0186] Embodiment 2. The cytokine designed according to Embodiment 1, wherein the polypeptide does not bind to IL-2 receptor alpha (IL-2Rα).

[0187] Embodiment 3. The polypeptide is (a) Sequences of sequence numbers 1 to 350, and (b) A designed cytokine according to Embodiment 1 or 2, comprising one or more sequences having at least 70% identity with the sequence of (a).

[0188] Embodiment 4. The cytokine designed according to Embodiment 1 or 2, wherein the polypeptide comprises the sequences of SEQ ID NOs: 1 to 38 or SEQ ID NOs: 150 to 350.

[0189] Embodiment 5. The designed cytokine according to Embodiment 1 or 2, wherein the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK.

[0190] Embodiment 6. The designed cytokine according to Embodiment 1 or 2, wherein the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0191] Embodiment 7. The designed cytokine according to Embodiment 1 or 2, wherein the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0192] Embodiment 8. The designed cytokine according to Embodiment 1 or 2, wherein the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTLTAGGSLSGDLKHLQNLSEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0193] Embodiment 9. The designed cytokine according to Embodiment 1 or 2, wherein the polypeptide comprises the sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSVDPEELAKELQKLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

[0194] Embodiment 10. A designed cytokine according to any one of Embodiments 1 to 9, wherein the polypeptide is operably linked to a targeting portion.

[0195] Embodiment 11. A cytokine designed according to any one of Embodiments 1 to 9, wherein the polypeptide includes a targeting moiety.

[0196] Embodiment 12. A cytokine designed according to any one of Embodiments 1 to 9, wherein the fusion protein comprises a polypeptide and a targeting moiety.

[0197] Embodiment 13. A cytokine designed according to any one of Embodiments 10 to 12, wherein the targeting portion binds to a component of the tumor microenvironment (TME).

[0198] Embodiment 14. A designed cytokine according to any one of Embodiments 10 to 13, wherein the targeting portion binds to one or more of the following: T cell surface glycoprotein CD8 (also known as differentiation antigen group 8), programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1; also known as differentiation antigen group 274 (CD274) or B7 homolog 1 (B7-H1) polypeptide), T cell immune receptor (TIGIT) having Ig and ITIM domains, cytotoxic T lymphocyte protein 4 (CTLA4), lymphocyte activation gene 3 protein (LAG3), and T cell immunoglobulin mucin receptor 3 (TIM3).

[0199] Embodiment 15. The targeting portion of the cytokine designed according to any one of Embodiments 10 to 13, wherein the targeting portion binds to CD8.

[0200] Embodiment 16. The targeting portion of the cytokine designed according to any one of Embodiments 10 to 13, wherein the targeting portion binds to PD-1.

[0201] Embodiment 17. A cytokine designed according to any one of Embodiments 10 to 13, wherein the targeting portion binds to PD-L1.

[0202] Embodiment 18. A designed cytokine according to any one of Embodiments 10 to 17, wherein the targeting portion comprises an antibody, an antibody mimetic, or a functional fragment thereof.

[0203] Embodiment 19. The cytokine designed according to Embodiment 18, wherein the targeting portion comprises one or more of the following: a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more heavy chain (VH) and light chain (VL) domains of an immunoglobulin (Ig) polypeptide or a gene encoding them, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and one or more nanobodies.

[0204] Embodiment 20. The cytokine designed according to Embodiment 18, wherein the targeting portion includes scFv.

[0205] Embodiment 21. The cytokine designed according to Embodiment 18, wherein the targeting portion includes VHH.

[0206] Embodiment 22. A designed cytokine according to any one of Embodiments 1 to 21, wherein the polypeptide is operably linked to a tether.

[0207] Embodiment 23. A cytokine designed according to any one of Embodiments 1 to 21, wherein the polypeptide includes a tether.

[0208] Embodiment 24. A designed cytokine according to any one of Embodiments 1 to 21, wherein the fusion protein comprises the polypeptide and a tether.

[0209] Embodiment 25. A cytokine designed according to any one of Embodiments 22 to 24, wherein the tether comprises one or more of a nucleic acid sequence, an amino acid sequence, or a small molecule.

[0210] Embodiment 26. A designed cytokine according to any one of Embodiments 22 to 25, wherein the tether comprises a sequence isolated from or derived from a transmembrane sequence.

[0211] Embodiment 27. The designed cytokine according to any one of Embodiments 22 to 26, wherein the tether comprises the sequence PLFIPVAVMVTAFSGLAFIIWLARRLKKGKK.

[0212] Embodiment 28. The designed cytokine according to any one of Embodiments 1 to 27, wherein the polypeptide is operably linked to a second cytokine or a second designed cytokine.

[0213] Embodiment 29. The designed cytokine according to any one of Embodiments 1 to 27, wherein the polypeptide comprises a second cytokine or a second designed cytokine.

[0214] Embodiment 30. The designed cytokine according to any one of Embodiments 1 to 27, wherein the fusion protein comprises the polypeptide and a second cytokine or a second designed cytokine.

[0215] Embodiment 31. The designed cytokine according to any one of Embodiments 28 to 30, wherein the second cytokine comprises a sequence isolated from or derived from one or more of the following: IL-2 polypeptide, IL-12 polypeptide, IL-15 polypeptide, IL-18 polypeptide, IL-21 polypeptide, IL-23 polypeptide, interferon alpha polypeptide, interferon beta polypeptide, interferon gamma polypeptide, and interferon omega polypeptide.

[0216] Embodiment 32. The designed cytokine according to Embodiment 31, wherein the polypeptide comprises a first targeting moiety and the second cytokine comprises a second targeting moiety.

[0217] Embodiment 33. The cytokine designed according to Embodiment 32, wherein the first targeting portion and the second targeting portion are identical.

[0218] Embodiment 34. The cytokine designed according to Embodiment 32, wherein the first targeting portion and the second targeting portion are not identical.

[0219] Embodiment 35. The designed cytokine according to any one of Embodiments 28 to 30, wherein the second designed cytokine comprises one or more sequences from SEQ ID NOs: 1 to 38 or 150 to 350.

[0220] Embodiment 36. The designed cytokine according to Embodiment 35, wherein the polypeptide comprises a first targeting moiety, and the second designed cytokine comprises a second targeting moiety.

[0221] Embodiment 37. The cytokine designed according to Embodiment 36, wherein the first targeting portion and the second targeting portion are identical.

[0222] Embodiment 38. The cytokine designed according to Embodiment 36, wherein the first targeting portion and the second targeting portion are not identical.

[0223] Embodiment 39. The designed cytokine according to Embodiment 31, wherein the polypeptide comprises a first tether and the second cytokine comprises a second tether.

[0224] Embodiment 40. The cytokine designed according to Embodiment 39, wherein the first tether and the second tether are identical.

[0225] Embodiment 41. The cytokine designed according to Embodiment 39, wherein the first tether and the second tether are not identical.

[0226] Embodiment 42. The designed cytokine according to Embodiment 35, wherein the polypeptide comprises a first tether and the second designed cytokine comprises a second tether.

[0227] Embodiment 43. The cytokine designed according to Embodiment 36, wherein the first tether and the second tether are identical.

[0228] Embodiment 44. The cytokine designed according to Embodiment 36, wherein the first tether and the second tether are not identical.

[0229] Embodiment 45. A nucleic acid encoding a fusion protein containing a designed cytokine as described in any one of Embodiments 1 to 44 or a designed cytokine as described in any one of Embodiments 1 to 44.

[0230] Embodiment 46. The nucleic acid according to Embodiment 45, further comprising a regulatory element capable of driving the expression of the designed cytokine.

[0231] Embodiment 47. The nucleic acid according to Embodiment 46, wherein the regulatory element includes a promoter.

[0232] Embodiment 48. The nucleic acid according to Embodiment 47, wherein the promoter includes a minimal promoter.

[0233] Embodiment 49. The nucleic acid according to Embodiment 48, wherein the minimal promoter comprises a sequence isolated from or derived from one or more of the following: minimal promoter-1 ("minP1"), YB-TATA, and human betaglobin.

[0234] Embodiment 50. The nucleic acid according to Embodiment 49, wherein minP1 comprises the sequence AGAGGGTATATAAAAGCTCGACTTCCAG.

[0235] Embodiment 51. The nucleic acid according to Embodiment 49, wherein the minimal promoter includes the sequence TAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC.

[0236] Embodiment 52. The nucleic acid according to Embodiment 49, wherein the minimal promoter comprises the sequence CTAGAGGGTATATAATGGGGGCCACTAGTCTACTACCAGAAAGCTTGGTACCGAGCTCGGATCCAGCCACC.

[0237] Embodiment 53. The nucleic acid according to any one of Embodiments 47 to 52, wherein the promoter is inducible.

[0238] Embodiment 54. The nucleic acid according to any one of Embodiments 47 to 53, wherein the regulatory element includes a response element.

[0239] Embodiment 55. The nucleic acid according to Embodiment 54, wherein the regulatory element comprises a non-coding or untranslated sequence isolated from or derived from one or more of NFAT, NFkB, REL, RELA, IRF2, GATA3, and ATF3.

[0240] Embodiment 56. The nucleic acid according to Embodiment 54, wherein the regulatory element comprises a non-coding or untranslated sequence isolated from or derived from the GATA3 gene, and optionally the GATA3 sequence comprises GTTATTCTCTCACGAGATCT.

[0241] Embodiment 57. The nucleic acid according to Embodiment 54, wherein the regulatory element comprises a non-coding or untranslated sequence isolated from or derived from RELA, and optionally the RELA sequence comprises GGGGATTTCCA.

[0242] Embodiment 58. The nucleic acid according to any one of Embodiments 47 to 57, wherein the response element includes a repeating sequence.

[0243] Embodiment 59. A vector comprising the nucleic acid described in any one of Embodiments 45 to 58.

[0244] Embodiment 60. The vector according to Embodiment 59, wherein the vector includes an expression vector.

[0245] Embodiment 61. The vector according to Embodiment 59, wherein the vector includes a delivery vector.

[0246] Embodiment 62. The vector according to any one of Embodiments 59 to 61, wherein the vector further comprises a sequence encoding an exogenous receptor.

[0247] Embodiment 63. The vector according to Embodiment 62, wherein the exogenous receptor includes an antigen-binding moiety.

[0248] Embodiment 64. The vector according to Embodiment 63, wherein the exogenous receptor comprises a T cell receptor (TCR).

[0249] Embodiment 65. The vector according to Embodiment 63, wherein the exogenous receptor comprises a chimeric antigen receptor (CAR).

[0250] Embodiment 66. A vector according to any one of Embodiments 62 to 65, wherein the antigen is expressed in or secreted within one or more of tumor cells, cancer cells, components of TME, and TME.

[0251] Embodiment 67. Cells containing the designed cytokine described in any one of Embodiments 1 to 44.

[0252] Embodiment 68. A cell containing nucleic acid as described in any one of Embodiments 45 to 58.

[0253] Embodiment 69. A cell containing the vector described in any one of Embodiments 59 to 66.

[0254] Embodiment 70. The cell according to any one of Embodiments 67 to 69, wherein the cell is a mammalian cell.

[0255] Embodiment 71. The cell according to Embodiment 70, wherein the cell is a human cell.

[0256] Embodiment 72. The cell according to any one of Embodiments 67 to 71, wherein the cell is a primary cell.

[0257] Embodiment 73. The cell according to any one of Embodiments 67 to 71, wherein the cell is a cultured cell.

[0258] Embodiment 74. The cells according to Embodiment 73, wherein the cultured cells are immortalized cells.

[0259] Embodiment 75. The cells according to any one of Embodiments 67 to 74, wherein the cells are present ex vivo or in vitro.

[0260] Embodiment 76. The cells according to any one of Embodiments 67 to 71, wherein the cells exist in vivo.

[0261] Embodiment 77. The cell according to any one of Embodiments 67 to 76, wherein the cell is an immune cell.

[0262] Embodiment 78. The cell according to Embodiment 77, wherein the cell is a stem cell or progenitor cell capable of producing the immune cell.

[0263] Embodiment 79. The cell according to Embodiment 78, wherein the stem cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), or a dedifferentiated immune cell.

[0264] Embodiment 80. The cells according to any one of Embodiments 67 to 79, wherein the immune cells are T lymphocytes (T cells), B lymphocytes (B cells), macrophages, or natural killer (NK) cells.

[0265] Embodiment 81. The cell according to any one of Embodiments 67 to 79, wherein the immune cell is a T cell.

[0266] Embodiment 82. The cell according to Embodiment 81, wherein the T cell is an alpha-beta T cell.

[0267] Embodiment 83. The cell according to Embodiment 81, wherein the T cell is a gamma delta T cell.

[0268] Embodiment 84. The cell according to any one of Embodiments 67 to 79, wherein the immune cell is an NK cell.

[0269] Embodiment 85. A composition comprising a cytokine designed according to any one of Embodiments 1 to 44.

[0270] Embodiment 86. A composition comprising the nucleic acid described in any one of Embodiments 45 to 58.

[0271] Embodiment 87. A composition comprising the vector described in any one of Embodiments 59 to 66.

[0272] Embodiment 88. A composition comprising the cells described in any one of Embodiments 67 to 84.

[0273] Embodiment 89. A pharmaceutical composition comprising (1) a cytokine designed according to any one of Embodiments 1 to 44, a nucleic acid according to any one of Embodiments 45 to 58, a vector according to any one of Embodiments 59 to 66, and a cell according to any one of Embodiments 67 to 84, and (2) a pharmaceutically acceptable carrier.

[0274] Embodiment 90. Use of a designed cytokine according to any one of Embodiments 1 to 44, a nucleic acid according to any one of Embodiments 45 to 58, a vector according to any one of Embodiments 59 to 66, a cell according to any one of Embodiments 67 to 84, or a pharmaceutical composition according to Embodiment 71 in the manufacture of a drug for the treatment of a disease or condition.

[0275] Embodiment 91. A cytokine designed according to any one of Embodiments 1 to 44, a nucleic acid according to any one of Embodiments 45 to 58, a vector according to any one of Embodiments 59 to 66, a cell according to any one of Embodiments 67 to 84, or a pharmaceutical composition according to Embodiment 89, for use in the treatment of a disease or condition.

[0276] Embodiment 92. The use according to Embodiment 90 or 91, wherein the disease or disorder includes cancer or a subtype thereof.

[0277] Embodiment 93. The use according to Embodiment 92, wherein the cancer or the subtype thereof includes liquid cancer.

[0278] Embodiment 94. The use according to Embodiment 92, wherein the cancer or its subtype includes hematological cancer.

[0279] Embodiment 95. The use according to Embodiment 92, wherein the cancer or its subtype includes solid tumors.

[0280] Embodiment 96. A method for treating a disease or disorder, comprising administering to a subject an effective amount of a designed cytokine according to any one of Embodiments 1 to 44, a nucleic acid according to any one of Embodiments 45 to 58, a vector according to any one of Embodiments 59 to 66, a cell according to any one of Embodiments 67 to 84, or a pharmaceutical composition according to Embodiment 89, wherein the severity of the signs or symptoms of the disease or disorder is reduced, thereby treating the disease or disorder.

[0281] Embodiment 97. A method for preventing a disease or disorder, comprising administering to a subject an effective amount of a designed cytokine according to any one of Embodiments 1 to 44, a nucleic acid according to any one of Embodiments 45 to 58, a vector according to any one of Embodiments 59 to 66, a cell according to any one of Embodiments 67 to 84, or a pharmaceutical composition according to Embodiment 89, wherein the onset or recurrence of signs or symptoms of the disease or disorder is delayed or suppressed, thereby preventing the disease or disorder.

[0282] Embodiment 98. The method according to Embodiment 96 or 97, wherein the disease or disorder includes cancer or a subtype thereof.

[0283] Embodiment 99. The method according to Embodiment 98, wherein the cancer or its subtype includes liquid cancer.

[0284] Embodiment 100. The method according to Embodiment 98, wherein the cancer or its subtype includes hematological cancer.

[0285] Embodiment 101. The method according to Embodiment 98, wherein the cancer or its subtype includes solid tumors. [Examples]

[0286] Example 1: General method Details of the ELISA experiment [Table 23] 50 micrograms (mg) of CD25-Fc (R&D1020-RL-050) were dissolved in 500 microliters of phosphate-buffered saline (PBS). This 100 mg / mL solution was frozen in 20 mL aliquots at -80°C.

[0287] 50 mg of CD25-His (R&D 10305-RL-050) was dissolved in 100 mL of PBS. This 500 mg / mL solution was frozen in 10 mL aliquots at -80°C.

[0288] Designed cytokines tagged with STII were captured from undiluted supernatant (100 mL / well) on streptactin plates at room temperature for 1 hour. The plates were washed with 4 × 200 mL of PBS-T.

[0289] Primary antibodies were prepared and added to plates: the dilution buffer contained casein block and PBS-T (1:1). 2 mg / mL of CD25-Fc was added to casein block + PBS-T (1:1), and 100 mL of 100 mg / mL of CD25-Fc was diluted in 5 mL of dilution buffer. 0.5 mg / mL of CD25-Fc was added to casein block + PBS-T (1:1), and then 25 mL of 100 mg / mL of CD25-Fc was diluted in 5 mL of dilution buffer. scIL12-anti-PDL1-scFv-STII+IL12Rb2-Fc was mixed at specific concentrations in casein block + PBS-T (1:1). The mixture was incubated at room temperature for 1 hour.

[0290] The plates were washed with PBS-T in 4 × 200 mL solutions.

[0291] Prepare the secondary antibody and add it to the plate. Prepare the secondary antibody composition by mixing protein G and HRP (1:5000) in casein block + PBS-T (1:1). Incubate at room temperature for 45 minutes.

[0292] The plates were washed with PBS-T in 4 x 250 mL solutions.

[0293] 75 mL of TMB-Ultra was added and incubated for approximately 5 minutes.

[0294] 75 mL of 2 M HCl was added.

[0295] Absorbance was measured at 450 nm (A450) using a plate reader with an ELISA program. HEK Blue - Experiment Details [Table 24] [Table 25]

[0296] Detach the cells using 10 mL of PBS / flask, count the cells, centrifuge at 300 × g for 5 minutes, seed 1e6 cells into a new flask, resuspend the cell pellet in DMEM + 10% FBS to a density of 280 K / mL, and plate 180 mL of cells / well into a flat-bottom 96-well plate.

[0297] Dilutions of the supernatant were prepared in a 96-well PCR plate:

[0298] Diluted supernatants were prepared in these plates. 120 mL / well of undiluted supernatant was added to the PCR plates.

[0299] For a 1:10 dilution, 20 mL of this solution was transferred to 180 mL of Expi293 medium.

[0300] For the final dilution of 1:50, 40 mL of 1:10 solution was transferred to 160 mL of Expi293 medium.

[0301] For a 1:100 dilution, 100 mL of 1:50 solution was transferred to 100 mL of Expi293.

[0302] To achieve a 1:200 dilution, 100 mL of 1:100 solution was transferred to 100 mL of Expi293.

[0303] For a 1:400 dilution, 100 mL of 1:200 solution was transferred to 100 mL of Expi293.

[0304] Designed cytokine preparation and dilution: 100 mM acetic acid was prepared from glacial acetic acid (17.4 M). 57.5 μL of glacial acetic acid was diluted in 10 mL of ultrapure water. Sterile filtration was performed. 100 mg of IL-2 (R&D 10453-IL-100) was dissolved in 200 mL of 100 mM acetic acid to prepare a 500 mg / mL solution.

[0305] Cytokines designed to range from 1,000 nanograms (ng / mL) to 0.01 ng / mL per milliliter were used.

[0306] IL2 preparation at 500 mg / mL. Diluted 1:50 in Expi293 medium, then diluted 5-fold serially (20 mL transferred to 80 mL of Expi293). For the assay, 20 mL of this was diluted to a final volume of 200 mL, and the concentration ranges were 1000 ng / mL > 200 ng / mL > 40 ng / mL > 8 ng / mL > 1.6 ng / mL > 0.32 ng / mL > 0.06 ng / mL > 0.012 ng / mL.

[0307] Plates were incubated overnight at 37°C with serial dilutions of the designed cytokine or WT IL-2. 180 mL of Quantiblue reagent was plated per well of a 96-well flat-bottom plate. 20 mL of culture supernatant was added to each well. Incubated at 37°C for 30 minutes. A640 was measured using a plate reader with the Quantiblue program.

[0308] Octet:Binding to IL2Ra Octet setup: ProA tip, Octet buffer (HBS-EP + 0.25% BSA), 2.6 mg / mL IL2Ra-Fc for capture, and a Zeba column (0.5 mL, 7 kDa) for buffer exchange of the protein to HBS-EP (without BSA).

[0309] Protein Octet dilution was prepared in Octet buffer (HBS-EP + 0.25% BSA).

[0310] During the experimental setup, the tips were hydrated in 200 mL of buffer / well for more than 10 minutes (one row of tips was required).

[0311] Assay plates (black 96-well plates) were prepared using protein diluents and buffer. 200 mL of sample / buffer was used per well (octet buffer was added in the baseline step). [Table 26]

[0312] Two 20 mL aliquots of 100 mg / mL IL2Ra-Fc were thawed. A 1.5 mL loading solution was prepared in Octet buffer, resulting in an IL2Ra-Fc concentration of 2.6 mg / mL.

[0313] For the designed cytokine columns, a 5-fold serial dilution was performed according to the layout table above.

[0314] A maximum concentration of 300 mL was prepared in the plate, 50 mL was transferred to 200 mL of buffer, and 50 mL was removed at the final concentration, so that the final volume is equal between samples.

[0315] For the 300 nM sample in row G, 20 mL of the highest concentration sample (3 mM, row A) was transferred to 180 mL of Octet buffer. Then, 30 mL of the 3 mM row A sample was further removed, so that the final volume was equal for the entire sample. [Table 27] [Table 28]

[0316] Octet: Binding to IL2Rb / g (beta / gamma) Octet setup information: ProA tip, Octet buffer (HBS-EP + 0.25% BSA), 12 ug / mL IL2Rb / g-Fc for capture, and a Zeba column (0.5 mL, 7 kDa) for buffer exchange of the protein to HBS-EP (without BSA).

[0317] Protein Octet dilution was prepared in Octet buffer (HBS-EP + 0.25% BSA).

[0318] During experimental setup, the tips were hydrated in 200 mL of buffer / well for over 10 minutes (5 rows of tips were required - a new set of tips for each construct).

[0319] Assay plates (black 96-well plates) were prepared using protein diluents and buffer. 200 mL of sample / buffer was used per well (octet buffer was added in the baseline step).

[0320] In this test, an Evap cover was used. [Table 29]

[0321] Two 15 mL aliquots of 600 mg / mL IL2Rb / g-Fc were thawed. A 1.5 mL loading solution was prepared in Octet buffer, resulting in an IL2Rb / g-Fc concentration of 12 mg / mL.

[0322] The designed cytokine columns were subjected to 3-fold serial dilutions according to the layout table above.

[0323] A 1 mL solution of the highest concentration was prepared in Eppendorf foam, and then 300 mL was transferred to the top well of the plate. For a 3-fold dilution, 100 mL was transferred to 200 mL of buffer, and 100 mL of the final concentration solution was removed, so that the final volumes were equal between samples. [Table 30] [Table 31] There was a problem with the Evap cover of the last two structures described above, and the experiment failed, so the inventors need to set up the experiment again for these structures.

[0324] During experimental setup, the tips were hydrated in 200 mL of buffer / well for over 10 minutes (two rows of tips are required - a new set of tips for each construct).

[0325] Designed cytokine proteins (in HBS-EP) with exchanged buffer were used.

[0326] The same settings as for repeated measures were used, except that an Evap cover was not used. [Table 32]

[0327] Autologous tryptophan fluorescence material: [Table 33]

[0328] Experiment details: The designed cytokine proteins were diluted to 0.2 mg / mL in Gibco PBS at pH 7.2 (with one exception, the gray area). [Table 34]

[0329] 25 μL of diluted protein was added to each well of a round-bottomed black 96-well plate.

[0330] As shown below, 75 μL of diluted GndHCl was added to 8 M GndHCl, and the concentrations before and after mixing are listed below. [Table 35]

[0331] Cover and incubate the plate at 37°C for 45 minutes.

[0332] Fluorescence is measured using a BioTek Synergy H1 plate reader as follows. ●37C ●Excitation at 280nm ● Orbital mixing 30 seconds ● Scans 310-450nm with a 5nm width.

[0333] Example 2: Designed cytokine signaling The designed cytokine was engineered to activate IL2R via IL2Rb and a common gamma chain receptor, but not by binding to CD25(IL2Ra). This study demonstrates the ability of the designed cytokine, expressed transiently in Expi293 cells, to bind to CD25(IL2Ra) and activate HEK-Blue reporter strains expressing either [IL2Ra, IL2Rb, and the gamma subunit common to IL-2 and IL-15] or [IL2Rb, and the gamma subunit common to IL-2 and IL-15] by ELISA.

[0334] Referring to Figure 5A and the designed cytokines having Sequence IDs 1-38, respectively, the data demonstrate that, compared to WT IL-2, the exemplary designed cytokines of this disclosure do not bind or signal via the alpha subunit of the IL-2 receptor (IL-2R).

[0335] Referring to Figure 5A and the designed cytokines having Sequence IDs 1-38, respectively, the data demonstrate that 33 of the 38 exemplary designed cytokines of this disclosure retain IL-2 / 15Rβγ signaling when compared to WT IL-2.

[0336] Based on this initial screening, we identified a number of engineered cytokines that 1) are expressed in Expi293 cells, 2) activate IL2R and CD122 / 132 only in the HEK-Blue assay, and 3) no longer bind to CD25 (despite relatively good expression). These designs were marked for further development. Among these, engineered cytokine No. 39 and engineered cytokine No. 40 appear particularly interesting because they are the proteins most highly expressed by anti-STII jess. Engineered cytokine No. 39 and engineered cytokine No. 40 showed good activation in the HEK-Blue assay, and they did not bind to CD25. The fact that these proteins are also closely related and therefore exhibit similar behavior supports a structure-function relationship.

[0337] Regarding binding to IL2Rb / g, the inventors compared the designed cytokine with WT IL2 (both "internal" IL-2 and commercially available IL2).

[0338] Referring to Figure 5B, the data demonstrate that the designed cytokines bind to IL2Rb / g with nanomolar affinity.

[0339] IL2Ra binding: No detectable binding of the designed cytokine to IL2Ra was observed, even at a maximum of 3 μM. In contrast, WT IL2 bound to IL2Ra and had a Kd of approximately 15 nM. Therefore, this experiment confirms that our WT IL2 bound to IL2Ra as expected, while the designed cytokine did not show binding.

[0340] IL2Rb / g binding: The engineered cytokines bind to IL2Rb / g with nanomolar affinity, similar to WT IL2 binding. Engineered cytokine No. 169 has a Kd of approximately 1.6 nM, compared to approximately 0.4 nM for WT IL2. Engineered cytokine No. 153 has a lower affinity of 3.9 nM, which is consistent with its lower activity in functional assays.

[0341] Example 3: The designed cytokine shows increased stability compared to WT IL-2. To test whether the designed cytokines were stable compared to WT IL-2, changes in autologous tryptophan fluorescence were measured at increased concentrations of the denaturant GndHCl. Tryptophan fluorescence is highly sensitive to its environment, and solvent exposure causes a "redshift" in the tryptophan emission spectrum upon excitation at 280 nm.

[0342] Regarding Figure 12, the designed cytokines exhibit lower autofluorescence compared to WT IL-12, and therefore higher stability upon contact with the denaturing agent.

[0343] Other Embodiments While this disclosure is described in some detail by illustrations and examples for the purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications can be made. References to “this disclosure,” etc., are intended to refer to any of the many diverse embodiments or aspects of this disclosure, and not to limit this disclosure to a single embodiment or aspect. Where used throughout this disclosure, the terms “aspect” and “embodiment” are interchangeable. Features discussed in relation to “certain,” “some,” or “other” aspects or embodiments of this disclosure may be found in any embodiment of this disclosure, but in these cases, the features may be considered preferred features in those emphasized embodiments.

[0344] The embodiments and examples for carrying out the invention should not be construed as limiting the scope of this disclosure to the embodiments and examples described herein, but rather as encompassing all modifications and substitutions that are included in the true scope and spirit of this disclosure.

Claims

1. It is a polypeptide, (a) A sequence comprising alpha helix H1, H2, H3, and H4, From the amino terminus to the carboxyl terminus, The first loop (L1) connects H1 and H4, The second loop (L2) connects H4 and H2, The third loop (L3) connects H2 and H3, The polypeptide has the sequence that binds to the IL-2 receptor beta-gamma (IL-2Rβγ), (b) The polypeptide comprising a targeting moiety that binds to a differentiation antigen group 8 (CD8) glycoprotein.

2. The polypeptide according to claim 1, wherein the polypeptide does not bind to IL-2 receptor alpha (IL-2Rα).

3. The arrangement in (a) above is (i) Sequences of sequence numbers 1 to 350, and (ii) A sequence having at least 70% identity with the sequence in (i), The polypeptide according to claim 1 or 2, comprising one or more of the above.

4. The polypeptide according to claim 1 or 2, wherein the sequence in (a) includes the sequence of sequence numbers 1 to 38 or 150 to 350.

5. The polypeptide according to claim 1 or 2, wherein the sequence of (a) comprises the sequence APTSSSTKKTQLQLEHLLLLDLQMILNGINNMNADPPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK.

6. The polypeptide according to claim 1 or 2, wherein the sequence of (a) comprises the sequence APTSSSTKKTQLQLEHLLLLDLQMILNGINNMNADPPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

7. The polypeptide according to claim 1 or 2, wherein the sequence of (a) comprises the sequence APTSSSTKKTQLQLEHLLLLDLQMILNGINNMNADPPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

8. The polypeptide according to claim 1 or 2, wherein the sequence of (a) comprises the sequence APTSSSTKKTQLQLEHLLLLDLQMILNGINNMNADPPELVEFFLNRWITFCQSIISTLTAGGSLSGDLKHLQNLSEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK.

9. The polypeptide according to claim 1 or 2, wherein the sequence of (a) comprises the sequence APTSSSTKKTQLQLEHLLLLDLQMILNGINNMNADPPELVEFLNRWITFCQSIISTGSVDPEELAKELQKLEEEELKPLEEVLNLAQSKNFHLRPRDLISINNIVVLELK.

10. The polypeptide according to claim 1 or 2, wherein the sequence of (a) comprises APTSSSSTKKTQLQLEHLLLLDLQMILNGINNMNADPPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLELK.

11. The polypeptide according to any one of claims 1 to 10, wherein the sequence of (a) is operably linked to the targeting portion of (b).

12. The polypeptide according to any one of claims 1 to 11, further comprising a linker that operably links the sequence of (a) to the targeting portion of (b).

13. The polypeptide according to claim 12, wherein the linker comprises deoxyribonucleic acid, ribonucleic acid, amino acids, or any combination thereof.

14. The polypeptide according to claim 12 or 13, wherein the linker comprises one or more of glycine (G) and serine (S).

15. The polypeptide according to any one of claims 12 to 14, wherein the linker comprises a GGGGS sequence or any number of repeats thereof.

16. The polypeptide according to any one of claims 1 to 15, wherein the targeting portion comprises an antibody, an antibody mimetic, a protein scaffold, or a functional fragment thereof.

17. The polypeptide according to any one of claims 1 to 16, wherein the targeting portion comprises a humanized form of antibody, an antibody mimetic, a protein scaffold, or a functional fragment thereof.

18. The polypeptide according to any one of claims 1 to 17, wherein the targeting portion comprises one or more of a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more heavy chain (VH) and light chain (VL) domains of an immunoglobulin (Ig) polypeptide or a gene encoding them, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and one or more nanobodies.

19. The polypeptide according to any one of claims 1 to 18, wherein the targeting portion comprises one or more humanized forms of a monoclonal antibody, an antigen-binding fraction (Fab), a single-chain variable fraction (scFv), a domain antibody, one or more heavy chain (VH) and light chain (VL) domains of an immunoglobulin (Ig) polypeptide or an encoding gene, a heavy chain antibody (VH or VHH), a camelid or camelid-like structured antibody, and one or more nanobodies.

20. The polypeptide according to any one of claims 1 to 19, wherein the targeting portion includes scFv.

21. The polypeptide according to any one of claims 1 to 19, wherein the targeting portion includes VHH.

22. The polypeptide according to any one of claims 1 to 19, wherein the targeting portion includes humanized VHH.

23. The polypeptide according to any one of claims 1 to 22, wherein the targeting portion includes one sequence from sequence numbers 9791 to 9800, or a sequence having at least 90% identity thereto.

24. The polypeptide according to any one of claims 1 to 22, wherein the targeting portion includes the sequence EVQLVESGGGLLVQPGGSLRLSCAASGFTFDDDYAMGWFRQAPGKGREGVSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYDMDYWGQGTQVTVSS, or a sequence having at least 90% identity thereto.

25. The polypeptide according to any one of claims 1 to 24, wherein the polypeptide comprises the sequence APTSSSSTKKTQLQLEHLLLLDLQMILNGINNMNADPELVEFLNRWITFCQSIISTGSLEDLKHLQALEEELKPLEEVLNLAQSKNFHLNPRDLISNINVLVLERKGGGGGSGGGGGSGGGGGSEVQLVESGGGGLVQPGGSLRLSCAASGFTFDDYAMGWFRQAPGKGREGVSSCIRVSDGSTYYADSVKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYCAAGSLYTCVQSIVVVPARPYYDMDYWGQGTQVTVSS.

26. The polypeptide according to any one of claims 1 to 25, further comprising a signal sequence.

27. The polypeptide according to claim 26, wherein the signal sequence comprises MVLQTQVFISLLLWISGAYG.

28. The polypeptide according to any one of claims 1 to 27, wherein the polypeptide is operably linked to a second polypeptide.

29. The polypeptide according to any one of claims 1 to 27, wherein the polypeptide further comprises a second polypeptide.

30. The polypeptide according to any one of claims 1 to 29, wherein the second polypeptide comprises the polypeptide according to any one of claims 1 to 27 or any component thereof.

31. The polypeptide according to any one of claims 1 to 29, wherein the second polypeptide comprises a sequence isolated from or derived from a cytokine or any nucleic acid sequence encoding the cytokine.

32. The cytokines include interleukin-1 polypeptide (IL-1), interleukin-2 polypeptide (IL-2), interleukin-3 polypeptide (IL-3), interleukin-4 polypeptide (IL-4), interleukin-5 polypeptide (IL-5), interleukin-6 polypeptide (IL-6), interleukin-7 polypeptide (IL-7), interleukin-8 polypeptide (IL-8), interleukin-9 polypeptide (IL-9), interleukin-10 polypeptide (IL-10), interleukin-11 polypeptide (IL-11), interleukin-12 polypeptide (IL-12), interleukin-13 polypeptide (IL-13), interleukin-14 polypeptide (IL-14), interleukin-15 polypeptide (IL-15), interleukin-16 polypeptide (IL-16), interleukin-17 polypeptide (IL-17), interleukin-18 polypeptide (IL-18), and interleukin-19 polypeptide (IL-16). The polypeptide according to claim 31, comprising one or more of the following: L-19), interleukin 20 polypeptide (IL-20), interleukin 21 polypeptide (IL-21), interleukin 22 polypeptide (IL-22), interleukin 23 polypeptide (IL-23), interleukin 24 polypeptide (IL-24), interleukin 25 polypeptide (IL-25), interleukin 26 polypeptide (IL-26), interleukin 27 polypeptide (IL-27), interleukin 28 polypeptide (IL-28), interleukin 29 polypeptide (IL-29), interleukin 30 polypeptide (IL-30), interleukin 31 polypeptide (IL-31), interleukin 32 polypeptide (IL-32), interleukin 33 polypeptide (IL-33), interleukin 34 polypeptide (IL-34), interleukin 35 polypeptide (IL-35), and interleukin 36 polypeptide (IL-36).

33. The polypeptide according to any one of claims 28 to 32, wherein the second polypeptide comprises a sequence isolated from or derived from an interferon or any nucleic acid sequence encoding the interferon.

34. The polypeptide according to claim 33, wherein the interferon comprises one or more of interferon type I, interferon type II, and interferon type III.

35. The polypeptide according to claim 33, wherein the interferon comprises one or more of interferon alpha (IFN-α), interferon beta (IFN-β), interferon epsilon (IFN-ε), interferon kappa (IFN-κ), interferon omega (IFN-ω), and interferon gamma (IFN-γ).

36. The polypeptide according to any one of claims 28 to 35, wherein the targeting portion of (b) is the first targeting portion, and the second polypeptide includes the second targeting portion.

37. The polypeptide according to claim 36, wherein the first targeting portion and the second targeting portion contain the same sequence.

38. The polypeptide according to claim 36, wherein the first targeting portion and the second targeting portion do not contain the same sequence.

39. A nucleic acid encoding a polypeptide according to any one of claims 1 to 38 or any functional component thereof.

40. The nucleic acid according to claim 39, further comprising a regulatory element, wherein when introduced into a mammalian cell, the regulatory element drives the expression of the nucleic acid.

41. The nucleic acid according to claim 40, wherein the regulatory element includes a promoter.

42. The nucleic acid according to claim 40, wherein the regulatory element includes a minimal promoter.

43. The nucleic acid according to claim 41 or 42, wherein the promoter comprises the sequence AGAGGGTATATATAGGAAGTCCGAG.

44. The nucleic acid according to any one of claims 40 to 43, wherein the regulatory element includes an inductive promoter.

45. The nucleic acid according to any one of claims 40 to 44, wherein the regulatory element includes a response element.

46. The nucleic acid according to claim 45, wherein the regulatory element comprises one or more repeats of the response element.

47. The nucleic acid according to any one of claims 40 to 46, wherein the regulatory error includes the sequence GGGGACTTCCCGCCTGGGGACTACTTCTCCCGCCTGGGGACTACTTCTCCCGCCTGGGGACTACTTCTCCCGCCTGGGGACTACTTCTCCCGCCTGGGGACTACTTCTCCCGCCTGGGGACTACTTCTCCCGCCTGGGGACTACTTCTCCCGCTAACGAGAGGTATAATAGGAAGTCGAATTCCAAG.

48. The nucleic acid according to any one of claims 40 to 47, wherein the regulatory element further comprises a sequence encoding one or more of the following: a five-prime untranslated region, a three-prime untranslated region, an intron, an exon, and an enhancer.

49. The nucleic acid according to any one of claims 40 to 47, wherein the regulatory element further comprises a sequence encoding a 5-prime untranslated region (5'UTR).

50. The nucleic acid according to claim 48 or 49, wherein the 5'UTR comprises the sequence ACTCTTCTGGTCCCCACAGAACTCAGAGAGAACCCCGCTAGCGCGCGCACC.

51. The nucleic acid according to any one of claims 40 to 50, wherein the regulatory element comprises a sequence isolated from or derived from a mammalian sequence.

52. The nucleic acid according to any one of claims 40 to 51, wherein the regulatory element comprises a sequence isolated from or derived from a human sequence.

53. The nucleic acid according to any one of claims 40 to 52, wherein the regulatory element includes a recombinant sequence.

54. A vector comprising the nucleic acid described in any one of claims 39 to 53.

55. The vector according to claim 54, wherein the vector includes an expression vector.

56. The vector according to claim 54, wherein the vector includes a delivery vector.

57. The vector according to claim 56, wherein the delivery vector is a non-viral vector.

58. The vector according to claim 56, wherein the delivery vector is a viral vector.

59. The vector according to claim 58, wherein the viral vector is a lentiviral vector.

60. The vector according to any one of claims 54 to 59, wherein the vector further comprises a sequence encoding an exogenous receptor.

61. The vector according to claim 60, wherein the exogenous receptor includes an antigen-binding moiety.

62. The vector according to claim 60 or 61, wherein the exogenous receptor comprises a T cell receptor (TCR) or a functional component thereof.

63. The vector according to claim 60 or 61, wherein the exogenous receptor comprises a chimeric antigen receptor (CAR) or a functional component thereof.

64. The vector according to any one of claims 61 to 63, wherein the antigen-binding portion binds to tumor cells, cancer cells, components of the tumor microenvironment (TME), or antigens expressed within or secreted by one or more TMEs.

65. The vector according to any one of claims 61 to 63, wherein the antigen-binding portion binds to an antigen expressed in one or more cancer cells or secreted therefrom.

66. The vector according to claim 65, wherein the liquid tumor comprises the cancer cells.

67. The vector according to claim 65, wherein the solid tumor comprises the cancer cells.

68. The vector according to any one of claims 61 to 67, wherein the antigen-binding portion is bound to a mesothelin (MSLN) polypeptide or a sequence thereof.

69. A cell comprising the polypeptide according to any one of claims 1 to 44.

70. A cell comprising nucleic acid according to any one of claims 45 to 58.

71. A cell comprising the vector according to any one of claims 59 to 66.

72. The cell according to any one of claims 67 to 69, wherein the cell is a mammalian cell.

73. The cell according to claim 70, wherein the cell is a human cell.

74. The cell according to any one of claims 67 to 71, wherein the cell is a primary cell.

75. The cell according to any one of claims 67 to 71, wherein the cell is a cultured cell.

76. The cell according to claim 73, wherein the cultured cell is an immortalized cell.

77. The cells according to any one of claims 67 to 74, wherein the cells exist ex vivo or in vitro.

78. The cell according to any one of claims 67 to 71, wherein the cell exists in vivo.

79. The cell according to any one of claims 67 to 76, wherein the cell is an immune cell.

80. The cell according to claim 77, wherein the cell is a stem cell or progenitor cell capable of producing the immune cell.

81. The cell according to claim 78, wherein the stem cell is a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), or a dedifferentiated immune cell.

82. The cell according to any one of claims 67 to 79, wherein the immune cell is a T lymphocyte (T cell), a B lymphocyte (B cell), a macrophage, or a natural killer (NK) cell.

83. The cell according to any one of claims 67 to 79, wherein the immune cell is a T cell.

84. The cell according to claim 81, wherein the T cell is an alpha-beta T cell.

85. The cell according to claim 81, wherein the T cell is a gamma delta T cell.

86. The cell according to any one of claims 67 to 79, wherein the immune cell is an NK cell.

87. A composition comprising the polypeptide according to any one of claims 1 to 44.

88. A composition comprising nucleic acid according to any one of claims 45 to 58.

89. A composition comprising the vector according to any one of claims 59 to 66.

90. A composition comprising the cells according to any one of claims 67 to 84.

91. A pharmaceutical composition comprising (1) a polypeptide according to any one of claims 1 to 38, a nucleic acid according to any one of claims 39 to 53, a vector according to any one of claims 54 to 68, and a cell according to any one of claims 69 to 86, and (2) a pharmaceutically acceptable carrier.

92. Use of a polypeptide according to any one of claims 1 to 38, a nucleic acid according to any one of claims 39 to 53, a vector according to any one of claims 54 to 68, a cell according to any one of claims 69 to 86, or a pharmaceutical composition according to claim 91 in the manufacture of a drug for the treatment of a disease or condition.

93. A polypeptide according to any one of claims 1 to 38, a nucleic acid according to any one of claims 39 to 53, a vector according to any one of claims 54 to 68, a cell according to any one of claims 69 to 86, or a pharmaceutical composition according to claim 91, for use in the treatment of a disease or condition.

94. The use according to claim 92 or 93, wherein the disease or disorder includes cancer or a subtype thereof.

95. The use according to claim 94, wherein the cancer or its subtype includes liquid cancer.

96. The use according to claim 94, wherein the cancer or its subtype includes hematological cancer.

97. The use according to claim 94, wherein the cancer or its subtype includes solid tumors.

98. A method for treating a disease or disorder, comprising administering to a subject an effective amount of a polypeptide according to any one of claims 1 to 38, a nucleic acid according to any one of claims 39 to 53, a vector according to any one of claims 54 to 68, a cell according to any one of claims 69 to 86, or a pharmaceutical composition according to claim 91, wherein the severity of the signs or symptoms of the disease or disorder is reduced, thereby treating the disease or disorder.

99. A method for preventing a disease or disorder, comprising administering to a target an effective amount of a polypeptide according to any one of claims 1 to 38, a nucleic acid according to any one of claims 39 to 53, a vector according to any one of claims 54 to 68, a cell according to any one of claims 69 to 86, or a pharmaceutical composition according to claim 91, wherein the onset or recurrence of signs or symptoms of the disease or disorder is delayed or suppressed, thereby preventing the disease or disorder.

100. The method according to claim 98 or 99, wherein the disease or disorder includes cancer or a subtype thereof.

101. The method according to claim 100, wherein the cancer or its subtype includes liquid cancer.

102. The method according to claim 100, wherein the cancer or its subtype includes hematological cancer.

103. The method according to claim 100, wherein the cancer or its subtype includes solid tumors.

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