Cytokine fusion protein and its use

Abstract

The present application provides a fusion protein comprising a cytokine fused to an albumin binding portion. The fusion protein may also include an antigen binding portion such as a therapeutic antibody. The present application also provides methods of preparing and using the fusion protein. The present application also provides a treatment method comprising administering a fusion protein and a second agent, wherein the fusion protein comprises a cytokine fused to a half-life extension domain.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 686,481, filed June 18, 2018, and U.S. Provisional Application No. 62 / 809,496, filed February 22, 2019. The above applications are incorporated herein by reference in their entirety for all purposes. Field of the Invention

[0003] The present application relates to fusion proteins, methods of making the same, and methods of treating diseases and disorders by administering the fusion proteins. Background of the Invention

[0005] Cytokine therapy is an effective strategy to stimulate the immune system to induce an immune response against a disease (e.g., cancer or infection). However, the cytokines administered to patients generally have a short half-life. For example, interleukin-21 can stimulate various immune cells (such as T cells, B cells, and NK cells) and enhance their anti-tumor activity. It is reported that the half-life of recombinant IL-21 after intravenous administration is only about one to three hours. See Schmidt H, Clin. Cancer Res. November 1, 2010; Vol. 16, No. 21: pp. 5312-5319.

[0006] Therefore, there is a need to develop new cytokine therapeutics that can effectively treat diseases.

[0007] The disclosures of all publications, patents, patent applications, and patent application publications cited herein are incorporated by reference in their entirety. Summary of the Invention

[0008] The present application provides a fusion protein comprising: a) a cytokine, and b) an albumin binding portion (such as an sdAb that binds to albumin). In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 that binds to IL-15Rα or a fragment thereof, IL-33, and IL-22. In some embodiments, the fusion protein further comprises an antigen binding portion.

[0009] The present application also provides a fusion protein comprising: a) a cytokine fused to an albumin binding portion ("cytokine-ALBBM"), and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33, and IL-22. In some embodiments, the antigen binding portion is fused to the C-terminus of the cytokine-ALBBM. In some embodiments, the antigen binding portion is fused to the N-terminus of the cytokine-ALBBM. In some embodiments, the antigen binding portion is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, protease, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NOs: 12-45 and 158-159. In some embodiments, the antigen binding portion binds to a tumor antigen. In some embodiments, the tumor antigen is selected from mesothelin ("MSLN"), GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA. In some embodiments, the antigen binding portion is an antibody or fragment thereof. In some embodiments, the antigen binding portion comprises a single domain antibody (sdAb). In some embodiments, the antigen binding portion comprises V H H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0010] In some embodiments according to any of the above fusion proteins, the cytokine is IL-21. In some embodiments, IL-21 comprises the amino acid sequence of SEQ ID NO: 1, 2, 126, 171 or 172, or a variant thereof, which has at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 1, 2, 126, 171 or 172.

[0011] In some embodiments according to any of the above fusion proteins, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA).

[0012] In some embodiments according to any of the above fusion proteins, the albumin binding moiety comprises an albumin binding domain (ABD).

[0013] In some embodiments according to any of the above fusion proteins, the albumin binding domain comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 3-11, or a variant thereof, which variant has at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11.

[0014] In some embodiments according to any of the above fusion proteins, the albumin binding moiety comprises a single domain antibody (sdAb). In some embodiments, the sdAb is V H H single domain antibody.

[0015] In some embodiments according to any of the above fusion proteins, the albumin binding moiety is fused to the C-terminus of the cytokine.

[0016] In some embodiments according to any of the above fusion proteins, the albumin binding moiety is fused to the N-terminus of the cytokine.

[0017] In some embodiments according to any of the above fusion proteins, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159.

[0018] The present application also provides a pharmaceutical composition comprising any of the above-mentioned fusion proteins.

[0019] The present application also provides a method for treating a disease or condition in an individual, comprising administering to the individual any of the above-mentioned fusion protein pharmaceutical compositions. In some embodiments, the method further comprises administering a second agent.

[0020] The present application also provides a method for treating a disease or condition in an individual, comprising administering to the individual a) a fusion protein comprising i) a cytokine and ii) a half-life-extending domain fused to the cytokine; and b) a second agent. In some embodiments, the half-life-extending domain is an albumin-binding moiety. In some embodiments, the half-life-extending domain is an albumin. In some embodiments, the half-life-extending domain is an Fc fragment. In some embodiments, the Fc fragment is selected from IgG1, IgG2, IgG3, and IgG4 Fc fragments or variants thereof. In some embodiments, the Fc fragment is an IgG1 Fc fragment or a variant thereof. In some embodiments, the IgG1 Fc fragment or a variant thereof comprises a mutation at position 297, wherein the amino acid mutation at position 297 is alanine, aspartic acid, or glycine. In some embodiments, the individual is human. In some embodiments, the disease or condition is selected from cancer, inflammatory conditions, and infection.

[0021] In some embodiments according to any of the above methods, the disease or condition is an inflammatory disease. In some embodiments, the cytokine is IL-22. In some embodiments, the disease is selected from ulcerative colitis, Crohn's disease, or ulcerative ileitis, and intestinal graft-versus-host disease.

[0022] In some embodiments according to any of the above methods, the disease or condition is cancer. In some embodiments, the cancer is a solid tumor or a liquid tumor. In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, and IL-33.

[0023] In some embodiments according to any of the methods above, the fusion protein is administered at a frequency of about once every three weeks to about twice per week.

[0024] In some embodiments according to any of the methods above, the amount of the fusion protein administered each time is about 100 ng / kg to about 10 mg / kg.

[0025] In some embodiments according to any of the above methods, the fusion protein is administered to the individual parenterally. In some embodiments, the fusion protein is administered to the individual intravenously or subcutaneously.

[0026] In some embodiments according to any of the methods above, the fusion protein is administered for at least about one week to six months per treatment cycle.

[0027] In some embodiments according to any one of the above methods, the second agent comprises a therapeutic antibody, an immune checkpoint inhibitor, a second cytokine, a chemotherapeutic agent, a tyrosine kinase inhibitor, or an immune cell. In some embodiments, the second agent is a therapeutic antibody. In some embodiments, the therapeutic antibody binds to a tumor antigen. In some embodiments, the tumor antigen is selected from mesothelin (MSLN), GPA33, Her-2 (ERBB2), EGFR, and CD20 (MS4A1). In some embodiments, the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA. In some embodiments, the tumor antigen is mesothelin. In some embodiments, the second agent is an anti-mesothelin antibody or a fragment thereof. In some embodiments, the anti-mesothelin antibody or a fragment thereof comprises a fragment thereof containing an anti-mesothelin heavy chain variable region (anti-MSLN V H ) of the single-chain antibody, wherein: a) anti-MSLN V H comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 48, or a variant comprising a total of up to 3, 2, or 1 amino acid substitutions in the CDRs; or b) anti-MSLN V HIncluding a CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 51, or a variant comprising a total of up to 3, 2, or 1 amino acid substitutions in the CDRs. In some embodiments, the second agent that binds to mesothelin is administered at a frequency of about once a month to about twice a week. In some embodiments, the amount of the second agent administered each time is about 100 ng / kg to about 100 mg / kg. In some embodiments, the second agent is an immune checkpoint regulator. In some embodiments, the immune checkpoint regulator is an immune checkpoint protein inhibitor selected from PD-L1, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, CD27, 4-1BB, and B7H4. In some embodiments, the immune checkpoint protein is PD-1. In some embodiments, the second agent is an anti-PD-1 antibody or a fragment thereof. In some embodiments, the amount of the second agent administered each time is from about 1 μg / kg to about 100 mg / kg. In some embodiments, the second agent is a second cytokine. In some embodiments, the cytokine in the fusion protein is IL-21, and wherein the second cytokine is selected from IL-7, IL-15, IL-15 combined with IL-15Rα, or a half-life extension variant thereof. In some embodiments, the second agent is an immune cell. In some embodiments, the immune cell includes a T cell or a NK cell. In some embodiments, the immune cell includes a T cell expressing a chimeric antigen receptor (CAR), a T cell expressing a modified T cell receptor (TCR), or a T cell isolated from a tumor. In some embodiments, the second agent is a tyrosine kinase inhibitor. In some embodiments, the second agent is administered to an individual parenterally or orally. In some embodiments, the second agent is administered to an individual parenterally. In some embodiments, the second agent is administered to an individual intravenously.

[0028] In some embodiments according to any of the methods above, the fusion protein and the second agent are administered to the individual simultaneously, concurrently, or sequentially. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Depicted are exemplary IL-21 fusion proteins provided herein.

[0030] Figure 2 Depicted is the assembly of an exemplary IL-21 fusion protein expression vector, wherein the albumin binding molecule is an anti-HSA antibody.

[0031] Figure 3Depicted is the assembly of an exemplary IL-21 fusion protein expression vector, wherein the albumin binding molecule is an ABD that binds to HSA.

[0032] Figure 4 The K values ​​of the anti-HSA antibody P367 when interacting with human, monkey or mouse serum albumin are shown. D P394-hgG1 Fc fusion protein was loaded onto a Protein A biosensor and immersed in human, monkey, or mouse serum albumin. The colored lines represent the binding response of serum albumin at different concentrations of 400 nM (dark blue), 200 nM (dark red), and 100 nM (light blue). The experimental data were analyzed using global fitting (red) to determine K D .

[0033] Figure 5 Shown are the signaling potencies of the anti-HSA IL-21 conjugates P390 and P394 compared to recombinant IL-21.

[0034] Figure 6A Shown are ADCC activities of PBMCs against N87 cells in the presence of P390, P394, and P461 / P462, as well as the anti-mesothelin antibody P303. Figure 6B Shown are ADCC activities of PBMCs against H226 cells in the presence of P390, P394 and P461 / P462, and the anti-mesothelin antibody P303. Figure 6C Shown are ADCC activities of PBMCs against N87 cells in the presence of different doses of P390, P394, P461 / P462, and P461 / P463, and 3 ng / mL P303. Figure 6D Shown are ADCC activities of PBMCs against H226 cells in the presence of different doses of P390, P394, P461 / P462, and P461 / P463, and 20 ng / mL P303.

[0035] Figure 7A STAT3 signaling in the presence of IL-21-anti-HSA fusion protein variants P394, P593, P636, P637, P744, P748, P750, P751, and P783 is shown. Figure 7B and Figure 7C Shown are NK cell-mediated ADCC against NCI-N87 tumor cells when IL-21-anti-HSA fusion protein variants P394, P593, P636, P637, P744, P748, P750, P751, and P783 were titrated with a fixed dose of anti-MSLN antibody (P303).

[0036] Figure 8Depicted is a comparison of the pharmacokinetics of 3 μg recombinant human IL-21, 3 μg P325 (human IL-21 non-related Nanobody), and 3 μg or 30 μg P394 (human IL-21-anti-HSA) in mice.

[0037] Figure 9A 、 Figure 9F and Figure 9G Depicted are SDS-PAGE photographs showing the staining status of IL-21-anti-HSA fusion proteins P394 (ie, AWT-P394), P593 (ie, AWT-P593), and P748. Figure 9F and Figure 9G In the figure, arrows indicate intact target proteins, and asterisks indicate cleaved proteins. Figure 9B Representative chromatograms of AWT-P394 IL-21-anti-HSA fusion protein are depicted. Figure 9C The T values ​​of AWT-P593 at pH 4.0 and pH 7.5 are plotted. onset comparison. Figure 9D The T of AWT-P394 and AWT-P593 are depicted. onset . Figure 9E Depicted is the binding of the human IL-21 receptor to the IL-21-anti-HSA fusion proteins AWT-P394 or AWT-P593.

[0038] Figure 10A The T values ​​of AWT-P593 at pH 4.0 and pH 7.5 are plotted. onset contrast. Figure 10B The T of AWT-P394 and AWT-P593 are depicted. onset contrast. Figure 10C Depicted are the binding of humanized anti-AWT-P610 (ie, P610) to human serum albumin or monkey serum albumin at pH 7.4 and pH 5.5. Figure 10D The T-terminal structure of the anti-albumin antibody AWT-P367 and its humanized form AWT-P494 is depicted. onset contrast. Figure 10E The T-axis of the anti-albumin antibody AWT-P342 and its humanized form AWT-P610 is depicted. onset contrast. Figure 10D Binding of the anti-HSA antibody AWT-367 and its humanized counterpart anti-AWT-P494 to human, monkey or mouse albumin is depicted. Figure 10E The K values ​​of AWT-P367 or AWT-494 binding to human, monkey, or mouse albumin are plotted. D .

[0039] Figure 11Depicted are the remaining cell numbers of N87 cells after treatment with NK cells alone or in combination with study drugs.

[0040] Figure 12 Depicted are the remaining cell numbers of N87 cells after treatment with NK cells alone or in combination with a) Herceptin alone, b) P303FF (ie, P303F) alone, c) Herceptin and P303FF, or d) Herceptin, P303FF, and IL-21-anti-HSA fusion protein P394.

[0041] Figure 13 Depicted are the percentages of dead Preiffer cells following treatment of NK cells with a) Retuxan, b) Retuxan and P394, c) Retuxan and IL-15 fusion protein P480, or d) Retuxan and IL-15.

[0042] Figure 14 Depicted are the remaining cell numbers of N87 cells after treatment with NK cells in combination with a) P303F, b) P303F and recombinant human IL-21 (ie, rhIL-21), c) P303F and P480, or d) P303F and recombinant human IL-15 (ie, rhIL-15).

[0043] Figure 15 Depicted are the remaining cell numbers of N87 cells after treatment with NK cells and a) P480, b) P597, or c) rIL-15 (top panel) and the IC values ​​of the three drugs. 50 (Picture below).

[0044] Figure 16 Depicted are the remaining cell numbers of H226 cells after treatment of NK cells with a) anti-mesothelin antibodies P129 (i.e., R2G12) and P126 (i.e., human IL-21-R2G12-IgG1 fusion), b) P129 and IL-21, c) P129 and P107 (human IL-21-IgG1 fusion), d) P129 and P325 (human IL-21-R2D2 fusion), or e) P129 and P286 / 288 (human IL-21-R3C7-IgG1-R2G12).

[0045] Figure 17 Depicted are the remaining cell numbers of N87 cells after treatment with NK cells in combination with a) P197 and P390, or b) P197 and P394.

[0046] 18A to 18B Depicts the secretion of IFN-γ ( Figure 18A ) and IL-6 ( Figure 18B ) level.

[0047] Figures 19A to 19B Depicted are changes in tumor volume in the MC38 mouse syngeneic tumor model following administration of anti-CTLA-4, IL-21-anti-HSA fusion protein (P394), anti-HSA-IL-15Rα / IL-15 fusion protein (P597), or a combination of these agents.

[0048] Figure 20 Depicted are changes in tumor volume in the MC38 mouse syngeneic tumor model following treatment with IL-21-anti-HSA fusion protein (P394), anti-HSA-IL-15Rα / IL-15 fusion protein (P597), or a combination of these agents.

[0049] Figure 21 Depicted are changes in tumor volume in animal models of the MC38 syngeneic model after treatment with a) 100 μg anti-PD-1 antibody, b) 25 μg P390, c) 100 μg anti-PD-1 antibody and 25 μg P390, or d) 100 μg anti-PD-1 antibody and 5 μg P390.

[0050] Figure 22 Depicted are changes in tumor volume in an animal model of N87 tumor-bearing NSG mice treated with a) 25 μg P394, b) 100 μg P303F, c) 100 μg P303F and 25 μg P394, or d) 100 μg P303F and 5 μg P394.

[0051] Figure 23 Depicted are changes in tumor volume in an animal model of N87 tumor-bearing NSG mice treated with a) 25 μg P394, b) 20 μg Herceptin, c) 20 μg Herceptin and 25 μg P394, or d) 20 μg Herceptin and 5 μg P394.

[0052] Figure 24 Depicted are changes in tumor volume in an animal model of N87 tumor-bearing SCID mice treated with 100 μg P303F alone or in combination with a) 25 μg P390, b) 5 μg P390, or c) 2.5 μg rIL-21.

[0053] Figure 25 Depicted are changes in tumor volume in MC38 syngeneic animal models following treatment with 25 μg P390 or 12.5 μg rmIL-21.

[0054] Figure 26 Depicted are changes in tumor volume in the CT-26 / MSLN animal model after treatment with anti-PD-1 antibody alone, P390 alone, or a combination of anti-PD-1 antibody and P390.

[0055] Figure 27 Depicted are changes in tumor volume in animal models of the MC38 syngeneic model following treatment with anti-PD-1 antibody alone, P394 alone, or a combination of anti-PD-1 antibody and P394.

[0056] Figure 28 Depicted are changes in tumor volume in the MC38 mouse syngeneic tumor model following treatment with anti-HSA-IL-33 fusion protein (P380).

[0057] Figures 29A to 29B Depicts the granzyme B-positive NK cells after P390 treatment ( Figure 29A ) and granzyme B-positive CD8 T cells ( Figure 29B ) percentage change.

[0058] Figures 30A to 30C Depicts the CD8 T cells ( Figure 30A ), CD4 T cells ( Figure 30B ) and NK cells ( Figure 30C ) Changes in IL-21 receptor expression levels in the human cervical stromal tuberculosis virus (CEV) and the cerebrospinal fluid (CSF) were detected.

[0059] Figure 31 Depicted are the cell numbers of IFN-γ-secreting immune cells in the spleen after treatment with anti-PD-1 antibody alone, P390 alone, a combination of anti-PD-1 antibody and P390, or a combination of anti-PD-1 antibody and rmIL-21.

[0060] Figure 32 Depicted are changes in tumor volume in the CT26 / MSLN mouse syngeneic tumor model following treatment with IL-21-anti-HSA-anti-MSLN fusion protein (P375).

[0061] Figure 33 Depicted are changes in tumor volume in an animal model of N87 tumor-bearing NSG mice treated with anti-MSLN-anti-HSA-IL-15Rα / IL-15 fusion protein (P669).

[0062] Figure 34 It was depicted that various IL-21 fusion proteins have similar signaling potency.

[0063] Figure 35 It was shown that various IL-21 fusion proteins have similar ADCC activities. DETAILED DESCRIPTION

[0064] The present application relates to fusion proteins comprising a cytokine and a half-life extension domain. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33, and IL-22. In some embodiments, the fusion protein comprises: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable.

[0065] The application also provides a method for treating a disease or condition (e.g., cancer or inflammatory disease), including administering a fusion protein as described above. In some embodiments, the method includes administering a) a fusion protein including i) a cytokine and ii) a half-life extension domain, and b) a second agent. Exemplary second agents include, but are not limited to, therapeutic agents, immune checkpoint inhibitors, second cytokines, tyrosine kinase inhibitors, chemotherapeutics, or immune cells.

[0066] The present application also provides compositions, kits, and articles of manufacture comprising the bispecific antibodies described herein, and methods for preparing the same.

[0067] I. Definition

[0068] The term "antibody" is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. The term "antibody portion" refers to a full-length antibody or an antigen-binding fragment thereof.

[0069] An "isolated" antibody (or construct) is one that has been identified, separated, and / or recovered from a component of its (e.g., natural or recombinant) production environment. Preferably, an isolated polypeptide is not associated with any other component of its production environment. Contaminating components of its production environment, such as those produced by recombinant transfected cells, are substances that would normally interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified to: (1) greater than 95% by weight of the antibody, as determined, for example, by the Lowry method, and in some embodiments, greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity as confirmed by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody (or construct) includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Typically, however, an isolated polypeptide, antibody, or construct will be prepared by at least one purification step.

[0070] A full-length antibody consists of two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains are referred to as “V H ” and “V L The variable regions in both chains typically contain three highly variable loops, termed complementarity determining regions (CDRs) (light chain (LC) CDRs, including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs, including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or delineated according to the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1989). 1991). The three CDRs of the heavy or light chain are inserted between flanking extensions called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0071] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially complete form, as opposed to an antibody fragment. Specifically, a full-length 4-chain antibody includes an antibody having a heavy chain and a light chain containing an Fc region. A full-length heavy chain antibody includes a heavy chain variable domain (such as a V H F) and Fc region. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, a complete antibody can have one or more effector functions.

[0072] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site. Only heavy chain antibodies from species of the family Camelidae have a single heavy chain variable region, referred to as "VL." H H". Therefore V H H is a special type of VH.

[0073] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain Fv (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody including one or more CDRs, camelized single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not include a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen as a parent antibody or parent antibody fragment (e.g., a parent scFv). In some embodiments, an antigen-binding fragment may include one or more CDRs from a specific human antibody grafted to the framework regions of one or more different human antibodies.

[0074] "Fv" is the smallest antibody fragment that contains a complete antigen recognition site and an antigen binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. These two domains fold to produce six hypervariable loops (three loops each on the heavy and light chains), which contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv that includes only three CDRs specific for a particular antigen) has the ability to recognize and bind to an antigen, but with a lower affinity than the entire binding site.

[0075] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a fragment comprising V sequences linked into a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H and V L The polypeptide linker between the domains enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, for example, Plückthun, The Pharmacology of Monoclonal Antibodies, edited by Rosenburg and Moore, Springer-Verlag, New York, volume 113, pages 269-315, 1994.

[0076] The term "heavy chain antibody" or "HCAb" refers to a functional antibody comprising a heavy chain but lacking the light chain typically found in 4-chain antibodies. Camelids (such as camels, llamas or alpacas) are known to produce HCAbs.

[0077] The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). An sdAb is capable of binding to an antigen alone without pairing with a corresponding CDR-containing polypeptide. In some cases, a single domain antibody is engineered from a camelid HCAb, and its heavy chain variable domain is referred to herein as a "V H H" (variable domain of the heavy chain of a heavy chain antibody). Camelid sdAbs are among the smallest known antigen-binding antibody fragments (see, e.g., Hammers-Casterman et al., Nature, vol. 363: 446-448, 1993; Greenberg et al., Nature, vol. 374: 168-173, 1995; Hassanzadeh-Ghassaeh et al., Nanomedicine (Lond), vol. 8: 1013-1026, 2013). Basic V H H has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0078] As used herein, the term "hypervariable region", "HVR" or "HV" refers to the region of an antibody variable domain that is highly variable in sequence and / or forms a structurally defined loop. Typically, a single-domain antibody comprises three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2) and HVR3 (or CDR3). HVR3 (or CDR3) is the most diverse of the three HVRs and is believed to play a unique role in conferring fine specificity to antibodies. See, for example, Hammers-Casterman et al., Nature, Vol. 363: 446-448, 1993; Sheriff et al., Nature Struct. Biol., Vol. 3: 733-736, 1996.

[0079] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem., Vol. 252: 6609-6616, 1977; Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest", 1991; Chothia et al., J. Mol. Biol., Vol. 196: 901-917, 1987; Al-Lazikani B. et al., J. Mol. Biol., Vol. 273: 927-948, 1997; MacCallum et al., J. Mol. Biol., Vol. 262: 732-745, 1996; Abhinandan and Martin, Mol. Immunol., Vol. 45: 3832-3839, 2008; Lefranc et al., J. Mol. Biol., Vol. 274: 927-948, 1997; MacCallum et al., J. Mol. Biol., Vol. 262: 732-745, 1996; Abhinandan and Martin, Mol. Immunol., Vol. 45: 3832-3839, 2008; Lefranc et al., J. Mol. Biol., Vol. 274: 927-948, 1997; MP et al., Dev. Comp. Immunol., Vol. 27: pp. 55-77, 2003; and Honegger and Plückthun, J. Mol. Biol., Vol. 309: pp. 657-670, 2001, wherein these definitions include overlapping or subsets of amino acid residues when compared to each other. However, the application of either definition to the CDRs of an antibody or grafted antibody or variant thereof is within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs defined in each of the references cited above are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., Vol. 45: pp. 3832-3839, 2008; Ehrmann F et al., Nucleic Acids Res., Vol. 38: pp. D301-D307, 2010; and Adolf-Bryfogle J et al., Nucleic Acids Res., Vol. 43: pp. D432-D438, 2015. The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and may be included in one or more claims herein.

[0080] Table 1: CDR Definition

[0081]

[0082] 1 The residue numbering follows the nomenclature of Kabat et al. (see above)

[0083] 2 The residue numbering follows the nomenclature of Chothia et al. (see above)

[0084] 3 Residue numbering follows the nomenclature of MacCallum et al. (see above)

[0085] 4 Residue numbering follows the nomenclature of Lefranc et al. (see above)

[0086] 5 Residue numbering follows the nomenclature of Honegger and Plückthun (see above)

[0087] The phrase "variable domain residues are numbered according to Kabat" or "amino acid positions are numbered according to Kabat" and variations thereof refer to the numbering system of Kabat et al. (supra) for compiling the heavy chain variable domain or light chain variable domain of an antibody. Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids corresponding to a shortening or insertion of a variable region FR or HVR. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to the Kabat system) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to the Kabat system). The Kabat residue numbering for a given antibody can be determined by aligning the antibody sequence with the "standard" Kabat numbering sequence for regions of homology.

[0088] Unless otherwise indicated, the numbering of residues in immunoglobulin heavy chains herein refers to the EU index numbering of Kabat et al. (supra). "EU index as of Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0089] "Framework" or "FR" residues are those variable region residues other than the CDR residues as herein defined.

[0090] The " humanization " form of non-human (such as rodent) antibody is a chimeric antibody containing a minimum sequence of non-human antibody sources. The major part of humanized antibodies is human immunoglobulin (receptor antibody), wherein the residues of receptor hypervariable region (HVR) are replaced by residues of non-human species (donor antibody) hypervariable region, non-human species such as mouse, rat, rabbit or non-human primate, and the hypervariable region of these species has desired antibody specificity, affinity and binding capacity. In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues not found in receptor antibody or donor antibody. These modifications are carried out to further improve antibody performance. Generally speaking, humanized antibodies will include substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulin, and all or substantially all of the FRs are the FRs of human immunoglobulin sequences. The humanized antibody optionally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, Vol. 321: 522-525, 1986; Riechmann et al., Nature, Vol. 332: 323-329, 1988; and Presta, Curr. Op. Struct. Biol., Vol. 2: 593-596, 1992.

[0091] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences mentioned herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after the sequences are aligned taking into account any conservative substitutions as part of the sequence identity. Alignment for determining percentage amino acid sequence identity can be achieved in a variety of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research, Vol. 32, No. 5: pp. 1792-1797, 2004; Edgar, RC, BMC Bioinformatics, Vol. 5, No. 1: p. 113, 2004).

[0092] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of two DNA molecules is occupied by adenine, then the two molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. For example, there is 50% homology between the DNA sequences ATTGCC and TATGGC. Typically, a comparison is made when two sequences are aligned for maximum homology.

[0093] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the variable domain, another portion of the immunoglobulin that contains the antigen-binding site. H 1. C H 2 and C H 3 domains (collectively referred to as C H ) and the CHL (or C L ) domain.

[0094] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0095] The "CH1 domain" of a human IgG Fc region (also called "C1" of the "H1" domain) typically stretches from about amino acid 118 to about amino acid 215 (EU numbering system).

[0096] The "hinge region" is generally defined as the region corresponding to Glu216 to Pro230 in human IgG1 (Burton, Molec. Immunol., Vol. 22: pp. 161-206, 1985). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain SS bond in the same position.

[0097] The "CH2 domain" (also called "C2" of the "H2" domain) of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is relatively unique in that it is not tightly paired with another domain. Instead, two N-linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that the sugar chains provide an alternative to domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol., Vol. 22: pp. 161-206, 1985.

[0098] The "CH3 domain" (also called a "C2" or "H3" domain) includes the stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically at amino acid residue 446 or 447 in IgG).

[0099] The term "Fc region" or "fragment crystallizable region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) in the Fc region can be removed, for example, during the production or purification process of the antibody, or removed by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, the composition of the intact antibody can include antibody populations in which all K447 residues are removed, antibody populations in which the K447 residue is not removed, and antibody populations with and without a mixture of antibodies containing the K447 residue. Suitable native sequence Fc regions for antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0100] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, a preferred FcR is one that binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors, FcγRII receptors including FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See FcRs are reviewed by Ravetch and Kinet, Annu. Rev. Immunol., Vol. 15: 203-234, 1997; Capel et al., Immunomethods, Vol. 4: 25-34, 1994; and de Haas et al., J. Lab. Clin. Med., Vol. 126: 330-341, 1995. Other FcRs, including those discovered in the future, are encompassed by the term "FcR" herein.

[0101] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. Two antibodies or antibody portions may bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0102] As used herein, a first antibody or fragment thereof "competes" for binding to a target antigen with a second antibody or fragment thereof when the first antibody or fragment thereof inhibits binding to the target antigen of a second antibody or fragment thereof at an equimolar concentration to the first antibody or fragment thereof by at least about 50% (such as at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), and vice versa. PCT Publication No. WO 03 / 48731 describes a high-throughput method for "binning" antibodies based on antibody cross-competition.

[0103] As used herein, the terms "specific binding," "specific recognition," and "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody or antibody portion, that is used to determine the presence of a target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to the target with greater affinity or avidity, more readily, and / or for a longer duration than it binds to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured by, for example, a radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a specific binding affinity of ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 The binding dissociation constant (K D). In some embodiments, the antibody specifically binds to a protein epitope that is conserved between proteins of different species. In some embodiments, specific binding may include but does not require exclusive binding. The binding specificity of an antibody or antigen binding domain can be determined experimentally by methods known in the art. These methods include, but are not limited to, Octet, Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE TM Detection and peptide scanning.

[0104] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair. Binding affinity can be expressed as K d , K off , K on or K a As used herein, the term "K off ” means the dissociation rate constant for the dissociation of an antibody (or antigen binding domain) from the antibody / antigen complex, as determined by the kinetic selection settings, expressed in s -1 As used herein, the term "K on " refers to the association rate constant of the antibody (or antigen binding domain) binding to the antigen to form the antibody / antigen complex, expressed as M -1 s -1 As used herein, the term equilibrium dissociation constant "K D ” or “K d ” refers to the binding dissociation constant for a specific antibody-antigen interaction and describes the concentration of antigen required to occupy half of all antibody binding domains present in a solution of antibody molecules at equilibrium and is equal to K off / K on , expressed in M. K d The measurement of assumes that all binding agent is in solution. In the case where the antibody is bound to the cell wall, such as in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC 50 , and K d Approximate affinity constant K a is the binding dissociation constant K d The reciprocal of M -1 The unit is expressed as . The binding dissociation constant (K D or K d) is used as an indicator of antibody affinity for antigen. For example, analysis can be easily performed by using the Scatchard method using antibodies labeled with various labeling reagents, or by using over-the-counter BiacoreX (manufactured by Amersham Biosciences), a measurement kit, or a similar kit according to the user manual and experimental operation method attached to the kit. K can be obtained using these methods. D The value is expressed in M ​​(mole). An antibody or antigen-binding fragment thereof that specifically binds to a target may have, for example, ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 The binding dissociation constant (K d ).

[0105] Half maximal inhibitory concentration (IC 50 ) is a measure of the effectiveness of a substance (such as an antibody) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, such as an antibody) is needed to inhibit half of a given biological process (e.g., the binding between albumin and CD155, or a component of a process, i.e., an enzyme, cell receptor, or microorganism). These values ​​are usually expressed as molar concentrations. For agonist drugs or other substances (such as antibodies), the IC 50 With "EC 50 "Equivalent.EC 50 It also represents the plasma concentration required to achieve 50% of the maximal effect in vivo. As used herein, "IC 50 IC is used to indicate the effective concentration of an antibody (such as an anti-albumin sdAb) required to neutralize 50% of the bioactivity of an antigen (such as albumin bioactivity) in vitro. 50 or EC 50 Inhibition of ligand binding can be measured by biological assays, such as by FACS analysis (competitive binding assay), cell-based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA).

[0106] As used herein, the term "cytokine" should be understood to refer to any protein or peptide, its analog or functional fragment, which is capable of stimulating or inducing a cytocidal immune response against a preselected cell type (e.g., cancer cells or virus-infected cells) in a mammal. Therefore, it is contemplated that a variety of cytokines may be incorporated into the present application. Useful cytokines include, for example, tumor necrosis factor (TNF), interleukin (IL), lymphokine (L), colony stimulating factor (CSF), interferon (IFN), including species variants and truncated analogs thereof that are capable of stimulating or inducing such a cytocidal immune response. Useful tumor necrosis factor includes, for example, TNFα. Useful lymphokines include, for example, LT. Useful colony stimulating factors include, for example, GM-CSF and M-CSF. Useful interleukins include, for example, IL-2, IL-4, IL-5, IL-7, IL-12, IL-15, IL-18, IL-21, IL22, and IL-33. Useful interferons include, for example, IFN-α, IFN-α and IFN-γ. The term "cytokine" should also be understood to include any variant of a wild-type cytokine (such as IL-21, IL-7, IL-15, etc.) that includes modifications and retains at least a significant portion (such as at least about 50%) of any desired function thereof.

[0107] As used herein, the term "truncated IL-21" refers to a protein or peptide comprising IL-21 that has one or more amino acids deleted at one or both of the C-terminus and N-terminus of wild-type IL-21. Fusion proteins comprising the truncated IL-21 described herein may have other moieties or domains, such as an antigen-binding portion, a linker, a signal sequence, or an albumin-binding portion, even though the IL-21 in the fusion protein is a truncated form having at least one amino acid missing from wild-type IL-21. In some embodiments, wild-type IL-21 has the amino acid sequence of SEQ ID NO: 1.

[0108] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof as described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule to which it is normally bound in the environment in which it is produced. Preferably, the isolated nucleic acid is not bound to all components associated with the production environment. The form of the isolated nucleic acid molecule encoding the polypeptides and antibodies described herein is different from the form or background in which it is found in nature. Thus, an isolated nucleic acid molecule is different from nucleic acids encoding the polypeptides and antibodies described herein that are naturally present in cells. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0109] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0110] A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, it is operably linked to that sequence; or if a ribosome binding site is positioned to facilitate translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, they are contiguous and in reading phase. However, enhancers do not have to be contiguous. Connection is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0111] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0112] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0113] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell but may include mutations. Mutant progeny that have the same function or biological activity as that screened or selected for in the original transformed cell are included herein.

[0114] As used herein, "treatment" or "treating" is a method for obtaining beneficial or desired results, including clinical results. In order to achieve the purpose of the present application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the degree of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing down the progress of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dosage of one or more other drugs required for treating the disease, delaying the progress of the disease, improving or improving the quality of life, improving weight gain and / or prolonging survival. "Treatment" also includes the reduction of the pathological results (e.g., tumor volume) of cancer. The method of the present application takes into account any one or more of these treatment aspects.

[0115] In the context of cancer, the term "treating" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0116] The term "inhibition" or "inhibit" refers to a reduction or cessation of any phenotypic characteristic, or a reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Reduce" or "inhibit" refers to reducing, lowering, or preventing an activity, function, and / or amount as compared to a reference. In certain embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or greater. In another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or greater. In yet another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or greater.

[0117] As used herein, "reference" refers to any sample, standard, or level used for comparison purposes. A reference can be obtained from a healthy and / or non-diseased sample. In some examples, a reference can be obtained from an untreated sample. In some examples, a reference is obtained from a non-diseased or untreated sample of an individual. In some examples, a reference is obtained from one or more healthy individuals who are not the individual or patient.

[0118] As used herein, "delaying the progression of a disease" means postponing, hindering, slowing, retarding, stabilizing, inhibiting, and / or delaying the progression of a disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or the individual being treated. It will be apparent to one skilled in the art that a sufficient or significant delay can actually include prevention, i.e., the individual will not develop the disease. For example, the progression of advanced cancer, such as metastasis, can be delayed.

[0119] As used herein, "prevention" includes providing protection against the occurrence or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease.

[0120] As used herein, "inhibiting" a function or activity means reducing the function or activity when compared to the same conditions except for the condition or parameter of interest, or alternatively, when compared to another condition. For example, an antibody that inhibits tumor growth reduces the growth rate of a tumor compared to the growth rate of the tumor in the absence of the antibody.

[0121] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to mammals, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0122] An "effective amount" of an agent is an amount effective to achieve the desired therapeutic or prophylactic result, at the dosage and for the required period of time. The term also applies to a dose that will provide an image for detection by any of the imaging methods described herein. The specific dose may vary depending on one or more of the following: the particular agent selected, the dosing regimen to be followed, whether the agent is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system carrying the agent.

[0123] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist of the present invention may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects of the substance / molecule, agonist, or antagonist outweigh any toxic or deleterious effects. A therapeutically effective amount may be delivered in one or more administrations.

[0124] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the dosage and time period required. Typically, but not necessarily, a prophylactic effective amount is less than a therapeutically effective amount because a prophylactic dose is used in a subject before or at an earlier stage of disease.

[0125] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation that is in such form that the biological activity of the active ingredient is effective and that contains no additional components that are unacceptably toxic to a subject to which the preparation would be administered. Such preparations may be sterile.

[0126] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation adjuvant, or vehicle conventionally used in the art for use with a therapeutic agent, which together constitute a "pharmaceutical composition" for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration employed and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation employed.

[0127] A "sterile" preparation is sterile or substantially free of viable microorganisms and their spores.

[0128] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and serial or sequential administration in any order.

[0129] As used herein, the term "concurrent" refers to the administration of two or more therapeutic agents wherein at least a portion of the administration overlaps in time, or wherein the administration of one therapeutic agent falls within a short time period relative to the administration of another therapeutic agent. For example, the time interval between the administration of the two or more therapeutic agents is no more than about 60 minutes, such as no more than about any of 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute.

[0130] As used herein, the term "sequentially" refers to the administration of two or more therapeutic agents, wherein the administration of one or more agents continues after the administration of one or more other agents has ceased. For example, the administration of the two or more therapeutic agents is administered at a time interval greater than about 15 minutes, such as about any of 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month, or longer.

[0131] As used herein, "in combination" refers to administering a treatment modality in addition to another treatment modality. Thus, "in combination" refers to administering a treatment modality before, during, or after administering one treatment modality to an individual.

[0132] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0133] An "article of manufacture" is any article of manufacture (e.g., a package or container) or kit comprising at least one pharmaceutical agent (e.g., a drug for treating a disease or condition (e.g., cancer)) or a probe for specifically detecting a biomarker described herein. In certain embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for practicing the methods described herein.

[0134] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0135] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0136] As used herein, reference to "not" a value or parameter generally refers to and describes a value or parameter "except for..." For example, the method is not used to treat type X cancer, which means that the method is used to treat other types of cancer other than type X.

[0137] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0138] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0139] II-A. Fusion proteins comprising an albumin binding moiety

[0140] Provided herein are fusion proteins comprising: a) a cytokine, and b) an albumin binding portion (such as an sdAb that binds to albumin). In some embodiments, the fusion protein comprises a) a cytokine selected from IL-21, IL-7, IL-15, IL-15 that binds to IL-15Rα or a fragment thereof, IL-33, and IL-22, and b) an albumin binding portion (such as an sdAb that binds to albumin). In some embodiments, the albumin binding portion comprises an albumin binding domain (ABD). In some embodiments, the albumin binding portion comprises a single domain antibody (sdAb). In some embodiments, the albumin binding portion is fused to the C-terminus of the cytokine. In some embodiments, the albumin binding portion is fused to the N-terminus of the cytokine. In some embodiments, the cytokine and the albumin binding portion are connected by a first linker (such as having about one to thirty amino acids). In some embodiments, the albumin binding portion is a single domain antibody (such as a V H H antibody). sdAbs with relatively small molecular weights may help increase cancer penetration of fusion proteins, making them more suitable for treating certain cancers, such as solid tumors.

[0141] In some embodiments, a fusion protein is provided, comprising: a) IL-21, and b) an albumin binding portion (such as an sdAb that binds to albumin). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding portion. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding portion. In some embodiments, the albumin binding portion is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding portion. In some embodiments, the albumin binding portion binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding portion comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises an anti-albumin antibody (such as a single domain antibody (sdAb), such as V H H single domain antibody). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, IL-21 is a truncated IL-21. In some embodiments, the truncated IL-21 comprises the amino acid sequence of SEQ ID NOs: 126, 171, or 172. In some embodiments, the first linker is a rigid linker. In some embodiments, the first linker is selected from SEQ ID NOs: 21, 22, and 24. In some embodiments, the first linker is a flexible linker. In some embodiments, the first linker is selected from SEQ ID NOs: 12-14.

[0142] In some embodiments, a fusion protein is provided comprising: a) IL-21, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-21 comprises the amino acid sequence of SEQ ID NO: 1, 2, 126, 171, or 172, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 1, 2, 126, 171, or 172, and wherein the albumin binding moiety comprises an albumin binding domain. In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is identical or different from the first cytokine is fused to the other end of the albumin binding portion. In some embodiments, the albumin binding portion is bound to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain includes an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with any one of SEQ ID NO: 3-11. In some embodiments, the cytokine and the albumin binding portion are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, IL-21 is a truncated IL-21. In some embodiments, the truncated IL-21 comprises the amino acid sequence of SEQ ID NO: 126, 171, or 172. In some embodiments, the first linker is a rigid linker. In some embodiments, the first linker is selected from SEQ ID NO: 21, 22, and 24. In some embodiments, the first linker is a flexible linker. In some embodiments, the first linker is selected from SEQ ID NO: 12-14.

[0143] In some embodiments, a fusion protein is provided comprising: a) IL-21, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-21 comprises the amino acid sequence of SEQ ID NO: 1, 2, 126, 171 or 172, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to SEQ ID NO: 1, 2, 126, 171 or 172, and wherein the albumin binding moiety comprises an anti-albumin antibody or fragment thereof (such as a single domain antibody, such as a V H H antibody). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, IL-21 is a truncated IL-21. In some embodiments, the truncated IL-21 comprises the amino acid sequence of SEQ ID NOs: 126, 171, or 172. In some embodiments, the first linker is a rigid linker. In some embodiments, the first linker is selected from SEQ ID NOs: 21, 22, and 24. In some embodiments, the first linker is a flexible linker.In some embodiments, the first linker is selected from SEQ ID NOs: 12-14.

[0144] In some embodiments, a fusion protein is provided, comprising: a) IL-7, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine, which is the same as or different from the first cytokine, is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises an anti-albumin antibody (such as a single domain antibody (sdAb), such as V H H single domain antibody). In some embodiments, the cytokine and albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, the first linker is a rigid linker. In some embodiments, the first linker is selected from SEQ ID NOs: 21, 22, and 24. In some embodiments, the first linker is a flexible linker. In some embodiments, the first linker is selected from SEQ ID NOs: 12-14.

[0145] In some embodiments, a fusion protein is provided comprising: a) IL-7, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-7 comprises the amino acid sequence of any one of SEQ ID NOs: 96-98, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 96-98, and wherein the albumin binding moiety comprises an albumin binding domain. In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine, which is the same as or different from the first cytokine, is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the cytokine and albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159.

[0146] In some embodiments, a fusion protein is provided comprising: a) IL-7, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-7 comprises the amino acid sequence of any one of SEQ ID NOs: 96-98, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 96-98, and wherein the albumin binding moiety comprises an anti-albumin antibody or fragment thereof (such as a single domain antibody, such as a V HH antibody). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159.

[0147] In some embodiments, a fusion protein is provided, comprising: a) IL-15, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises an anti-albumin antibody (such as a single domain antibody (sdAb), such as V HH single domain antibody). In some embodiments, the cytokine and the albumin binding portion are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, the cytokine is IL-15Rα or IL-15 that binds to IL-15Rα or a fragment thereof, and wherein IL-15Rα or IL-15 that binds to IL-15Rα or a fragment thereof includes the amino acid sequence of any one of SEQ ID NOs: 101-108 or a variant thereof, the variant having at least about 80% sequence identity with any one of SEQ ID NOs: 101-108. In some embodiments, the cytokine includes IL-15 and IL-15Rα. In some embodiments, IL-15 and IL-15Rα are connected by a linker ("linker between IL-15 and IL-15Rα"). In some embodiments, the linker between IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between IL-15 and IL-15Rα is non-cleavable.

[0148] In some embodiments, a fusion protein is provided comprising: a) IL-15, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15 comprises the amino acid sequence of any one of SEQ ID NOs: 99, 100, or 127, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 99, 100, or 127, and wherein the albumin binding moiety comprises an albumin binding domain. In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine, which is the same as or different from the first cytokine, is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to any one of SEQ ID NO: 3-11. In some embodiments, the cytokine and albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the cytokine is IL-15Rα or IL-15 bound to IL-15Rα or a fragment thereof, and wherein IL-15Rα or IL-15 bound to IL-15Rα or a fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108 or a variant thereof, the variant having at least about 80% sequence identity with any one of SEQ ID NOs: 101-108. In some embodiments, the cytokine comprises IL-15 and IL-15Rα. In some embodiments, IL-15 and IL-15Rα are connected by a linker ("linker between IL-15 and IL-15Rα"). In some embodiments, the linker between IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between IL-15 and IL-15Rα is non-cleavable.

[0149] In some embodiments, a fusion protein is provided comprising: a) IL-15, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15 comprises the amino acid sequence of any one of SEQ ID NOs: 99, 100, or 127, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 99, 100, or 127, and wherein the albumin binding moiety comprises an anti-albumin antibody or fragment thereof (such as a single domain antibody, such as a V H H antibody). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, the cytokine is IL-15Rα or IL-15 bound to IL-15Rα or a fragment thereof, and wherein IL-15Rα or IL-15 bound to IL-15Rα or a fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108 or a variant thereof, the variant having at least about 80% sequence identity with any one of SEQ ID NOs: 101-108. In some embodiments, the cytokine comprises IL-15 and IL-15Rα. In some embodiments, IL-15 and IL-15Rα are connected by a linker ("linker between IL-15 and IL-15Rα"). In some embodiments, the linker between IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between IL-15 and IL-15Rα is non-cleavable.

[0150] In some embodiments, a fusion protein is provided, comprising: a) a cytokine comprising an IL-15R sushi domain, and b) an albumin binding portion (such as an sdAb that binds to albumin). In some embodiments, the IL-15R sushi domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 127-128, or a variant thereof, which variant has at least 80% (such as at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 127-128. In some embodiments, the fusion protein does not comprise IL-15. In some embodiments, the fusion protein further comprises IL-15. In some embodiments, the IL-15 and IL-15R sushi domains are connected by a linker ("linker between IL-15 and IL-15R sushi domains"). In some embodiments, the linker between IL-15 and the IL-15R sushi domain is cleavable. In some embodiments, the linker between IL-15 and the IL-15R sushi domain is non-cleavable.

[0151] In some embodiments, a fusion protein is provided, comprising: a) IL-15 bound to IL-15Rα, the IL-15 comprising IL-15 and IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the albumin binding moiety is fused to one of IL-15 or IL-15Rα. In some embodiments, the albumin binding moiety is fused to both IL-15 and IL-15Rα. In some embodiments, the albumin binding moiety is fused to the N-terminus and / or C-terminus of IL-15Rα. In some embodiments, the albumin binding moiety is fused to the N-terminus and / or C-terminus of IL-15. In some embodiments, the albumin binding moiety is fused to the C-terminus of IL-15 and / or IL-15Rα. In some embodiments, the albumin binding moiety is fused to the N-terminus of IL-15 and / or IL-15Rα. In some embodiments, IL-15 is non-covalently bound to IL-15Rα. In some embodiments, IL-15 is fused to IL-15Rα. In some embodiments, IL-15 is fused to the N-terminus of IL-15Rα. In some embodiments, IL-15 is fused to the C-terminus of IL-15Rα. In some embodiments, the albumin binding portion is also fused to the N-terminus or C-terminus of the second cytokine. In some embodiments, the albumin binding portion is bound to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding portion includes an albumin binding domain (ABD). In some embodiments, the albumin binding domain includes an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to any one of SEQ ID NO: 3-11. In some embodiments, the albumin binding moiety comprises an anti-albumin antibody (such as a single domain antibody (sdAb), such as V HH single domain antibody). In some embodiments, the cytokine and the albumin binding portion are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, IL-15 and IL-15Rα are fused via a second linker. In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is selected from SEQ ID NOs: 27-45. In some embodiments, the second linker has the sequence of SEQ ID NO: 27. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 bound to IL-15Rα or a fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 101-108 or a variant thereof having at least about 80% sequence identity with any one of SEQ ID NOs: 101-108. In some embodiments, IL-15 and IL-15Rα are connected by a linker ("a linker between IL-15 and IL-15Rα"). In some embodiments, the linker between IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between IL-15 and IL-15Rα is non-cleavable.

[0152] In some embodiments, a fusion protein is provided, comprising: a) IL-15 bound to IL-15Rα, the IL-15 comprising IL-15 and IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the fusion protein comprises the IL-15, IL-15Rα, and albumin binding moiety from N-terminus to C-terminus in an order selected from the group consisting of: (1) albumin binding moiety, IL-15, IL-15Rα; (2) albumin binding moiety, IL-15Rα, IL-15; (3) IL-15, IL-15Rα, albumin binding moiety; (4) IL-15Rα, IL-15, albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises an anti-albumin antibody (such as a single domain antibody (sdAb), such as V HH single domain antibody). In some embodiments, the cytokine (IL-15 or IL-15Rα) and the albumin binding portion are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, IL-15 and IL-15Rα are fused via a second linker. In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is selected from SEQ ID NOs: 27-45. In some embodiments, the second linker has the sequence of SEQ ID NO: 27. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 bound to IL-15Rα or a fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 101-108 or a variant thereof having at least about 80% sequence identity with any one of SEQ ID NOs: 101-108. In some embodiments, IL-15 and IL-15Rα are connected by a linker ("a linker between IL-15 and IL-15Rα"). In some embodiments, the linker between IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between IL-15 and IL-15Rα is non-cleavable.

[0153] In some embodiments, a fusion protein is provided, comprising: a) IL-15 that binds to IL-15Rα, the IL-15 comprising IL-15 and IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15 comprises the amino acid sequence of any one of SEQ ID NOs: 99, 100, or 127, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 99, 100, or 127, and wherein the albumin binding moiety comprises an albumin binding domain. In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is identical or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain includes an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with any one of SEQ ID NO: 3-11. In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, IL-15 is non-covalently bound to IL-15Rα. In some embodiments, IL-15 is fused to IL-15Rα. In some embodiments, IL-15 is fused to the N-terminus of IL-15Rα. In some embodiments, IL-15 is fused to the C-terminus of IL-15Rα. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the IL-15, IL-15Rα, and the albumin binding portion in an order selected from the group consisting of: (1) albumin binding portion, IL-15, IL-15Rα; (2) albumin binding portion, IL-15Rα, IL-15; (3) IL-15, IL-15Rα, albumin binding portion; (4) IL-15Rα, IL-15, albumin binding portion. In some embodiments, IL-15 and IL-15Rα are fused via a second linker.In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is selected from SEQ ID NO: 27-45. In some embodiments, the second linker has the sequence of SEQ ID NO: 27. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the IL-15 bound to IL-15Rα or a fragment thereof comprises an amino acid sequence of any one of SEQ ID NO: 101-108 or a variant thereof, the variant having at least about 80% sequence identity with any one of SEQ ID NO: 101-108. In some embodiments, IL-15 and IL-15Rα are connected by a linker ("linker between IL-15 and IL-15Rα"). In some embodiments, the linker between IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between IL-15 and IL-15Rα is non-cleavable.

[0154] In some embodiments, a fusion protein is provided comprising: a) IL-15 that binds to IL-15Rα, the IL-15 comprising IL-15 and IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15 comprises the amino acid sequence of any one of SEQ ID NO: 99, 100, or 127, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NO: 99, 100, or 127, and wherein the albumin binding moiety comprises an anti-albumin antibody or fragment thereof (such as a single domain antibody, such as a V HH antibody). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, IL-15 is non-covalently bound to IL-15Rα. In some embodiments, IL-15 is fused to IL-15Rα. In some embodiments, IL-15 is fused to the N-terminus of IL-15Rα. In some embodiments, IL-15 is fused to the C-terminus of IL-15Rα. In some embodiments, the fusion protein comprises the IL-15, IL-15Rα, and albumin binding portion in an order selected from the group consisting of from N-terminus to C-terminus: (1) albumin binding portion, IL-15, IL-15Rα; (2) albumin binding portion, IL-15Rα, IL-15; (3) IL-15, IL-15Rα, albumin binding portion; (4) IL-15Rα, IL-15, albumin binding portion. In some embodiments, IL-15 and IL-15Rα are fused via a second linker. In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 27-45. In some embodiments, the second linker has the sequence of SEQ ID NO: 27. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 that binds to IL-15Rα or a fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 101-108, or a variant thereof having at least about 80% sequence identity with any one of SEQ ID NOs: 101-108. In some embodiments, IL-15 and IL-15Rα are connected by a linker ("a linker between IL-15 and IL-15Rα"). In some embodiments, the linker between IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between IL-15 and IL-15Rα is non-cleavable.

[0155] In some embodiments, a fusion protein is provided, comprising: a) IL-15 that binds to IL-15Rα, the IL-15 comprising IL-15 and IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15Rα comprises the amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104), or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104), and wherein the albumin binding moiety comprises an albumin binding domain. In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is identical or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain includes an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with any one of SEQ ID NO: 3-11. In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, IL-15 is non-covalently bound to IL-15Rα. In some embodiments, IL-15 is fused to IL-15Rα. In some embodiments, IL-15 is fused to the N-terminus of IL-15Rα. In some embodiments, IL-15 is fused to the C-terminus of IL-15Rα. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the IL-15, IL-15Rα, and the albumin binding portion in an order selected from the group consisting of: (1) albumin binding portion, IL-15, IL-15Rα; (2) albumin binding portion, IL-15Rα, IL-15; (3) IL-15, IL-15Rα, albumin binding portion; (4) IL-15Rα, IL-15, albumin binding portion.In some embodiments, IL-15 and IL-15Rα are fused via a second linker. In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is selected from SEQ ID NOs: 27-45. In some embodiments, the second linker has the sequence of SEQ ID NO: 27. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is selected from SEQ ID NOs: 12-26 and 158-159.

[0156] In some embodiments, a fusion protein is provided comprising: a) IL-15 that binds to IL-15Rα, the IL-15 comprising IL-15 and IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15Rα comprises the amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104), or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104), and wherein the albumin binding moiety comprises an anti-albumin antibody or fragment thereof (such as a single domain antibody, such as a V HH antibody). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, IL-15 is non-covalently bound to IL-15Rα. In some embodiments, IL-15 is fused to IL-15Rα. In some embodiments, IL-15 is fused to the N-terminus of IL-15Rα. In some embodiments, IL-15 is fused to the C-terminus of IL-15Rα. In some embodiments, the fusion protein comprises the IL-15, IL-15Rα, and albumin binding portion in an order selected from the group consisting of from N-terminus to C-terminus: (1) albumin binding portion, IL-15, IL-15Rα; (2) albumin binding portion, IL-15Rα, IL-15; (3) IL-15, IL-15Rα, albumin binding portion; (4) IL-15Rα, IL-15, albumin binding portion. In some embodiments, IL-15 and IL-15Rα are fused via a second linker. In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 27-45. In some embodiments, the second linker has the sequence of SEQ ID NO: 27. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 12-26 and 158-159.

[0157] In some embodiments, a fusion protein is provided, comprising: a) IL-15 that binds to IL-15Rα, the IL-15 comprising IL-15 and IL-15Rα, and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the IL-15 comprises the amino acid sequence of any one of SEQ ID NOs: 99, 100, or 127, or a variant thereof, which variant has at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 99, 100, or 127, wherein the IL-15Rα comprises the amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104), or a variant thereof, which variant has at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: NOs: 103-104) have at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity, and wherein the albumin binding portion comprises an anti-albumin antibody or fragment thereof (such as a single domain antibody, such as a V HH antibody). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, IL-15 is non-covalently bound to IL-15Rα. In some embodiments, IL-15 is fused to IL-15Rα. In some embodiments, IL-15 is fused to the N-terminus of IL-15Rα. In some embodiments, IL-15 is fused to the C-terminus of IL-15Rα. In some embodiments, the fusion protein comprises the IL-15, IL-15Rα, and albumin binding portion in an order selected from the group consisting of from N-terminus to C-terminus: (1) albumin binding portion, IL-15, IL-15Rα; (2) albumin binding portion, IL-15Rα, IL-15; (3) IL-15, IL-15Rα, albumin binding portion; (4) IL-15Rα, IL-15, albumin binding portion. In some embodiments, IL-15 and IL-15Rα are fused via a second linker. In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 27-45. In some embodiments, the second linker has the sequence of SEQ ID NO: 27. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 12-26 and 158-159.

[0158] In some embodiments, a fusion protein is provided, comprising: a) IL-15 that binds to IL-15Rα, the IL-15 comprising IL-15 and IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15, IL-15Rα, and albumin binding moiety are arranged from N-terminus to C-terminus in the order of albumin binding moiety, IL-15, IL-15Rα, wherein the IL-15 comprises the amino acid sequence of any one of SEQ ID NOs: 99, 100, or 127, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 99, 100, or 127, wherein the IL-15Rα comprises SEQ ID NOs: The amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104), or a variant thereof, which variant has at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104), and wherein the albumin binding moiety comprises an anti-albumin antibody or fragment thereof (such as a single domain antibody, such as a V H H antibody). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, IL-15 and IL-15Rα are fused via a second linker. In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is selected from SEQ ID NOs: 27-45. In some embodiments, the second linker has the sequence of SEQ ID NO: 27. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is selected from SEQ ID NOs: 12-26 and 158-159.

[0159] In some embodiments, a fusion protein is provided, comprising: a) IL-33, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine, which is the same as or different from the first cytokine, is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises an anti-albumin antibody (such as a single domain antibody (sdAb), such as V H H single domain antibody). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, there is no linker between the cytokine and the albumin binding moiety.

[0160] In some embodiments, a fusion protein is provided comprising: a) IL-33, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-33 comprises the amino acid sequence of any one of SEQ ID NOs: 109 and 155-157, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 109 and 155-157, and wherein the albumin binding moiety comprises an albumin binding domain. In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine, which is the same as or different from the first cytokine, is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding portion binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with any one of SEQ ID NO: 3-11. In some embodiments, the cytokine and the albumin binding portion are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, there is no linker between the cytokine and the albumin binding portion.

[0161] In some embodiments, a fusion protein is provided comprising: a) IL-33, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-33 comprises the amino acid sequence of any one of SEQ ID NOs: 109 and 155-157, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 109 and 155-157, and wherein the albumin binding moiety comprises an anti-albumin antibody or fragment thereof (such as a single domain antibody, such as a V HH antibody). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, there is no linker between the cytokine and the albumin binding moiety.

[0162] In some embodiments, a fusion protein is provided, comprising: a) IL-22, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises an anti-albumin antibody (such as a single domain antibody (sdAb), such as V H H single domain antibody). In some embodiments, the cytokine and albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159.

[0163] In some embodiments, a fusion protein is provided comprising: a) IL-22, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-22 comprises the amino acid sequence of any one of SEQ ID NOs: 109-110, or a variant thereof, which variant has at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 99, 100, or 127, and wherein the albumin binding moiety comprises an albumin binding domain. In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine, which is the same as or different from the first cytokine, is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to any one of SEQ ID NO: 3-11. In some embodiments, the cytokine and albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159.

[0164] In some embodiments, a fusion protein is provided comprising: a) IL-22, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-22 comprises the amino acid sequence of any one of SEQ ID NOs: 109-110, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 109-110, and wherein the albumin binding moiety comprises an anti-albumin antibody or fragment thereof (such as a single domain antibody, such as a V HH antibody). In some embodiments, the cytokine is fused to the C-terminus of the albumin binding moiety. In some embodiments, the cytokine is fused to the N-terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety is fused to both the N-terminus and the C-terminus of the cytokine. In some embodiments, a second cytokine that is the same as or different from the first cytokine is fused to the other end of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159.

[0165] Fusion proteins comprising antigen binding moieties

[0166] Also provided herein is a fusion protein comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable. In some embodiments, the antigen binding moiety binds to a tumor antigen. Such fusion proteins provide a variety of advantages. For example, in some embodiments, the antigen binding moiety binding to a tumor antigen enables local delivery of the cytokine near the cancer, resulting in lower off-target toxicity and increased efficacy.

[0167] Such fusion protein provides certain advantages due to its tertiary structure and overall configuration design. For example, in some embodiments, cytokine (such as IL-21) is fused to the C-terminal of albumin binding portion by cleavable joint (such as MMP sensitive joint), and antigen binding moiety is fused to the N-terminal of albumin binding portion. The cytokine (such as IL-21) in the fusion protein can be temporarily blocked from interacting with cytokine receptor (such as IL-21R), because their interaction may need its N-terminal (near C-terminal in tertiary structure). When fusion protein is combined with tumor antigen, because known cancer cell secretes multiple MMP, if joint can be cut by MMP and become active, then cytokine (such as IL-21) can be discharged from fusion protein. MMP sensitive joint ensures that cancer with higher MMP activity usually has higher exposure to active cytokines. Therefore, the unnecessary toxicity and side effect of cytokine (such as IL-21) can be avoided. Furthermore, the efficiency of cancer delivery of fusion proteins can be enhanced by preventing the interaction between cytokines (such as IL-21) and cytokine receptors (such as IL-21Rα) on peripheral immune cells.

[0168] In some embodiments, the albumin binding moiety is a single domain antibody (such as a V H H antibody). In some embodiments, the antigen binding portion is a single domain antibody (such as a V H H antibody). In some embodiments, both the albumin binding portion and the antigen binding portion are single domain antibodies (such as V H H antibody).

[0169] In some embodiments, a fusion protein is provided comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable, wherein the albumin binding moiety is fused to the N-terminus of the cytokine, and wherein the antigen binding moiety is linked to the N-terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises a single domain antibody (sdAb). In some embodiments, the sdAb is a V HH single domain antibody. In some embodiments, the cytokine and albumin binding moieties are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the antigen binding moiety is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, protease, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NO: 12-45 and 158-159. In some embodiments, the antigen binding moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from mesothelin (MSLN), GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA. In some embodiments, the antigen binding portion is an antibody or a fragment thereof. In some embodiments, the antigen binding portion comprises a single domain antibody (sdAb). In some embodiments, the antigen binding portion comprises V H H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0170] In some embodiments, a fusion protein is provided comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable, wherein the albumin binding moiety is fused to the N-terminus of the cytokine, and wherein the antigen binding moiety is linked to the C-terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises a single domain antibody (sdAb). In some embodiments, the sdAb is a V H H single domain antibody. In some embodiments, the cytokine and albumin binding moieties are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the antigen binding moiety is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, protease, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NO: 12-45 and 158-159. In some embodiments, the antigen binding moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from mesothelin (MSLN), GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA. In some embodiments, the antigen binding portion is an antibody or a fragment thereof. In some embodiments, the antigen binding portion comprises a single domain antibody (sdAb). In some embodiments, the antigen binding portion comprises VH H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0171] In some embodiments, a fusion protein is provided comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable, wherein the albumin binding moiety is fused to the C-terminus of the cytokine, and wherein the antigen binding moiety is attached to the C-terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises a single domain antibody (sdAb). In some embodiments, the sdAb is a V HH single domain antibody. In some embodiments, the cytokine and albumin binding moieties are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the antigen binding moiety is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, protease, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NO: 12-45 and 158-159. In some embodiments, the antigen binding moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from mesothelin (MSLN), GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA. In some embodiments, the antigen binding portion is an antibody or a fragment thereof. In some embodiments, the antigen binding portion comprises a single domain antibody (sdAb). In some embodiments, the antigen binding portion comprises V H H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0172] In some embodiments, a fusion protein is provided comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable, wherein the albumin binding moiety is fused to the C-terminus of the cytokine, and wherein the antigen binding moiety is linked to the N-terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises a single domain antibody (sdAb). In some embodiments, the sdAb is a V H H single domain antibody. In some embodiments, the cytokine and albumin binding moieties are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the antigen binding moiety is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, protease, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NO: 12-45 and 158-159. In some embodiments, the antigen binding moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from mesothelin (MSLN), GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA. In some embodiments, the antigen binding portion is an antibody or a fragment thereof. In some embodiments, the antigen binding portion comprises a single domain antibody (sdAb). In some embodiments, the antigen binding portion comprises VH H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0173] In some embodiments, a fusion protein is provided comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable, wherein the albumin binding moiety is an albumin binding domain (ABD), and wherein the antigen binding moiety is a single domain antibody that binds to a tumor antigen (such as a V that binds to a tumor antigen). H H antibody). In some embodiments, the albumin binding portion is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the antigen binding portion is connected to the N-terminus or C-terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33 and IL-22. In some embodiments, the albumin binding portion binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain includes an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to any one of SEQ ID NO: 3-11. In some embodiments, the cytokine and the albumin binding portion are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the antigen binding portion is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, proteinase, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NO: 12-45 and 158-159. In some embodiments, the tumor antigen is selected from mesothelin (MSLN), GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA. In some embodiments, the antigen binding portion includes V HH single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0174] In some embodiments, a fusion protein is provided comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable, and wherein the albumin binding moiety is a single domain antibody ("anti-albumin dsAb," such as anti-albumin V H H antibody), and wherein the antigen binding portion is a single domain antibody that binds to a tumor antigen (such as a V H H antibody). In some embodiments, the albumin binding portion is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the antigen binding portion is attached to the N-terminus or C-terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33 and IL-22. In some embodiments, the albumin binding portion binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the anti-albumin sdAb is V H H single domain antibody. In some embodiments, the cytokine and albumin binding moieties are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the antigen binding moiety is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, protease, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NO: 12-45 and 158-159. In some embodiments, the tumor antigen is selected from mesothelin (MSLN), GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from the group consisting of CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA. In some embodiments, the antigen binding portion comprises V H H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0175] In some embodiments, a fusion protein is provided comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable, wherein the albumin binding moiety is an albumin binding domain or a single domain antibody ("anti-albumin dsAb," such as anti-albumin V H H antibody), and wherein the antigen binding portion is an anti-mesothelin single domain antibody (such as anti-MSLN V H H antibody). In some embodiments, the albumin binding portion is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the antigen binding portion is connected to the N-terminus or C-terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33 and IL-22. In some embodiments, the albumin binding portion binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain includes an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with any one of SEQ ID NO: 3-11. In some embodiments, the albumin binding moiety comprises a single domain antibody (sdAb) comprising a V H H single domain antibody. In some embodiments, the cytokine and albumin binding moieties are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, the antigen binding moiety is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, protease, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NOs: 12-45 and 158-159.

[0176] In some embodiments, a fusion protein is provided comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable, wherein the albumin binding moiety is an albumin binding domain or a single domain antibody ("anti-albumin dsAb," such as anti-albumin V H H antibody), and wherein the antigen binding portion is a single domain antibody that binds to a tumor antigen, wherein the cytokine is IL-21. In some embodiments, the albumin binding portion is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the antigen binding portion is attached to the N-terminus or C-terminus of the cytokine-ALBBM. In some embodiments, the albumin binding portion binds to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NO: 3-11 or a variant thereof, the variant having at least about 80% (such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with any one of SEQ ID NO: 3-11. In some embodiments, the albumin binding portion comprises a single domain antibody (sdAb) comprising V H H single domain antibody. In some embodiments, the cytokine and albumin binding moieties are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the antigen binding moiety is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, protease, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NO: 12-45 and 158-159. In some embodiments, the tumor antigen is selected from mesothelin (MSLN), GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from the group consisting of CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA. In some embodiments, the antigen binding portion comprises V H H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0177] In some embodiments, a fusion protein is provided comprising: a) a cytokine fused to an albumin binding moiety ("cytokine-ALBBM"), and b) an antigen binding moiety, wherein the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable, wherein the albumin binding moiety is an albumin binding domain or a single domain antibody ("anti-albumin dsAb," such as anti-albumin V H H antibody), and wherein the antigen binding portion comprises an anti-mesothelin single domain antibody comprising an anti-mesothelin heavy chain variable region (anti-MSLN V H ), wherein: a) anti-MSLNV H comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 46 or a variant comprising up to 3, 2 or 1 substitutions in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 47 or a variant comprising up to 3, 2 or 1 substitutions in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 48 or a variant comprising up to 3, 2 or 1 substitutions in CDR3; or b) anti-MSLN V H In some embodiments, the albumin binding portion is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the antigen binding portion is connected to the N-terminus or C-terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33 and IL-22. In some embodiments, the albumin binding portion is bound to human serum albumin (HSA) and / or cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from SEQ ID NOs: 3-11 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 3-11. In some embodiments, the albumin binding moiety comprises a single domain antibody (sdAb) comprising V HH single domain antibody. In some embodiments, the cytokine and albumin binding moieties are connected by a first linker. In some embodiments, the first linker has a length of about one to thirty amino acids. In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159. In some embodiments, the antigen binding moiety is fused to the cytokine-ALBBM via a second linker. In some embodiments, the second linker has a length of about one to thirty amino acids. In some embodiments, the second linker is cleavable. In some embodiments, the cleavable linker is a matrix metalloproteinase, legumin, protease, or urokinase-sensitive linker. In some embodiments, the second linker is selected from SEQ ID NOs: 12-45 and 158-159.

[0178] In some embodiments, the fusion protein includes the amino acid sequence of any one of SEQ ID NOs: 120-125, 129-154, and 160-167, or a variant thereof, which has at least about 80% (such as, at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 120-125, 129-154, and 160-167.

[0179] A. Fusion Protein Characteristics

[0180] 1 Serum half-life

[0181] In some embodiments, when administered to an individual, the serum half-life of the fusion protein is at least about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 24 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour. The fusion protein can be administered by various routes, such as intravenous, oral, subcutaneous, or intraperitoneal administration.

[0182] In some embodiments, the serum half-life of the fusion protein is longer than the serum half-life of the reference protein (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% longer). In some embodiments, the serum half-life of the fusion protein is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold or 50-fold longer than the serum half-life of the reference protein. In some embodiments, the reference protein comprises the same cytokine and / or the same antigen binding portion, but does not have an albumin binding portion. In some embodiments, the same cytokine and the same antigen binding portion are fused in the same order as the fusion protein and / or through the same linker.

[0183] 2 Stability

[0184] In some embodiments, the fusion protein has greater stability than the reference protein. In some embodiments, the reference protein comprises the same cytokine and / or the same antigen binding portion, but does not have an albumin binding portion. In some embodiments, the same cytokine and the same antigen binding portion are fused in the same order and / or via the same linker as the fusion protein.

[0185] In some embodiments, stability comprises thermal stability.

[0186] In some embodiments, stability is assessed by the extent to which the fusion protein retains an acceptable degree of chemical structure or biological function after storage under defined conditions. In some embodiments, a fusion protein has high stability even if it does not retain 100% of its chemical structure or biological function after storage for a defined amount of time. In some embodiments, a fusion protein that retains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function as described herein after storage for a defined amount of time can be considered to have high stability.

[0187] Stability can be measured by determining the percentage of natural (non-aggregation or non-degradation) fusion protein retained in the formulation (liquid or reconstructed) after storing a defined amount of time at a defined temperature. The percentage of natural fusion protein can be determined by size exclusion chromatography (for example, size exclusion high performance liquid chromatography [SE-HPLC]), and the natural means non-aggregation and non-degradation. In some embodiments, after storing a defined amount of time at a given temperature, at least about 90% (such as, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the natural form of the fusion protein can be detected in the formulation. In some embodiments, at least about 90% (such as at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the native form of the fusion protein is detectable in the formulation after at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, at least about 32 hours, at least about 34 hours, at least about 36 hours, at least about 38 hours, at least about 40 hours, at least about 42 hours, at least about 44 hours, at least about 46 hours, or at least about 48 hours at room temperature (about 25°C).

[0188] Stability can be measured by determining the percentage of fusion protein formed in the form of aggregates in a formulation (liquid or reconstituted) after a defined amount of time stored at a defined temperature, wherein stability is inversely proportional to the percentage of aggregates formed. The percentage of aggregated fusion protein can be determined by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]). In some embodiments, after a defined amount of time is stored at a given temperature, there is less than about 10% (preferably less than about 5%) of the fusion protein in the form of aggregates in the formulation. In some embodiments, the fusion proteins described herein are substantially free of aggregation, for example, no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the fusion protein is detected in the formulation as aggregates after storage at a given temperature for a defined amount of time, for example, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, at least about 32 hours, at least about 34 hours, at least about 36 hours, at least about 38 hours, at least about 40 hours, at least about 42 hours, at least about 44 hours, at least about 46 hours, or at least about 48 hours at room temperature (about 25°C).

[0189] Measuring the binding affinity of fusion protein and its target can also be used for assessing stability.For example, if after the time (for example, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours) of storing a limited amount at, for example, room temperature (about 25 DEG C), the affinity that cytokine and / or antigen-binding domains have is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even higher of the binding affinity of antibody before described storage, then the fusion protein of the application can be considered to be stable.Binding affinity can be determined by any method, such as ELISA or plasma resonance.Fusion protein can be directly measured in combination with this cell, for example, by FACS analysis.

[0190] 3 Clinical characteristics

[0191] In some embodiments, the fusion proteins described herein have improved clinical properties relative to the reference protein. In some embodiments, the fusion protein exhibits improved cytotoxic activity compared to the reference protein. In some embodiments, the fusion protein exhibits higher anti-tumor effects (such as, reducing tumor burden, improving survival rate, etc.) compared to the reference protein. In some embodiments, the reference protein includes the same cytokine and / or the same antigen binding portion, but does not have an albumin binding portion. In some embodiments, the same cytokine and the same antigen binding portion are fused in the same order and / or through the same linker as the fusion protein.

[0192] Cytotoxicity

[0193] The cytotoxicity (such as ADCC activity) of the fusion proteins described herein to cells can be tested using a variety of assays. For example, cancer cell lines expressing antigens that can be recognized by the fusion protein and effector cells (e.g., PBMC cells) are mixed together in a 96-well plate. Different concentrations of the fusion protein are added to each well. After incubation, the EC 50 (represents ADCC activity).

[0194] In some embodiments, the fusion protein exhibits improved ADCC activity against cells compared to a reference protein. In some embodiments, the EC of the cell-specific fusion protein 50In some embodiments, the cell is a tumor cell. In some embodiments, the tumor cell is derived from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the reference protein comprises the same cytokine and / or the same antigen binding portion, but does not have an albumin binding portion. In some embodiments, the same cytokine and the same antigen binding portion are fused in the same order and / or via the same linker as the fusion protein.

[0195] Treating cancer

[0196] In some embodiments, the fusion protein treats cancer in an individual (e.g., by inhibiting tumor growth). In some embodiments, the fusion protein exhibits an enhanced anti-tumor effect for cancer compared to a reference protein. For example, in some embodiments, the administration of the fusion protein reduces tumor burden (such as, tumor volume is reduced by at least about 10%, 20%, 30%, 40% or 50%) compared to a reference protein. In some embodiments, cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, esophageal cancer, gastric cancer and colorectal cancer. In some embodiments, the reference protein includes the same cytokine and / or the same antigen binding portion, but does not have an albumin binding portion. In some embodiments, the same cytokine and the same antigen binding portion are fused in the same order as the fusion protein and / or by the same linker.

[0197] B.Connector

[0198] In some embodiments, the fusion proteins described herein include a first linker between the cytokine and the albumin binding moiety. In some embodiments, the fusion proteins described herein include a second linker between the cytokine fused to the albumin binding moiety ("cytokine-ALBBM") and the antigen binding moiety.

[0199] In some embodiments, the first linker is a rigid linker.In some embodiments, the first linker is selected from SEQ ID NO: 21, 22 and 24.

[0200] In some embodiments, the first linker is a flexible linker.In some embodiments, the first linker is selected from SEQ ID NOs: 12-14.

[0201] In some embodiments, the first linker is a non-cleavable linker.

[0202] In some embodiments, the first linker has a length of about one to forty (such as one to thirty-five, one to thirty, one to twenty-five, one to twenty, four to twenty, or four to sixteen) amino acids.

[0203] In some embodiments, the first linker is selected from SEQ ID NOs: 12-26 and 158-159.

[0204] In some embodiments, the second linker is a rigid linker.In some embodiments, the second linker is selected from SEQ ID NO: 21, 22 and 24.

[0205] In some embodiments, the second linker is a flexible linker.In some embodiments, the second linker is selected from SEQ ID NOs: 12-14.

[0206] In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is a matrix metalloproteinase, legumin, matriptase, or urokinase-sensitive linker.

[0207] In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 12-45 and 158- 159. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 27-45.

[0208] In some embodiments, the second linker has a length of about one to forty (such as one to thirty-five, one to thirty, one to twenty-five, one to twenty, four to twenty, four to sixteen, four to twelve, or five to nine) amino acids.

[0209] In some embodiments, the first and / or second linker does not include a Gly-Gly-Gly-Gly-Ser sequence. In some embodiments, the first and / or second linker is not a GS linker.

[0210] The length, flexibility and / or other properties of the first and / or second linker used in the fusion protein may have some impact on the following properties, including but not limited to the affinity, specificity or avidity of one or more components (such as cytokines, albumin binding molecules and / or antigen binding portions) binding to their targets. For example, a longer linker can be selected to ensure that two adjacent domains do not interfere with each other in space. In some embodiments, the linker (such as a peptide linker) includes flexible residues (such as glycine and serine) so that adjacent domains can move freely relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker.

[0211] Other linker considerations include the effect on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, ability to be incorporated into micelles or liposomes, etc.

[0212] Non-peptide linkers

[0213] The coupling of the above components can be achieved by any chemical reaction that will bind the two molecules, as long as both components retain their respective activity, i.e., bind to cytokine receptors, albumin, or target antigens, respectively. This connection can include a variety of chemical mechanisms, such as covalent binding, affinity binding, embedding, coordinate binding, and complexing. In some embodiments, the binding is covalent. Covalent binding can be achieved by directly condensing existing side chains or by incorporating external bridging molecules. In this case, many divalent or multivalent linking agents can be used to couple protein molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various coupling agents known in the art, but is illustrative of the more common coupling agents (see Killen and Lindstrom, Jour. Immun., Vol. 133: pp. 1335-2549, 1984; Jansen et al., Immunological Reviews, Vol. 62: pp. 185-216, 1982; and Vitetta et al., Science, Vol. 238: p. 1098, 1987).

[0214] Linkers useful in the present application are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res., Vol. 44: pp. 201-208, 1984), describing the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). In some embodiments, the non-peptide linker used herein includes: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene: Pierce Chem. Co., catalog number 21558G); (iii) SPDP (succinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoic acid: Pierce Chem. Co., catalog number 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6-[3-(2-pyridyldithio)-propionamido]hexanoic acid: Pierce Chem. Co., catalog number 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., catalog number 24510).

[0215] The components contained in the above-mentioned linkers have different properties and may therefore result in fusion proteins with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylic acids are more stable than sulfo-NHS esters of aromatic carboxylic acids. Linkers containing NHS-esters have lower solubility than sulfo-NHS esters. In addition, the linker SMPT contains sterically hindered disulfide bonds and can form antibody fusion proteins with increased stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in less available antibody fusion protein. Sulfo-NHS can especially enhance the stability of carbodiimide conjugates. When carbodiimide conjugates (such as EDC) are used in combination with sulfo-NHS, esters are formed that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

[0216] Peptide linker

[0217] The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain antibody can be used as a linker. See, for example, WO 1996 / 34103.

[0218] The peptide linker can have any suitable length. In some embodiments, the length of the peptide linker is at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. In some embodiments, the length of the peptide linker is no more than about any of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less amino acids. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids in length, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0219] The basic technical feature of this peptide linker is that the peptide linker does not have any polymerization activity. The characteristics of peptide linkers, including the absence of promotion of secondary structure, are known in the art and are described in, for example, Dall'Acqua et al. (Biochem., 1998, Vol. 37, pp. 9266-9273), Cheadle et al. (Mol Immunol, 1992, Vol. 29, pp. 21-30), and Raag and Whitlow (FASEB, 1995, Vol. 9, No. 1, pp. 73-80). With regard to "peptide linkers", a particularly preferred amino acid is Gly. In addition, a peptide linker that does not promote any secondary structure is preferred. The connection between domains can be provided by, for example, genetic engineering. Methods for preparing fused and operably linked fusion protein components and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY, 1989 and 1994, or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0220] The peptide linker may be a stable linker that is not cleavable by proteases, particularly matrix metalloproteinases (MMPs).

[0221] The linker can also be a flexible linker. Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , (GSGS) n (SEQ ID NO: 19), (GGSG) n (SEQ ID NO: 20), (GGGGS) n (SEQ ID NO: 14), wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can be used as neutral tethers between components. Glycine even enters significantly more phi-psi space than alanine and is much less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem., Vol. 11, pp. 173-142, 1992). One of ordinary skill will recognize that the design of the antibody fusion protein may include a fully or partially flexible linker, such that the linker may include a flexible linker portion and one or more portions that impart a less flexible structure to provide the desired antibody fusion protein structure.

[0222] C. Cytokines

[0223] The fusion proteins described herein include cytokines. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33, and IL-22.

[0224] IL-21

[0225] IL-21 is a type I cytokine produced by T cells and natural killer T cells that has pleiotropic effects on a variety of immune and non-immune cell types. This cytokine has different effects on a variety of cell types, including but not limited to CD4 + T cells and CD8 + T cells, B cells, macrophages, monocytes, and dendritic cells (DCs). The functional receptor for IL-21 consists of the IL-21 receptor (IL-21R) and the common cytokine receptor gamma chain (γc), which is also a subunit of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors.

[0226] NK cells and CD8 +Activation of the cytotoxic program in T cells is key to cancer immunotherapy, and early studies have provided compelling evidence that IL-21 is a promising immunotherapeutic agent for this disease. IL-21 promotes maturation, enhances cytotoxicity, and induces IFN-γ and perforin production by NK cells. Correspondingly, IL-21-induced cytolytic activity significantly inhibits the growth of B16 melanoma. Furthermore, IL-21, together with IL-15, expands antigen-specific CD8 + T cell numbers and their effector functions lead to tumor regression. Leonard et al., F1000 Res., 2016 Feb 26;5.pii:F1000 FacultyRev-224.

[0227] In some embodiments, the cytokine is IL-21. In some embodiments, IL-21 is wild-type IL-21. In some embodiments, IL-21 is derived from human IL-21. In some embodiments, IL-21 is human wild-type IL-21. In some embodiments, IL-21 is truncated IL-21.

[0228] In some embodiments, IL-21 comprises the amino acid sequence set forth in SEQ ID NO: 1, 2, 126, 171, or 172, or a variant thereof, which variant has at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 1, 2, 126, 171, or 172.

[0229] In some embodiments, the IL-21 is a truncated IL-21. In some embodiments, the truncated IL-21 comprises the amino acid sequence of SEQ ID NO: 126, 171 or 172.

[0230] In some embodiments, the IL-21 variant lacks one or more amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any amino acid between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any two amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any three amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any four amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any five amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any six amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any seven amino acids between L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any eight amino acids between L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any nine amino acids between L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any ten amino acids between L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks all eleven amino acids between L123 and S133 at the C-terminus.

[0231] In some embodiments, the IL-21 variant lacks 11 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 10 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 9 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 8 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 7 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 6 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 5 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 4 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 3 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 2 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 1 amino acid at the C-terminus of SEQ ID NO: 1.

[0232] In some embodiments, the IL-21 variant lacks 14, 13, or 12 amino acids at the C-terminus of SEQ ID NO: 1.

[0233] In some embodiments, the IL-21 variant lacks at least 9-10, 9-11, 10-12, or 12-15 consecutive amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks at least 9-14, 10-14, 11-14, or 12-14 consecutive amino acids at the C-terminus of SEQ ID NO: 1.

[0234] In some embodiments, the IL-21 variant provided herein has the amino acid sequence of SEQ ID NO: 2, which lacks 10 amino acids at the C-terminus and represents the sequence of Q1 to L123 of SEQ ID NO: 1.

[0235] IL-7

[0236] IL-7 is a member of the IL-2 superfamily. The IL-2 superfamily includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. It binds to a receptor that shares a common γ chain subunit. In addition to the common γ chain subunit, the IL-7 receptor (IL-7R) also requires the IL-7R α chain for binding. See Lin et al., Anticancer Res., March 2017, Vol. 37, No. 3: pp. 963-967.

[0237] Interleukin-7 (IL-7) is essential for T cell development in mice and humans and is produced by stromal tissues rather than activated lymphocytes. Under normal conditions, IL-7 is a restricted source of T cells, but it accumulates during lymphocytopenia. IL-7 signals through a heterodimeric receptor composed of the IL-7 receptor alpha chain (IL-7Rα) and the common cytokine receptor gamma chain (γc). IL-7 has also recently been shown to regulate lymphoid tissue inducer (LTi) cells, which induce the development of secondary lymphoid organs and can induce tertiary lymphoid tissues in the setting of chronic inflammation after birth. In animals, IL-7 therapy enhances the effectiveness of adoptive immunotherapy for cancer, enhances vaccine responses, and enhances viral clearance in acute and chronic infections. See Mackall et al., Nature Reviews Immunology, Vol. 11, pp. 330-342, 2011.

[0238] In some embodiments, the cytokine is IL-7. In some embodiments, the IL-7 is wild-type IL-7. In some embodiments, the IL-7 is derived from human IL-7. In some embodiments, the IL-7 is human wild-type IL-7.

[0239] In some embodiments, IL-7 comprises the amino acid sequence of any one of SEQ ID NOs: 96-98, or a variant thereof having at least 80% (such as at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 96-98.

[0240] IL-15, IL-15Rα, and IL-15 bound to IL-15Rα

[0241] The heterotrimeric receptor for IL-15 shares the IL-2R / IL-15Rβ (CD122) and a common gamma (γc) chain (CD132) with the IL-2 receptor. IL-15 and IL-2 share certain functions, including stimulation of T cell proliferation, generation of cytotoxic T lymphocytes, stimulation of B cell immunoglobulin synthesis, and generation and maintenance of NK cells. However, IL-2 and IL-15 also have distinct and often competing roles in many adaptive immune responses. Unlike IL-2, IL-15 is not required for the maintenance of regulatory T cells (Tregs) that can attenuate anti-tumor immune responses. In contrast to IL-15, IL-2 inhibits T cell responses through activation-induced cell death (AICD) of CD8+ effector T cells. However, IL-15 is required for the differentiation of NK cells, effector CD8+ cells, and memory phenotype CD8+ T cells. Furthermore, based on preclinical studies, their toxicities appear to be distinct, with less vascular capillary leakage observed with IL-15 compared to IL-2. In summary, IL-15 primarily stimulates the proliferation and cytotoxic function of CD8 T cells and NK cells, leading to enhanced anti-tumor responses. However, although IL-15 initially showed promise as a cancer therapeutic, its efficacy was limited by its short in vivo half-life. Steel et al., Trends Pharmacol Sci., January 2012; Vol. 33, No. 1: pp. 35-41. See Robinson et al., Immunol Lett., October 2017; Vol. 190: pp. 159-168.

[0242] In some cases, the efficacy of IL-15 as a therapeutic agent is limited by the availability of IL-15Rα, which plays an integral role in stabilizing and increasing the biological activity of IL-15. Because unbound IL-15 does not naturally occur in vivo, IL-15 bound to IL-15Rα resembles the physiological form of IL-15 and has a higher affinity for IL-15Rβ / γC than free IL-15. See Robinson et al., Immunol Lett., October 2017; Vol. 190: pp. 159-168.

[0243] In some embodiments, the cytokine is or includes IL-15. In some embodiments, the IL-15 is wild-type IL-15. In some embodiments, the IL-15 is derived from human IL-15. In some embodiments, the IL-15 is human wild-type IL-15.

[0244] In some embodiments, the cytokine is or includes IL-15Rα. In some embodiments, IL-15Rα is wild-type IL-15Rα. In some embodiments, IL-15Rα is derived from human IL-15Rα. In some embodiments, IL-15Rα is human wild-type IL-15Rα. In some embodiments, IL-15Rα is the sushi domain of the soluble IL-15 receptor (i.e., IL-15R sushi domain). In some embodiments, the IL-15R sushi domain includes or consists of the amino acid sequence of any one of SEQ ID NO: 127-128 or a variant thereof, which has at least 80% (such as, at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with any one of SEQ ID NO: 127-128.

[0245] In some embodiments, the cytokine is IL-15 that binds to IL-15Rα or a fragment thereof. In some embodiments, the IL-15 that binds to IL-15Rα or a fragment thereof includes wild-type IL-15 and / or wild-type IL-15Rα. In some embodiments, the IL-15 that binds to IL-15Rα or a fragment thereof includes IL-15 derived from human IL-15 and / or IL-15Rα derived from human IL-15Rα. In some embodiments, the IL-15 that binds to IL-15Rα or a fragment thereof includes human wild-type IL-15 and / or human wild-type IL-15Rα. In some embodiments, the IL-15 that binds to IL-15Rα or a fragment thereof includes IL-15 derived from mouse IL-15 and / or IL-15Rα derived from mouse IL-15Rα. In some embodiments, the IL-15 that binds to IL-15Rα or a fragment thereof includes mouse wild-type IL-15 and / or mouse wild-type IL-15Rα. In some embodiments, the IL-15 that binds to IL-15Rα or a fragment thereof is the sushi domain of the soluble IL-15 receptor (i.e., an IL-15R sushi domain). In some embodiments, the IL-15R sushi domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 127-128, or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 127-128.

[0246] In some embodiments, IL-15 is non-covalently bound to IL-15Rα. In some embodiments, IL-15 is fused to IL-15Rα. In some embodiments, IL-15 is fused to the N-terminus of IL-15Rα. In some embodiments, IL-15 is fused to the C-terminus of IL-15Rα. In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the IL-15, IL-15Rα, and albumin binding moiety in an order selected from the group consisting of: (1) albumin binding moiety, IL-15, IL-15Rα; (2) albumin binding moiety, IL-15Rα, IL-15; (3) IL-15, IL-15Rα, albumin binding moiety; (4) IL-15Rα, IL-15, albumin binding moiety. In some embodiments, IL-15 and IL-15Rα are fused via a second linker. In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 27-45. In some embodiments, the second linker has the sequence of SEQ ID NO: 27. In some embodiments, the second linker is a non-cleavable linker. In some embodiments, the second linker is selected from SEQ ID NO: 12-26 and 158-159.

[0247] In some embodiments, IL-15 or IL-15 bound to IL-15Rα comprises human IL-15 comprising the amino acid sequence of any one of SEQ ID NOs: 99, 100, or 127, or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 99, 100, or 127.

[0248] In some embodiments, IL-15 or IL-15 bound to IL-15Rα comprises human IL-15 comprising an N72D mutation. See Han et al., Cytokine., December 2011; Vol. 56, No. 3: pp. 804-810.

[0249] In some embodiments, IL-15Rα or IL-15 that binds to IL-15Rα comprises human IL-15Rα comprising the amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104), or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104).

[0250] IL-33

[0251] Interleukin-33 (IL-33) is a member of the IL-1 family. It was originally described as an inducer of type 2 immune responses, activating T helper 2 (T H 2) cells and mast cells. Now, there is increasing evidence that IL-33 also effectively stimulates group 2 innate lymphoid cells (ILC2), regulatory T (T reg ) cells, T H 1 cell, CD8 + This pleiotropic effect is reflected in the role of IL-33 in tissue and metabolic homeostasis, infection, inflammation, cancer, and central nervous system diseases.

[0252] IL-33 is widely expressed in stromal and barrier tissues, making it a ubiquitous key immunomodulator of type 1, type 2, and regulatory immune responses. Despite lacking a secretory sequence and being sequestered in the nucleus, IL-33 is released and processed by various proteases into a highly active form. IL-33 contributes to a cytokine network that not only controls pathogen removal but also supports tissue repair mediated by group 2 innate lymphoid cells and regulatory T cells. The role of IL-33 is expected to continue to expand, regulating both protective and pathological immune responses. Delivery or blockade of IL-33 is emerging as a promising therapeutic strategy for maintaining immune homeostasis and preventing infectious and inflammatory diseases. See Liew, Nature Reviews Immunology, Vol. 16, pp. 676–689, 2016.

[0253] In some embodiments, the cytokine is IL-33. In some embodiments, the IL-33 is wild-type IL-33. In some embodiments, the IL-33 is derived from human IL-33. In some embodiments, the IL-33 is wild-type human IL-33.

[0254] In some embodiments, IL-33 comprises the amino acid sequence of any one of SEQ ID NOs: 109 and 155-157, or a variant thereof having at least 80% (such as at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 109 and 155-157.

[0255] In some embodiments, IL-33 comprises one or more mutations selected from C208S, C227S, C232S, and C259S. See Cohen et al., Nature Communications, Vol. 6, Article No. 8327 (2015) IL-22

[0256] Interleukin-22 (IL-22) is a recently described cytokine of the IL-10 family that is produced by T-helper (Th)-17 cells, γδ T cells, NKT cells, and newly described innate lymphoid cells (ILCs). The human IL22 gene is located on chromosome 12q15 near the genes encoding IFN-γ and IL-26. The active secreted form of the cytokine is a 146-amino acid protein.

[0257] The IL-22 receptor (IL-22R) is a type 2 cytokine receptor and a member of the IL-10 receptor family, as well as a receptor for IL-10, IL-19, IL-20, IL-24, IL-26, IL-28, and IL-29. It is composed of two heterodimeric subunits, IL-22R1 and IL-10R2. Studies have shown that initial binding of IL-22 to the IL-22R1 subunit enables secondary binding to the IL-10R2 subunit, thereby enabling downstream signaling.

[0258] IL-22 has multiple functions, most notably its trophic effects on non-hematopoietic cells, particularly epithelial cells. IL-22 participates in epithelial regeneration and pathologies in multiple organs, depending on the context and / or cytokine milieu. Its involvement in a wide range of diseases makes it an attractive target for clinical development. See Dudakov et al., Annu Rev Immunol., Mar 21, 2015; Vol. 33: 747–785.

[0259] In some embodiments, the cytokine is IL-22. In some embodiments, the IL-22 is wild-type IL-22. In some embodiments, the IL-22 is derived from human IL-22. In some embodiments, the IL-22 is human wild-type IL-22.

[0260] In some embodiments, IL-22 comprises the amino acid sequence of any one of SEQ ID NOs: 110-111, or a variant thereof having at least 80% (such as at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 110-111.

[0261] Cytokine variants

[0262] In some embodiments, cytokine variants may be in fusion proteins provided herein. Mutation may be the replacement, deletion or insertion of one or more codons encoding a cytokine polypeptide, resulting in a change in the amino acid sequence compared to a human wild-type cytokine protein. Amino acid substitution may be the result of replacing an amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, for example, a conservative amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertion or deletion may optionally be within the range of about 1 to 10 amino acids. In certain embodiments, relative to the original molecule, substitution, deletion or insertion include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions. In some embodiments, substitution is a conservative amino acid substitution carried out at one or more predicted non-essential amino acid residues. The variation allowed can be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

[0263] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to multiple residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0264] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similarly charged side chain. Families of amino acid residues with similarly charged side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity (e.g., binding to a cytokine receptor) to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined.

[0265] Conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) can be made so as to maintain or not significantly alter the properties of the cytokine. Amino acids can be grouped according to the similarity of their side chain properties (see, e.g., Lehninger, Biochemistry, pp. 73-75 (2nd ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).

[0266] Alternatively, naturally occurring residues can be divided into the following groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0267] Methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis, can be used to perform mutations. Cloned DNA can be subjected to site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J., Vol. 237: 1-7; and Zoller et al., 1982, Nucl. Acids Res., Vol. 10: 6487-6500), cassette mutagenesis (see, e.g., Wells et al., 1985, Gene, Vol. 34: 315-323), or other known techniques to produce polypeptides.

[0268] D. Albumin binding fraction

[0269] Fusion proteins described herein include albumin binding molecules. Albumin binding molecules and methods of linking them to proteins of interest are described, for example, in WO 1991 / 01743, WO 2001 / 45746, WO 2002 / 076489, WO 2004 / 041865 or US20070269422A1, the contents of which are incorporated herein by reference.

[0270] The albumin binding molecule may be any of the albumin binding molecules described, for example, in WO1991 / 01743, WO2001 / 45746, WO2002 / 076489, WO2004 / 041865, US20070269422A1, US20160152686A1; Dennis et al., 2002, JBC, Vol. 277, No. 38: pp. 35035-35043.

[0271] In some embodiments, the albumin binding molecule binds to human serum albumin (HSA), cynomolgus monkey serum albumin (CMSA), and / or mouse serum albumin (MSA).

[0272] In some embodiments, the albumin binding moiety is synthesized with a K between about 1-1000 nM, such as about 1-900 nM, 1-800 nM, 1-700 nM, 1-600 nM, 1-500 nM, 1-400 nM, 1-300 nM, 1-200 nM, 1-100 nM, 1-50 nM, 1-25 nM, 0.1-1 nM. D Binds to albumin (such as HAS). In some embodiments, the albumin binding protein includes the albumin binding domain (ABD) of Streptococcus protein G (SPG). See, for example, Nygren et al., J. Mol. Recogn., 1988, Vol. 1, No. 2: pp. 69-74.

[0273] 1Albumin binding domain (ABD)

[0274] In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding molecule comprises the ABD of SPG strain G148. In some embodiments, the albumin binding molecule comprises the C-terminal albumin binding domain 3 (ABD3) of SPG strain G148. See, e.g., Nilvebrant and Hober, 2013, Comput. Struct. Biotechol. J., Vol. 6: e201303009.

[0275] In some embodiments, the ABD has

[0276] The amino acid sequence of LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP (SEQ ID NO: 3) is D It is about 1.2nM.

[0277] In some embodiments, an ABD having a relatively lower affinity for HSA is preferred compared to the ABD of SEQ ID NO: 3. Thus, variants of SEQ ID NO: 3 having a lower affinity for HSA are encompassed by the present disclosure.

[0278] In some specific embodiments, the ABD has the amino acid sequence of any one of SEQ ID NOs: 4-11.

[0279] The variation can be a substitution, deletion, or insertion of one or more codons encoding an ABD polypeptide of any one of SEQ ID NOs: 3-11 that results in an altered amino acid sequence. An amino acid substitution can be the result of replacing one amino acid with another amino acid having similar or different structural and / or chemical properties. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that result in amino acid substitutions.

[0280] The variation allowed can be determined by systematically inserting, deleting or substituting amino acids in the sequence and testing the activity of the resulting variants, and in some embodiments, selecting variants with lower affinity for HSA. Certain such variants are exemplified in Table 3.

[0281] 2 Anti-albumin antibodies or their fragments

[0282] According to the present invention, the albumin binding moiety may also be an anti-albumin antibody or an antigen-binding fragment thereof. In some embodiments, the anti-albumin antibody or an antigen-binding fragment thereof is an anti-HSA antibody or an antigen-binding fragment thereof.

[0283] Some isoforms of HSA are listed in Table 2 below (see, eg, UniProtKB-P02768 (ALBU_HUMAN)). In some embodiments, the anti-albumin antibody or antigen-binding fragment thereof binds to any one of SEQ ID NOs: 52-55.

[0284] Table 2.

[0285]

[0286] Anti-albumin antibodies or fragments thereof can be derived from any animal source, including birds and mammals (e.g., humans, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens). In certain embodiments, the antibodies are human or humanized monoclonal antibodies. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or from a mouse expressing antibodies derived from a human gene.

[0287] In certain embodiments, the anti-albumin antibody is a fully human antibody, such as a fully human antibody that immunospecifically binds to a cancer antigen. Such fully human antibodies would be preferred over fully mouse (or other fully or partially non-human species antibodies), humanized antibodies, or chimeric antibodies to minimize the development of unwanted or undesirable side effects, such as an immune response against a non-fully human antibody when administered to a subject.

[0288] The anti-albumin antibodies provided herein can be monospecific, bispecific, trispecific, or have greater multispecificity. Multispecific antibodies can be specific for different epitopes of a polypeptide, or can be specific for both the polypeptide and a heterologous epitope, such as a heterologous polypeptide or solid support material. In some embodiments, the antibodies provided herein are monospecific for a given epitope of a polypeptide and do not immunospecifically bind to other epitopes.

[0289] The anti-albumin antibodies provided herein may be monoclonal antibodies or derived from monoclonal antibodies. The anti-albumin antibodies may be, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies or humanized forms thereof, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0290] In particular, the anti-albumin antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to albumin (such as HSA). The immunoglobulin molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. In some embodiments, the anti-albumin antibody is an IgG antibody, such as an IgG1 antibody.

[0291] The present disclosure also includes variants and derivatives of anti-albumin antibodies, including antibody fragments that retain the ability to specifically bind to albumin epitopes. Exemplary fragments include Fab fragments (antibody fragments that contain an antigen binding domain and include a portion of a light chain and a heavy chain bridged by a disulfide bond); Fab' (antibody fragments that contain a single anti-binding domain that includes Fab and another portion of a heavy chain via a hinge region); F(ab')2 (two Fab' molecules linked by an interchain disulfide bond at the hinge region of the heavy chain; the Fab' molecules can be directed against the same or different epitopes); bispecific Fab (Fab molecule with two antigen binding domains, each of which can be directed against a different epitope); single-chain Fab chain comprising a variable region, also known as sFv (variable antigen binding determining regions of a single light chain and a heavy chain of an antibody linked together by a chain of 10-25 amino acids); disulfide-linked Fv or dsFv (antibody fragments linked together by disulfide bonds). The variable antigen-binding determining region of a single light chain and a heavy chain); Camelized VH (the variable antigen-binding determining region of a single heavy chain of an antibody, wherein some amino acids at the VH interface are those found in the heavy chain of a naturally occurring camel antibody); Bispecific sFv (sFv or dsFv molecule with two antigen-binding domains, wherein each domain can be directed against a different epitope); Diabody (a dimerized sFv formed when the VH domain of a first sFv is assembled with the VL domain of a second sFv and the VL domain of the first sFv is assembled with the VH domain of the second sFv; the two antigen-binding regions of the diabody can be directed against the same or different epitopes); and triabody (trimeric sFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are produced in a single complex; the three antigen-binding domains can be directed against the same or different epitopes). Derivatives of antibodies also include one or more CDR sequences of the antibody combining site. When two or more CDR sequences are present, the CDR sequences can be linked together on a scaffold. In certain embodiments, the anti-HSA antibodies provided herein comprise single-chain Fvs ("scFvs"). ScFvs are antibody fragments that comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, scFv polypeptides also include a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore, eds., Springer-Verlag, New York, Vol. 113, pp. 269-315, 1994.

[0292] In some cases, the use of anti-albumin antibody fragments is advantageous over the use of whole antibodies. The smaller size of the fragments allows for rapid clearance and can result in improved access to cells, tissues, or organs. For a review of certain antibody fragments, see Hudson et al., 2003, Nature Med., Vol. 9: pp. 129-134.

[0293] A variety of techniques for producing antibody fragments have been developed. Traditionally, these fragments are derived by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., 1992, J. Biochem. Biophys. Methods, Vol. 24: pp. 107-117; and Brennan et al., 1985, Science, Vol. 229: pp. 81-83). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed in E. coli (E. coli) or yeast cells and secreted therefrom, so these fragments can be easily produced in large quantities. Antibody fragments can be isolated from the above-mentioned antibody phage libraries. Alternatively, Fab'-SH fragments can be recovered directly from E. coli (E. coli) and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology, Vol. 10: pp. 163-167). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments containing salvage receptor binding epitope residues with increased in vivo half-life are described in, for example, U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to skilled practitioners. In certain embodiments, the antibody is a single-chain Fv fragment (scFv) (see, for example, WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458). Fv and scFv have complete binding sites lacking constant regions; therefore, they may be suitable for reducing nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce fusions of effector proteins at the amino or carboxyl termini of scFv (see, for example, Borrebeck, ed., supra). Antibody fragments can also be "linear antibodies", for example, as described in the references cited above. Such linear antibodies can be monospecific or multispecific, such as bispecific.

[0294] Single domain antibodies (sdAbs)

[0295] In some embodiments, the antibody fragment is a single domain antibody (sdAb).

[0296] In some embodiments, the sdAb is V H H single domain antibody.

[0297] In some embodiments, the sdAb is expressed with a K between about 1-1000 nM, such as about 1-900 nM, 1-800 nM, 1-700 nM, 1-600 nM, 1-500 nM, 1-400 nM, 1-300 nM, 1-200 nM, 1-100 nM, 1-50 nM, 1-25 nM, or 0.1-1 nM. D In other embodiments, the sdAb binds to albumin (such as HSA). In other embodiments, the sdAb binds to albumin (such as HSA) with a K of between about 10-800 nM (such as between about 20-500 nM, 50-300 nM, or 100-200 nM). D Bound to albumins such as HSA.

[0298] In some embodiments, the sdAb is expressed at a pH of about 5.5 and / or at a pH of about 7.5 with a K between about 1-1000 nM, such as about 1-900 nM, 1-800 nM, 1-700 nM, 1-600 nM, 1-500 nM, 1-400 nM, 1-300 nM, 1-200 nM, 1-100 nM, 1-50 nM, 1-25 nM, or 0.1-1 nM. D Binds to albumin (such as HSA). In some embodiments, the sdAb binds to albumin at a pH of about 5.5 and at a pH of about 7.5 with a K between about 1-200 nM. D Bound to albumins such as HSA.

[0299] In some embodiments, the single domain antibody or fragment thereof comprises a CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 69, 72, 75, 78, 81, 84, 87, 90, and 93, or a variant thereof comprising up to about 3 amino acid substitutions, a CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 70, 73, 76, 79, 82, 85, 88, 91, and 94, or a variant thereof comprising up to about 3 amino acid substitutions, and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 71, 74, 77, 80, 83, 86, 89, 92, and 95, or a variant thereof comprising up to about 3 amino acid substitutions.

[0300] In some embodiments, the single domain antibody or fragment thereof comprises any of the following: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 70, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71, or a variant thereof comprising up to about 3 amino acid substitutions; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 72, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 73, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 74, or a variant thereof comprising up to about 3 amino acid substitutions; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 75, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 76, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: NO:77, or a variant thereof comprising up to about 3 amino acid substitutions; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO:78, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO:79, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO:80, or a variant thereof comprising up to about 3 amino acid substitutions; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO:81, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO:82, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO:83, or a variant thereof comprising up to about 3 amino acid substitutions; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO:84, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO:85, or a variant thereof comprising up to about 3 amino acid substitutions NO:85, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO:86, or a variant thereof comprising up to about 3 amino acid substitutions; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO:87, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO:88, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO:89, or a variant thereof comprising up to about 3 amino acid substitutions;(8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 90, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 91, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92, or a variant thereof comprising up to about 3 amino acid substitutions; or (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 93, or a variant thereof comprising up to about 3 amino acid substitutions; a CDR2 comprising the amino acid sequence of SEQ ID NO: 94, or a variant thereof comprising up to about 3 amino acid substitutions; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 95, or a variant thereof comprising up to about 3 amino acid substitutions.

[0301] In some embodiments, the single domain antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-68 and 168-170. In some embodiments, the single domain antibody or fragment thereof comprises an amino acid sequence having about or at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-68 and 168-170. In some embodiments, the single domain antibody or fragment thereof comprises a V H An H domain comprising the amino acid sequence of any one of SEQ ID NOs: 60-68 and 168-170, or a variant thereof, which variant has at least about 80% (such as at least about any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NOs: 60-68 and 168-170.

[0302] In some embodiments, the single domain antibody or fragment thereof that binds to albumin comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a heavy chain variable domain having the sequence set forth in any one of SEQ ID NOs: 60-68 and 168-170.

[0303] E. Antigen Binding Portion

[0304] In some embodiments, the fusion protein further comprises an antigen binding moiety, and wherein the antigen binding moiety is fused to the N-terminus or C-terminus of the cytokine fused to the albumin binding moiety ("cytokine-ALBBM").

[0305] In some embodiments, the antigen binding portion binds to a tumor antigen. In some embodiments, the tumor is a solid tumor or a liquid tumor. In some embodiments, the tumor is a cancer selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer expresses high levels of tumor antigens. For example, in some embodiments, the level of cancer expression is at least 2 times, 5 times, 10 times, 20 times, 30 times, 40 times, or 50 times that of a reference tissue. In some embodiments, the reference tissue is a tissue that does not include cancer cells in the same individual.

[0306] In some embodiments, the tumor antigen is selected from mesothelin ("MSLN"), GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA.

[0307] In some embodiments, the antigen binding portion is an antibody (such as a full-length antibody) or an antigen binding fragment thereof. In some embodiments, the antibodies or antigen binding fragments provided herein can be immunospecifically bound to an epitope of a polypeptide, polypeptide fragment or antigen expressed on a cancer cell. In one embodiment, the antibody binds to a human cancer antigen. In some embodiments, the antibodies or antigen binding fragments provided herein bind to the extracellular domain (ECD) of a cancer antigen. In certain embodiments, the antibody binds to an epitope in the ECD of a cancer antigen. In some embodiments, the cancer antigen is expressed on solid tumors or liquid tumor cancer cells.

[0308] The antibodies that bind to the cancer antigens provided herein can be, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabody, single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies or humanized variants thereof, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0309] In some embodiments, provided herein are antibodies including immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing antigen binding sites that are immunospecifically bound to cancer antigens (e.g., solid tumor or liquid tumor cancer antigens). Provided herein are immunoglobulin molecules that can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), classification (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. In a specific embodiment, provided herein are antibodies that are IgG antibodies, such as IgG1 antibodies.

[0310] The present disclosure also includes variants and derivatives of antibodies, including antibody fragments that retain the ability to specifically bind to cancer antigen epitopes. Exemplary fragments include Fab fragments; Fab'; F(ab')2; bispecific Fab; single-chain Fab chains including variable regions, also referred to as sFv; disulfide-linked Fv or dsFv; camelized VH; bispecific sFv; diabodies; and triabodies. Derivatives of antibodies also include one or more CDR sequences of the antibody binding site. When two or more CDR sequences are present, the CDR sequences may be linked together on a scaffold. In certain embodiments, the antibodies provided herein include single-chain Fv ("scFv"). As briefly described in the above section, a variety of techniques for producing antibody fragments have been developed.

[0311] In some embodiments, the antigen binding moiety is a single variable domain antibody (sdAb) (such as a V H H antibody). Certain types of organisms (camelids and cartilaginous fish) have a single high-affinity V-like domain that is installed on an Fc equivalent domain structure as part of their immune system. (Woolven et al., 1999, Immunogenetics, Vol. 50: 98-101; and Stretsov et al., 2004, Proc Natl Acad Sci USA., Vol. 101: 12444-12449). The V-like domain (called V in camelids) H VH domains (called V-NARs in sharks) typically display long surface loops that allow penetration into the cavity of the target antigen. They also stabilize the isolated VH domain by masking a hydrophobic surface patch.

[0312] Anti-mesothelin single domain antibody (anti-MSLN dsAb)

[0313] In some embodiments, the antigen binding moiety is an anti-mesothelin single domain antibody ("anti-MSLN dsAb").

[0314] The anti-MSLN antibodies (e.g., sdAbs) provided herein can bind to any isoform of mesothelin or any fragment thereof, such as any one of SEQ ID NOs: 56-59. In some embodiments, the anti-MSLN antibodies provided herein bind to any one of SEQ ID NOs: 56-59 or a fragment thereof.

[0315] In some embodiments, an anti-MSLN dsAb comprises an anti-mesothelin heavy chain variable region (anti-MSLN V H ), wherein: a) anti-MSLN V H comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 48, or a variant comprising a total of up to 5, 4, 3, 2, or 1 amino acid substitutions in the CDRs; or b) an anti-MSLN V H A variant comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 51, or comprising a total of up to 5, 4, 3, 2, or 1 amino acid substitutions in the CDRs.

[0316] In some embodiments, an anti-MSLN dsAb comprises an anti-mesothelin heavy chain variable region (anti-MSLN V H ), wherein: a) anti-MSLN V H comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 46 or a variant comprising up to 3, 2 or 1 substitutions in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 47 or a variant comprising up to 3, 2 or 1 substitutions in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 48 or a variant comprising up to 3, 2 or 1 substitutions in CDR3; or b) anti-MSLN V H comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 49 or a variant comprising up to 3, 2, or 1 substitutions in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 50 or a variant comprising up to 3, 2, or 1 substitutions in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 51 or a variant comprising up to 3, 2, or 1 substitutions in CDR3. In some embodiments, the anti-MSLN dsAb comprises an anti-mesothelin heavy chain variable region (anti-MSLN V H ), wherein: a) anti-MSLN V HThe anti-MSLN dsAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 187 or a variant comprising up to 3, 2, or 1 substitutions in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 188 or a variant comprising up to 3, 2, or 1 substitutions in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 189 or a variant comprising up to 3, 2, or 1 substitutions in CDR3. In some embodiments, the anti-MSLN dsAb comprises an anti-mesothelin heavy chain variable region (anti-MSLN V H ), wherein: a) anti-MSLN V H The anti-MSLN dsAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 190 or a variant comprising up to 3, 2, or 1 substitutions in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 191 or a variant comprising up to 3, 2, or 1 substitutions in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 192 or a variant comprising up to 3, 2, or 1 substitutions in CDR3. In some embodiments, the anti-MSLN dsAb comprises an anti-mesothelin heavy chain variable region (anti-MSLN V H ), wherein: a) anti-MSLN V H comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 193 or a variant comprising up to 3, 2 or 1 substitutions in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 194 or a variant comprising up to 3, 2 or 1 substitutions in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 195 or a variant comprising up to 3, 2 or 1 substitutions in CDR3.

[0317] In some embodiments, the anti-MSLN sdAb comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of CDR1, CDR2, and CDR3, respectively, within a heavy chain variable domain having the sequence set forth in any one of SEQ ID NOs: 173-186.

[0318] F. Fusion protein variants

[0319] In some embodiments, the amino acid sequence variants of the fusion rotein provided herein are considered.For example, it may be desirable to improve the binding affinity and / or other biological properties of the fusion rotein in the integral body or any component of the fusion rotein. The amino acid sequence variants of the fusion rotein can be prepared by introducing suitable modifications or by peptide synthesis to the nucleotide sequence encoding the fusion rotein. Such modification includes, for example, the disappearance and / or insertion and / or replacement of the residue in the amino acid sequence of the fusion rotein. Any combination of disappearance, insertion and replacement can be carried out to obtain the final construct, provided that the final construct has the desired feature.

[0320] 1Substitutions, insertions, deletions and variants

[0321] In some embodiments, fusion protein variants having one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVRs and FRs of the albumin binding molecule and / or antigen binding portion. Conservative substitutions are shown in Table 3. More substantial changes are provided under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into components of the fusion protein and screened for products having the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. See also subsection "1. Amino Acid Sequence Variants" under section "V. Preparation Methods."

[0322] Table 3: Amino acid substitutions

[0323]

[0324]

[0325] Amino acids can be grouped according to common side chain properties:

[0326] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0327] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0328] (3) Acidic: Asp, Glu;

[0329] (4) Basic: His, Lys, Arg;

[0330] (5) Residues that affect chain orientation: Gly, Pro;

[0331] (6) Aromatic: Trp, Tyr, Phe.

[0332] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0333] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., humanized or human antibody). Typically, the resulting variant selected for further study will have modifications (e.g., improvements) and / or will substantially retain certain biological properties of the parent antibody in terms of certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently produced, for example, using affinity maturation techniques based on phage display (such as those described herein). In short, one or more HVR residues are mutated, the variant antibody is displayed on phage, and screened for specific biological activity (e.g., binding affinity).

[0334] Changes (e.g., substitutions) can be made in the HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots," i.e., residues encoded by codons that undergo high-frequency mutation during somatic maturation (see, e.g., Chodwhury, Methods Mol. Biol., Vol. 207: pp. 179-196, 2008) and / or SDRs (a-CDRs), and the resulting variant VH or VL is tested for binding affinity. Affinity maturation is performed by constructing secondary libraries and reselecting from them, as described, for example, in Hoogenboom et al., Methods in Molecular Biology, Vol. 178: pp. 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves an HVR-directed approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues that participate in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are particularly often targeted.

[0335] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in an HVR. Such changes may be outside of an HVR "hotspot" or CDR.

[0336] A useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells, 1989, Science, Vol. 244: pp. 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at the amino acid position, indicating functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0337] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

[0338] 2 derivatives

[0339] In some embodiments, the fusion protein provided herein can be further modified to contain other non-protein parts known in the art and easily obtained. Suitable parts for derivatizing fusion proteins include but are not limited to water-soluble polymers. Non-limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (such as glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. Polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the fusion protein can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the fusion protein to be improved, whether the fusion protein derivative will be used therapeutically under defined conditions, etc.

[0340] In some embodiments, conjugates of a fusion protein and a non-protein moiety that can be selectively heated by exposure to radiation are provided. In some embodiments, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, Vol. 102: pp. 11600-11605, 2005). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein moiety to a temperature that kills cells in proximity to the non-protein moiety of the fusion protein.

[0341] II-B. Fusion proteins including truncated IL-21

[0342] The application also provides a fusion protein including a human IL-21 variant, the variant including a truncated human IL-21. Without being bound by theory, it was found that the fusion protein of the IL-21 including a truncated form lacking 1-11 amino acids at the C-terminus has improved stability compared to the wild-type counterpart and the truncated counterpart lacking 12 or more amino acids at the C-terminus. It is expected that the fusion protein including the truncated IL-21 as described herein is not limited to the fusion protein including the anti-albumin binding portion.

[0343] In some embodiments, a fusion protein is provided, comprising: a) a truncated IL-21 lacking approximately 1-11 amino acids at the C-terminus of wild-type IL-21, and b) a second portion (such as a single-domain antibody portion). In some embodiments, the IL-21 is of human origin. In some embodiments, a fusion protein is provided, comprising: a) a truncated IL-21 comprising the amino acid sequence of SEQ ID NO: 126, 171, or 172, and b) a second portion. In some embodiments, the truncated IL-21 lacks one or more amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the truncated IL-21 lacks any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, or all eleven amino acids between and including L123 and S133 at the C-terminus of the IL-21 having the sequence shown in SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the truncated IL-21 lacks about 5-11, 6-11, 7-11, 8-11, 9-11, or 10-11 amino acids (e.g., contiguous amino acids) at the C-terminus of SEQ ID NO: 1.

[0344] In some embodiments, the fusion protein has a molecular weight of at least about 15kDa, 18kDa, 20kDa, 22kDa, 25kDa, 28kDa. In some embodiments, the fusion protein has a molecular weight of no more than about 1000kDa, 500kDa, 250kDa, 100kDa, 70kDa, 50kDa, 40kDa, or 30kDa. In some embodiments, the fusion protein has a molecular weight of about 15kDa to about 1000kDa, about 15kDa to about 500kDa, about 15kDa to about 100kDa, about 15kDa to about 70kDa, about 20kDa to about 50kDa, about 25kDa to about 30kDa, or about 28kDa.

[0345] In some embodiments, the second portion comprises a half-life extending portion. In some embodiments, the half-life extending portion is an albumin binding portion (e.g., an albumin binding antibody portion, e.g., a single domain albumin binding antibody portion). In some embodiments, the half-life extending portion is an Fc domain (e.g., an IgG1 Fc domain).

[0346] In some embodiments, the second portion comprises an antigen binding portion. In some embodiments, the antigen binding portion has a molecular weight of less than about 50 kDa, 40 kDa, 30 kDa, 20 kDa, or 15 kDa. In some embodiments, the antigen binding portion comprises a single domain antibody portion. In some embodiments, the antigen binding portion comprises a single domain antibody portion that specifically binds to albumin.

[0347] In some embodiments, the second portion is fused to the N-terminus of the truncated IL-21. In some embodiments, the second portion is fused to the C-terminus of the truncated IL-21.

[0348] In some embodiments, the second portion is fused to the truncated IL-21 (which may be adjacent to IL-21 or not adjacent to IL-21) via a linker. In some embodiments, the second portion is fused to the N-terminus or C-terminus of the truncated IL-21 via a linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a flexible linker.

[0349] III. Pharmaceutical Compositions

[0350] The present application also provides pharmaceutical compositions comprising any of the fusion proteins described herein and an optional pharmaceutically acceptable carrier. The pharmaceutical compositions can be prepared by mixing the fusion proteins described herein having a desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed., 1980) in the form of a lyophilized formulation or an aqueous solution.

[0351] The pharmaceutical composition is preferably stable, wherein the fusion protein described herein substantially maintains its physical and chemical stability and integrity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, pp. 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, 1991, and Jones, A., Adv. Drug Delivery Rev., Vol. 10: pp. 29-90, 1993. Stability can be measured at a selected temperature and over a selected time period. For rapid screening, the formulation can be kept at 40°C for 2 weeks to 1 month, at which point stability is measured. When the formulation is to be stored at 2°C to 8°C, the formulation should generally be stable at 30°C or 40°C for at least 1 month and / or at 2°C to 8°C for at least 2 years. When the formulation is to be stored at 30°C, the formulation should generally be stable at 30°C for at least 2 years and / or at 40°C for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability.In some embodiments, a stable formulation of a fusion protein described herein can include less than about 10% (preferably less than about 5%) of the fusion protein present in the formulation as aggregates.

[0352] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers; antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives; isotonicity agents (e.g., sodium chloride); stabilizers; metal complexes (e.g., Zn-protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0353] Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM , polyethylene glycol (PEG) and PLURONICS TM or polyethylene glycol (PEG).

[0354] Buffers are used to control pH within the range of optimizing therapeutic effects, particularly when stability depends on pH. The buffer is preferably present in a concentration within the range of about 50 mM to about 250 mM. Suitable buffers for the present application include organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. In addition, the buffer may include histidine and trimethylamine salts such as Tris.

[0355] Preservatives are added to retard microbial growth, and these preservatives are typically present in the range of 0.2% to 1.0% (w / v). The addition of preservatives can, for example, facilitate the production of multi-use (multi-dose) formulations. Suitable preservatives for use herein include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; halogenated (e.g., chlorinated, brominated, iodinated) benzalkonium, benzethonium chloride; thimerosal, phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0356] There are tonicity agents, sometimes referred to as "stabilizers," to adjust or maintain the tension of the liquid in the composition. When tonicity agents are used with large charged biomolecules such as proteins and antibodies, they are generally referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the possibility of intermolecular and intramolecular interactions. Taking into account the relative amounts of the other ingredients, the tonicity agent can be present in any amount between 0.1% and 25% by weight, preferably between 1% and 5% by weight. Preferred tonicity agents include polyols, preferably trivalent or higher-valent sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0357] Additional excipients include agents that can act as one or more of the following: (1) fillers, (2) solubilizers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the walls of the container. Such excipients include: polysaccharide alcohols (such as those listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, inositol, inositol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), Polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioacetate, thioglycerol, α-thioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0358] A nonionic surfactant or detergent (also called a "wetting agent") is present to help dissolve the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation. It also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant is present in a range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0359] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), polyols, Polyoxyethylene sorbitan monoether ( Examples of the present invention include glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0360] In order to use the pharmaceutical compositions for in vivo administration, these pharmaceutical compositions must be sterile. The pharmaceutical compositions can be sterilized by filtration through a sterile filtration membrane. The pharmaceutical compositions herein are typically placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle.

[0361] The route of administration is according to known and recognized methods, such as by single or multiple boluses or infusions over a longer period of time in a suitable manner, for example, by injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical application, inhalation or by sustained release or delayed release means. In some embodiments, the pharmaceutical composition is administered locally, such as intratumorally or intravitreally.

[0362] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antagonist in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT®, and the like. TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

[0363] The pharmaceutical compositions herein may also contain more than one active compound necessary to treat a particular indication, preferably those having complementary activities that do not adversely affect each other. Alternatively or additionally, the composition may include a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitory agent. Such molecules are appropriately combined in amounts effective for the intended purpose.

[0364] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. These techniques are disclosed in "Remington's Pharmaceutical Sciences," 18th edition.

[0365] The antibody fusion proteins disclosed herein can be formulated into immunoliposomes. Liposomes containing the antibody fusion protein can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, Vol. 82: p. 3688, 1985; Hwang et al., Proc. Natl Acad. Sci. USA, Vol. 77: p. 4030, 1980; and U.S. Patents 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Patent 5,013,556.

[0366] Particularly useful liposomes can be generated by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and phosphatidylethanolamine derivatized with polyethylene glycol (PEG-PE).The liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0367] In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multiple-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is stored frozen.

[0368] IV. Treatment Methods

[0369] One aspect of the present application provides a method for treating a disease or condition in an individual using a fusion protein or pharmaceutical composition as described herein. For example, the fusion protein described herein includes: a) a cytokine, and b) an albumin binding portion (such as an sdAb that binds to albumin). In some embodiments, the fusion protein includes a) a cytokine selected from IL-21, IL-7, IL-15, IL-15, IL-33, and IL-22 that bind to IL-15Rα or a fragment thereof, and b) an albumin binding portion (such as an sdAb that binds to albumin). In some embodiments, the albumin binding portion includes an albumin binding domain as described herein or a single domain antibody (sdAb) that binds to albumin. In some embodiments, the method further includes administering a second agent.

[0370] Another aspect of the present application provides a method for treating a disease or condition in an individual, comprising administering to the individual a) a fusion protein comprising i) a cytokine and ii) a half-life extension domain fused to the cytokine; and b) a second agent. In some embodiments, the half-life extension domain is fused to the C-terminus of the cytokine. In some embodiments, the half-life extension domain is fused to the N-terminus of the cytokine. In some embodiments, the cytokine is connected to the half-life extension domain via a joint. In some embodiments, the joint has a length of one to forty (such as one to thirty-five, one to thirty, one to twenty-five, one to twenty, four to twenty, or four to sixteen) amino acids. In some embodiments, the joint is selected from SEQ ID NO: 12-26 and 158-159. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15, IL-33, and IL-22 bound to IL-15Rα or a fragment thereof. In some embodiments, the cytokine is IL-21. In some embodiments, the half-life extension domain is an albumin binding moiety (such as an albumin binding domain or an anti-albumin single domain antibody). In some embodiments, the half-life extension domain is albumin. In some embodiments, the half-life extension domain is an Fc fragment. In some embodiments, the Fc fragment is selected from IgG1, IgG2, IgG3 and IgG4 Fc fragments or variants thereof. In some embodiments, the Fc fragment is an IgG1 Fc fragment or a variant thereof. In some embodiments, the IgG1 Fc fragment or a variant thereof includes a mutation at position 297, wherein the amino acid mutation at position 297 is alanine, aspartic acid or glycine. In some embodiments, the individual is human.

[0371] In some embodiments, the second agent comprises a therapeutic antibody, an immune checkpoint inhibitor, a second cytokine, a chemotherapeutic agent, a tyrosine kinase inhibitor, or an immune cell.

[0372] In some embodiments, the second agent is a therapeutic antibody. In some embodiments, the therapeutic antibody binds to a tumor antigen. In some embodiments, the tumor antigen is selected from mesothelin, GPA33, Her-2, EGFR, and CD20. In some embodiments, the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, and BCMA.

[0373] In some embodiments, the tumor antigen is mesothelin. In some embodiments, the second agent is an anti-mesothelin antibody or fragment thereof. In some embodiments, the anti-mesothelin antibody or fragment thereof comprises an antibody comprising an anti-mesothelin heavy chain variable region (anti-MSLN V H) of a single-chain antibody, wherein the anti-MSLN V H Comprising CDR1, CDR2 and CDR3, wherein: a) CDR1 comprising the amino acid sequence of SEQ ID NO: 46, CDR2 comprising the amino acid sequence of SEQ ID NO: 47 and CDR3 comprising the amino acid sequence of SEQ ID NO: 48, or variants comprising a total of up to 3, 2 or 1 amino acid substitutions in the CDRs; or b) CDR1 comprising the amino acid sequence of SEQ ID NO: 49, CDR2 comprising the amino acid sequence of SEQ ID NO: 50 and CDR3 comprising the amino acid sequence of SEQ ID NO: 51, or variants comprising a total of up to 3, 2 or 1 amino acid substitutions in the CDRs.

[0374] In some embodiments, the second agent is an immune checkpoint regulator. In some embodiments, the immune checkpoint regulator is an inhibitor of an immune checkpoint protein selected from PD-L1, CTLA4, PD-L2, PD-1, CD47, TIGIT, GITR, TIM3, LAG3, 4-1BB, CD27, and B7H4. In some embodiments, the immune checkpoint protein is PD-1. In some embodiments, the second agent is an anti-PD-1 antibody or a fragment thereof.

[0375] In some embodiments, the second agent is a second cytokine. In some embodiments, the cytokine in the fusion protein is IL-21, and wherein the second cytokine is selected from IL-7, IL-15, IL-15 bound to IL-15Rα, or a half-life extended variant thereof.

[0376] In some embodiments, the second agent is an immune cell. In some embodiments, the immune cell includes a T cell or a NK cell. In some embodiments, the immune cell includes a T cell expressing a chimeric antigen receptor (CAR), a T cell expressing a modified T cell receptor (TCR), or a T cell isolated from a tumor.

[0377] In some embodiments, the second agent is a tyrosine kinase inhibitor.

[0378] In some embodiments, the second agent is a chemotherapeutic agent (such as sorafenib).

[0379] In some embodiments, a method for treating a disease or condition in an individual is provided, comprising administering to the individual a) a fusion protein comprising i) IL-21 and ii) a half-life extension domain fused to a cytokine; and b) a second agent. In some embodiments, the half-life extension domain is fused to the N-terminus of the cytokine. In some embodiments, the half-life extension domain is fused to the C-terminus of the cytokine. In some embodiments, the second agent comprises a therapeutic antibody (such as a therapeutic antibody that binds to a tumor antigen such as CD20 or mesothelin), an immune checkpoint inhibitor (such as an anti-PD-1 antibody), a second cytokine, a chemotherapeutic agent, a tyrosine kinase inhibitor, or an immune cell. In some embodiments, the half-life extension domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the fusion protein further comprises an antigen binding portion (such as described above). In some embodiments, the antigen binding portion binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma (non-Hodgkin's lymphoma), leukemia (such as acute myeloid leukemia), head and neck cancer, liver cancer, kidney cancer, renal cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0380] In some embodiments, a method for treating a disease or condition in an individual is provided, comprising administering to the individual a) a fusion protein comprising i) IL-7 and ii) a half-life extension domain fused to a cytokine; and b) a second agent. In some embodiments, the half-life extension domain is fused to the N-terminus of the cytokine. In some embodiments, the half-life extension domain is fused to the C-terminus of the cytokine. In some embodiments, the second agent comprises a therapeutic antibody (such as a therapeutic antibody that binds to a tumor antigen such as CD20 or mesothelin), an immune checkpoint inhibitor (such as an anti-PD-1 antibody), a second cytokine, a chemotherapeutic agent, a tyrosine kinase inhibitor, or an immune cell. In some embodiments, the half-life extension domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the fusion protein further comprises an antigen binding portion (such as described above). In some embodiments, the antigen binding portion binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma (non-Hodgkin's lymphoma), leukemia (such as acute myeloid leukemia), head and neck cancer, liver cancer, kidney cancer, renal cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0381] In some embodiments, a method for treating a disease or condition in an individual is provided, comprising administering to the individual a) a fusion protein comprising i) IL-15 and ii) a half-life extension domain fused to a cytokine; and b) a second agent. In some embodiments, the half-life extension domain is fused to the N-terminus of the cytokine. In some embodiments, the half-life extension domain is fused to the C-terminus of the cytokine. In some embodiments, the second agent comprises a therapeutic antibody (such as a therapeutic antibody that binds to a tumor antigen such as CD20 or mesothelin), an immune checkpoint inhibitor (such as an anti-PD-1 antibody), a second cytokine, a chemotherapeutic agent, a tyrosine kinase inhibitor, or an immune cell. In some embodiments, the half-life extension domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the fusion protein further comprises an antigen binding portion (such as described above). In some embodiments, the antigen binding portion binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma (non-Hodgkin's lymphoma), leukemia (such as acute myeloid leukemia), head and neck cancer, liver cancer, kidney cancer, renal cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0382] In some embodiments, a method for treating a disease or condition in an individual is provided, comprising administering to the individual a) a fusion protein comprising i) IL-15 bound to IL-15Rα or a fragment thereof and ii) a half-life extension domain fused to a cytokine; and b) a second agent. In some embodiments, the half-life extension domain is fused to the N-terminus of the cytokine. In some embodiments, the half-life extension domain is fused to the C-terminus of the cytokine. In some embodiments, the second agent comprises a therapeutic antibody (such as a therapeutic antibody bound to a tumor antigen such as CD20 or mesothelin), an immune checkpoint inhibitor (such as an anti-PD-1 antibody), a second cytokine, a chemotherapeutic agent, a tyrosine kinase inhibitor, or an immune cell. In some embodiments, the half-life extension domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the fusion protein further comprises an antigen binding portion (such as described above). In some embodiments, the antigen binding portion binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma (non-Hodgkin's lymphoma), leukemia (such as acute myeloid leukemia), head and neck cancer, liver cancer, kidney cancer, renal cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0383] In some embodiments, a method for treating a disease or condition in an individual is provided, comprising administering to the individual a) a fusion protein comprising i) IL-33 and ii) a half-life extension domain fused to a cytokine; and b) a second agent. In some embodiments, the half-life extension domain is fused to the N-terminus of the cytokine. In some embodiments, the half-life extension domain is fused to the C-terminus of the cytokine. In some embodiments, the second agent comprises a therapeutic antibody (such as a therapeutic antibody that binds to a tumor antigen such as CD20 or mesothelin), an immune checkpoint inhibitor (such as an anti-PD-1 antibody), a second cytokine, a chemotherapeutic agent, a tyrosine kinase inhibitor, or an immune cell. In some embodiments, the half-life extension domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the fusion protein further comprises an antigen binding portion (such as described above). In some embodiments, the antigen binding portion binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma (non-Hodgkin's lymphoma), leukemia (such as acute myeloid leukemia), head and neck cancer, liver cancer, kidney cancer, renal cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0384] In some embodiments, there is provided a method for treating cancer in an individual, comprising administering to the individual a fusion protein comprising IL-21 as described above. For example, in some embodiments, the fusion protein comprises a) IL-21 and b) an albumin binding portion (such as an sdAb bound to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of IL-21 or its variant. In some embodiments, the fusion protein comprises a) IL-21 fused to an albumin binding portion ("IL-21-ALBBM") and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the antigen binding portion is bound to a tumor antigen (such as mesothelin). In some embodiments, there is provided a method for treating cancer in an individual, comprising administering to the individual a fusion protein comprising i) IL-21 and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of IL-21. In some embodiments, the method further comprises a second agent (such as a therapeutic antibody bound to a tumor antigen such as CD20 or mesothelin, a chemotherapeutic agent such as sorafenib, an immunomodulator such as an anti-PD-1 antibody). In some embodiments, the cancer expresses a high level of tumor antigens. In some embodiments, the half-life extension domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma (non-Hodgkin's lymphoma), leukemia (such as acute myeloid leukemia), head and neck cancer, liver cancer, kidney cancer, renal cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0385] In some embodiments, a method for treating cancer in an individual is provided, comprising administering to the individual a fusion protein comprising IL-7 as described above. For example, in some embodiments, the fusion protein comprises a) IL-7 and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of IL-7 or its variant. In some embodiments, the fusion protein comprises a) IL-7 fused to an albumin binding portion ("IL-7-ALBBM") and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the antigen binding portion is bound to a tumor antigen (such as mesothelin). In some embodiments, a method for treating cancer in an individual is provided, comprising administering to the individual a fusion protein comprising i) IL-7 and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of IL-7. In some embodiments, the method further comprises a second agent. In some embodiments, the cancer expresses high levels of tumor antigens. In some embodiments, the half-life extending domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0386] In some embodiments, a method for treating cancer in an individual is provided, comprising administering to the individual a fusion protein comprising IL-15 as described above. For example, in some embodiments, the fusion protein comprises a) IL-15 and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of IL-15 or its variant. In some embodiments, the fusion protein comprises a) IL-15 fused to an albumin binding portion ("IL-15-ALBBM") and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the antigen binding portion is bound to a tumor antigen (such as mesothelin). In some embodiments, a method for treating cancer in an individual is provided, comprising administering to the individual a fusion protein comprising i) IL-15 and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of IL-15. In some embodiments, the method further comprises a second agent. In some embodiments, the cancer expresses high levels of tumor antigens. In some embodiments, the half-life extending domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0387] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a fusion protein comprising IL-15 bound to IL-15Rα as described above. For example, in some embodiments, the fusion protein comprises a) IL-15 bound to IL-15Rα and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of IL-15 that binds to IL-15Rα or a variant thereof. In some embodiments, the fusion protein comprises a) IL-15 bound to IL-15Rα fused to an albumin binding portion ("IL-15 bound to IL-15Rα-ALBBM") and b) an antigen binding portion, wherein the linkage between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the antigen binding portion binds to a tumor antigen (such as mesothelin). In some embodiments, a method for treating cancer in an individual is provided, comprising administering to the individual a fusion protein comprising i) IL-15 that binds to IL-15Rα and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of the IL-15 that binds to IL-15Rα. In some embodiments, the method further comprises a second agent. In some embodiments, the cancer expresses high levels of tumor antigens. In some embodiments, the half-life extension domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is human.

[0388] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a fusion protein comprising IL-33 as described above. For example, in some embodiments, the fusion protein comprises a) IL-33 and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of IL-33 or a variant thereof. In some embodiments, the fusion protein comprises a) IL-33 fused to an albumin binding portion ("IL-33-ALBBM") and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the antigen binding portion binds to a tumor antigen (such as mesothelin). In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a fusion protein comprising i) IL-33 and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of IL-33. In some embodiments, the method further comprises a second agent. In some embodiments, the cancer expresses high levels of a tumor antigen. In some embodiments, the half-life extending domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0389] In some embodiments, a method for treating an inflammatory disease in an individual is provided, comprising administering a fusion protein to the individual, wherein the fusion protein comprises a) IL-22 and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of IL-22 or its variant. In some embodiments, the albumin binding portion is an albumin binding domain or an anti-albumin single domain antibody, such as those described herein. In some embodiments, a method for treating an inflammatory disease in an individual is provided, comprising administering a fusion protein to the individual, wherein the fusion protein comprises a) IL-22 and ii) a half-life extending domain. In some embodiments, the half-life extending domain is an antibody or fragment thereof, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the disease is selected from ulcerative colitis, Crohn's disease, or ulcerative ileitis and intestinal graft-versus-host disease. In some embodiments, the individual is human.

[0390] In some embodiments, a method of treating mesothelioma in an individual is provided, comprising administering to the individual a fusion protein comprising a cytokine as described above. For example, in some embodiments, the fusion protein comprises a) a cytokine and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the fusion protein comprises a) a cytokine fused to an albumin binding portion ("cytokine-ALBBM") and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or a fragment thereof, IL-33, and IL-22. In some embodiments, the antigen binding portion binds to a tumor antigen (such as mesothelin). In some embodiments, a method of treating mesothelioma in an individual is provided, comprising administering to the individual a fusion protein comprising i) a cytokine and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the method further comprises a second agent (such as an anti-mesothelin antibody). In some embodiments, the half-life extension domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence.

[0391] In some embodiments, a method for treating lung cancer in an individual is provided, comprising administering to the individual a fusion protein comprising a cytokine as described above. For example, in some embodiments, the fusion protein comprises a) a cytokine and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the fusion protein comprises a) a cytokine fused to an albumin binding portion ("cytokine-ALBBM") and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15, IL-33, and IL-22 bound to IL-15Rα or a fragment thereof. In some embodiments, the antigen binding portion binds to a tumor antigen. In some embodiments, a method for treating lung cancer in an individual is provided, comprising administering to the individual a fusion protein comprising i) a cytokine and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the method further comprises a second agent.In some embodiments, the half-life extending domain is an antibody or fragment thereof, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence as described herein.

[0392] In some embodiments, a method for treating ovarian cancer in an individual is provided, comprising administering to the individual a fusion protein comprising a cytokine as described above. For example, in some embodiments, the fusion protein comprises a) a cytokine and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the fusion protein comprises a) a cytokine fused to an albumin binding portion ("cytokine-ALBBM") and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15, IL-33, and IL-22 bound to IL-15Rα or a fragment thereof. In some embodiments, the antigen binding portion binds to a tumor antigen. In some embodiments, a method for treating ovarian cancer in an individual is provided, comprising administering to the individual a fusion protein comprising i) a cytokine and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the method further comprises a second agent.In some embodiments, the half-life extending domain is an antibody or fragment thereof, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence as described herein.

[0393] In some embodiments, a method for treating gastric cancer in an individual is provided, comprising administering to the individual a fusion protein comprising a cytokine as described above. For example, in some embodiments, the fusion protein comprises a) a cytokine and b) an albumin binding portion (such as an sdAb bound to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the fusion protein comprises a) a cytokine fused to an albumin binding portion ("cytokine-ALBBM") and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15, IL-33, and IL-22 bound to IL-15Rα or a fragment thereof. In some embodiments, the antigen binding portion binds to a tumor antigen. In some embodiments, a method for treating gastric cancer in an individual is provided, comprising administering to the individual a fusion protein comprising i) a cytokine and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of the cytokine. In some embodiments, the method further comprises a second agent.In some embodiments, the half-life extending domain is an antibody or fragment thereof, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence as described herein.

[0394] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a) a fusion protein comprising IL-21 as described above, and b) a second cytokine selected from IL-7, IL-15, IL15 bound to IL15Rα or an extended half-life variant thereof. For example, in some embodiments, the fusion protein comprises a) IL-21 and b) an albumin binding portion (such as an sdAb that binds to albumin), wherein the albumin binding portion is fused to the N-terminus or C-terminus of IL-21 or a variant thereof. In some embodiments, the fusion protein comprises a) IL-21 fused to an albumin binding portion ("IL-21-ALBBM") and b) an antigen binding portion, wherein the connection between the cytokine-ALBBM and the antigen binding portion is optionally cleavable. In some embodiments, the antigen binding portion binds to a tumor antigen (such as mesothelin). In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual a) a fusion protein comprising i) IL-21 and ii) a half-life extension domain, wherein the half-life extension domain is fused to the N-terminus or C-terminus of IL-21; b) a second agent selected from IL-7, IL-15, IL15 bound to IL15Rα or a half-life extension variant thereof. In some embodiments, the half-life extension domain is an antibody or fragment thereof as described herein, albumin, a binding protein (such as an albumin binding protein or an IgG binding protein), an antibody derivative, or a polyamino sequence. In some embodiments, the first half-life extension cytokine is fused to the second half-life extension cytokine via a peptide linker, which is optionally cleavable by a protease. In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma (non-Hodgkin lymphoma), leukemia (such as acute myeloid leukemia), head and neck cancer, liver cancer, kidney cancer, renal cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer, and gastric cancer.In some embodiments, the individual is a human.

[0395] Fusion proteins for treating diseases

[0396] In some embodiments, the fusion protein for treating a disease or illness includes any one of the fusion proteins described herein (such as Section II). In some embodiments, the fusion protein includes i) a cytokine and ii) a half-life extension domain fused to the cytokine. In some embodiments, the half-life extension domain is fused to the C-terminus of the cytokine. In some embodiments, the half-life extension domain is fused to the N-terminus of the cytokine. In some embodiments, the cytokine is connected to the half-life extension domain via a joint. In some embodiments, the joint may be any joint described herein (such as Section II-B). In some embodiments, the cytokine is selected from IL-21, IL-7, IL-15, IL-15, IL-33 and IL-22 bound to IL-15Rα or its fragment. In some embodiments, the cytokine is IL-21.

[0397] Half-life extension domain

[0398] In some embodiments, a fusion protein for treating a disease or condition as described herein includes a half-life extending domain.

[0399] In some embodiments, the fusion proteins provided herein include a half-life extension domain selected from antibodies and fragments thereof, albumin, albumin binding proteins, IgG binding proteins, and polyamino acid sequences. It is contemplated that other mechanisms available in the art for extending the half-life of fusion proteins may also be used.

[0400] a) Antibodies and their fragments

[0401] By linking a cytokine to an antibody or fragment thereof capable of FcRn-mediated recycling, clearance of the cytokine from the subject can be reduced or otherwise delayed, thereby extending the half-life of the administered cytokine.

[0402] In some embodiments, the half-life extension domain comprises an antibody or a fragment thereof. In some embodiments, the antibody or fragment thereof is any antibody or fragment thereof capable of FcRn-mediated recycling, such as any heavy chain polypeptide or portion thereof (e.g., Fc domain or fragment thereof) capable of FcRn-mediated recycling. It is known in the art that FcRn-mediated recycling requires binding of the FcRn receptor to the Fc region of the antibody or fragment thereof. For example, studies have shown that residues I253, S254, H435, and Y436 (numbered according to the Kabat EU index numbering system) are important for the interaction between the human Fc region and the human FcRn complex. See, for example, Firan, M. et al., Int. Immunol., Vol. 13, pp. 993-1002, 2001; Shields, RL et al., J. Biol. Chem., Vol. 276, pp. 6591-6604, 2001. Various mutants of residues 248-259, 301-317, 376-382, and 424-437 (numbered according to the Kabat EU index numbering system) were also examined and reported. Yeung, YA et al., J. Immunol., Vol. 182, pp. 7667-7671, 2009.

[0403] In some embodiments, the antibody or its fragment comprises a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or its fragment comprises a portion of a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or its fragment comprises an Fc domain or a fragment thereof. In some embodiments, the antibody or its fragment comprises a CH2 and CH3 domains or a fragment thereof. In some embodiments, the antibody or its fragment comprises a constant domain of a heavy chain polypeptide. In some embodiments, the antibody or its fragment comprises a constant domain of a light chain polypeptide. In some embodiments, the antibody or its fragment comprises a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). In some embodiments, the antibody or its fragment comprises a light chain polypeptide.

[0404] In some embodiments, the antibody or its fragment includes an Fc domain or its fragment. In some embodiments, the Fc fragment is selected from IgG1, IgG2, IgG3 and IgG4 Fc fragments or variants thereof. In some embodiments, the Fc fragment is an IgG1 Fc fragment or a variant thereof. In some embodiments, the IgG1 Fc fragment or a variant thereof includes a mutation at position 297. In some embodiments, the amino acid at position 297 is asparagine. In some embodiments, the amino acid at position 297 (e.g., asparagine) is mutated to alanine, aspartic acid or glycine.

[0405] In some embodiments, the cytokines of the fusion protein form dimers through disulfide bonds between one copy of the half-life extending domain of the cytokine and the corresponding half-life extending domain of a second copy of the cytokine.

[0406] b) Albumin

[0407] Albumin is a natural carrier protein that, due to its size and susceptibility to FcRn-mediated recycling, has an extended serum half-life of approximately three weeks, which prevents intracellular degradation. Therefore, linking cytokines to albumin can significantly extend their half-life. This approach has been used to extend the plasma half-life of therapeutically beneficial proteins. See, for example, WO 2001 / 079271 A1 and WO 2003 / 59934 A2, the contents of which are incorporated herein by reference. Some isoforms of HSA are listed in Table 2.

[0408] In some embodiments, the fusion protein includes a half-life extension domain comprising an albumin polypeptide or a fragment or variant thereof (hereinafter referred to as "albumin" or "albumin polypeptide"). As used herein, the terms "albumin" and "albumin polypeptide" include fragments of albumin and variants of albumin. The albumin polypeptide includes an amino terminus and a carboxyl terminus. The albumin polypeptide can be any albumin polypeptide, including any fragment or variant thereof, such as any albumin polypeptide described in WO 2001 / 079271A1, WO 2003 / 59934A2, US20160152686A1, WO 2012 / 059486, WO 2011 / 124718, US20070048282, the contents of which are incorporated herein by reference. In some embodiments, the albumin polypeptide is HSA.

[0409] c) Binding protein

[0410] Another strategy for extending the half-life of cytokines or their variants in serum includes linking the cytokines to certain binding proteins, such as the albumin binding proteins or IgG binding proteins described above. The binding protein can be any protein that binds to a serum protein with an extended half-life, such as albumin or IgG. Albumin and IgG are polypeptides known to have long half-lives in serum.

[0411] In some embodiments, the half-life extension domain comprises an albumin binding protein.In some embodiments, the fusion protein comprises more than one albumin binding protein, each of which can be any of the albumin binding proteins described herein.

[0412] In some embodiments, the albumin binding protein is a single domain antibody or fragment thereof, such as a nanobody, which binds to or otherwise associates with albumin, such as those described herein. See, for example, WO2004041865A2 and US20070269422A1, the contents of which are incorporated herein by reference.

[0413] Another example of a binding protein is an IgG binding protein. IgG binding proteins have been reported. For a review of IgG binding proteins, including specific IgG binding proteins and their applications, see, for example, Choe et al., Materials, 2016, Vol. 9, No. 12: p. 994, the contents of which are incorporated herein by reference.

[0414] d) Antibody derivatives

[0415] The cytokines described herein may alternatively be linked to various antibody derivatives, including but not limited to scFv, scFc, dual variable domains (DVD), and antibody derivatives based on the CrossMab approach. See, for example, Klein et al., 2012, MAbs, Vol. 4, No. 6: pp. 653-663; US20070071675A1. Antibody derivatives include antibody derivatives engineered into bispecific antibodies or fragments thereof. Thus, in some embodiments, the half-life extension domain may include any antibody derivative, variant, or fusion product thereof, including but not limited to scFv, scFc, dual variable domains (DVD), antibody derivatives based on the CrossMab approach, and bispecific antibodies or fragments thereof.

[0416] e) Polyamino acid sequence

[0417] Another strategy for extending the half-life of a fusion protein in serum is to connect the fusion protein to a polyamino acid sequence. Thus, in some embodiments, the half-life extension domain comprises a polyamino acid sequence. The polyamino acid sequence can be any polyamino acid sequence that can extend the half-life of the fusion protein in serum when connected to the fusion protein. Examples of polyamino acid sequences include PAS polypeptides and XTEN polypeptides.

[0418] f) PEGylation and glycosylation

[0419] Additional strategies for extending the half-life of the cytokines provided herein include pegylation and engineering of additional glycosylation sites. Each of these strategies will be discussed in further detail below.

[0420] "PEGylation" refers to the process of covalently or non-covalently attaching or fusing polyethylene glycol (PEG) polymer chains to molecules and macrostructures, such as any of the drugs, therapeutic proteins, polypeptides, antibodies, antibody fragments, antibody derivatives, or fusion proteins provided herein, or components thereof (e.g., a half-life extension domain of a fusion protein and / or a cytokine or functional fragment thereof). The benefits of PEGylation include, for example, (1) significantly extending the in vivo circulation half-life by escaping renal clearance due to the polymer increasing the apparent size of the molecule above the glomerular filtration limit and / or by escaping cellular clearance mechanisms, (2) reducing the antigenicity and immunogenicity of the PEG-attached molecule, (3) improving pharmacokinetics, (4) increasing solubility, (5) improving formulation and dosing options, (6) increasing bioavailability by reducing loss at the subcutaneous injection site, and (7) increasing the thermal and mechanical stability of the PEGylated molecule.

[0421] Methods for pegylation of various molecules and macrostructures are well known in the art. See, for example, US20140256636A1; Fee and Damodaran, 2010, European Pharmaceutical Review, Vol. 15, No. 1: pp. 18-26; Chapman et al., 1999, Nature Biotechnol., Vol. 17: pp. 780-783; Yang et al., 2003, Protein Eng., Vol. 16, No. 10: pp. 761-770; Chapman, Adv. Drug. Deliv. Rev., 2002, Vol. 54, No. 4: pp. 531-545, the contents of which are incorporated herein by reference.

[0422] "Glycosylation" refers to the addition of carbohydrates or glycosyl groups to a polypeptide. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine (NXS) and asparagine-X-threonine (NXT) are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain, where X is any amino acid except proline (P). Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the carbohydrates (e.g., N-acetylgalactosamine, galactose, or xylose) to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0423] Naturally occurring glycosylation has been shown to increase the molecular stability of proteins. See, for example, Sola et al., 2007, Cell. Mol. Life Sci., Vol. 64, No. 16: pp. 2133-2152. Engineering of additional glycosylation sites has also been shown to stabilize a variety of protein therapeutics against most major biochemical instabilities. See, for example, Sola and Griebenow, 2009, Pharm. Sci., Vol. 98, No. 4: pp. 1223-1245. Among the pharmaceutically relevant protein instabilities that have been shown to be ameliorated by glycosylation are, for example, oxidation, cross-linking, pH-, chemical-, heat-, and freeze-induced denaturation / unfolding, precipitation, kinetic activation, and aggregation. Id.

[0424] By altering the amino acid sequence, one or more of the above tripeptide sequences are generated in the amino acid sequence of the fusion protein (e.g., in the amino acid sequence of the half-life extension domain and / or the cytokine or its functional fragment) (for N-linked glycosylation sites), thereby conveniently adding glycosylation sites to the fusion protein. The alteration can also be made by adding or substituting one or more serine or threonine residues in the amino acid sequence of the fusion protein (e.g., in the amino acid sequence of the half-life extension domain and / or the cytokine or its functional fragment) (for O-linked glycosylation sites).

[0425] g) Heterodimerization modification

[0426] The half-life extension domains described herein can include one or more modifications that promote heterodimerization of two different half-life extension domains. In some embodiments comprising a first half-life extension domain and a second half-life extension domain, it is desirable to promote heterodimerization of the first and second half-life extension domains so that a fusion protein in the correct heterodimeric form is effectively produced. Thus, one or more amino acid modifications can be made to the first half-life extension domain and one or more amino acid modifications can be made to the second half-life extension domain using any strategy available in the art, including any strategy described in Klein et al., 2012, MAbs, Vol. 4, No. 6: pp. 653-663.

[0427] One strategy for promoting heterodimerization of two different half-life extension domains is a method called "knot-in-knot". In some embodiments, the fusion protein includes a first half-life extension domain and a second half-life extension domain, each of which includes a CH3 domain. In some embodiments, the half-life extension domain including the CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). The CH3 domains of the two half-life extension domains can be altered by the "knot-in-knot" technique, which is described in detail in several examples, such as WO 1996 / 027011; Ridgway, JB et al., Protein Eng., 1996, Vol. 9, No. 7: pp. 617-621; Merchant, AM et al., Nat. Biotechnol., 1998, Vol. 16, No. 7: pp. 677-681. See also Klein et al., 2012, MAbs, Vol. 4, No. 6: pp. 653-663. A knot-in-knob approach is used to alter the interaction surfaces of the two CH3 domains to increase heterodimerization of two half-life extension domains containing two altered CH3 domains. This occurs by introducing bulky residues into the CH3 domain of one of the half-life extension domains, which acts as a "knob." Then, to accommodate the bulky residues, a "knob" is formed in the other half-life extension domain that can accommodate the knot. Any one of the altered CH3 domains can be a "knob" and the other can be a "knob." The introduction of disulfide bonds further stabilizes the heterodimer (Merchant, AM et al., Nat. Biotechnol., 1998, Vol. 16, No. 7; Atwell, S. et al., J. Mol. Biol., 1997, Vol. 270, No. 1: pp. 26-35) and increases yield. Exemplary sequences that contribute to the "knot" and "buckle" effects are disclosed, for example, in the sequences included in SEQ ID NOs: 164-167. In some embodiments, the CH3 domain has one or more mutations selected from Y349C, T366S, L368A, Y407V, S354C, T366W.

[0428] Another strategy to promote heterodimerization of two different half-life extension domains is to stabilize ionic interactions that favor heterodimerization by changing charged residues. In some embodiments, the fusion protein includes a first half-life extension domain and a second half-life extension domain, each of which includes a CH3 domain. In some embodiments, the half-life extension domain including the CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). It has been observed that changing the charge polarity between the two different Fc domains can lead to ionic interactions, thereby favoring heterodimerization and inhibiting homodimerization. See, for example, WO 2006 / 106905A1; Gunasekaran et al., 2010, Vol. 285, No. 25: pp. 19637-19646. For example, the negatively charged E356 of an Fc domain is observed to pair with the positively charged K439 of another Fc domain, and the negatively charged E357, E357, and D399 of a first Fc domain are observed to pair with the positively charged K439, K370, and K409 of a second Fc domain, respectively. See WO2006 / 106905A1; Gunasekaran et al., 2010, Vol. 285, No. 25: pp. 19637-19646. Thus, by introducing at least two of the E356K, E357K, and D399K mutations into a first Fc domain and introducing the K370E, K409D, and K439E mutations into a second Fc domain, efficient heterodimerization can be achieved while suppressing homodimer formation. Id. Efficient heterodimerization was achieved by introducing K392D and K409D mutations into the first Fc chain and D399K and E356K mutations into the second Fc chain. Gunasekaran et al., 2010, Vol. 285, No. 25: pp. 19637-19646.

[0429] Another strategy for promoting heterodimerization of two different half-life extension domains is to use structure- and sequence-based methods to identify changes that promote heterodimerization and / or inhibit homodimerization. Among the approaches to identifying changes that promote heterodimerization is to perform structural calculations to determine the energetics of paired variant combinations of residues that interact across the CH3-CH3 dimer interface, such as the method employed in Moore et al., 2011, Vol. 3, No. 6: pp. 546-557, the contents of which are incorporated herein by reference. Moore et al. identified pairs predicted to have lower energies in the heterodimer form relative to the homodimer form as starting points for further analysis. An 89% heterodimer yield was observed by introducing S364H and F405A mutations into the first Fc domain and Y349T and T394F mutations into the second Fc domain. Id.

[0430] Disease or condition

[0431] The methods described herein can be used to treat a disease or condition. In some embodiments, the disease or condition is selected from cancer, inflammatory disorders, and infection.

[0432] In some embodiments, the disease or disorder is an inflammatory disease. In some embodiments, the disease is selected from ulcerative colitis, Crohn's disease or ulcerative ileitis and intestinal graft-versus-host disease.

[0433] In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a solid tumor or a liquid tumor. In some embodiments, the cancer is selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma (non-Hodgkin's lymphoma), leukemia (such as acute myeloid leukemia), head and neck cancer, liver cancer, kidney cancer, renal cancer, esophageal cancer, gastric cancer and colorectal cancer. In some embodiments, the cancer is selected from mesothelioma, lung cancer, ovarian cancer and gastric cancer.

[0434] Dosage regimen

[0435] The fusion protein and / or the second agent can be administered to an individual using any suitable dosage and route of administration. In some embodiments, the fusion protein and / or the second agent is administered to an individual parenterally. The route of administration is according to known and recognized methods, such as by single or multiple boluses or infusions over a period of time in a suitable manner, for example, by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional, intraarticular, intratumoral, or oral route injection or infusion.

[0436] In some embodiments, the fusion protein and the second agent are administered to the subject simultaneously, concurrently, or sequentially.

[0437] Determining the appropriate dosage or route of administration is well known to those of ordinary skill in the art. Animal studies provide reliable guidance for determining effective doses for human diagnostic applications. Interspecies scaling of effective doses can be performed according to the principles of "The Use of Interspecies Scaling in Toxicokinetics" as formulated by Mordenti, J. and Chappell, W., in Toxicokinetics and New Drug Development, ed. Yacobi et al., Pergamon Press, New York, 1989, pp. 42-46.

[0438] In some embodiments, the fusion protein is administered at a frequency of about once every three weeks to about twice a week (such as about once every three weeks to about once every two weeks, about once every two weeks to about once a week, about once a week to about twice a week). In some embodiments, the fusion protein is administered at a frequency of no less than about once every three weeks, about once every two weeks, about once a week, about twice a week. In some embodiments, the fusion protein is administered at a frequency of no more than about once every three weeks, about once every two weeks, about once a week, about twice a week. In some embodiments, the fusion protein is administered at a frequency of about once every three weeks, about once every two weeks, about once a week, about twice a week.

[0439] In some embodiments, the fusion protein is administered for at least about one to six months per treatment cycle (such as one to two, three, or four weeks, one to one, two, three, four, five, or six months, one to two, three, four, five, or six months, three to four, five, or six months).

[0440] In some embodiments, the amount of the fusion protein administered per administration is from about 100 ng / kg to about 10 mg / kg (e.g., from about 100 ng / kg to about 500 ng / kg, from about 500 ng / kg to about 1 μg / kg, from about 1 μg / kg to about 5 μg / kg, from about 5 μg / kg to about 10 μg / kg, from about 10 μg / kg to about 50 μg / kg, from about 50 μg / kg to about 100 μg / kg, from about 100 μg / kg to about 500 μg / kg, from about 500 μg / kg to about 1 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 5 mg / kg to about 10 mg / kg).

[0441] In some embodiments, the second agent (such as a therapeutic antibody that binds to mesothelin or an inhibitor of PD-1) is administered at a frequency of about once a month to about twice a week (such as about once a month to twice a month, three times a month, or four times a month, about once every two weeks, about once every three weeks, about once a week, or twice a week).

[0442] In some embodiments, the amount of the second agent (such as a therapeutic antibody that binds to mesothelin or an inhibitor of PD-1) administered per dose is from about 100 ng / kg to about 100 mg / kg (e.g., from about 100 ng / kg to about 500 ng / kg, from about 500 ng / kg to about 1 μg / kg, from about 1 μg / kg to about 5 μg / kg, from about 5 μg / kg to about 10 μg / kg, from about 10 μg / kg to about 50 μg / kg, from about 50 μg / kg to about 100 μg / kg, from about 100 μg / kg to about 500 μg / kg, from about 500 μg / kg to about 1 mg / kg, from about 1 mg / kg to about 5 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 10 mg / kg to about 50 mg / kg, or from about 50 mg / kg to about 100 mg / kg).

[0443] V. Preparation Method

[0444] The fusion proteins described herein, as well as components of the fusion proteins described herein (such as albumin binding portions, cytokines or variants thereof, antigen binding portions), can be prepared by any of the protein expression and purification methods known in the art.

[0445] In some embodiments, the present application provides an isolated nucleic acid encoding one or more polypeptide chains of any one of a fusion protein, an albumin binding portion, a cytokine or variant thereof, or an antigen binding portion. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID NOs: 46-51 and 60-95.

[0446] In some embodiments, the isolated nucleic acid is inserted into a vector such as an expression vector, a viral vector, or a cloning vector. To express the nucleic acid, the vector can be introduced into a host cell to allow the nucleic acid to be expressed within the host cell. The expression vector can contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. These elements can be appropriately selected by one of ordinary skill in the art. For example, a promoter sequence can be selected to promote transcription of the polynucleotide in the vector. Suitable promoter sequences include but are not limited to T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. Enhancer sequences can be selected to enhance transcription of the nucleic acid. Selectable markers can be selected to allow host cells inserted with the vector to be distinguished from host cells not inserted with the vector. For example, a selectable marker can be a gene that confers antibiotic resistance. A signal sequence can be selected to allow the expressed polypeptide to be transported outside the host cell. In some embodiments, the isolated nucleic acid also includes a nucleic acid sequence encoding a signal peptide.

[0447] In some embodiments, isolated host cells containing the above-described vectors are provided. The host cells containing the vectors can be used to express or clone the isolated nucleic acids. Suitable host cells may include, but are not limited to, prokaryotes, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Expression of antibodies and antigen-binding fragments in prokaryotic cells such as Escherichia coli is well established in the art. For a review, see, for example, Pluckthun, A., BioTechnology, Vol. 9: pp. 545-551, 1991. Expression in cultured eukaryotic cells is also available to those skilled in the art as an option for producing antibodies or antigen-binding fragments thereof, see recent reviews, for example, Ref, ME, 1993, Curr. Opinion Biotech., Vol. 4: pp. 573-576; Trill JJ et al., 1995, Curr. Opinion Biotech., Vol. 6: pp. 553-560. Higher eukaryotic cells, particularly those derived from multicellular organisms, can be used for expression of glycosylated polypeptides. Suitable higher eukaryotic cells include, but are not limited to, invertebrate cells and insect cells, as well as vertebrate cells.

[0448] The vector can be introduced into the host cell using any suitable method known in the art, including but not limited to diethylaminoethyl dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, gene guns, receptor-mediated gene delivery, polylysine, histone, chitosan and peptide-mediated delivery. Standard methods for transfecting and transforming cells to express the target vector are well known in the art. In some embodiments, the host cell comprises a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In some embodiments, the host cell comprises a single vector comprising isolated nucleic acids encoding the first polypeptide and the second polypeptide.

[0449] In some embodiments, the present application provides a method for expressing any of the fusion proteins, albumin binding portions, cytokines or variants thereof, or antigen binding portions described herein, comprising culturing isolated host cells containing a vector and recovering the fusion protein, albumin binding portion, cytokine or variant thereof, or antigen binding portion from the cell culture. The isolated host cells are cultured under conditions that allow expression of the isolated nucleic acid inserted into the vector. Suitable conditions for expressing the polynucleotide may include, but are not limited to, a suitable culture medium, a suitable density of host cells in the culture medium, the presence of essential nutrients, the presence of supplemental factors, suitable temperature and humidity, and the absence of microbial contaminants. One of ordinary skill in the art can select suitable conditions for the purpose of expression.

[0450] Any suitable method can be used to collect the expressed polypeptide. The polypeptide can be expressed intracellularly, in the periplasmic space, or secreted extracellularly into the culture medium. If the polypeptide is expressed intracellularly, the host cells containing the polypeptide can be lysed and the polypeptide can be isolated from the lysate by removing unwanted debris by centrifugation or ultrafiltration. If the polypeptide is secreted into the periplasmic space of E. coli, the cell slurry can be thawed for about 30 minutes in the presence of agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF), and cell debris can be removed by centrifugation (Carter et al., BioTechnology, Vol. 10: pp. 163-167, 1992). If the polypeptide is secreted into the culture medium, the supernatant of the cell culture can be collected and concentrated using a commercially available protein concentration filter (e.g., Amincon or Millipore Pellicon ultrafiltration unit). The collection and concentration steps can include protease inhibitors and / or antibiotics to inhibit protein degradation and / or the growth of contaminating microorganisms.

[0451] The expressed polypeptide can be further purified by suitable methods such as, but not limited to, affinity chromatography, hydroxyapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion exchange column, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin sepharose chromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (for review, see Bonner, PL, Protein purification, Taylor & Francis, 2007; Janson, JC et al., Protein purification: principles, high resolution methods and applications, Wiley-VCH, 1998).

[0452] In some embodiments, the polypeptide can be purified by affinity chromatography. In some embodiments, protein A chromatography or protein A / G (a fusion protein of protein A and protein G) chromatography can be used to purify polypeptides including those derived from antibody CH2 domains and / or CH3 domains and / or VH and / or VH domains. HH components (Lindmark et al., J. Immunol. Meth., Vol. 62: 1-13, 1983); Zettlit, KA, Antibody Engineering, Part V, pp. 531-535, 2010; Fridy et al., 2015, Analytical Biochemistry, Vol. 477, pp. 92-94; Henry et al., 2016, PLoS One., 2016, Vol. 11, No. 9: e0163113). In some embodiments, protein G chromatography can be used to purify polypeptides and / or polypeptide complexes including IgG γ3 heavy chains (Guss et al., EMBO J., Vol. 5: 1567-1575, 1986). In some embodiments, protein L chromatography can be used to purify polypeptides and / or polypeptide complexes comprising kappa light chains (Sudhir, P., Antigen engineering protocols, Chapter 26, Humana Press, 1995; Nilson, BHK et al., J. Biol. Chem., Vol. 267, pp. 2234-2239, 1992). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices may also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. When the antibody comprises a CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) can be used for purification.

[0453] VI. Products and Kits

[0454] In some embodiments of the present invention, a product containing a material for treating a disease or condition in an individual (such as cancer or inflammatory disease) is provided for administering the fusion protein to the individual. The product may include a container and a label or package insert about or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from various materials such as glass or plastic. Typically, the container holds a composition that is effective for treating a disease or condition as described herein and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a fusion protein as described herein. The label or package insert indicates that the composition is used to treat a specific condition. The label or package insert will also include instructions for administering the fusion protein to the patient. Products and kits comprising the combination therapies described herein are also contemplated.

[0455] Package insert refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concernin...

Claims

1. A fusion protein comprising: a) a cytokine, and b) an albumin binding moiety, wherein the cytokine is selected from IL-21, IL-33, IL-15 bound to IL-15Rα or a fragment thereof; Wherein, the albumin binding portion comprises a single domain antibody that specifically binds to albumin, and the single domain antibody that specifically binds to albumin comprises: 1) CDR1 consisting of the amino acid sequence SEQ ID NO: 90; CDR2 consisting of the amino acid sequence SEQ ID NO: 91; A CDR3 consisting of the amino acid sequence of SEQ ID NO: 92; or 2) an amino acid sequence selected from any one of SEQ ID NO: 67 or 168.

2. A fusion protein comprising: a) a cytokine fused to an albumin binding moiety as claimed in claim 1, and b) an antigen binding moiety, wherein the cytokine is selected from IL-21, IL-33, IL-15 bound to IL-15Rα or a fragment thereof.

3. The fusion protein according to claim 1 or 2, wherein the cytokine is selected from IL-21 and IL-33. The fusion protein according to claim 3 , wherein the cytokine is IL-21.

5. The fusion protein according to claim 4, wherein the amino acid sequence of IL-21 is shown in any one of SEQ ID NO: 1, 2, 126, 171 or 172. The fusion protein of claim 3 , wherein the cytokine is IL-33.

7. The fusion protein according to any one of claims 1-2, 4-6, wherein the albumin binding moiety is fused to the C-terminus of the cytokine.

8. The fusion protein according to any one of claims 1-2, 4-6, wherein the albumin binding moiety is fused to the N-terminus of the cytokine.

9. The fusion protein according to any one of claims 1-2, 4-6, wherein the cytokine and the albumin binding moiety are connected by a first linker.

10. The fusion protein of claim 9, wherein the first linker is selected from SEQ ID NO: 12-26 or 158-159. The fusion protein according to claim 10 , wherein the first linker is a flexible linker. The fusion protein of claim 10 , wherein the first linker is a rigid linker.

13. The fusion protein according to any one of claims 1-2, 4-6, 10-12, wherein the amino acid sequence of the fusion protein is shown in any one of SEQ ID NOs: 120-122, 129-132, 134, 135, 137-154 or 160-164.

14. The fusion protein of any one of claims 2, wherein the antigen binding moiety is fused to the cytokine fused to the albumin binding moiety via a second linker.

15. The fusion protein of any one of claim 2, wherein the antigen binding portion binds to a tumor antigen.

16. The fusion protein of claim 15, wherein the tumor antigen is selected from mesothelin, GPA33, Her-2, EGFR or CD20.

17. The fusion protein of claim 15, wherein the tumor antigen is selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA or BCMA.

18. A pharmaceutical composition comprising the fusion protein according to any one of claims 1 to 17.

19. Use of the fusion protein according to any one of claims 1 to 17 or the pharmaceutical composition according to claim 18 in the preparation of a drug for treating a disease, wherein the disease is cancer, and the cancer is selected from lung cancer, lymphoma, gastric cancer or colon cancer.

20. The use according to claim 19, wherein the medicament further comprises a second agent.

21. The use according to claim 20, wherein the second agent comprises a therapeutic antibody, a second cytokine or a chemotherapeutic agent.

22. The use according to claim 20, wherein the second agent comprises an immune checkpoint inhibitor, a tyrosine kinase inhibitor or an immune cell.

23. The use according to any one of claims 19 to 22, wherein the cancer is selected from lung cancer or gastric cancer.

24. The use according to any one of claims 19 to 22, wherein the cytokine in the fusion protein is selected from IL-21, IL-33, IL-15 bound to IL-15Rα or a fragment thereof.

25. The use of claim 24, wherein the medicament comprises a second agent, and wherein the second agent is a therapeutic antibody.

26. The use according to claim 25, wherein the therapeutic antibody binds to a tumor antigen selected from mesothelin, GPA33, Her-2, EGFR or CD20.

27. The use according to claim 25, wherein the therapeutic antibody binds to a tumor antigen selected from CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA or BCMA.

28. The use according to claim 26, wherein the therapeutic antibody binds to mesothelin.

29. The use according to claim 26, wherein the therapeutic antibody binds to Her-2.

30. The use of claim 24, wherein the medicament comprises a second agent, and wherein the second agent is an immune checkpoint inhibitor.

31. The use according to claim 30, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint protein selected from PD-L1, CTLA4, PD-L2, PD-1, 4-1BB, CD47, TIGIT, GITR, TIM3, LAG3, CD27 or B7H4.

32. The use according to claim 31, wherein the immune checkpoint inhibitor is an antibody that binds to PD-1.

33. The use according to claim 31, wherein the immune checkpoint inhibitor is an antibody that binds to CTLA4.

34. The use of claim 24, wherein the medicament comprises a second agent, and wherein the second agent is a second cytokine.

35. The use according to claim 34, wherein the cytokine in the fusion protein is IL-21, and wherein the second cytokine is selected from IL-7 and IL-15 bound to IL-15Rα.

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