Cytokine fusion proteins and uses thereof

Abstract

The present application provides fusion proteins that comprise a cytokine fused to an albumin binding moiety. The fusion proteins may further comprise an antigen binding moiety such as a therapeutic antibody. The present application also provides methods of making and using the fusion proteins. The present application also provides methods of treatment comprises administering a fusion protein comprising a cytokine fused to a half-life extending domain and a second agent.

Description

CYTOKINE FUSION PROTEINS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of United States Provisional Application Serial No. 62 / 686,481 filed June 18, 2018, and United States Provisional Application Serial No. 62 / 809,496 filed February 22, 2019. FIELD OF THE APPLICATION

[0002] The present application relates to fusion proteins, methods of making thereof, and methods of treating a disease or disorder by administering a fusion protein. BACKGROUND OF THE APPLICATION

[0003] Cytokine therapy is an effective strategy for stimulating the immune system to induce immune response against a disease (such as a cancer or infection). However, cytokines that are administered to patients generally have a short half-life. For example, interleukin-2 1 stimulates various immune cells (such as T, B and NK cells) and enhances anti-turnor activity. It was reported that a recombinant IL-21 has a half-life of about one to three hours following intravenous administration. See Schmidt H, Clin Cancer Res. 2010 Nov 1;16 (21):53 12-9.

[0004] Therefore, there is a need for developing new cytokine therapeutics that effectively treating a disease.

[0005] [Intentionally left blank]. BRIEF SUMMARY OF THE APPLICATION

[0006] The present application provides fusion proteins comprising: a) a cytokine, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL- 15Ra or fragment thereof, IL-33, and IL-22. In some emboidments, the fusion protein further comprises an antigen binding moiety.

[0007] The present application also provides fusion proteins 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 cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Ra or fragment thereof, IL-33, and IL- 22. In some embodiments, the antigen binding moiety is fused to the C-terminus of the cytokine-ALBBM. In some embodiments, the antigen binding moiety is fused to the N- terminus of the cytokine-ALBBM. 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 metalloprotease, legumain, matriptase, or urokinase sensitive. 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 antigen binding moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of 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 moiety is an antibody or fragment thereof. In some embodiments, the antigen binding moiety comprises a single domain antibody (sdAb). In some embodiments, antigen binding moiety comprises a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0008] In some embodiments according to any one of the fusion proteins described above, the cytokine is IL-21. In some embodiments, the IL-21 comprises an amino acid sequence of SEQ ID NO: 1, 2, 126, 171, or 172 or a variant thereof comprising 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.

[0009] In some embodiments according to any one of the fusion proteins described above, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA).

[0010] In some embodiments according to any one of the fusion proteins described above, the albumin binding moiety comprises an albumin binding domain (ABD).

[0011] In some embodiments according to any one of the fusion proteins described above, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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.

[0012] In some embodiments according to any one of the fusion proteins described above, the albumin binding moiety comprises a single domain antibody (sdAb). In some embodiments, the sdAb is a V<com:Sub>H< / com:Sub>H single domain antibody.

[0013] In some embodiments according to any one of the fusion proteins described above, the albumin binding moiety is fused to the C-terminus of the cytokine.

[0014] In some embodiments according to any one of the fusion proteins described above, the albumin binding moiety is fused to the N-terminus of the cytokine.

[0015] In some embodiments according to any one of the fusion proteins described above, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159.

[0016] The present application also provides pharmaceutical compositions comprising any of the fusion protein described above.

[0017] The present application also provides methods of treating a disease or condition in an individual comprising administering to the individual any of the fusion proteins pharmaceutical compositions described above. In some embodiments, the method further comprises administering a second agent.

[0018] The present application also provides methods of 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 embodimetris, the Fc fragment is selected from the group consisting of an IgG1, IgG2, IgG3, and IgG4 Fc fragments or a variant thereof. In some embodiments, the Fc fragment is an IgG1 Fc fragment or variant thereof. In some embodiments, the IgG1 Fc fragment or variant thereof comprises a mutation at position 297, wherein the amino acid at position 297 is mutated to alanine, aspartic acid or glycine. In some embodiments, the individual is a human. In some embodiments, the disease or condition is selected from the group consisting of a cancer, an inflammatory condition, and an infection.

[0019] In some embodiments according to any one of the methods described above, the disease or condition is an inflammatory disease. In some embodiments, the cytokine is IL-22. In some embodiments, the disease is selected from the group consisting of ulcerative colitis, Crohn's disease, or ulcerative ileitis, and intestinal graft vs host disease.

[0020] In some embodiments according to any one of the methods described above, the disease or condition is a cancer. In some embodiments, the cancer is a solid or liquid tumor. In some embodiments, the cancer is selected from the group consisting of 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 the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, and IL- 33.

[0021] In some embodiments according to any one of the methods described above, the fusion protein is administered about once every three weeks to about twice a week.

[0022] In some embodiments according to any one of the methods described above, the amount of fusion protein for each administration is about 100 ng / kg to about 10 mg / kg.

[0023] In some embodiments according to any one of the methods described above, the fusion protein is administered parenterally into the individual. In some embodiments, the fusion protein is administered intravenously or subcutaneously into the individual.

[0024] In some embodiments according to any one of the methods described above, the fusion protein is administered for at least about one week to six months for each treatment cycle.

[0025] In some embodiments according to any one of the methods described above, 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 the group consisting of mesothelin (MSLN), GPA33, Her-2 (ERBB2), EGFR, and CD20 (MS4A1). 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 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 a single chain antibody comprising an anti-mesothelin heavy chain variable region (anti-MSLN V<com:Sub>H< / com:Sub>), wherein: a) the anti-MSLN V<com:Sub>H< / com:Sub> comprises 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 thereof comprising up to a total of 3, 2, or 1 amino acid substitutions in the CDRs; or b) the anti- MSLN V<com:Sub>H< / com:Sub> comprises 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 thereof comprising up to a total of 3, 2, or 1 amino acid substitutions in the CDRs. In some embodiments, the second agent that binds to mesothelin is administered about once per month to about twice per week. In some embodiments, the amount of the second agent for each administration is about 100 ng / kg to about 100 mg / kg. In some embodiments, the second agent is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an inhibitor of an immune checkpoint protein selected from the group consisting of 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 fragment thereof. In some embodiments, the amount of the second agent for each administration is 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 the group consisting of IL-7, IL-15, IL15 bound to IL15Rα or half-life extended variants thereof. In some embodiments, the second agent is an immune cell. In some embodiments, the immune cell comprises T cells or NK cells. In some embodiments, the immune cell comprises T cells expressing a chimeric antigen receptor (CAR), T cells expressing a modified T cell receptor (TCR), or T cells isolated from a tumor. In some embodiments, the second agent is a tyrosine kinase inhibitor. In some embodiments, the second agent is administered parenterally or orally into the individual. In some embodiments, the second agent is administered parenterally into the individual. In some embodiments, the second agent is administered intravenously into the individual.

[0026] In some embodiments according to any one of the methods described above, the fusion protein and the second agent are administered simultaneously, concurrently or sequentially into the individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 depicts exemplary IL-21 fusion proteins provided herein.

[0028] FIG. 2 depicts assembly of an exemplary IL-21 fusion protein expression vector wherein the albumin binding molecule is an anti-HSA antibody.

[0029] FIG. 3 depicts assembly of an exemplary IL-21 fusion protein expression vector wherein the albumin binding molecule is an ABD that binds to HSA.

[0030] FIG. 4 shows KD of anti-HSA antibody P367 when interacting with human, monkey or mouse serum albumin. P394-hgG1 Fc fusion protein was loaded onto protein A biosensor and dip into human, monkey or mouse serum albumin. Colored lines represent the binding response for different concentration of serum albumin at 400 nM (dark blue), 200 nM (dark red) and 100 nM (light blue). Experimental data was analyzed with global fitting (red) to determine KD.

[0031] FIG. 5 shows anti-HSA IL-21 conjugate P390 and P394 signaling potency as compared to recombinant IL-21.

[0032] FIG. 6A shows ADCC activities of PBMCs against N87 cells in the presence of P390, P394, and P461 / P462 in combination with anti-mesothelin antibody P303. FIG. 6B shows ADCC activities of PBMCs against H226 cells in the presence of P390, P394, and P461 / P462 in combination with anti-mesothelin antibody P303 against H226 cells. FIG. 6C shows ADCC activities of PBMCs against N87 cells in the presence of P390, P394, P461 / P462, and P461 / P463 at different dosages in combination with 3 ng / mL P303. FIG. 6D shows ADCC activities of PBMCs against H226 cells in the presence of P390, P394, P461 / P462, and P461 / P463 at different dosages in combination with 20 ng / mL P303.

[0033] FIG. 7A shows STAT3 signaling by IL-21-anti-HSA fusion protein variants P394, P593, P636, P637, P744, P748, P750, P751 and P783. FIG. 7B and FIG. 7C show NK cell mediated ADCC on NCI-N87 tumor cells when titrating IL-21-anti-HSA fusion protein variants P394, P593, P636, P637, P744, P748, P750, P751 and P783 at a fixed dose of anti- MSLN antibody (P303).

[0034] FIG. 8 shows depicts a comparison of pharamacokinetics in mice of 3 µg of recombinant human IL-21, 3 μg of P325 (human IL-21-irrelevant nanobody) and 3 or 30 μg of P394 (human IL-21-anti-HSA)

[0035] FIG. 9A, 9F, and 9G depict SDS-PAGE pictures that shows the statining of IL-21- anti-HSA fusion protein P394 (i.e., AWT-P394) and P593 (i.e., AWT-P593) and P748. In FIGS. 9F and 9G, arrows indicate intact target protein, and asterick indicates cleaved protein. FIG. 9B depicts a representative chromatogram of AWT-P394 IL21-anti HSA fusion protein. FIG. 9C depicts comparison of T<com:Sub>onset< / com:Sub> of AWT-P593 at pH 4.0 and pH 7.5. FIG. 9D depicts of the T<com:Sub>onset< / com:Sub> of AWT-P394 and AWT-P593. FIG. 9E depicts binding of human IL021 receptor to IL-21-anti-HSA fusion protein AWT-P394 or AWT-P593.

[0036] FIG. 10A depicts comparison of the Tonset of AWT-P593 at pH 4.0 and pH 7.5. FIG. 10B depicts comparison of the Tonset of AWT-P394 and AWT-P593. FIG. 10C depicts the binding of humanized anti-AWT-P610 (i.e., P610) with human serum albumin or monkey serum albumin at pH 7.4 and pH 5.5. FIG. 10D depicts comparison of the Tonset of anti- albumin antibody AWT-P367 and its humanized version AWT-P494. FIG. 10E depicts comparison of the Tonset of anti-albumin antibody AWT-P342 and its humanized version AWT-P610. FIG. 10D depicts the binding of anti-HSA antibody AWT-367 and its humanized anti-AWT-P494 with human, monkey or mouse albumin. FIG. 10E depicts KD of the binding of AWT-P367 or AWT-494 with human, monkey or mouse albumin.

[0037] FIG. 11 depicts remaining cell numbers of N87 cells after treatment of NK cells alone or in combination with study drug as shown in the figure.

[0038] FIG. 12 depicts remaining cell numbers of N87 cells after treatment of NK cells alone or in combination with a) Herceptin alone, b) P303FF (i.e., P303F) alone, c) Herceptin and P303FF, or d) Herceptin, P303FF, and IL-21-anti-HSA fusion protein P394.

[0039] FIG. 13 depicts percentage of dead Preiffer cells after treatment of NK cells in combination with a) retuxan, b) retuxan and P394, c) retuxan and IL-15 fusion protein P480, or d) retuxan and IL-15.

[0040] FIG. 14 depicts remaining cell numbers of N87 cells after treatment of NK cells in combination with a) P303F, b) P303F and recombinanant human IL-21 (i.e., rhIL-21), c) P303F and P480, or d) P303F and recombination human IL-15 (i.e., rhIL-15).

[0041] FIG. 15 depicts remaning cell numbers of N87 cells after treatment of NK cells in combination with a) P480, b) P597, or c) rIL-15 (upper panel) and IC50 of three drugs (lower panel).

[0042] FIG. 16 depicts remaning cell numbers of H226 cells after treatment of NK cells in combination with a) anti-mesothelin antibody 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).

[0043] FIG. 17 depicts remaning cell numbers of N87 cells after treatment of NK cells in combination with a) P197 and P390; or b) P197 and P394.

[0044] FIGS. 18A-18B depict levels of IFN-gamma (FIG. 18A) and IL-6 (FIG. 18B) secreted by PBMC after incubation with different drugs as shown.

[0045] FIGS. 19A-19B depict depict the change in tumor volume in an MC38 mouse syngeneic tumor model when dosed with anti-CTLA-4, IL-21-anti-HSA fusion protein (P394), anti-HSA- IL-15Ra / IL-15 fusion protein (P597) or combinations of these agents.

[0046] FIG. 20 depicts the change in tumor volume in an MC38 syngeneic mouse tumor model after treatment with IL-21-anti-HSA fusion protein (P394), anti-HSA-IL-15Ra / IL-15 fusion protein (P597) or a combination of these agents.

[0047] FIG. 21 depicts change of tumor volume in animal model of MC38 syngeneic model after treatment with a) 100 μg of anti- PD-1 antibody, b) 25 μg of P390, c) 100 μg of anti-PD-1 antibody and 25 μg of P390 or d) 100 μg of anti-PD-1 antibody and 5 μg of P390.

[0048] FIG. 22 depicts change of tumor volume in animal model of NSG mice with N87 tumors after treatment with a) 25 µg of P394, b) 100 µg of P303F, c) 100 µg of P303F and 25 μg of P394, or d) 100 μg of P303F and 5 μg of P394.

[0049] FIG. 23 depicts change of tumor volume in animal model of NSG mice with N87 tumors after treatment with a) 25 µg of P394, b) 20 µg of Herceptin, c) 20 µg of Herceptin and 25 µg of P394, or d) 20 µg of Herceptin and 5 µg of P394.

[0050] FIG. 24 depicts change of tumor volume in animal model of SCID mice with N87 tumors after treatment with 100 µg of P303F alone or in combination with a) 25 µg of P390 b) 5 μg of P390, or c) 2.5 μg of rmIL-21.

[0051] FIG. 25 depicts change of tumor volume in animal model of MC38 syngeneic model after treatment of 25 µg of P390 or 12.5 µg of rmIL-21.

[0052] FIG. 26 depicts change of tumor volume in animal model of CT-26 / MSLN after treatment of anti-PD-1 antibody alone, P390 alone, or a combination of anti-PD-1 antibody and P390.

[0053] FIG. 27 depicts change of tumor volume in animal model of MC38 syngeneic model after treatment of anti-PD-1 antibody alone, P394 alone, or a combination of anti-PD-1 antibody and P394.

[0054] FIG. 28 depicts change in tumor volume in mouse MC38 syngeneic tumor model after treatment with anti-HSA-IL-33 fusion protein (P380).

[0055] FIGS. 29A-29B depicts percentage change of granzyme B positive NK cells (FIG. 29A) and CD8 T cells (FIG. 29B) after treatment with P390.

[0056] FIGS. 30A-30C depicts change of expression levels of IL-21 receptor in CD8 T cells (FIG. 32A), CD4 T cell (FIG. 32B) and NK cells (FIG. 32C) after treatment with anti- PD-1 antibody alone or anti-PD-1 antibody in combination with P390.

[0057] FIG. 31 depicts cell number of IFN-gamma secreting immune cells in 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.

[0058] FIG. 32 depicts change in tumor volume in mouse syngeneic CT26 / MSLN tumor model using IL-21-anti-HSA-anti-MSLN fusion protein (P375).

[0059] FIG. 33 depicts change of tumor volume in animal model of NSG mice with N87 tumors after treatment with anti-MSLN-anti-HSA-IL-15Ra / IL15 fusion protein (P669).

[0060] FIG. 34 depicts various IL-21 fusion proteins have similar signaling potency.

[0061] FIG. 35 depicts various IL-21 fusion proteins have similar ADCC activity. DETAILED DESCRIPTION OF THE APPLICATION

[0062] The present application is related to fusion proteins that comprise a cytokine and a half-life extending domain. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or 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; the linkage between the cytokine-ALBBM and the antigen binding moiety is optionally cleavable.

[0063] The present application further provides methods of treating diseases or disorders (such as a cancer or an inflammatory disease) comprising administering a fusion protein as described above. In some embodiments, the method comprises administering a) a fusion protein comprising i) a cytokine, and ii) a half-life extending domain, and b) a second agent. Exemplary second agents include, and are not limited to, a therapeutic agent, an immune checkpoint inhibitor, a second cytokine, a tyrosine kinase inhibitor, a chemotherapeutic agent, or an immune cell.

[0064] Also provided are compositions, kits and articles of manufacture comprising the bispecific antibodies described herein and methods of making thereof. I. Definitions

[0065] 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 moiety" refers to a full-length antibody or an antigen-binding fragment thereof.

[0066] An "isolated" antibody (or construct) is one that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N- terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Blue or, preferably, silver stain. 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. Ordinarily, however, an isolated polypeptide, antibody, or construct will be prepared by at least one purification step.

[0067] A full-length antibody comprises 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 chain and light chain may be referred to as "V<com:Sub>H< / com:Sub>" and "V<com:Sub>L< / com:Sub>", respectively. The variable regions in both chains generally contain three highly variable loops called the 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). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al- Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chains are interposed between flanking stretches known as 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 assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>δ<annotation encoding="application / x-tex">\delta< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ϵ<annotation encoding="application / x-tex">\epsilon< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math> heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as lgG1 (γ1 heavy chain), lgG2 (γ2 heavy chain), lgG3 (γ3 heavy chain), lgG4 (γ4 heavy chain), lgA1 (<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>1 heavy chain), or lgA2 (<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>2 heavy chain).

[0068] The terms "full-length antibody", "intact antibody", or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, full-length 4-chain antibodies include those with heavy and light chains including an Fc region. Full-length heavy-chain only antibodies include the heavy chain variable domain (such as V<com:Sub>H< / com:Sub>H) and an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0069] The "variable region" or "variable domain" of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy-chain only antibodies from the Camelid species have a single heavy chain variable region, which is referred to as "V<com:Sub>H< / com:Sub>H". V<com:Sub>H< / com:Sub>H is thus a special type of VH.

[0070] The term "antigen-binding fragment" as used herein refers to an antibody fragment including, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0071] "Fv" is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0072] "Single-chain Fy," also abbreviated as "sFy" or "scFy," are antibody fragments that comprise the V<com:Sub>H< / com:Sub> and V<com:Sub>L< / com:Sub> antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the V<com:Sub>H< / com:Sub> and V<com:Sub>L< / com:Sub> domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0073] The term "heavy chain-only antibody" or "HCAb" refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain antibodies. Camelid animals (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0074] The term "single-domain antibody," "single domain antibody," or "sdAb" refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains are referred herein as "V<com:Sub>H< / com:Sub>Hs" (Variable domain of the heavy chain of the Heavy chain antibody). Camelid sdAb is one of the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh- Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). A basic V<com:Sub>H< / com:Sub>H has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3.

[0075] The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, single-domain antibodies comprise three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) displays the most diversity of the three HVRs, and is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363:446- 448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0076] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U.S. Dept of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al, J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). TABLE 1: CDR DEFINITIONS [Image disponible dans le document PDF, Image available in the PDF document] Residue numbering follows the nomenclature of Kabat et al, supra <com:Sup>2< / com:Sup>Residue numbering follows the nomenclature of Chothia et al., supra Residue numbering follows the nomenclature of MacCallum et al, supra <com:Sup>4< / com:Sup>Residue numbering follows the nomenclature of Lefranc et al, supra <com:Sup>5< / com:Sup>Residue numbering follows the nomenclature of Honegger and Pluckthun, supra

[0077] The expression "variable-domain residue-numbering as in Kabat" or "amino-acid- position numbering as in Kabat," and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

[0078] Unless indicated otherwise herein, the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra. The "EU index" as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0079] "Framework" or "FR" residues are those variable-domain residues other than the CDR residues as herein defined.

[0800] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0081] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0082] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of 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 times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0083] The term "constant domain" refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the C<com:Sub>H< / com:Sub>1, C<com:Sub>H< / com:Sub>2 and C<com:Sub>H< / com:Sub>3 domains (collectively, C<com:Sub>H< / com:Sub>) of the heavy chain and the CHL (or C<com:Sub>L< / com:Sub>) domain of the light chain.

[0084] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>") and lambda ("<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>"), based on the amino acid sequences of their constant domains.

[0085] The "CH1 domain" of a human IgG Fc region (also referred to as "C1" of "H1" domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0086] "Hinge region" is generally defined as a region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions.

[0087] The "CH2 domain" of a human IgG Fc region (also referred to as "C2" of "H2" domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985).

[8800] The "CH3 domain" (also referred to as "C2" or "H3" domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).

[0089] The term "Fc region" or "fragment crystallizable region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0090] "Fc receptor" or "FcR" describes a receptor that binds the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Daëron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein.

[0091] The term "epitope" as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.

[0092] 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 the target antigen binding of the second antibody of fragment thereof by at least about 50% (such as at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolar concentration of the first antibody or fragment thereof, or vice versa. A high throughput process for "binning" antibodies based upon their cross-competition is described in PCT Publication No. WO 03 / 48731.

[0093] As use herein, the terms "specifically binds," "specifically recognizing," and "is specific for" refer to measurable and reproducible interactions, such as binding between a target and an antibody or antibody moiety, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody or antibody moiety that specifically recognizes a target (which can be an epitope) is an antibody or antibody moiety that binds this target with greater affinity, avidity, more readily, and / or with greater duration than its bindings 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, e.g., by a radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds a target has a dissociation constant (<com:Math><semantics>KD<annotation encoding="application / x-tex">K_D< / annotation>< / semantics>< / com:Math>) of <com:Math><semantics>≤10−5<annotation encoding="application / x-tex">\leq 10^{-5}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−6<annotation encoding="application / x-tex">\leq 10^{-6}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−7<annotation encoding="application / x-tex">\leq 10^{-7}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−8<annotation encoding="application / x-tex">\leq 10^{-8}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−9<annotation encoding="application / x-tex">\leq 10^{-9}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−10<annotation encoding="application / x-tex">\leq 10^{-10}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−11<annotation encoding="application / x-tex">\leq 10^{-11}< / annotation>< / semantics>< / com:Math> M, or <com:Math><semantics>≤10−12<annotation encoding="application / x-tex">\leq 10^{-12}< / annotation>< / semantics>< / com:Math> M. In some embodiments, an antibody specifically binds an epitope on a protein that is conserved among the protein from different species. In some embodiments, specific binding can include, but does not require exclusive binding. Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Octet, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE<com:Sup>TM< / com:Sup> -tests and peptide scans.

[0094] "Binding affinity" generally refers to the strength of the sum total 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 indicated otherwise, 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 indicated by K<com:Sub>d< / com:Sub>, K<com:Sub>off< / com:Sub>, K<com:Sub>on< / com:Sub>, or K<com:Sub>a< / com:Sub>. The term "K<com:Sub>off< / com:Sub>", as used herein, is intended to refer to the off rate constant for dissociation of an antibody (or antigen-binding domain) from the antibody / antigen complex, as determined from a kinetic selection set up, expressed in units of s<com:Sup>-1< / com:Sup>. The term "K<com:Sub>on< / com:Sub>", as used herein, is intended to refer to the on rate constant for association of an antibody (or antigen-binding domain) to the antigen to form the antibody / antigen complex, expressed in units of M<com:Sup>-1< / com:Sup>s<com:Sup>-1< / com:Sup>. The term equilibrium dissociation constant "K<com:Sub>D< / com:Sub>" or "K<com:Sub>d< / com:Sub>", as used herein, refers to the dissociation constant of a particular antibody-antigen interaction, and describes the concentration of antigen required to occupy one half of all of the antibody-binding domains present in a solution of antibody molecules at equilibrium, and is equal to <com:Math><semantics>Koff / Kon<annotation encoding="application / x-tex">K_{off} / K_{on}< / annotation>< / semantics>< / com:Math>, expressed in units of M. The measurement of K<com:Sub>d< / com:Sub> presupposes that all binding agents are in solution. In the case where the antibody is tethered to a cell wall, e.g., in a yeast expression system, the corresponding equilibrium rate constant is expressed as EC50, which gives a good approximation of <com:Math><semantics>Kd<annotation encoding="application / x-tex">K_d< / annotation>< / semantics>< / com:Math>. The affinity constant, K<com:Sub>a< / com:Sub>, is the inverse of the dissociation constant, K<com:Sub>d< / com:Sub>, expressed in units of M<com:Sup>-1< / com:Sup>. The dissociation constant <com:Math><semantics>(KD or Kd)<annotation encoding="application / x-tex">(K_D \text{ or } K_d)< / annotation>< / semantics>< / com:Math> is used as an indicator showing affinity of antibodies to antigens. For example, easy analysis is possible by the Scatchard method using antibodies marked with a variety of marker agents, as well as by using BiacoreX (made by Amersham Biosciences), which is an over-the-counter, measuring kit, or similar kit, according to the user's manual and experiment operation method attached with the kit. The K<com:Sub>D< / com:Sub> value that can be derived using these methods is expressed in units of M (Mols). An antibody or antigen- binding fragment thereof that specifically binds to a target may have a dissociation constant <com:Math><semantics>(Kd)<annotation encoding="application / x-tex">(K_d)< / annotation>< / semantics>< / com:Math> of, for example, <com:Math><semantics>≤10−5<annotation encoding="application / x-tex">\leq 10^{-5}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−6<annotation encoding="application / x-tex">\leq 10^{-6}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−7<annotation encoding="application / x-tex">\leq 10^{-7}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−8<annotation encoding="application / x-tex">\leq 10^{-8}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−9<annotation encoding="application / x-tex">\leq 10^{-9}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−10<annotation encoding="application / x-tex">\leq 10^{-10}< / annotation>< / semantics>< / com:Math> M, <com:Math><semantics>≤10−11<annotation encoding="application / x-tex">\leq 10^{-11}< / annotation>< / semantics>< / com:Math> M, or <com:Math><semantics>≤10−12 M.<annotation encoding="application / x-tex">\leq 10^{-12} \text{ M}.< / annotation>< / semantics>< / com:Math>

[0095] Half maximal inhibitory concentration (IC<com:Sub>50< / com:Sub>) 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 a given biological process (e.g., the binding between albumin and CD155, or component of a process, i.e. an enzyme, cell, cell receptor or microorganism) by half. The values are typically expressed as molar concentration. IC<com:Sub>50< / com:Sub> is comparable to an "EC<com:Sub>50< / com:Sub>" for agonist drug or other substance (such as an antibody). EC<com:Sub>50< / com:Sub> also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo. As used herein, an "IC<com:Sub>50< / com:Sub>" is used to indicate the effective concentration of an antibody (such as an anti-albumin sdAb) needed to neutralize 50% of the antigen bioactivity (such as albumin bioactivity) in vitro. IC<com:Sub>50< / com:Sub> or EC<com:Sub>50< / com:Sub> can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay), cell based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA).

[0096] As used herein, the term "cytokine" is understood to mean any protein or peptide, analog or functional fragment thereof, which is capable of stimulating or inducing a cytocidal immune response against a preselected cell-type, for example, a cancer cell or a virally- infected cell, in a mammal. Accordingly, it is contemplated that a variety of cytokines can be incorporated into this application. Useful cytokines include, for example, tumor necrosis factors (TNFs), interleukins (ILs), lymphokines (Ls), colony stimulating factors (CSFs), interferons (IFNs) including species variants, truncated analogs thereof which are capable of stimulating or inducing such cytocidal immune responses. Useful tumor necrosis factors include, 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-<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, IFN-<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> and IFN-<com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>. The term "cytokine" is also understood to encompass any variant of a wildtype cytokine (such as IL-21, IL-7, IL-15, etc.) that comprises modification and maintains at least a significant portion (such as at least about 50%) of any of its desired function.

[0097] The term "truncated IL-21", as used herein, refers to a protein or peptide comprising an IL-21 that has a deletion of one or more amino acids at one or both of C- and N- terminus of a wildtype IL-21. A fusion protein comprising a truncated IL-21 described herein can have other moieties or domains such as an antigen-binding moiety, a linker, a signal sequence, or an albumin-binding moiety, although the IL-21 in the fusion protein is a truncated form that has at least one amino acid less than a wildtype IL-21. In some embodiments, the wildtype IL-21 has an amino acid sequence of SEQ ID NO: 1.

[0098] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen- binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0099] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0100] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0101] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has 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."

[0102] The term "transfected" or "transformed" or "transduced" as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0103] 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 a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0104] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival. Also encompassed by "treatment" is a reduction of pathological consequence of cancer (such as, for example, tumor volume). The methods of the application contemplate any one or more of these aspects of treatment.

[0105] In the context of cancer, the term "treating" includes any or all of: inhibiting growth of cancer cells, inhibiting replication of cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease.

[0106] The terms "inhibition" or "inhibit" refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To "reduce" or "inhibit" is to decrease, reduce or arrest an activity, function, and / or amount as compared to that of a reference. In certain embodiments, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 20% or greater. In another embodiment, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.

[0107] A "reference" as used herein, refers to any sample, standard, or level that is used for comparison purposes. A reference may be obtained from a healthy and / or non-diseased sample. In some examples, a reference may be obtained from an untreated sample. In some examples, a reference is obtained from a non-diseased on non-treated sample of an individual. In some examples, a reference is obtained from one or more healthy individuals who are not the individual or patient.

[0108] As used herein, "delaying development of a disease" means to defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0109] "Preventing," as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.

[0110] As used herein, to "suppress" a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.

[0111] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0112] An "effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

[0113] A "therapeutically effective amount" of a substance / molecule of the application, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, agonist or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations.

[0114] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0115] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to an individual to which the formulation would be administered. Such formulations may be sterile.

[0116] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a "pharmaceutical" composition" for administration to an individual. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.

[0117] A "sterile" formulation is aseptic or essentially free from living microorganisms and their spores.

[0118] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.

[0119] The term "concurrently" is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent. For example, the two or more therapeutic agents are administered with a time separation of no more than about 60 minutes, such as no more than about any of 30, 15, 10, 5, or 1 minutes.

[0120] The term "sequentially" is used herein to refer to administration of two or more therapeutic agents where the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s). For example, administration of the two or more therapeutic agents are administered with a time separation of more than about 15 minutes, such as about any of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month, or longer.

[0121] As used herein, "in conjunction with" refers to administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.

[0122] 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.

[0123] An "article of manufacture" is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0124] It is understood that embodiments of the application described herein include "consisting" and / or "consisting essentially of" embodiments.

[0125] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0126] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0127] The term "about X-Y" used herein has the same meaning as "about X to about Y."

[0128] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. II-A. Fusion proteins comprising an albumin binding moiety

[0129] Provided herein are fusion proteins comprising: a) a cytokine, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the fusion ptotein comprises a) a cytokine selected from the group consisting of IL-21, IL-7, IL-15, IL- 15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin binding moiety comprises a single domain antibody (sdAb). In some embodiments, the albumin binding moiety is fused to the C-terminus of the cytokine. In some embodiments, the albumin binding moiety is fused to the N-terminus of the cytokine. In some embodiments, the cytokine and the albumin binding moiety are connected via a first linker (such as of about one to thirty amino acids). In some embodiments, the albumin binding moiety is a single domain antibody (such as a V<com:Sub>H< / com:Sub>H antibody). An sdAb with relatively small molecular weight may help increase cancer cancer penetration of the fusion protein, thereby making it better suited to treat certain cancers, e.g., solid tumors.

[0130] In some embodiments, there is provided a fusion protein comprising: a) an IL-21, 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a V<com:Sub>H< / com:Sub>H single domain antibody). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-21 is a truncated IL-21. In some embodiments, the truncated IL-21 comprises an 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 the group consisting of 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 the group consisting of SEQ ID NO: 12-14.

[0131] In some embodiments, there is provided a fusion protein comprising: a) an IL-21, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-21 comprises an amino acid sequence of SEQ ID NO: 1, 2, 126, 171, or 172 or a variant thereof comprising 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 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-21 is a truncated IL-21. In some embodiments, the truncated IL-21 comprises an 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 the group consisting of 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 the group consisting of SEQ ID NO: 12-14.

[0132] In some embodiments, there is provided a fusion protein comprising: a) an IL-21, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-21 comprises an amino acid sequence of SEQ ID NO: 1, 2, 126, 171, or 172 or a variant thereof comprising 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 an V<com:Sub>H< / com:Sub>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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-21 is a truncated IL-21. In some embodiments, the truncated IL-21 comprises an 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 the group consisting of 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 the group consisting of SEQ ID NO: 12-14.

[0133] In some embodiments, there is provided a fusion protein comprising: a) an 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a V<com:Sub>H< / com:Sub>H single domain antibody). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 the group consisting of 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 the group consisting of SEQ ID NO: 12- 14.

[0134] In some embodiments, there is provided a fusion protein comprising: a) an IL-7, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-7 comprises an amino acid sequence of any one of SEQ ID NOs: 96-98 or a variant thereof comprising 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 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159.

[0135] In some embodiments, there is provided a fusion protein comprising: a) an IL-7, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-7 comprises an amino acid sequence of any one of SEQ ID NOs: 96-98 or a variant thereof comprising 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 an V<com:Sub>H< / com:Sub>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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159.

[0136] In some embodiments, there is provided a fusion protein comprising: a) an 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a V<com:Sub>H< / com:Sub>H single domain antibody). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the cytokine is IL- 15Rα or IL-15 bound to IL-15Rα or fragment thereof, and wherein the IL-15Rα or IL-15 bound to IL-15Ra or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108, or a variant thereof comprising at least about 80% sequence identity to any one of SEQ ID NOs: 101-108. In some embodiments, the cytokine comprises IL-15 and IL- 15Rα. In some embodiments, the IL-15 and IL-15Rα are connected via a linker ("linker between the IL-15 and IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>"). In some embodiments, the linker between the IL-15 and IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> is cleavable. In some embodiments, the linker between the IL-15 and IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> is non-cleavable.

[0137] In some embodiments, there is provided a fusion protein comprising: a) an IL-15, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15 comprises an amino acid sequence of any one of SEQ ID NO: 99, 100, or 127 or a variant thereof comprising 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 NO: 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the cytokine is IL-15Rα or IL-15 bound to IL-15Rα or fragment thereof, and wherein the IL-15Rα or IL-15 bound to IL-15Rα or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108, or a variant thereof comprising at least about 80% sequence identity to any one of SEQ ID NOs: 101-108. In some embodiments, the cytokine comprises IL-15 and IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the IL-15 and IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> are connected via a linker ("linker between the IL-15 and IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>"). In some embodiments, the linker between the IL-15 and IL-15Ra is cleavable. In some embodiments, the linker between the IL-15 and IL-15Rα is non-cleavable.

[0138] In some embodiments, there is provided a fusion protein comprising: a) an IL-15, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15 comprises an amino acid sequence of any one of SEQ ID NO: 99, 100, or 127 or a variant thereof comprising 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 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 an V<com:Sub>H< / com:Sub>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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the cytokine is IL-15Rα or IL-15 bound to IL-15Rα or fragment thereof, and wherein the IL-15Rα or IL-15 bound to IL-15Rα or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108, or a variant thereof comprising at least about 80% sequence identity to any one of SEQ ID NOs: 101-108. In some embodiments, the cytokine comprises IL-15 and IL-15Rα. In some embodiments, the IL-15 and IL-15Rα are connected via a linker ("linker between the IL-15 and IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>"). In some embodiments, the linker between the IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between the IL-15 and IL-15Rα is non-cleavable.

[0139] In some embodiments, there is provided a fusion protein comprising a) a cytokine comprising an IL-15R sushi domain, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the IL-15R sushi domain comprises or consists of an amino acid sequence of any one of SEQ ID NO: 127-128 or a variant thereof comprising 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 NO: 127-128. In some embodiments, the fusion protein does not comprise an IL-15. In some embodiments, the fusion protein further comprises an IL-15. In some embodiments, the IL-15 and IL-15R sushi domain are connected via a linker ("linker between the IL-15 and IL-15R sushi domain"). In some embodiments, the linker between the IL-15 and IL-15R sushi domain is cleavable. In some embodiments, the linker between the IL-15 and IL-15 sushi domain is non-cleavable.

[0140] In some embodiments, there is provided a fusion protein comprising: a) an IL-15 bound to IL-15Rα comprising an IL-15 and an 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 the IL-15 or the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the albumin binding moiety is fused to both the IL-15 and the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the albumin binding moiety is fused to the N- and / or C-terminus of IL-15Ra. In some embodiments, the albumin binding moiety is fused to the N- 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-15Ra. In some embodiments, the albumin binding moiety is fused to the N-terminus of IL-15 and / or IL- 15Rα. In some embodiments, the IL-15 is non-convalently bound to the IL-15Rα. In some embodiments, the IL-15 is fused to the IL-15Rα. In some embodiments, the IL-15 is fused to the N-terminus of the IL-15Ra. In some embodiments, the IL-15 is fused to the C-terminus of the IL-15Rα. In some embodiments, the albumin binding moiety is further fused to the N- or C-terminus of a second cytokine. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a <com:Math><semantics>VHH<annotation encoding="application / x-tex">V_HH< / annotation>< / semantics>< / com:Math> single domain antibody). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 and the 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 a 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, IL-15 bound to IL-15Rα or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108, or a variant thereof comprising at least about 80% sequence identity to any one of SEQ ID NOs: 101-108. In some embodiments, the IL-15 and IL-15Rα are connected via a linker ("linker between the IL-15 and IL-15Rα"). In some embodiments, the linker between the IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between the IL-15 and IL-15Rα is non-cleavable.

[0141] In some embodiments, there is provided a fusion protein comprising: a) an IL-15 bound to IL-15Rα comprising an IL-15 and an IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the fusion protein comprises said IL-15, IL- 15Rα and albumin binding moiety from N-terminal to C-terminal in an order selected from the group consisting of (1) the albumin binding moiety, the IL-15, the IL-15Rα; (2) the albumin binding moiety, the IL-15Rα, the IL-15; (3) the IL-15, the IL-15Rα, the albumin binding moiety; (4) the IL-15Rα, the IL-15, the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a V<com:Sub>H< / com:Sub>H single domain antibody). In some embodiments, the cytokine (IL-15 or IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>) and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 and the IL-15Rα are fused viα 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 a 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, IL-15 bound to IL- 15Rα or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108, or a variant thereof comprising at least about 80% sequence identity to any one of SEQ ID NOs: 101-108. In some embodiments, the IL-15 and IL-15Rα are connected via a linker ("linker between the IL-15 and IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>"). In some embodiments, the linker between the IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between the IL-15 and IL-15Rα is non-cleavable.

[0142] In some embodiments, there is provided a fusion protein comprising: a) an IL-15 bound to IL-15Rα comprising an IL-15 and an IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15 thereof comprises an amino acid sequence of any one of SEQ ID NO: 99, 100, or 127 or a variant thereof comprising 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 NO: 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 is non-convalently bound to the IL-15Rα. In some embodiments, the IL-15 is fused to the IL- 15Rα. In some embodiments, the IL-15 is fused to the N-terminus of the IL-15Rα. In some embodiments, the IL-15 is fused to the C-terminus of the IL-15Rα. In some embodiments, the fusion protein comprises said IL-15, IL-15Rα and albumin binding moiety from N-terminal to C-terminal in an order selected from the group consisting of (1) the albumin binding moiety, the IL-15, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>; (2) the albumin binding moiety, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, the IL-15; (3) the IL-15, the IL-15Rα, the albumin binding moiety; (4) the IL-15Rα, the IL-15, the albumin binding moiety. In some embodiments, the IL-15 and the 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 a 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, IL-15 bound to IL-15Rα or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108, or a variant thereof comprising at least about 80% sequence identity to any one of SEQ ID NOs: 101-108. In some embodiments, the IL-15 and IL-15Rα are connected via a linker ("linker between the IL-15 and IL-15Rα"). In some embodiments, the linker between the IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between the IL-15 and IL-15Rα is non-cleavable.

[0143] In some embodiments, there is provided a fusion protein comprising: a) an IL-15 bound to IL-15Rα comprising an IL-15 and an IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15 comprises an amino acid sequence of any one of SEQ ID NO: 99, 100, or 127 or a variant thereof comprising 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 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 an <com:Math><semantics>VHH<annotation encoding="application / x-tex">V_HH< / annotation>< / semantics>< / com:Math> 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 is non-convalently bound to the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the IL-15 is fused to the IL-15Rα. In some embodiments, the IL-15 is fused to the N-terminus of the IL-15Rα. In some embodiments, the IL-15 is fused to the C-terminus of the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the fusion protein comprises said IL-15, IL-15Rα and albumin binding moiety from N-terminal to C-terminal in an order selected from the group consisting of (1) the albumin binding moiety, the IL-15, the IL-15Rα; (2) the albumin binding moiety, the IL- 15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, the IL-15; (3) the IL-15, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, the albumin binding moiety; (4) the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, the IL-15, the albumin binding moiety. In some embodiments, the IL-15 and the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> 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 a 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, IL-15 bound to IL-15Rα or fragment thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108, or a variant thereof comprising at least about 80% sequence identity to any one of SEQ ID NOs: 101-108. In some embodiments, the IL-15 and IL-15Rα are connected via a linker ("linker between the IL-15 and IL-15Rα"). In some embodiments, the linker between the IL-15 and IL-15Rα is cleavable. In some embodiments, the linker between the IL-15 and IL-15Rα is non-cleavable.

[0144] In some embodiments, there is provided a fusion protein comprising: a) an IL-15 bound to IL-15Rα comprising an IL-15 and an IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15Rα thereof comprises an amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104) or a variant thereof comprising 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 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 is non-convalently bound to the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the IL-15 is fused to the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the IL-15 is fused to the N-terminus of the IL-15Rα. In some embodiments, the IL-15 is fused to the C-terminus of the IL-15Ra. In some embodiments, the fusion protein comprises said IL-15, IL-15Rα and albumin binding moiety from N-terminal to C-terminal in an order selected from the group consisting of (1) the albumin binding moiety, the IL-15, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>; (2) the albumin binding moiety, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, the IL-15; (3) the IL-15, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, the albumin binding moiety; (4) the IL-15Rα, the IL-15, the albumin binding moiety. In some embodiments, the IL-15 and the 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 a 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.

[0145] In some embodiments, there is provided a fusion protein comprising: a) an IL-15 bound to IL-15Rα comprising an IL-15 and an IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the <com:Math><semantics>IL−15Rα<annotation encoding="application / x-tex">IL-15R\alpha< / annotation>< / semantics>< / com:Math> comprises an amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104) or a variant thereof comprising 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 an V<com:Sub>H< / com:Sub>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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 is non-convalently bound to the IL- 15Rα. In some embodiments, the IL-15 is fused to the IL-15Rα. In some embodiments, the IL-15 is fused to the N-terminus of the IL-15Rα. In some embodiments, the IL-15 is fused to the C-terminus of the IL-15Ra. In some embodiments, the fusion protein comprises said IL- 15, IL-15Rα and albumin binding moiety from N-terminal to C-terminal in an order selected from the group consisting of (1) the albumin binding moiety, the IL-15, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>; (2) the albumin binding moiety, the IL-15Rα, the IL-15; (3) the IL-15, the IL-15Rα, the albumin binding moiety; (4) the IL-15Rα, the IL-15, the albumin binding moiety. In some embodiments, the IL-15 and the 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 a 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.

[0146] In some embodiments, there is provided a fusion protein comprising: a) an IL-15 bound to IL-15Rα comprising an IL-15 and an IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-15 comprises an amino acid sequence of any one of SEQ ID NO: 99, 100, or 127 or a variant thereof comprising 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 NO: 99, 100, or 127, wherein the IL-15Rα comprises an amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104) or a variant thereof comprising 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 an <com:Math><semantics>VHH<annotation encoding="application / x-tex">V_HH< / annotation>< / semantics>< / com:Math> 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 is non-convalently bound to the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the IL-15 is fused to the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the IL-15 is fused to the N-terminus of the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the IL-15 is fused to the C-terminus of the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the fusion protein comprises said IL-15, IL-15Rα and albumin binding moiety from N-terminal to C-terminal in an order selected from the group consisting of (1) the albumin binding moiety, the IL-15, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>; (2) the albumin binding moiety, the IL- 15Rα, the IL-15; (3) the IL-15, the IL-15Rα, the albumin binding moiety; (4) the IL-15Rα, the IL-15, the albumin binding moiety. In some embodiments, the IL-15 and the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> 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 a 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.

[0147] In some embodiments, there is provided a fusion protein comprising: a) an IL-15 bound to IL-15Rα comprising an IL-15 and an IL-15Rα, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein said IL-15, IL-15Rα and albumin binding moiety from N-terminal to C-terminal in an order of the albumin binding moiety, the IL-15, the IL-15Rα, wherein the IL-15 comprises an amino acid sequence of any one of SEQ ID NO: 99, 100, or 127 or a variant thereof comprising 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 NO: 99, 100, or 127, wherein the IL-15Rα comprises an amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104) or a variant thereof comprising 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 an V<com:Sub>H< / com:Sub>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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the IL-15 and the 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 a 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.

[0148] In some embodiments, there is provided a fusion protein comprising: a) an 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a V<com:Sub>H< / com:Sub>H single domain antibody). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, there is no linker between the cytokine and the albumin binding moiety.

[0149] n some embodiments, there is provided a fusion protein comprising: a) an IL-33, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-33 comprises an amino acid sequence of any one of SEQ ID NO: 109 and 155-157 or a variant thereof comprising 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 NO: 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, there is no linker between the cytokine and the albumin binding moiety.

[0150] In some embodiments, there is provided a fusion protein comprising: a) an IL-33, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-33 comprises an amino acid sequence of any one of SEQ ID NO: 109 and 155-157 or a variant thereof comprising 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 NO: 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 an V<com:Sub>H< / com:Sub>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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, there is no linker between the cytokine and the albumin binding moiety.

[0151] In some embodiments, there is provided a fusion protein comprising: a) an 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a V<com:Sub>H< / com:Sub>H single domain antibody). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159.

[0152] In some embodiments, there is provided a fusion protein comprising: a) an IL-22, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-22 comprises an amino acid sequence of any one of SEQ ID NOs: 109-110 or a variant thereof comprising 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 NO: 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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159.

[0153] In some embodiments, there is provided a fusion protein comprising: a) an IL-22, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the IL-22 comprises an amino acid sequence of any one of SEQ ID NOs: 109-110 or a variant thereof comprising 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 an V<com:Sub>H< / com:Sub>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, either the same as the first cytokine or different, is fused to the other terminus of the albumin binding moiety. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of SEQ ID NOs: 12-26 and 158-159. Fusion proteins comprising an antigen binding moiety

[0154] Also provided herein are fusion proteins 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 various advantages. For example, in some embodiments, the antigen binding moiety that binds to a tumor antigen enables the local delivery of the cytokine to cancer proximity, leading to lower off-target toxicity and increased efficacy.

[0155] Certain advantages are offered by such fusion proteins due to their tertiary structure and overall configuration design. For example, in some embodiments, the cytokine (such as IL-21) is fused to the C-terminus of the albumin binding moiety via a cleavable linker (such as an MMP sensitive linker), and the antigen binding moiety is fused to the N-terminus of the albumin binding moiety. The cytokine (such as IL-21) in the fusion protein may be temporarilty blocked from interacting with the cytokine receptor (such as IL-21R) since its N-terminus (close to C-terminus in teritiary structure) may be required for their interaction. When the fusion protein binds to a tumor antigen, the cytokine (such as IL-21) can be released from the fusion protein if the linker is cleavable by MMP and become active, since cancer cell are known to secret various MMPs. An MMP sensitive linker ensures that cancers which typically have higher MMP activities have higher exposure to the active cytokine. Thus, unnecessary toxicity and side effects of the cytokine (such as IL-21) can be avoided. In addition, by preventing the interaction between the cytokine (such as IL-21) and the cytokine receptor (such as IL-21Rα) on peripheral immune cells, the efficiency of cancer delivery of the fusion protein can be increased.

[0156] In some embodiments, the albumin binding moiety is a single domain antibody (such as a <com:Math><semantics>VHH<annotation encoding="application / x-tex">V_HH< / annotation>< / semantics>< / com:Math> antibody). In some embodiments, the antigen binding moiety is a single domain antibody (such as a V<com:Sub>H< / com:Sub>H antibody). In some embodiments, the albumin binding moiety and the antigen binding moiety are both single domain antibodies (such as V<com:Sub>H< / com:Sub>H antibodies).

[0157] In some embodiments, there is provided 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, 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 the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 metalloprotease, legumain, matriptase, or urokinase sensitive. 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 antigen binding moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of 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 moiety is an antibody or fragment thereof. In some embodiments, the antigen binding moiety comprises a single domain antibody (sdAb). In some embodiments, antigen binding moiety comprises a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0158] In some embodiments, there is provided 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, 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 the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 metalloprotease, legumain, matriptase, or urokinase sensitive. 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 antigen binding moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of 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 moiety is an antibody or fragment thereof. In some embodiments, the antigen binding moiety comprises a single domain antibody (sdAb). In some embodiments, antigen binding moiety comprises a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0159] In some embodiments, there is provided 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, wherein the albumin binding moiety is fused to the C-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 the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 metalloprotease, legumain, matriptase, or urokinase sensitive. 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 antigen binding moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of 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 moiety is an antibody or fragment thereof. In some embodiments, the antigen binding moiety comprises a single domain antibody (sdAb). In some embodiments, antigen binding moiety comprises a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0160] In some embodiments, there is provided 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, 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 the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a 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 the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 metalloprotease, legumain, matriptase, or urokinase sensitive. 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 antigen binding moiety binds to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of 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 moiety is an antibody or fragment thereof. In some embodiments, the antigen binding moiety comprises a single domain antibody (sdAb). In some embodiments, antigen binding moiety comprises a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0161] In some embodiments, there is provided 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, 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<com:Sub>H< / com:Sub>H antibody that binds to a tumor antigen). In some embodiments, the albumin binding moiety is fused to the N- or C-terminus of the cytokine. In some embodiments, the antigen binding moiety is linked to the N- or C- terminus of the cytokine- ALBBM. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 metalloprotease, legumain, matriptase, or urokinase sensitive. 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 tumor antigen is selected from the group consisting of 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, antigen binding moiety comprises a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0162] In some embodiments, there is provided 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, wherein the albumin binding moiety is a single domain antibody ("anti- albumin dsAb", such as an anti-albumin V<com:Sub>H< / com:Sub>H antibody), and wherein the antigen binding moiety is a single domain antibody that binds to a tumor antigen (such as a V<com:Sub>H< / com:Sub>H antibody that binds to a tumor antigen). In some embodiments, the albumin binding moiety is fused to the N- or C-terminus of the cytokine. In some embodiments, the antigen binding moiety is linked to the N- or C- terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the anti-albumin sdAb is a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 metalloprotease, legumain, matriptase, or urokinase sensitive. 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 tumor antigen is selected from the group consisting of 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, antigen binding moiety comprises a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0163] In some embodiments, there is provided 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, wherein the albumin binding moiety is an albumin binding domain or a single domain antibody ("anti-albumin dsAb", such as an anti-albumin V<com:Sub>H< / com:Sub>H antibody), and wherein the antigen binding moiety is an anti-mesothelin single domain antibody (such as an anti-MSLN V<com:Sub>H< / com:Sub>H antibody). In some embodiments, the albumin binding moiety is fused to the N- or C-terminus of the cytokine. In some embodiments, the antigen binding moiety is linked to the N- or C- terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Ra or fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 metalloprotease, legumain, matriptase, or urokinase sensitive. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 12-45 and 158-159.

[0164] In some embodiments, there is provided 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, wherein the albumin binding moiety is an albumin binding domain or a single domain antibody ("anti-albumin dsAb", such as an anti-albumin V<com:Sub>H< / com:Sub>H antibody), and wherein the antigen binding moiety is a single domain antibody that binds to a tumor antigen, wherein the cytokine is IL-21. In some embodiments, the albumin binding moiety is fused to the N- or C-terminus of the cytokine. In some embodiments, the antigen binding moiety is linked to the N- or C- terminus of the cytokine-ALBBM. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 metalloprotease, legumain, matriptase, or urokinase sensitive. 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 tumor antigen is selected from the group consisting of 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, antigen binding moiety comprises a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the sdAb binds to mesothelin.

[0165] In some embodiments, there is provided 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, wherein the albumin binding moiety is an albumin binding domain or a single domain antibody ("anti-albumin dsAb", such as an anti-albumin V<com:Sub>H< / com:Sub>H antibody), and wherein the antigen binding moiety comprises an anti-mesothelin single domain antibody comprising an anti-mesothelin heavy chain variable region (anti-MSLN V<com:Sub>H< / com:Sub>), wherein: a) the anti-MSLN V<com:Sub>H< / com:Sub> comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID: NO: 48 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR3; or b) the anti-MSLN V<com:Sub>H< / com:Sub> comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 50 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID: NO: 51 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR3. In some embodiments, the albumin binding moiety is fused to the N- or C-terminus of the cytokine. In some embodiments, the antigen binding moiety is linked to the N- or C- terminus of the cytokine-ALBBM. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the albumin binding moiety binds to a human serum albumin (HSA) and / or a cynomolgus monkey serum albumin (CMSA). In some embodiments, the albumin binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-11 or a variant thereof comprising 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 a V<com:Sub>H< / com:Sub>H single domain antibody. In some embodiments, the cytokine and the albumin binding moiety are connected via 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 the group consisting of 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 metalloprotease, legumain, matriptase, or urokinase sensitive. In some embodiments, the second linker is selected from the group consisting of SEQ ID NOs: 12-45 and 158-159.

[0166] In some embodiments, the fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 120-125, 129-154, and 160-167, or a variant thereof comprising 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: 120-125, 129-154, and 160-167. A. Fusion protein properties 1 Serum half-life

[0167] In some embodiments, 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 hrs, about 24 hrs, about 20 hrs, about 18 hrs, about 16 hrs, about 14 hrs, about 12 hrs, about 10 hrs, about 8 hrs, about 6 hrs, about 4 hrs, about 3 hrs, about 2 hrs, or about 1 hr when administered to an individual. The fusion protein can be administered via various routes, for example, intravenously, orally, subcutaneously or intraperitoneously.

[0168] In some embodiments, the serum half-life of the fusion protein is longer (such as at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% longer) than that of a reference protein. 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 of that of the reference protein. In some embodiments, the reference protein comprises the same cytokine and / or the same antigen binding moiety but does not have the albumin binding moiety. In some embodiments, the same cytokine and the same antigen binding moiety is fused in same order as the fusion protein and / or via the same linker. 2 Stability

[0169] In some embodiments, the fusion protein has a higher stability than a reference protein. In some embodiments, the reference protein comprises the same cytokine and / or the same antigen binding moiety but does not have the albumin binding moiety. In some embodiments, the same cytokine and the same antigen binding moiety is fused in same order as the fusion protein and / or via the same linker.

[0170] In some embodiments, the stability comprises a thermal stability.

[0171] In some embodiments, the 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, the fusion protein has a high stability even if it does not maintain 100% of its chemical structure or biological function after storage for a defined amount of time. In some embodiments, maintenance of about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of structure or function of a fusion protein as described herein after storage for a defined amount of time may be regarded as having a high stability.

[0172] Stability can be measured, inter alia, by determining the percentage of native (non- aggregated or degraded) fusion protein that remains in the formulation (liquid or reconstituted) after storage for a defined amount of time at a defined temperature. The percentage of native fusion protein can be determined by, inter alia, size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]), such that native means non- aggregated and non-degraded. 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 can be detected in the formulation after storage for a defined amount of time at a given temperature. 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 can be detected in the formulation after at least about 6 hrs, at least about 8 hrs, at least about 10 hrs, at least about 12 hrs, at least about 14 hrs, at least about 16 hrs, at least about 18 hrs, at least about 20 hrs, at least about 22 hrs, at least about 24 hrs, at least about 26 hrs, at least about 28 hrs, at least about 30 hrs, at least about 32 hrs, at least about 34 hrs, at least about 36 hrs, at least about 38 hrs, at least about 40 hrs, at least about 42 hrs, at least about 44 hrs, at least about 46 hrs, or at least about 48 hrs under room temperature (about 25°C).

[0173] Stability can be measured, inter alia, by determining the percentage of fusion protein that forms in an aggregate within the formulation (liquid or reconstituted) after storage for a defined amount of time at a defined temperature, wherein stability is inversely proportional to the percent aggregate that is formed. The percentage of aggregated fusion protein can be determined by, inter alia, size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]). In some embodiment, there is less than about 10% (preferably less than about 5%) of the fusion protein present as an aggregate in the formulation after storage for a defined amount of time at a given temperature. In some embodiments, the fusion protein descried herein has substantially no aggregation, for example, at most about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the fusion protein can be detected in an aggregate in the formulation after storage for a defined amount of time at a given temperature, for example, after at least about 6 hrs, at least about 8 hrs, at least about 10 hrs, at least about 12 hrs, at least about 14 hrs, at least about 16 hrs, at least about 18 hrs, at least about 20 hrs, at least about 22 hrs, at least about 24 hrs, at least about 26 hrs, at least about 28 hrs, at least about 30 hrs, at least about 32 hrs, at least about 34 hrs, at least about 36 hrs, at least about 38 hrs, at least about 40 hrs, at least about 42 hrs, at least about 44 hrs, at least about 46 hrs, or at least about 48 hrs under room temperature (about 25°C).

[0174] Measuring the binding affinity of the fusion protein to its target(s) may also be used to assess stability. For example, a fusion protein of the present application may be regarded as stable if, after storage at e.g., room temperature (about 25°C) for a defined amount of time (e.g., 6 hrs, 12 hrs, 24 hrs, 36 hrs, 48 hrs), the cytokine and / or the antigen-binding domain have an affinity that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even more of the binding affinity of the antibody prior to said storage. Binding affinity may be determined by any method, such as e.g., ELISA or plasmon resonance. The binding of the fusion protein to such a cell may be measured directly, such as via FACS analysis. 3 Clinical properties

[0175] In some embodiments, the fusion protein described herein has improved clinical properties relative to a reference protein. In some embodiments, the fusion protein exhibit improved cytotoxicity activity, compared to that of the reference protein. In some embodiments, the fusion protein exhibits higher anti-tumor effects (such as reducing tumor burden, improving survival, etc.), compared to that of the reference protein. In some embodiments, the reference protein comprises the same cytokine and / or the same antigen binding moiety but does not have the albumin binding moiety. In some embodiments, the same cytokine and the same antigen binding moiety is fused in same order as the fusion protein and / or via the same linker. Cytotoxicity

[0176] Cytotoxicity (such as ADCC activity) of the fusion protein described herein against a cell can be tested with many assays. For example, cancer cell line expressing the antigen that can be recognized by the fusion protein and effector cells (e.g., PBMC cells) are mixed together in a 96-well plate. Varying concentrations of fusion protein is added into each well. After incubation, EC50 (representing ADCC activity) can be calculated.

[0177] In some embodiments, the fusion protein exhibits improved ADCC activity against a cell, compared to that of the reference protein. In some embodiments, the EC50 of the fusion protein specific for the cell is no more than about 50%, 40%, 30%, 20%, 10%, or less than the reference protein. In some embodiments, the cell is a tumor cell. In some embodiments, the tumor cell is derived from a 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 moiety but does not have the albumin binding moiety. In some embodiments, the same cytokine and the same antigen binding moiety is fused in same order as the fusion protein and / or via the same linker. Treating a cancer

[0178] In some embodiments, the fusion protein treats a cancer (for example, by inhibiting tumor growth) in an individual. In some embodiments, the fusion exhibited enhanced anti- tumor effect against a cancer, compared to that of a reference protein. For example, in some embodiments, the administration of the fusion protein resulted in a reduced tumor burden (such as at least about 10%, 20%, 30%, 40% or 50% less tumor volume) as compared to that of the reference protein. In some embodiments, the cancer is selected from the group comprising a 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 moiety but does not have the albumin binding moiety. In some embodiments, the same cytokine and the same antigen binding moiety is fused in same order as the fusion protein and / or via the same linker. B. Linker

[0179] In some embodiments, the fusion proteins described herein comprise a first linker between the cytokine and the albumin binding moiety. In some embodiments, the fusion proteins described herein comprise a second linker between the cytokine fused to an albumin binding moiety ("cytokine-ALBBM") and the antigen binding moiety.

[0180] In some embodiments, the first linker is a rigid linker. In some embodiments, the first linker is selected from the group consisting of SEQ ID NO: 21, 22, and 24.

[0181] In some embodiments, the first linker is a flexible linker. In some embodiments, the first linker is selected from the group consisting of SEQ ID NO: 12-14.

[0182] In some embodiments, the first linker is a non-cleavable linker.

[0183] 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.

[0184] In some embodiments, the first linker is selected from the group consisting of SEQ ID NOs: 12-26 and 158-159.

[0185] In some embodiments, the second linker is a rigid linker. In some embodiments, the second linker is selected from the group consisting of SEQ ID NO: 21, 22, and 24.

[0186] In some embodiments, the second linker is a flexible linker. In some embodiments, the second linker is selected from the group consisting of SEQ ID NO: 12-14.

[0187] In some embodiments, the second linker is a cleavable linker. In some embodiments, the second linker is a matrix metalloprotease, legumain, matriptase, or urokinase sensitive.

[0188] 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.

[0189] 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.

[0190] In some embodiments, the first and / or second linker does not comprise a Gly-Gly- Gly-Gly-Ser sequence. In some embodiments, the first and / or second linker is not a GS linker.

[0191] The length, the degree of flexibility and / or other properties of the first and / or second linker(s) used in the fusion proteins may have some influence on properties, including but not limited to the affinity, specificity or avidity for one or more components (such as the cytokine, the albumin-binding molecule, and / or the antigen binding moiety) to bind its target. For example, longer linkers may be selected to ensure that two adjacent domains do not sterically interfere with one another. In some embodiment, a linker (such as peptide linker) comprises flexible residues (such as glycine and serine) so that the adjacent domains are free to move 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.

[0192] Other linker considerations include the effect on 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, the ability to be incorporated into a micelle or liposome, and the like. Non-peptide linkers

[0193] Coupling of the components described above may be accomplished by any chemical reaction that will bind the two molecules so long as both components retain their respective activities, i.e. binding to cytokine receptor, albumin, or the target antigen, respectively. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. In some embodiments, the binding is covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents may be useful in coupling protein molecules in this context. For example, representative coupling agents can include organic compounds such as thioesters, carbodimide, succinimide esters, diisocyanate, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)).

[0194] Linkers the can be applied in the present application are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). In some embodiments, non- peptide linkers used herein include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2- pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC- SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0195] The linkers described above contain components that have different attributes, thus may lead to fusion proteins with differing physio-chemical properties. For example, sulfo- NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form antibody fusion protein with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less antibody fusion protein available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone. Peptide linkers

[0196] The peptide linker may have a naturally occurring sequence, or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of heavy chain only antibodies may be used as the linker. See, for example, WO1996 / 34103.

[0197] The peptide linker can be of any suitable length. In some embodiments, 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 long. In some embodiments, 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 fewer amino acids long. In some embodiments, the length of the peptide linker is any of about 1 amino acid to about 10 amino acids, about 1 amino acids 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 long, 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.

[0198] An essential technical feature of such peptide linker is that said peptide linker does not comprise any polymerization activity. The characteristics of a peptide linker, which comprise the absence of the promotion of secondary structures, are known in the art and described, e.g., in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9(1), 73-80). A particularly preferred amino acid in context of the "peptide linker" is Gly. Furthermore, peptide linkers that also do not promote any secondary structures are preferred. The linkage of the domains to each other can be provided by, e.g., genetic engineering. Methods for preparing fused and operatively 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, N. Y. 1989 and 1994 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001).

[0199] The peptide linker can be a stable linker, which is not cleavable by protease, especially by Matrix metalloproteinases (MMPs).

[0200] The linker can also be a flexible linker. Exemplary flexible linkers include glycine polymers (G)<com:Sub>n< / com:Sub>, glycine-serine polymers (including, for example, (GS)<com:Sub>n< / com:Sub>, (GSGS)<com:Sub>n< / com:Sub> (SEQ ID NO: 19), (GGSG)<com:Sub>n< / com:Sub> (SEQ ID NO: 20), (GGGGS)<com:Sub>n< / com:Sub> (SEQ ID NO: 14), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). The ordinarily skilled artisan will recognize that design of an antibody fusion protein can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired antibody fusion protein structure. C. Cytokines

[0201] The fusion proteins described herein comprise a cytokine. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL- 15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> or fragment thereof, IL-33, and IL-22. IL-21

[0202] IL-21 is a type I cytokine produced by T cells and natural killer T cells that has pleiotropic actions on a wide range of immune and non-immune cell types. This cytokine has diverse effects on a broad range of cell types including, but not limited to, CD4 + and CD8 + T cells, B cells, macrophages, monocytes, and dendritic cells (DCs). The functional receptor for IL-21 is composed of the IL-21 receptor (IL-21R) and the common cytokine receptor γ chain (γ c), which is also a subunit of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15.

[0203] Activation of the cytotoxic programs in NK cells and CD8 + T cells is key for cancer immunotherapy, and consequently early studies provided compelling evidence that IL- 21 is a promising immunotherapeutic agent for this disease. IL-21 promotes maturation, enhances cytotoxicity, and induces production of IFN- y and perforin by NK cells. Correspondingly, cytolytic activity induced by IL-21 significantly inhibits the growth of B16 melanoma. Moreover, IL-21 together with IL-15 expands antigen-specific CD8 + T-cell numbers and their effector function, resulting in tumor regression. Leonard et al. F1000Res. 2016 Feb 26;5. pii: F1000 Faculty Rev-224.

[0204] In some embodiments, the cytokine is IL-21. In some embodiments, the IL-21 is a wild-type IL-21. In some embodiments, the IL-21 is derived from a human IL-21. In some embodiments, the IL-21 is a human wildtype IL-21. In some embodiments, the IL-21 is a truncated IL-21.

[0205] In some embodiments, the IL-21 comprises an amino acid sequence of SEQ ID NO: 1, 2, 126, 171, or 172 or a variant thereof comprising 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.

[0206] In some embodiments, the IL-21 is a truncated IL-21. In some embodiments, the truncated IL-21 comprises an amino acid sequence of SEQ ID NO: 126, 171, or 172.

[0207] 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 one 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 and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any eight amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any nine amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks any ten amino acids between and including L123 and S133 at the C-terminus. In some embodiments, the IL-21 variant lacks all eleven amino acids between and including L123 and S133 at the C-terminus.

[0208] In some embodiments, the IL-21 variant lacks the 11 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 10 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 9 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 8 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 7 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 6 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 5 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 4 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 3 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 2 amino acids at the C-terminus of SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 1 amino acid at the C- terminus of SEQ ID NO: 1.

[0209] In some embodiments, the IL-21 variant lacks the 14, 13, or 12 amino acids at the C-terminus of SEQ ID NO: 1.

[0210] 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.

[0211] In some embodiments, the IL-21 variant provided herein has an amino acid sequence of SEQ ID NO: 2, which lacks the 10 amino acids at the C-terminus and represents a sequence of Q1 to L123 of SEQ ID NO: 1. <com:Math><semantics>IL−7<annotation encoding="application / x-tex">IL-7< / annotation>< / semantics>< / com:Math>

[0212] IL-7 is one of the members of IL-2 superfamily. IL-2 superfamily includes IL-2, IL- 4, IL-7, IL-9, IL-15 and IL-21. It binds to receptors with a common <com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math> chain subunit. In addition to a common <com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math> chain subunit, the receptor for IL-7 (IL-7R) requires an IL-7R <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> chain in order for binding to take place. See Lin et al., Anticancer Res. 2017 Mar;37(3):963- 967.

[0213] Interleukin-7 (IL-7) is required 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 limiting resource for T cells, but it accumulates during lymphopenic conditions. IL-7 signals through a heterodimeric receptor consisting of the IL-7 receptor <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>-chain (IL-7R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>) and the common cytokine receptor <com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>-chain (<com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>c). IL-7 has also been recently demonstrated to regulate lymphoid tissue inducer (LTi) cells, which induce the development of secondary lymphoid organs and can induce tertiary lymphoid tissue postnatally in settings of chronic inflammation. In animals, IL-7 therapy enhances the effectiveness of adoptive immunotherapy for cancer, enhances vaccine responses and enhances viral clearance in the setting of acute and chronic infections. See Mackall et al., Nature Reviews Immunology volume 11, pages 330–342 (2011)

[0214] In some embodiments, the cytokine is IL-7. In some embodiments, the IL-7 is a wild-type IL-7. In some embodiments, the IL-7 is derived from a human IL-7. In some embodiments, the IL-7 is a human wildtype IL-7.

[0215] In some embodiments, the IL-7 comprises an amino acid sequence of any one of SEQ ID NOs: 96-98 or a variant thereof comprising 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: 96-98. IL-15, IL-15 Ra, and IL-15 bound to IL-15Ra

[0216] The heterotrimeric receptor of IL-15 shares the IL-2R / IL-15Rβ (CD122) and common gamma (γc) chain (CD132) with the IL-2 receptor. IL-15 and IL-2 share certain functions that include the stimulation of T cell proliferation, the generation of cytotoxic T lymphocytes, stimulation of immunoglobulin synthesis by B cells and the generation and persistence of NK cells. However, in many adaptive immune responses, IL-2 and IL-15 also have distinct and often competing roles. Unlike IL-2, IL-15 is not required for the maintenance of T regulatory cells (Tregs) that can attenuate antitumor immune responses. IL- 2 in contrast to IL-15 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, effector CD8+ and memory phenotype CD8+ T cells. In addition, based on pre-clinical studies, their toxicities appear to be different, with little vascular capillary leak observed with IL-15 in contrast to IL-2. In summary, IL-15 primarily stimulates the proliferation and cytotoxic functions of CD8 T cells and NK cells leading to enhanced anti-tumor responses. However, while initially showing promise as a cancer therapeutic, the efficacy of IL-15 was limited by its short in vivo half-life. Steel et al., Trends Pharmacol Sci. 2012 Jan; 33(1): 35-41. See Robinson et al, Immunol Lett. 2017 Oct; 190: 159–168.

[0217] In some cases, the efficacy of IL-15 as a treatment is limited by the availability of IL-15Rα, which plays an integral part in stabilizing and increasing the biological activity of IL-15. Since unassociated IL-15 isn't found naturally in vivo, IL-15 bound to IL-15Rα resembles the physiological form of IL-15 and has a higher affinity for IL-15R<com:Math><semantics>β / γ<annotation encoding="application / x-tex">\beta / \gamma< / annotation>< / semantics>< / com:Math>C than free IL-15. See Robinson et al, Immunol Lett. 2017 Oct; 190: 159–168.

[0218] In some embodiments, the cytokine is or comprises IL-15. In some embodiments, the IL-15 is a wild-type IL-15. In some embodiments, the IL-15 is derived from a human IL- 15. In some embodiments, the IL-15 is a human wildtype IL-15.

[0219] In some embodiments, the cytokine is or comprises IL-15Rα. In some embodiments, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> is a wild-type IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> is derived from a human IL-15Rα. In some embodiments, the IL-15Rα is a human wildtype IL-15Rα. In some embodiments, the IL-15Rα is a sushi domain of soluble IL-15 receptor (i.e. IL-15R sushi domain). In some embodiments, the IL-15R sushi domain comprises or consists of an amino acid sequence of any one of SEQ ID NO: 127-128 or a variant thereof comprising 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 NO: 127-128.

[0220] In some embodiments, the cytokine is IL-15 bound to IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> or fragment thereof. In some embodiments, the IL-15 bound to IL-15Rα or fragment thereof comprises a wild- type IL-15 and / or a wildtype IL-15Rα. In some embodiments, the IL-15 bound to IL-15Rα or fragment thereof comprises an IL-15 derived from a human IL-15 and / or an IL-15Rα derived from a human IL-15Rα. In some embodiments, the IL-15 bound to IL-15Rα or fragment thereof comprises a human wildtype IL-15 and / or a human wildtype IL-15Rα. In some embodiments, the IL-15 bound to IL-15Rα or fragment thereof comprises an IL-15 derived from a mouse IL-15 and / or an IL-15Rα derived from a mouse IL-15Rα. In some embodiments, the IL-15 bound to IL-15Rα or fragment thereof comprises a mouse wildtype IL-15 and / or a mouse wildtype IL-15Rα. In some embodiments, the IL-15 bound to IL-15Rα or fragment thereof is a sushi domain of soluble IL-15 receptor (i.e. IL-15R sushi domain). In some embodiments, the IL-15R sushi domain comprises or consists of an amino acid sequence of any one of SEQ ID NO: 127-128 or a variant thereof comprising 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 NO: 127-128.

[0221] In some embodiments, the IL-15 is non-convalently bound to the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>. In some embodiments, the IL-15 is fused to the IL-15Rα. In some embodiments, the IL-15 is fused to the N-terminus of the IL-15Ra. In some embodiments, the IL-15 is fused to the C-terminus of the IL-15Rα. In some embodiments, the fusion protein comprises said IL-15, IL-15Rα and albumin binding moiety from N-terminal to C-terminal in an order selected from the group consisting of (1) the albumin binding moiety, the IL-15, the IL-15Rα; (2) the albumin binding moiety, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, the IL-15; (3) the IL-15, the IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, the albumin binding moiety; (4) the IL-15Rα, the IL-15, the albumin binding moiety. In some embodiments, the IL-15 and the 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 a 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.

[0222] In some embodiments, the IL-15 or IL-15 bound to IL-15Rα comprises a human IL- 15 comprising an amino acid sequence of any one of SEQ ID NO: 99, 100, or 127 or a variant thereof comprising 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 NO: 99, 100, or 127.

[0223] In some embodiments, the IL-15 or IL-15 bound to IL-15Rα comprises a human IL- 15 comprising a N72D mutation. See Han et al., Cytokine. 2011 Dec; 56(3): 804–810.

[0224] In some embodiments, the IL-15Rα or the IL-15 bound to IL-15Rα comprises a human IL-15Rα comprising an amino acid sequence of any one of SEQ ID NOs: 101-108 (such as SEQ ID NOs: 103-104) or a variant thereof comprising 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: 101-108 (such as SEQ ID NOs: 103-104). IL-33

[0225] 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<com:Sub>H< / com:Sub>2) cells and mast cells. Now, evidence is accumulating that IL-33 also potently stimulates group 2 innate lymphoid cells (ILC2s), regulatory T (T<com:Sub>reg< / com:Sub>) cells, T<com:Sub>H< / com:Sub>1 cells, CD8<com:Sup>+< / com:Sup> T cells and natural killer (NK) cells. This pleiotropic nature is reflected in the role of IL-33 in tissue and metabolic homeostasis, infection, inflammation, cancer and diseases of the central nervous system.

[0226] IL-33 has a broad expression in stromal and barrier tissue, which renders it a ubiquitous and crucial immune modulator that shapes type 1, type 2 and regulatory immune responses. Although lacking a secretion sequence and sequestered in the nucleus, IL-33 is released and processed into highly active forms by various proteases. IL-33 contributes to cytokine networks that not only control pathogen removal but also support tissue repair mediated by group 2 innate lymphoid cells and regulatory T cells. The role of IL-33 is expected to continue to expand, modulating both protective and pathological immune responses. Delivering or blocking IL-33 is emerging as a promising therapeutic strategy for maintaining immune homeostasis and protecting against infectious and inflammatory diseases. See Liew, Nature Reviews Immunology volume 16, pages 676–689 (2016)

[0227] In some embodiments, the cytokine is IL-33. In some embodiments, the IL-33 is a wild-type IL-33. In some embodiments, the IL-33 is derived from a human IL-33. In some embodiments, the IL-33 is a human wildtype IL-33.

[0228] In some embodiments, the IL-33 comprises an amino acid sequence of any one of SEQ ID NO: 109 and 155-157 or a variant thereof comprising 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 NO: 109 and 155-157.

[0229] In some embodiments, the IL-33 comprises one or more mutations selected from C208S, C227S, C232S and C259S. See Cohen et al., Nature Communications volume 6, Article number: 8327 (2015) IL-22

[0230] Interleukin-22 (IL-22) is a recently described IL-10 family cytokine that is produced by T-helper (Th)-17 cells, <com:Math><semantics>γδ<annotation encoding="application / x-tex">\gamma\delta< / annotation>< / semantics>< / com:Math> T cells, NKT cells and newly described innate lymphoid cells (ILCs). The human IL22 gene is located at chromosome 12q15 in the vicinity of the genes encoding IFN-y and IL-26. The active, secreted form of the cytokine is a 146 amino acid protein.

[0231] The IL-22 receptor (IL-22R) is a Type 2 cytokine receptor and member of the IL-10 family of receptors along with the receptors 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 suggest that initial binding of IL-22 to the IL-22R1 subunit enables secondary binding of the IL-10R2 subunit, thereby enabling downstream signaling.

[0232] IL-22 has a variety of functions, most notably its trophic effect on non- hematopoietic cells, especially epithelial cells. IL-22 is involved in epithelial regeneration and pathology in several organs depending on the context and / or cytokine milieu. Its involvement in a variety of diseases makes it an attractive target for clinical development. See Dudakov et al., Annu Rev Immunol. 2015 Mar 21; 33: 747–785.

[0233] In some embodiments, the cytokine is IL-22. In some embodiments, the IL-22 is a wild-type IL-22. In some embodiments, the IL-22 is derived from a human IL-22. In some embodiments, the IL-22 is a human wildtype IL-22.

[0234] In some embodiments, the IL-22 comprises an amino acid sequence of any of SEQ ID NOs: 110-111 or a variant thereof comprising at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any of SEQ ID NOs: 110-111. Cytokine variants

[0235] In some embodiments, a cytokine variant can be in the fusion protein provided herein. Variations may be a substitution, deletion, or insertion of one or more codons encoding the cytokine polypeptide that results in a change in the amino acid sequence as compared with the human wide-type cytokine protein. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 10 amino acids. In certain embodiments, the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In some embodiments, the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues. The variation allowed may 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.

[0236] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue or multiple residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0237] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity (e.g., binding to 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.

[0238] Conservative (e.g., within an amino acid group with similar properties and / or side chains) substitutions may be made, so as to maintain or not significantly change the properties of the cytokine. Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) non-polar: 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).

[0239] Alternatively, naturally occurring residues may be divided into 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 that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0240] The variations can be made using methods known in the art such as oligonucleotide- mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J. 237:1-7; and Zoller et al, 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (see, e.g., Wells et al, 1985, Gene 34:315-23), or other known techniques can be performed on the cloned DNA to produce a polypeptide. D. Albumin binding moiety

[0241] The fusion proteins described herein comprise an albumin-binding molecule. Albumin-binding molecules and methods by which they are linked to proteins of interest are described, for example, in WO 1991 / 01743, WO 2001 / 45746, WO 2002 / 076489, WO 2004 / 041865, or US20070269422A1.

[0242] The albumin-binding molecule can be any of the albumin-binding molecule described, for instance, in WO1991 / 01743, WO2001 / 45746, W02002 / 076489, W02004 / 041865, US20070269422A1; US20160152686A1; Dennis et al. (2002), JBC 277(38): 35035-35043.

[0243] In some embodiments, the albumin-binding molecule binds to a human serum albumin (HSA), a cynomolgus monkey serum albumin (CMSA), and / or a mouse serum albumin (MSA).

[0244] In some embodiments, the albumin binding moiety binds to an albumin (such as an HAS) with a K<com:Sub>D< / com:Sub> of between about 1-1000nM (such as between about 1-900nM, 1-800nM, 1-700nM, 1-600ηM, 1-500ηM, 1-400ηM, 1-300ηM, 1-200ηM, 1-100ηM, 1-50ηM, 1-25ηM, O.l-lnM. In some embodiments, the albumin-binding protein comprises an albumin-binding domain (ABD) of Streptococcal protein G (SPG). See, e.g., Nygren et al. J. Mol. Recogn. (1988) 1(2): 69-74. 1 Albumin binding domain (ABD)

[0245] In some embodiments, the albumin binding moiety comprises an albumin binding domain (ABD). In some embodiments, the albumin-binding molecule comprises an 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., 6: e201303009.

[0246] In some specific embodiments, the ABD has an amino acid sequence of LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP (SEQ ID NO: 3), which has a K<com:Sub>D< / com:Sub> to HSA of about 1.2 nM.

[0247] In some embodiments, an ABD having relatively lower affinity to HSA than the ABD of SEQ ID NO: 3 is preferred. Accordingly, variants of SEQ ID NO: 3 that have lower affinity to HSA are included in the present disclosure.

[0248] In some specific embodiments, the ABD has an amino acid sequence of any one of SEQ ID NOs: 4-11.

[0249] Variations may be a substitution, deletion, or insertion of one or more codons encoding the ABD polypeptide of any one of SEQ ID NOs: 3-11 that results in a change in the amino acid sequence. Amino acid substitutions 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 of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule provided herein, including, for example, site- directed mutagenesis and PCR-mediated mutagenesis which results in amino acid substitutions.

[0250] The variation allowed may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity and in some embodiments, variants having a lower affinity to HSA are selected. Certain such kind of variants are exemplified in Table 3. 2 Anti-albumin antibody or fragment thereof

[0251] According to the present invention, the albumin binding moiety can also be anti- albumin antibody or antigen binding fragment thereof. In some embodiments, the anti- albumin antibody or antigen binding fragment thereof is an anti-HSA antibody or antigen binding fragment thereof.

[0252] A few isoforms of HSA are listed in Table 2 below (see for example, UniProtKB - P02768 (ALBU_HUMAN)). In some embodiments, the anti-albumin antibody or antigen binding fragment thereof binds to any one of SEQ ID NO: 52-55. Table 2 [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document]

[0253] The anti-albumin antibodies or fragments thereof may be from any animal origin including birds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken). 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 human immunoglobulin libraries or from mice that express antibodies from human genes.

[0254] In certain embodiments, the anti-albumin antibodies are fully human antibodies, such as fully human antibodies that immunospecifically bind a cancer antigen. Such fully human antibodies would be advantageous over fully mouse (or other full or partial non- human species antibodies), humanized antibodies, or chimeric antibodies to minimize the development of unwanted or unneeded side effects, such as immune responses directed toward non-fully human antibodies when administered to the subject.

[0255] The anti-albumin antibodies provided herein may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide or may be specific for both a polypeptide as well as for 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.

[0256] The anti-albumin antibodies provided herein may be monoclonal antibodies or derived from monoclonal antibodies. The anti-albumin antibodies can be, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies or their humanized versions, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0257] 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 an albumin (such as an 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 molecule. In some embodiments, the anti-albumin antibody is an IgG antibody, such as an IgG1 antibody.

[0258] Variants and derivatives of anti-albumin antibodies including antibody fragments that retain the ability to specifically bind to an epitope of albumin are also included in the present disclosure. Exemplary fragments include Fab fragments (an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab' (an antibody fragment containing a single anti-binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab')<com:Sub>2< / com:Sub> (two Fab' molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab' molecules may be directed toward the same or different epitopes); a bispecific Fab (a Fab molecule having two antigen binding domains, each of which may be directed to a different epitope); a single chain Fab chain comprising a variable region, also known as, a sFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); a disulfide-linked Fv, or dsFv (the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a camelized VH (the variable, antigen-binding determinative region of a single heavy chain of an antibody in which some amino acids at the VH interface are those found in the heavy chain of naturally occurring camel antibodies); a bispecific sFv (a sFv or a dsFv molecule having two antigen- binding domains, each of which may be directed to a different epitope); a diabody (a dimerized sFv formed when the VH domain of a first sFv assembles with the VL domain of a second sFv and the VL domain of the first sFv assembles with the VH domain of the second sFv; the two antigen-binding regions of the diabody may be directed towards the same or different epitopes); and a triabody (a trimerized sFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen binding domains may be directed towards the same or different epitopes). Derivatives of antibodies also include one or more CDR sequences of an antibody combining site. The CDR sequences may be linked together on a scaffold when two or more CDR sequences are present. In certain embodiments, anti-HSA antibody provided herein comprises a single- chain Fv ("scFv"). scFvs are antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

[0259] In certain circumstances there are advantages of using anti-albumin antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to cells, tissues, or organs. For a review of certain antibody fragments, see Hudson et al., 2003, Nature Med. 9:129-34.

[0260] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')<com:Sub>2< / com:Sub> fragments (Carter et al., 1992, Bio / Technology 10:163-67). According to another approach, F(ab')<com:Sub>2< / com:Sub> fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')<com:Sub>2< / com:Sub> fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described in, for example, U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv) (see, e.g., WO 93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,587,458). Fv and scFv have intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv (See, e.g., Borrebaeck ed., supra). The antibody fragment may also be a "linear antibody," for example, as described in the references cited above. Such linear antibodies may be monospecific or multi-specific, such as bispecific. Single domain antibody (sdAb)

[0261] In some embodiments, the antibody fragment is a single domain antibody (sdAb).

[0262] In some embodiments, the sdAb is a <com:Math><semantics>VHH<annotation encoding="application / x-tex">V_HH< / annotation>< / semantics>< / com:Math> single domain antibody.

[0263] In some embodiments, the sdAb binds to the albumin (such as an HSA) with a K<com:Sub>D< / com:Sub> of between about 1-1000nM (such as between about 1-900nM, 1-800nM, 1-700nM, 1- 600nM, 1-500nM, 1-400nM, 1-300nM, 1-200nM, 1-100nM, 1-50nM, 1-25nM, or 0.1-1nM). In other embodiments, the sdAb binds to the albumin (such as an HSA) with a K<com:Sub>D< / com:Sub> of between about 10-800nM (such as between about 20-500nM, 50-300nM or 100-200nM).

[0264] In some embodiments, the sdAb binds to the albumin (such as an HSA) with a K<com:Sub>D< / com:Sub> of between about 1-1000nM (such as between about 1-900nM, 1-800nM, 1-700nM, 1- 600nM, 1-500nM, 1-400nM, 1-300nM, 1-200nM, 1-100nM, 1-50nM, 1-25nM, or 0.1-1nM) at a pH of about 5.5 and / or at a pH of about 7.5. In some embodiments, the sdAb binds to the albumin (such as an HSA) with a K<com:Sub>D< / com:Sub> of between about 1-200 nM at a pH of about 5.5 and at a pH of about 7.5.

[0265] 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.

[0266] In some embodiments, the single-domain antibody or fragment thereof comprises any one 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: 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 3amino 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; 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.

[0267] 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 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 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<com:Sub>H< / com:Sub>H domain comprising the amino acid sequence of any one of SEQ ID NOs: 60-68 and 168-170, 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: 60-68 and 168-170.

[0268] In some embodiments, the single-domain antibody or fragment thereof that binds to albumin comprises a CDR1, a CDR2, a CDR3, respectively comprising the amino acid sequence of a CDR1, a CDR2, and a CDR3 within a heavy chain variable domain having the sequence set forth in any of SEQ ID NOs: 60-68 and 168-170. E. Antigen binding moiety

[0269] In some embodiments, the fusion protein further comprises an antigen binding moiety, and wherein the antigen binding moiety is fused to the N- or C-terminus of the cytokine fused to the albumin binding moiety ("cytokine-ALBBM").

[0270] In some embodiments, the antigen binding moiety binds to a tumor antigen. I some embodiments, the tumor is a solid or liquid tumor. In some embodiments, the tumor is a cancer selected from the group consisting of 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 a high level of the tumor antigen. For example, in some embodiments, the cancer expresses a level of at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold of that of a reference tissue. In some embodiments, the reference tissue is a tissue that does not comprise a cancer cell in the same individual.

[0271] In some embodiments, the tumor antigen is selected from the group consisting of 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.

[0272] In some embodiments, the antigen binding moiety is an antibody (such as a full length antibody) or antigen binding fragment thereof. In some embodiments, the antibodies or antigen binding fragments thereof provided herein can immunospecifically bind to a polypeptide, a polypeptide fragment, or an epitope of an antigen expressed on a cancer cell. In one embodiment, the antibodies bind to a human cancer antigen. In some embodiments, the antibodies or antigen binding fragments thereof provided herein bind to the extracellular domain (ECD) of a cancer antigen. In certain embodiments, the antibodies bind to an epitope in the ECD of a cancer antigen. In some embodiments, the cancer antigen is expressed on a solid or liquid tumor cancer cell.

[0273] Antibodies that bind to a cancer antigen provided herein can be, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies or their humanized variants, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

[0274] In some embodiments, 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 a cancer antigen (e.g., a solid or liquid tumor cancer antigen). 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 molecule. In a specific embodiment, an antibody provided herein is an IgG antibody, such as an IgG1 antibody.

[0275] Variants and derivatives of antibodies including antibody fragments that retain the ability to specifically bind to an epitope of a cancer antigen are also included in the present disclosure. Exemplary fragments include Fab fragments; Fab'; F(ab')2; a bispecific Fab; a single chain Fab chain comprising a variable region, also known as, a sFv; a disulfide-linked Fy, or dsFy; a camelized VH; a bispecific sFy; a diabody; and a triabody. Derivatives of antibodies also include one or more CDR sequences of an antibody combining site. The CDR sequences may be linked together on a scaffold when two or more CDR sequences are present. In certain embodiments, an antibody provided herein comprises a single-chain Fv ("scFv"). Various techniques have been developed for the production of antibody fragments as briefly described in the above section.

[0276] In some embodiments, the antigen binding moiety is a single variable domain antibodies (sdAbs) (such as a V<com:Sub>H< / com:Sub>H antibody) that bind to a tumor antigen. Certain types of organisms, the camelids and cartilaginous fish, possess high affinity single V-like domains mounted on an Fc equivalent domain structure as part of their immune system. (Woolven et al., 1999, Immunogenetics 50: 98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49). The V-like domains (called <com:Math><semantics>VHH<annotation encoding="application / x-tex">V_HH< / annotation>< / semantics>< / com:Math> in camelids and V-NAR in sharks) typically display long surface loops, which allow penetration of cavities of target antigens. They also stabilize isolated VH domains by masking hydrophobic surface patches. Anti-mesothelin single domain antibody (anti-MSLN dsAb)

[0277] In some embodiments, the antigen binding moiety is an anti-mesothelin single domain antibody ("anti-MSLN dsAb").

[0278] The anti-MSLN antibodies (e.g., sdAbs) provided herein can bind to any of the isoforms of mesothelin or any fragments thereof (such as any one of SEQ ID NOs 56-59). In some embodiments, the anti-MSLN antibody provided herein binds to any one of SEQ ID NOs: 56-59 or a fragment thereof.

[0279] In some embodiments, the anti-MSLN dsAb comprises an anti-mesothelin heavy chain variable region (anti-MSLN V<com:Sub>H< / com:Sub>), wherein: a) the anti-MSLN V<com:Sub>H< / com:Sub> comprises 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 thereof comprising up to a total of 5, 4, 3, 2, or 1 amino acid substitutions in the CDRs; or b) the anti-MSLN V<com:Sub>H< / com:Sub> comprises 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 thereof comprising up to a total of 5, 4, 3, 2, or 1 amino acid substitutions in the CDRs.

[0280] In some embodiments, the anti-MSLN dsAb comprises an anti-mesothelin heavy chain variable region (anti-MSLN V<com:Sub>H< / com:Sub>), wherein: a) the anti-MSLN V<com:Sub>H< / com:Sub> comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID: NO: 48 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR3; or b) the anti-MSLN V<com:Sub>H< / com:Sub> comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 50 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID: NO: 51 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR3. In some embodiments, the anti-MSLN dsAb comprises an anti- mesothelin heavy chain variable region (anti-MSLN V<com:Sub>H< / com:Sub>), wherein: a) the anti-MSLN V<com:Sub>H< / com:Sub> comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 187 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 188 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID: NO: 189 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR3. In some embodiments, the anti- MSLN dsAb comprises an anti-mesothelin heavy chain variable region (anti-MSLN V<com:Sub>H< / com:Sub>), wherein: a) the anti-MSLN V<com:Sub>H< / com:Sub> comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 190 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 191 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID: NO: 192 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR3. In some embodiments, the anti-MSLN dsAb comprises an anti-mesothelin heavy chain variable region (anti-MSLN V<com:Sub>H< / com:Sub>), wherein: a) the anti-MSLN V<com:Sub>H< / com:Sub> comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 193 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 194 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR2, and a CDR3 comprising the amino acid sequence of SEQ ID: NO: 195 or a variant thereof comprising up to 3, 2, or 1 substitution in CDR3.

[0281] In some embodiments, the anti-MSLN sdAb comprises a CDR1, a CDR2, a CDR3, respectively comprising the amino acid sequence of a CDR1, a CDR2, and a CDR3 within a heavy chain variable domain having the sequence set forth in any of SEQ ID NOs: 173-186. F. Fusion protein variants

[0282] In some embodiments, amino acid sequence variants of the fusion proteins provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the fusion protein in a whole or any component(s) of the fusion protein. Amino acid sequence variants of a fusion protein may be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the fusion protein, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the fusion protein. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Substitution, insertion, deletion and variants

[0283] In some embodiments, fusion protein variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs of albumin-binding molecule and / or antigen binding moiety. Conservative substitutions are shown in Table 3. More substantial changes are provided under the heading of "exemplary substitutions," and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into the component of the fusion protein and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Also see subsection "1. Amino acid sequence variants" under section "V. Methods of preparation." Table 3. Amino acid substitutions [Image disponible dans le document PDF, Image available in the PDF document] [Image disponible dans le document PDF, Image available in the PDF document]

[0284] Amino acids may be grouped according to 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 that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0285] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0286] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).

[0287] Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen 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 method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0288] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR "hotspots" or CDRs.

[0289] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0290] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. 2 Derivatives

[0291] In some embodiments, a fusion protein provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the fusion protein 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), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3- dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide co- polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the fusion protein may vary, and if more than one polymer are 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 particular properties or functions of the fusion protein to be improved, whether the fusion protein derivative will be used in a therapy under defined conditions, etc.

[0292] In some embodiments, conjugates of a fusion protein and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In some embodiments, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA) 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the fusion protein nonproteinaceous moiety are killed. П-В. Fusion proteins comprising a truncated IL-21

[0293] The present application also provides a fusion protein comprising a human IL-21 variant that comprises a truncated human IL-21. Without being bound to theory, it is discovered that a fusion protein comprising a truncated form of IL-21 that lack 1-11 amino acids at the C-terminus has an improved stability than the wildtype counterpart and a truncated counterpart that lack 12 or more amino acids at the C-terminus. It is comtemplated that fusion proteins comprising a truncated IL-21 as described herein are not limited to fusion proteins that comprise an anti-albumin binding moiety.

[0294] In some embodiments, there is provided a fusion protein that comprises a) a truncated IL-21 that lacks about 1-11 amino acids at the C-terminus of the wildtype IL-21, and b) a second moiety (such as a single domain antibody moiety). In some embodiments, the IL-21 is derived from human. In some embodiments, there is provided a fusion protein that comprises a) a truncated IL-21 that comprises an amino acid sequence of SEQ ID NO: 126, 171, or 172, and b) a second moiety. 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 the eleven amino acids between and including L123 and S133 at the C-terminus of the IL-21 having a sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-21 variant lacks the 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid 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., consecutive amino acids) at the C-terminus of SEQ ID NO: 1.

[0295] In some embodiments, the fusion protein has a molecular weight of at least about 15 kDa, 18 kDa, 20 kDa, 22 kDa, 25 kDa, 28 kDa. In some embodiments, the fusion protein has a molecular weight of no more than about 1000 kDa, 500 kDa, 250 kDa, 100 kDa, 70 kDa, 50 kDa, 40 kDa, or 30 kDa. In some embodiments, the fusion protein has a molecular weight of about 15 kDa to about 1000 kDa, about 15 kDa to about 500 k Da, about 15 kDa to about 100 kDa, about 15 kDa to about 70 kDa, about 20 kDa to about 50 kDa, about 25 kDa to about 30 kDa, or about 28 kDa.

[0296] In some embodiments, the second moiety comprises a half-life extending moiety. In some embodiments, the half-life extending moiety is an albumin binding moiety (e.g., an albumin binding antibody moiety, e.g., a single domain albumin binding antibody moiety). In some embodiments, the half-life extending moiety is an Fc domain (e.g., an IgG1 Fc domain).

[0297] In some embodiments, the second moiety comprises an antigen binding moiety. In some embodiments, the antigen binding moiety 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 moiety comprises a single domain antibody moiety. In some embodiments, the antigen binding moiety comprises a single domain antibody moiety that specifically binds to albumin.

[0298] In some embodiments, the second moiety is fused to the N-terminus of the truncated IL-21. In some embodiments, the second moiety is fused to the C-terminus of the truncated IL-21.

[0299] In some embodiments, the second moiety is fused to the truncated IL-21 via a linker (can be either adjacent to IL-21 or not adjacent to IL-21). In some embodiments, the second moiety is fused to N- 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. III. Pharmaceutical compositions

[0300] Further provided by the present application are pharmaceutical compositions comprising any one of fusion proteins described herein, and optionally a pharmaceutically acceptable carrier. Pharmaceutical compositions can be prepared by mixing a fusion protein described herein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.

[0301] The pharmaceutical composition is preferably to be stable, in which the fusion protein described herein essentially retains 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, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation may be kept at 40°C for 2 weeks to 1 month, at which time stability is measured. Where the formulation is to be stored at 2-8°C, generally the formulation should be stable at 30°C or 40°C for at least 1 month, and / or stable at 2-8°C for at least 2 years. Where the formulation is to be stored at 30°C, generally the formulation should be stable for at least 2 years at 30°C, and / or stable at 40°C for at least 6 months. For example, the extent of aggregation during storage can be used as an indicator of protein stability. In some embodiments, the stable formulation of fusion proteins described herein may comprise less than about 10% (preferably less than about 5%) of the fusion protein present as an aggregate in the formulation.

[0302] 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, isotonicifiers (e.g. sodium chloride), stabilizers, metal complexes (e.g. Zn-protein complexes); chelating agents such as EDTA and / or non-ionic surfactants.

[0303] 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 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) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; 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<com:Sup>TM< / com:Sup>, polyethylene glycol (PEG), and PLURONICS<com:Sup>TM< / com:Sup> or polyethylene glycol (PEG).

[0304] Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use in the present application include both organic and inorganic acids and salts thereof. For example, citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

[0305] Preservatives are added to retard microbial growth, and are typically present in a range from 0.2%-1.0% (w / v). The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation. Suitable preservatives for use in the present application include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0306] Tonicity agents, sometimes known as "stabilizers" are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1% to 25% by weight, preferably 1% to 5%, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0307] Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall. Such excipients include: polyhydric sugar alcohols (enumerated 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, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thio sulfate; 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.

[0308] Non-ionic surfactants or detergents (also known as "wetting agents") are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non- ionic surfactants are 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.

[0309] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl celluose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0310] In order for the pharmaceutical compositions to be used for in vivo administration, they must be sterile. The pharmaceutical composition may be rendered sterile by filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0311] The route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained release or extended-release means. In some embodiments, the pharmaceutical composition is administered locally, such as intratumorally, or intravitreally.

[0312] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), 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 the LUPRON DEPOT<com:Sup>TM< / com:Sup> (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0313] The pharmaceutical compositions herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise a cytotoxic agent, chemotherapeutic agent, cytokine, immunosuppressive agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0314] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition.

[0315] The antibody fusion protein disclosed herein can be formulated as immunoliposomes. Liposomes containing the antibody fusion protein are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0316] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

[0317] 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 multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved. IV. Methods of Treatments

[0318] One aspect of the present application provides methods of treating a disease or condition in an individual using the fusion proteins or pharmaceutical compositions described herein. For example, the fusion proteins described herein comprise: a) a cytokine, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the fusion ptotein comprises a) a cytokine selected from the group consisting of IL-21, IL-7, IL- 15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22, and b) an albumin binding moiety (such as an sdAb that binds to albumin). In some embodiments, the albumin binding moiety comprises an albumin binding domain or a single domain antibody (sdAb) that binds to albumin as described herein. In some embodiments, the method further comprises administering a second agent.

[0319] Another aspect of the present application provides methods of 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 fused to the C- terminus of the cytokine. In some embodiments, the half-life extending domain is fused to the N-terminus of the cytokine. In some embodiments, the cytokine and the half-life extending domain are connected via a linker. In some embodiments, the linker 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 linker is selected from the group consisting of SEQ ID NOs: 12-26 and 158-159. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the cytokine is IL-21. In some embodiments, the half-life extending 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 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 the group consisting of an IgG1, IgG2, IgG3, and IgG4 Fc fragments or a variant thereof. In some embodiments, the Fc fragment is an IgG1 Fc fragment or variant thereof. In some embodiments, the IgG1 Fc fragment or variant thereof comprises a mutation at position 297, wherein the amino acid at position 297 is mutated to alanine, aspartic acid or glycine. In some embodiments, the individual is a human.

[0320] 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.

[0321] 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 the group consisting of mesothelin, 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.

[0322] 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 a single chain antibody comprising an anti-mesothelin heavy chain variable region (anti-MSLN V<com:Sub>H< / com:Sub>), wherein the anti-MSLN V<com:Sub>H< / com:Sub> comprises a CDR1, a CDR2, and a CDR3, wherein: a) the CDR1 comprising the amino acid sequence of SEQ ID NO: 46, the CDR2 comprising the amino acid sequence of SEQ ID NO: 47, and the CDR3 comprising the amino acid sequence of SEQ ID: NO: 48, or a variant thereof comprising up to a total of 3, 2, or 1 amino acid substitutions in the CDRs; or b) the CDR1 comprising the amino acid sequence of SEQ ID NO: 49, the CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and the CDR3 comprising the amino acid sequence of SEQ ID: NO: 51, or a variant thereof comprising up to a total of 3, 2, or 1 amino acid substitutions in the CDRs.

[0323] In some embodiments, the second agent is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is an inhibitor of an immune checkpoint protein selected from the group consisting of 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 fragment thereof.

[0324] 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 the group consisting of IL-7, IL-15, IL15 bound to IL15Ra or half-life extended variants thereof.

[0325] In some embodiments, the second agent is an immune cell. In some embodiments, the immune cell comprises T cells or NK cells. In some embodiments, the immune cell comprises T cells expressing a chimeric antigen receptor (CAR), T cells expressing a modified T cell receptor (TCR), or T cells isolated from a tumor.

[0326] In some embodiments, the second agent is a tyrosine kinase inhibitor.

[0327] In some embodiments, the second agent is a chemotherapeutic agent (such as sorafenib).

[0328] In some embodiments, there is provided a method of treating a disease or condition in an individual comprising administering to the individual a) a fusion protein comprising i) IL-21 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 fused to N-terminus of the cytokine. In some embodments, the half-life extending domain is fused to 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 extending domain is an antibody of fragment thereof, an 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. In some embodiments, the fusion protein further comprises an antigen binding moiety (such as described above). In some embodiments, the antigen binding moiety binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from the group consisting of 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, renal cancer, kidney cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0329] In some embodiments, there is provided a method of treating a disease or condition in an individual comprising administering to the individual a) a fusion protein comprising i) IL-7 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 fused to N-terminus of the cytokine. In some embodments, the half-life extending domain is fused to 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 extending domain is an antibody of fragment thereof, an 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. In some embodiments, the fusion protein further comprises an antigen binding moiety (such as described above). In some embodiments, the antigen binding moiety binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from the group consisting of 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, renal cancer, kidney cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0330] In some embodiments, there is provided a method of treating a disease or condition in an individual comprising administering to the individual a) a fusion protein comprising i) IL-15 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 fused to N-terminus of the cytokine. In some embodments, the half-life extending domain is fused to 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 extending domain is an antibody of fragment thereof, an 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. In some embodiments, the fusion protein further comprises an antigen binding moiety (such as described above). In some embodiments, the antigen binding moiety binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from the group consisting of 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, renal cancer, kidney cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0331] In some embodiments, there is provided a method of treating a disease or condition in an individual 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 extending domain fused to the cytokine; and b) a second agent. In some embodiments, the half-life extending domain is fused to N-terminus of the cytokine. In some embodments, the half-life extending domain is fused to 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 extending domain is an antibody of fragment thereof, an 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. In some embodiments, the fusion protein further comprises an antigen binding moiety (such as described above). In some embodiments, the antigen binding moiety binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from the group consisting of 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, renal cancer, kidney cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0332] In some embodiments, there is provided a method of treating a disease or condition in an individual comprising administering to the individual a) a fusion protein comprising i) IL-33 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 fused to N-terminus of the cytokine. In some embodments, the half-life extending domain is fused to 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 extending domain is an antibody of fragment thereof, an 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. In some embodiments, the fusion protein further comprises an antigen binding moiety (such as described above). In some embodiments, the antigen binding moiety binds to a tumor antigen (such as mesothelin). In some embodiments, the cancer is selected from the group consisting of 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, renal cancer, kidney cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0333] In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising an IL-21 as described above. For example in some embodiments, the fusion protein comprises a) IL-21, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the IL-21 or the variant thereof. In some embodiments, the fusion protein comprises a) IL-21 fused to an albumin binding moiety ("IL-21-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 as mesothelin). In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising i) IL-21, and ii) a half-life extending domain, wherein the half-life extending domain is fused to the N- or C- terminus of IL-21. In some embodiments, the method further comprises a second agent (such as a therapeutic antibody that binds 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 a tumor antigen. In some embodiments, the half-life extending domain is an antibody of fragment thereof, an 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. In some embodiments, the cancer is selected from the group consisting of 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, renal cancer, kidney cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0334] In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising an IL-7 as described above. For example in some embodiments, the fusion protein comprises a) IL-7, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the IL-7 or the variant thereof. In some embodiments, the fusion protein comprises a) IL-7 fused to an albumin binding moiety ("IL-7-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 as mesothelin). In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising i) IL-7, and ii) a half- life extending domain, wherein the half-life extending domain is fused to the N- or C- terminus of IL-7. In some embodiments, the method further comprises a second agent. In some embodiments, the cancer expresses a high level of a tumor antigen. In some embodiments, the half-life extending domain is an antibody of fragment thereof, an 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. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0335] In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising an IL-15 as described above. For example in some embodiments, the fusion protein comprises a) IL-15, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the IL-15 or the variant thereof. In some embodiments, the fusion protein comprises a) IL-15 fused to an albumin binding moiety ("IL-15-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 as mesothelin). In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising i) IL-15, and ii) a half-life extending domain, wherein the half-life extending domain is fused to the N- or C- terminus of IL-15. In some embodiments, the method further comprises a second agent. In some embodiments, the cancer expresses a high level of a tumor antigen. In some embodiments, the half-life extending domain is an antibody of fragment thereof, an 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. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0336] In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising an IL-15 bound to IL-15R<com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math> as described above. For example in some embodiments, the fusion protein comprises a) IL-15 bound to IL-15Ra, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the IL-15 bound to IL-15Ra or the variant thereof. In some embodiments, the fusion protein comprises a) IL-15 bound to IL-15Rα fused to an albumin binding moiety ("IL-15") bound to IL-15Rα-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 as mesothelin). In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising i) IL-15 bound to IL- 15Rα, and ii) a half-life extending domain, wherein the half-life extending domain is fused to the N- or C- terminus of IL-15 bound to IL-15Rα. In some embodiments, the method further comprises a second agent. In some embodiments, the cancer expresses a high level of a tumor antigen. In some embodiments, the half-life extending domain is an antibody of fragment thereof, an 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. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0337] In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising an IL-33 as described above. For example in some embodiments, the fusion protein comprises a) IL-33, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the IL-33 or the variant thereof. In some embodiments, the fusion protein comprises a) IL-33 fused to an albumin binding moiety ("IL-33-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 as mesothelin). In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a fusion protein comprising i) IL-33, and ii) a half-life extending domain, wherein the half-life extending domain is fused to the N- or C- terminus of IL-33. In some embodiments, the method further comprises a second agent. In some embodiments, the cancer expresses a high level of a tumor antigen. In some embodiments, the half-life extending domain is an antibody of fragment thereof, an 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. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human.

[0338] In some embodiments, there is provided a method of treating an inflammatory disease in an individual comprising administering to the individual a fusion protein, wherein the fusion protein comprises a) IL-22, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the IL-22 or the variant thereof. In some embodiments, the albumin binding moiety is an albumin binding domain or an anti-albumin single domain antibody such as those described herein. In some embodiments, there is provided a method of treating an inflammatory disease in an individual comprising administering to the individual a fusion protein, 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 of fragment thereof, an 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 the group consisting of ulcerative colitis, Crohn's disease, or ulcerative ileitis, and intestinal graft vs host disease. In some embodiments, the individual is a human.

[0339] In some embodiments, there is provided a method of treating a mesothelioma in an individual 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 moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the cytokine. 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. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15R\alpha or fragment thereof, IL-33, and IL-22. In some embodiments, the antigen binding moiety binds to a tumor antigen (such as mesothelin). In some embodiments, there is provided a method of treating a mesothelioma in an individual comprising administering to the individual a fusion protein comprising i) cytokine, and ii) a half-life extending domain, wherein the half-life extending domain is fused to the N- or C- terminus of cytokine. In some embodiments, the method further comprises a second agent (such as an anti-mesothelin antibody). In some embodiments, the half-life extending domain is an antibody of fragment thereof, an 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.

[0340] In some embodiments, there is provided a method of treating a lung cancer in an individual 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 moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the cytokine. 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. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the antigen binding moiety binds to a tumor antigen. In some embodiments, there is provided a method of treating a lung cancer in an individual comprising administering to the individual a fusion protein comprising i) cytokine, and ii) a half-life extending domain, wherein the half-life extending domain is fused to the N- or C- terminus of cytokine. In some embodiments, the method further comprises a second agent. In some embodiments, the half-life extending domain is an antibody of fragment thereof, an 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.

[0341] In some embodiments, there is provided a method of treating an ovarian cancer in an individual 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 moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the cytokine. 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. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL- 15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the antigen binding moiety binds to a tumor antigen. In some embodiments, there is provided a method of treating an ovarian cancer in an individual comprising administering to the individual a fusion protein comprising i) cytokine, and ii) a half-life extending domain, wherein the half-life extending domain is fused to the N- or C- terminus of cytokine. In some embodiments, the method further comprises a second agent. In some embodiments, the half- life extending domain is an antibody of fragment thereof, an 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.

[0342] In some embodiments, there is provided a method of treating a gastric cancer in an individual 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 moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the cytokine. 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. In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15R\alpha or fragment thereof, IL-33, and IL-22. In some embodiments, the antigen binding moiety binds to a tumor antigen. In some embodiments, there is provided a method of treating a gastric cancer in an individual comprising administering to the individual a fusion protein comprising i) cytokine, and ii) a half-life extending domain, wherein the half- life extending domain is fused to the N- or C- terminus of cytokine. In some embodiments, the method further comprises a second agent. In some embodiments, the half-life extending domain is an antibody of fragment thereof, an 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.

[0343] In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a) a fusion protein comprising an IL-21 as described above and b) a second cytokine selected from the group consisting of IL-7, IL- 15, IL15 bound to IL15Rα or half-life extended variants thereof. For example, in some embodiments, the fusion protein comprises a) IL-21, and b) an albumin binding moiety (such as an sdAb that binds to albumin), wherein the albumin binding moiety is fused to the N- or C- terminus of the IL-21 or the variant thereof. In some embodiments, the fusion protein comprises a) IL-21 fused to an albumin binding moiety ("IL-21-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 as mesothelin). In some embodiments, there is provided a method of treating a cancer in an individual comprising administering to the individual a) a fusion protein comprising i) IL-21, and ii) a half-life extending domain, wherein the half-life extending domain is fused to the N- or C- terminus of IL-21; b) a second agent selected from the group consisting of IL-7, IL-15, IL15 bound to IL15Rα or half-life extended variants thereof. In some embodiments, the half-life extending domain is an antibody of fragment thereof, an 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. In some embodiments, the first extended half-life cytokine is fused to the second extended half-life cytokine via a peptide linker, which is optionally protease cleavable. In some embodiments, the cancer is selected from the group consisting of 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, renal cancer, kidney cancer, esophageal cancer, gastric cancer, and colorectal cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, ovarian cancer, and gastric cancer. In some embodiments, the individual is a human. Fusion proteins for treating a disease

[0344] In some embodiments, the fusion protein for treating a disease or condition comprises any of the fusion proteins described herein (such as in Section II). In some embodiments, the fusion protein comprises i) a cytokine and ii) a half-life extending domain fused to the cytokine. In some embodiments, the half-life extending domain is fused to the C- terminus of the cytokine. In some embodiments, the half-life extending domain is fused to the N-terminus of the cytokine. In some embodiments, the cytokine and the half-life extending domain are connected via a linker. In some embodiments, the linker can be any linker described herein (such as in Section II-B). In some embodiments, the cytokine is selected from the group consisting of IL-21, IL-7, IL-15, IL-15 bound to IL-15Rα or fragment thereof, IL-33, and IL-22. In some embodiments, the cytokine is IL-21. Half-life extending domain

[0345] In some embodiments, the fusion proteins for treating a disease or condition as described herein comprise a half-life extending domain.

[0346] In some embodiments, the fusion protein provided herein comprises a half-life extension domain selected from the group consisting of antibodies and fragments thereof, albumin, albumin-binding proteins, IgG-binding proteins, and polyamino acid sequences. It is contemplated that other mechanisms for extending the half-life of the fusion protein available in the art may also be employed. a) Antibodies and Fragments Thereof

[0347] By linking a cytokine to an antibody or fragment thereof that is capable of FcRn- mediated recycling, clearance of the cytokine from a subject can be reduced or otherwise delayed, thereby prolonging the half-life of the administered cytokine.

[0348] In some embodiments, the half-life extension domain comprises an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof is any antibody or fragment thereof that is capable of FcRn-mediated recycling, such as any heavy chain polypeptide or portion thereof (e.g., Fc domain or fragment thereof) that is capable of FcRn- mediated recycling. It is recognized in the art that FcRn-mediated recycling requires binding of the FcRn receptor to the Fc region of the antibody or fragment thereof. For instance, studies have shown that residues I253, S254, H435, and Y436 (numbering according to the Kabat EU index numbering system) are important for the interaction between the human Fc region and the human FcRn complex. See, e.g., Firan, M., et al., Int. Immunol. 13 (2001) 993-1002; Shields, R.L., et al, J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248-259, 301-317, 376-382, and 424-437 (numbering according to the Kabat EU index numbering system) have also been examined and reported. Yeung, Y.A., et al. (J. Immunol. 182 (2009) 7667-7671.

[0349] In some embodiments, the antibody or fragment thereof comprises either a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a portion of either a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises an Fc domain or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a CH2 and CH3 domain or a fragment thereof. In some embodiments, the antibody or fragment thereof comprises the constant domain of the heavy chain polypeptide. In some embodiments, the antibody or fragment thereof comprises the constant domain of the light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a heavy chain polypeptide or fragment thereof (e.g., an Fc domain or fragment thereof). In some embodiments, the antibody or fragment thereof comprises a light chain polypeptide.

[0350] In some embodiments, the antibody of fragment thereof comprises an Fc domain or fragment thereof. In some embodiments, the Fc fragment is selected from the group consisting of an IgGl, IgG2, IgG3, and IgG4 Fc fragments or a variant thereof. In some embodiments, the Fc fragment is an IgGl Fc fragment or variant thereof. In some embodiments, the IgGl Fc fragment or variant thereof comprises 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.

[0351] In some embodiments, the cytokine of the fusion protein forms a dimer by the half- life extension domain of one copy of the cytokine forming a disulfide bond with the corresponding half-life extension domain of a second copy of the cytokine. b) Albumin

[0352] Albumin is a natural carrier protein that has an extended serum half-life of approximately three weeks due to its size and its susceptibility to FcRn-mediated recycling, which prevents intracellular degradation. Thus, linking a cytokine to albumin can greatly extend the half-life of the cytokine. This approach has been taken to extend the plasma half- life of therapeutically beneficial proteins. See, e.g., WO 2001 / 079271A1 and WO 2003 / 59934A2. A few isoforms of HSA were listed in Table 2.

[0353] In some embodiments, the fusion protein comprises a half-life extension domain that comprises 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" includes fragments of albumin as well as variants of albumin. The albumin polypeptide comprises an amino-terminus and a carboxy-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 201 1 / 124718; US20070048282. In some embodiments, the albumin polypeptide is HSA. c) Binding Proteins

[0354] Additional strategies for extending the half-life of the cytokines or variants thereof in serum include linking the cytokine to certain binding proteins, such as albumin-binding proteins as described above or IgG-binding proteins. The binding proteins can be any protein that binds to a serum protein having a prolonged half-life, such as albumin or IgG. Albumin and IgG are polypeptides that are known to have long half-lives in serum.

[0355] 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.

[0356] In some embodiments, the albumin-binding protein is a single-domain antibody or fragment thereof, such as a Nanobody, that binds to or otherwise associates with albumin, such as those described herein. See, e.g., WO 2004041865A2 and US20070269422A1.

[0357] Another example of a binding protein is an IgG-binding protein. IgG-binding proteins have been reported. For an overview of IgG-binding proteins, including specific IgG-binding proteins and their applications, see, e.g., Choe et al. (2016) Materials 9(12): 994. d) Antibody Derivatives

[0358] The cytokines described herein may alternatively be linked to various antibody derivatives including, but not limited to, an scFv, an scFc, a dual-variable domain (DVD), and antibody derivatives based on the CrossMab approach. See, e.g., Klein et al. (2012), MAbs, 4(6): 653-663; US20070071675A1. The antibody derivatives include antibody derivatives engineered as bispecific antibodies or fragments thereof. As such, in some embodiments, a half-life extension domain can comprise any antibody derivative, variant, or fusion product thereof including, but not limited to an scFv, an scFc, a dual-variable domain (DVD), antibody derivatives based on the CrossMab approach, and bispecific antibodies or fragments thereof. e) Polyamino acid sequences

[0359] An additional strategy for extending the half-life of fusion proteins in serum is by linking the fusion protein to a polyamino acid sequence. As such, in some embodiments, the half-life extension domain comprises a polyamino acid sequence. The polyamino acid sequence can be any polyamino acid sequence capable of extending the half-life of the fusion protein in serum when it is linked to the fusion protein. Examples of polyamino acid sequences include PAS polypeptides and XTEN polypeptides. f) PEGylation and Glycosylation

[0360] Additional strategies for extending the half-life of the cytokines provided herein include PEGylation and the engineering of additional glycosylation sites. Each of these strategies is discussed in further detail below.

[0361] "PEGylation" refers to a process of covalent or non-covalent attachment or amalgamation of polyethylene glycol (PEG) polymer chains to molecules and macrostructures, such as a drug, therapeutic protein, polypeptide, antibody, antibody fragment, antibody derivative, or to any of the fusion proteins or components thereof provided herein (e.g., the half-life extension domain of a fusion protein and / or the cytokine or functional fragment thereof of the fusion protein). The benefits of PEGylation include, for example, (1) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and / or through evasion of cellular clearance mechanisms, (2) reduced antigenicity and immunogenicity of the molecule to which PEG is attached, (3) improved pharmacokinetics, (4) improved solubility, (5) improved formulation and dosing options, (6) improved bioavailability via reduced losses at subcutaneous injection sites, (7) improved thermal and mechanical stability of the PEGylated molecule.

[0362] Methods for the pegylation of various molecules and macrostructures are well known in the art. See, e.g., ETS20140256636A1; Fee and Damodaran (2010) European Pharmaceutical Review, 15(1): 18-26; Chapman et al. (1999) Nature Biotechnok, 17: 780- 783; Yang et al. (2003), Protein Eng., 16(10): 761-770; Chapman, Adv. Drug. Deliv. Rev. (2002), 54(4): 531-545.

[0363] "Glycosylation" refers to the addition of saccharides 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 carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine (N-X-S) and asparagine-X-threonine (N-X-T), where X is any amino acid except proline (P), are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars (e.g., N-aceylgalactosamine, galactose, or xylose) to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0364] Naturally-occurring glycosylation has been shown to increase the molecular stability of proteins. See, e.g., Sola et al. (2007), Cell. Mol. Life Sci., 64(16): 2133-2152. It has also been shown that the engineering of additional glycosylation sites can stabilize a variety of protein therapeutics against most major physiochemical instabilities. See, e.g., Sola and Griebenow (2009), J. Pharm. Sci., 98(4): 1223-1245. Among the pharmaceutically relevant protein instabilities that have been shown to be improved by glycosylation are, for example, oxidation; cross-linking; pH-, chemical-, thermal-, and freezing-induced denaturation / unfolding; precipitation; kinetic activation; and aggregation. Id.

[0365] Addition of glycosylation sites to the fusion protein is conveniently accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences (for N-linked glycosylation sites) is created 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 functional fragment thereof). The alteration may also be made by the addition to, or substitution of, 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 functional fragment thereof) (for O-linked glycosylation sites). g) Heterodimerization Modifications

[0366] The half-life extension domains described herein may 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 such that production of the fusion protein in its correct heterodimeric form is produced efficiently. As such, 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 as described in K...

Claims

1. A fusion protein comprising: a) an IL-21 and b) an albumin binding moiety, wherein the albumin binding moiety comprises a single domain antibody (sdAb) comprising: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 69; a CDR2 comprising the amino acid sequence of SEQ ID NO: 70; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 71; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 72; a CDR2 comprising the amino acid sequence of SEQ ID NO: 73; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 74; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 75; a CDR2 comprising the amino acid sequence of SEQ ID NO: 76; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 77; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 78; a CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 80; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 81; a CDR2 comprising the amino acid sequence of SEQ ID NO: 82; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 83; (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 84; a CDR2 comprising the amino acid sequence of SEQ ID NO: 85; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 86; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 87; a CDR2 comprising the amino acid sequence of SEQ ID NO: 88; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 89; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 90; a CDR2 comprising the amino acid sequence of SEQ ID NO: 91; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92; or (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 93; a CDR2 comprising the amino acid sequence of SEQ ID NO: 94; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

95. The fusion protein of claim 1, wherein the IL-21 comprises an amino acid sequence of SEQ ID NO: 1, 2, 126, 171, or 172.

3. The fusion protein of claim 1, wherein the IL-21 comprises an amino acid sequence that is at least 80% sequence identity to SEQ ID NO: 1, 2, 126, 171, or 172.

4. The fusion protein of claim 1, wherein the IL-21 comprises an amino acid sequence of SEQ ID NO: 126, 171, or 172.

5. The fusion protein of claim 1, wherein the IL-21 comprises an amino acid sequence of SEQ ID NO: 126.

6. The fusion protein of claim 1, wherein the IL-21 comprises an amino acid sequence of SEQ ID NO: 171.

7. The fusion protein of claim 1, wherein the IL-21 comprises an amino acid sequence of SEQ ID NO: 172.

8. The fusion protein of any one of claims 1 to 7, wherein the sdAb comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 90; a CDR2 comprising the amino acid sequence of SEQ ID NO: 91; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92.

9. The fusion protein of any one of claims 1 to 8, wherein the sdAb comprises an amino acid sequence of SEQ ID NO: 67 or 168.

10. The fusion protein of any one of claims 1 to 9, wherein the sdAb comprises an amino acid sequence of SEQ ID NO: 67.

11. The fusion protein of any one of claims 1 to 9, wherein the sdAb comprises an amino acid sequence of SEQ ID NO: 168.

12. The fusion protein of any one of claims 1 to 11, wherein the sdAb has a dissociation constant (KD) between 1 and 200 nM at pH 5.5 or at pH 7.5.

13. The fusion protein of any one of claims 1 to 12, wherein the albumin binding moiety is fused to the C-terminus of the IL-21.

14. The fusion protein of any one of claims 1 to 12, wherein the albumin binding moiety is fused to the N-terminus of the IL-21.

15. The fusion protein of any one of claims 1 to 14, wherein the IL-21 and the albumin binding moiety are connected via a first linker.

16. The fusion protein of claim 15, wherein the first linker is selected from the group consisting of SEQ ID NOs: 12-26 and 158-159.

17. The fusion protein of claim 15 or 16, wherein the first linker is a flexible linker.

18. The fusion protein of claim 15 or 16, wherein the first linker is a rigid linker.

19. The fusion protein of any one of claims 1 to 18, wherein the fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 120, 121, 129 to 132, 134, 135, 137 to 150, 163, and 164.

20. The fusion protein of any one of claims 1 to 19, wherein the fusion protein comprises an amino acid sequence of any one of SEQ ID NOs: 132, 134, 135, and 137 to 150.

21. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

22. The fusion protein of any one of claims 1 to 20 for use in treating cancer.

23. The fusion protein for use of claim 22, wherein the fusion protein is for use in combination with a second agent that comprises a therapeutic antibody, an immune checkpoint inhibitor, a second cytokine, a chemotherapeutic agent, a tyrosine kinase inhibitor, or an immune cell.

24. The fusion protein for use of any one of claim 22 or 23, wherein the cancer is mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, esophageal cancer, gastric cancer, or colorectal cancer.

25. The fusion protein for use of claim 24, wherein the cancer is mesothelioma, lung cancer, ovarian cancer, or gastric cancer.

26. The fusion protein for use of any one of claims 23 to 25, wherein the second agent is a therapeutic antibody.

27. The fusion protein for use of claim 26, wherein the therapeutic antibody binds to a tumor antigen that is mesothelin (MSLN), GPA33, Her-2 (ERBB2), EGFR, or CD20 (MS4A1).

28. The fusion protein for use of claim 26, wherein the therapeutic antibody binds to a tumor antigen that is CEA, MUC16, MUC1, AFP, EPCAM, CD19, CD21, CD22, CD30, CD33, CD37, CD45, PSMA, or BCMA.

29. The fusion protein for use of claim 27, wherein the therapeutic antibody binds to mesothelin.

30. The fusion protein for use of claim 27, wherein the therapeutic antibody binds to Her2.

31. The fusion protein for use of any one of claims 23 to 24, wherein the second agent is an immune checkpoint modulator.

32. The fusion protein for use of claim 31, wherein the immune checkpoint modulator is an inhibitor of an immune checkpoint protein that is PD-L1, CTLA4, PD-L2, PD-1, 4-1BB, CD47, TIGIT, GITR, TIM3, LAG3, CD27, or B7H4.

33. The fusion protein for use of claim 31, wherein the immune checkpoint modulator is an antibody that binds to PD-1.

34. The fusion protein for use of claim 31, wherein the immune checkpoint modulator is an antibody that binds to CTLA4.

35. The fusion protein for use of any one of claims 23 to 25, wherein the second agent is a second cytokine.

36. The fusion protein for use of claim 35, wherein the second cytokine is IL-7, IL-15, or IL-15 bound to IL15R α \alpha or half-life extended variants thereof.