Masked il-2 fusion proteins

Abstract

Provided herein are novel IL-2 or IL-15 fusion proteins that include (i) an anti-PD1 moiety, and (ii) an IL-2 or an IL-15 moiety attached by a protease sensitive linker (PSL) to (iii) to a masking moiety that includes an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof. In some embodiments, the protease sensitive linker is cleavable by a protease in the tumor microenvironment. Also provided herein are novel IL-2 fusion proteins that include (i) an anti- PD1 moiety attached by a linker to (ii) an IL-2 moiety. Such IL-2 fusion proteins are useful, for example, for the treatment of IL-13Rα2 expressing cancers. Also provided herein are novel fusion proteins that include a cytokine or antibody moiety, a protease-sensitive linker, and a masking moiety or an IL-13 superkine.

Description

Attorney Docket No. 117802-5021 -WOFUSION PROTEINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 738,711, filed December 24, 2024; and U.S. Provisional Application No. 63 / 796,608, filed April 29, 2025, which are herein incorporated by reference in their entireties.BACKGROUND

[0002] Interleukin 2 (IL-2) is a pluripotent cytokine produced primarily by activated CD4+T cells, which plays a crucial role in producing a normal immune response. IL-2 promotes proliferation and expansion of activated T lymphocytes, potentiates B cell growth, and activates monocytes and natural killer cells. It was by virtue of these activities that IL-2 was tested and is used as an approved treatment of cancer (aldesleukin, Proleukin®). In eukaryotic cells, human IL-2 is synthesized as a precursor polypeptide of 153 amino acids, from which 20 amino acids are removed to generate mature secreted IL-2 (Taniguchi 1983). Recombinant human IL-2 has been produced in E. coli (Rosenberg 1984), in insect cells (Smith 1985) and in mammalian COS cells (Taniguchi 1983).

[0003] lnterleukin-2 (IL-2) is a four a-helical bundle type I cytokine first identified as a T cell growth factor (Morgan et al., Science 193: 1007 (1976)) but subsequently shown to have broad actions. IL-2 promotes CD4+T helper differentiation (Zhu et al., Annual review of immunology 28: 445 (2010); Liao et al., Nat Immunol 9: 1288 (2008); and Liao et al., Nat Immunol 12: 551 (2011 )) and the development of regulatory T (Treg) cells (Cheng et al., Immunol Rev 241 : 63 (2011)), induces natural killer and cytotoxic CD8+T cells (Liao et al., Immunity 38: 13 (2013)), and mediates activation-induced cell death (AICD) (Lenardo et al., Nature 353: 858 (1991)).

[0004] IL-2 works by interacting with three different receptors: the interleukin 2 receptor alpha (I L-2Ra; CD25), the interleukin 2 receptor beta (IL-2R ; CD122), and the interleukin 2 receptor gamma (IL-2Ry;CD132; common gamma chain). The first receptor to be identified was the I L-2Roc, which is a 55 kD polypeptide (p55) that appears upon T cell activation and was originally called Tac (for T activation) antigen. The I L-2Roc binds IL-2 with a Kd of approximately 108M and is also known as the “high affinity” IL-2 receptor. Binding of IL-2 to cells expressing only the I L-2Roc does not lead to any detectable biologic response. In most circumstances, IL-2 works through three different receptors: the I L-2Ra, the IL-2R , andAttorney Docket No. 117802-5021 -WOthe IL-2Ry. Most cells, such as resting T cells, are not responsive to IL-2 since they only express the IL-2Rp, and the I L-2Ry, which have low affinity for IL-2. Upon stimulation, resting T cells express the relatively high affinity IL-2 receptor I L-2Ra. Binding of IL-2 to the I L-2Ra causes this receptor to sequentially engage the IL-2RP, and the IL-2Ry, bringing aboutT cell activation. IL-2 “superkines” with augmented action due to enhanced binding affinity for IL-2RJ3 were previously developed (Levin et al., Nature 484: 529 (2012)).

[0005] Despite the great potential of IL-2 for use in cancer therapies, its clinical application remains relatively restricted due in part to the severe toxicity associated with IL-2 administered at high doses. As IL-2 has a short serum half-life of several minutes, high doses of IL-2 are typically needed to achieve an optimal immunomodulatory effect. Such high doses, however, inevitably result in severe toxicities, including vascular leak syndrome (VLS), pulmonary edema, hypotension, and heart toxicities. Thus, there remains a need for novel effective IL-2 cancer therapies that minimize IL-2 associated toxicity.BRIEF SUMMARY

[0006] Provided herein is a fusion protein comprising: (a) a cytokine or an antibody moiety; (b) a protease sensitive linker (PSL); and (c) a masking moiety; wherein the PSL attaches the masking moiety to the cytokine or an antibody moiety.

[0007] In embodiments, the masking moiety comprises an IL-13, an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody, or an antigen-binding fragment thereof, wherein the masking moiety is capable of binding to IL-13Ra2, and does not bind to I L-13Ra1.

[0008] In embodiments, the masking moiety comprises an IL-13, an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody, or an antigen-binding fragment thereof, wherein the masking moiety is capable of binding to IL-13Ra2 and does bind to IL-13Ra1.

[0009] In embodiments, the masking moiety comprises an IL-13, an IL-13 mutein, or type II IL-4 receptor antagonist thereof, wherein the masking moiety binds with higher affinity to I L-13Ra1 than wild type IL-13 (SEQ ID NO:200), and binds with lower affinity to IL-13Ra2 than wild type IL-13.

[0010] In embodiments, the masking moiety comprises an IL-13 mutein that has the amino acid sequence of SEQ ID NO:201 or 202.

[0011] In embodiments, the fusion protein further comprises a protein binding domain.Attorney Docket No. 117802-5021 -WO

[0012] In embodiments, the protein binding domain comprises an albumin binding domain (ABD), a transferrin binding domain (TBD), a CD122 domain, a CD25 domain, a CD132 domain, an IL-13Ra2 domain, an IL-13Ra1 domain, or an IL-4Ra domain.

[0013] In embodiments, the cytokine or an antibody moiety is an IL-2, an IL-2 mutein, an IL-12, an IL-12 mutein, an IL-15, an IL-18, an IL-4, an IL-7, an IL-13, and IL-13 mutein, an anti-PD-1 antibody, an anti-VEGF antibody, an anti-PDL1 antibody, an anti-PSMA antibody, an anti-EGFR antibody, an anti-CTLA4 antibody, an anti-CD3 antibody, or a hybrid of two or more of an anti-VEGF antibody, an anti-PSMA antibody, an anti-PD-1 antibody, and anti-PDL1 antibody, an anti-CD3 antibody, and an anti-CTLA4 antibody.

[0014] In embodiments, the cytokine or an antibody moiety comprises an IL-2 mutein having the amino acid sequence of SEQ ID NO:5, SEQ ID NO 51, SEQ ID NO:52, or SEQ ID NO:53.

[0015] In embodiments, the PSL is capable of being cleaved in the tumor microenvironment.

[0016] In embodiments, the PSL has the amino acid sequence of PLGLWA (SEQ ID NQ:300) or PAGLIG (SEQ ID NQ:301).

[0017] In embodiments, the activity of the cytokine or antibody is reduced by at least two-fold in comparison to an isolated counterpart in the absence of protease cleavage.

[0018] In embodiments, provided herein is a pharmaceutical composition comprising a fusion protein disclosed herein, and a pharmaceutically acceptable carrier.

[0019] In embodiments, provided herein is a nucleic acid composition that encodes a fusion protein disclosed herein.

[0020] In embodiments, provided herein is an expression vector composition comprising a nucleic acid composition disclosed herein.

[0021] In embodiments, provided herein is a method of making a fusion protein disclosed herein, comprising culturing a nucleic acid composition disclosed herein or an expression vector composition disclosed herein under conditions wherein the fusion protein is expressed, and recovering the fusion protein.Attorney Docket No. 117802-5021 -WO

[0022] In embodiments, provided herein is a method of treating a disease or disorder in a subject in need thereof comprising administering to the subject a fusion protein disclosed herein, or a pharmaceutical composition disclosed herein.

[0023] In embodiments, provided herein are novel fusion proteins that include (i) an anti-PD 1 moiety, and (ii) an IL-2 moiety or an IL-15 moiety attached by a protease sensitive linker (PSL) to (iii) at least one masking and / or targeting moiety comprising an IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof.

[0024] In embodiments, the IL-13 mutein, I L-13Ra2 binding mutein or IL-13Ra2 antibody or antigenbinding fragment or type II IL-4 receptor antagonist thereof is capable of binding to IL-13Ra2 (e.g., an anti-IL-13Ra antibody), and does not bind to IL-13Ra1 , and the protease sensitive linker is cleavable by a protease in the tumor microenvironment. In embodiments, the IL-13 mutein, IL-13Ra2 binding mutein or IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof is capable of binding to IL-13Ra2 (e.g., an anti-IL-13Ra antibody), and also binds to IL-13Ra1 , and the protease sensitive linker is cleavable by a protease in the tumor microenvironment. When the IL-13 mutein or I L-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof is attached to the IL-2 moiety by the PSL, the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof of the fusion proteins described herein associates with, obstructs, and / or masks activity of the IL-2 moiety. In addition, the IL-13 mutein or I L-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof allows the fusion protein to bind to IL-13Ra2 expressing tumors. Once localized to the tumor microenvironment, the PSL of the fusion protein undergoes proteolytic cleavage, thereby releasing the IL-13 mutein or I L-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof and “unmasking” the IL-2 moiety. The unmasked IL-2 moiety can then provide anti-tumor activity at the localized tumor site. Thus, in embodiments, the fusion proteins provided herein advantageously exhibit reduced IL-2 associated toxicity while allowing for targeted activity at IL-13Ro2 expressing tumors. Such fusion proteins are particularly useful for the treatment of IL-13Ro2 expressing cancers.

[0025] In embodiments, provided herein is a cytokine fusion protein comprising: (a) an anti-PD 1 antibody; (b) an IL-2 or an IL-15 moiety comprising an IL-2, an IL-2 mutein, an IL-15, or an IL-15 mutein; (c) a protease sensitive linker (PSL); and (d) a masking moiety; wherein the PSL attaches the masking moiety to the IL-2 moiety, the IL-15 moiety, or the anti-PD1 antibody.Attorney Docket No. 117802-5021 -WO

[0026] In embodiments, the masking moiety comprises an IL-13 mutein that comprises the following amino acid substitutions compared to a wild-type human IL-13 (SEQ ID NO:200): (a) L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T; or (b) L10H, E15R, R86T, D87G, T88R, R108K, and Q111.

[0027] In embodiments, the masking moiety comprises an IL-13, an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody, or an antigen-binding fragment thereof, wherein the masking moiety is capable of binding to IL-13Ra2, and does not bind to IL-13Ra1.

[0028] In embodiments, the masking moiety comprises an IL-13, an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody, or an antigen-binding fragment thereof, wherein the masking moiety is capable of binding to IL-13Ra2, and does bind to IL-13Ra1.In embodiments, the masking moiety comprises an IL-13, an IL-13 mutein, or type II IL-4 receptor antagonist thereof, wherein the masking moiety binds with higher affinity to IL-13Ra1 than wild type IL-13 (SEQ ID NQ:200), and binds with lower affinity to IL-13Ra2 than wild type IL-13.

[0029] In embodiments, the masking moiety comprises an IL-13 mutein that has the amino acid sequence of SEQ ID NOs: 201 or 202.

[0030] In embodiments, the IL-2 moiety comprises an IL-2 mutein having the amino acid sequence of SEQ ID NO:5, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53.

[0031] In embodiments, the anti-PD1 antibody is engineered as “knob-in-hole” (KiH) with mutations in constant region 3 (CH3) in its heavy chains.

[0032] In embodiments, the fusion protein comprises: (a) a first polypeptide comprising an IL-2 or an IL-2 mutein, optionally an IL-2 fusion attached to the second and third constant regions (CH2 and CH3) of the “knob” heavy chain of the anti-PD1 antibody (KiH); (b) a second polypeptide comprising the “hole” heavy chain of the anti-P D 1 antibody (KiH); and (c) a third polypeptide comprising the light chain of the anti-PD 1 antibody (KiH).

[0033] In embodiments, the fusion protein comprises: (a) a first polypeptide comprising the “knob” heavy chain of the anti-PD1 antibody (KiH) attached to an IL-2 or an IL-2 mutein, optionally an IL-2 fusion; (b) a second polypeptide comprising the “hole” heavy chain of the anti-PD1 antibody (KiH); (c) a third polypeptide comprising the light chain of the anti-PD 1 antibody (KiH).Attorney Docket No. 117802-5021 -WO

[0034] In embodiments, the fusion protein comprises: (a) a first polypeptide comprising the “knob” heavy chain of the anti-PD1 antibody (KiH); (b) a second polypeptide comprising the “hole” heavy chain of the anti-PD 1 antibody (KiH) attached to an IL-2 or an IL-2 mutein or an IL-2 mutein fusion; (c) a third polypeptide comprising the light chain of the anti-PD1 antibody (KiH).

[0035] In embodiments, the masking moiety is attached to the IL-2 or the IL-2 mutein. In embodiments, the masking moiety is attached to the antibody heavy chain that is not attached to the IL-2 or the IL-2 mutein. In embodiments, the masking moiety is attached to an additional masking moiety.

[0036] In embodiments, the PSL is capable of being cleaved in the tumor microenvironment.

[0037] In embodiments, the PSL has the amino acid sequence of PLGLWA (SEQ ID NO:300) or PAGLIG (SEQ ID NQ:301).

[0038] In embodiments, provided herein is a cytokine fusion protein comprising sequences selected from the group consisting of: (a) SEQ ID NOs: 6, 7, and 8; (b) SEQ ID NOs: 9, 10, and 11; (c) SEQ ID NOs: 12, 13, and 14; (d) SEQ ID NOs: 15, 16, and 17; (e) SEQ ID NOs: 18, 19, and 20; (f) SEQ ID NOs: 21, 22, and 23; (g) SEQ ID NOs: 24, 25, and 26; (h) SEQ ID NOs: 27, 28, and 29; (i) SEQ ID NOs: 30, 31, and 32; 0) SEQ ID NOs: 33, 34, and 35; (k) SEQ ID NOs: 36, 37, and 38; (I) SEQ ID NOs: 39, 40, and 41; (m) SEQ ID NOs: 42, 43, and 44; (n) SEQ ID NOs: 45, 46, and 47; (o) SEQ ID NOs: 48, 49, and 50; (p) SEQ ID NOs: 71, 72 and 73; (q) SEQ ID NOs: 74, 75 and 76; (r) SEQ ID NOs: 77, 78, and 79; (s) SEQ ID NOs: 80, 81, and 82; (t) SEQ ID NOs: 83, 84, and 85; (u) SEQ ID NOs: 86, 87, and 88; (v) SEQ ID NOs: 89, 90, and 91; and (w) SEQ ID NOs: 92, 93, and 94.

[0039] In embodiments, provided herein is a pharmaceutical composition comprising a cytokine fusion protein disclosed herein, and a pharmaceutically acceptable carrier.

[0040] In embodiments, provided herein is a nucleic acid composition that encodes a cytokine fusion protein disclosed herein.

[0041] In embodiments, provided herein is an expression vector composition comprising a nucleic acid composition disclosed herein.

[0042] In embodiments, provided herein is a method of making a cytokine fusion protein disclosed herein, comprising culturing a nucleic acid composition disclosed herein or an expression vector composition disclosed herein under conditions wherein the cytokine fusion protein is expressed, and recovering the cytokine fusion protein.Attorney Docket No. 117802-5021 -WO

[0043] In embodiments, provided herein is a method of treating an IL-13Ra2 expressing cancer in a subject in need thereof comprising administering to the subject a cytokine fusion protein disclosed herein, or a pharmaceutical composition disclosed herein.

[0044] In embodiments, the cytokine fusion protein comprises one or more of SEQ ID NOs:6-50 or 71-94.

[0045] In embodiments, the cytokine fusion protein is administered as a neo-adjuvant prior to a surgery to remove a tumor.

[0046] In embodiments, the cytokine fusion protein is administered up to one week prior to the surgery, optionally up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 1 day, or less than 1 day prior to the surgery, or wherein the fusion protein is administered up to 9 weeks prior to the surgery, optionally up to 8 weeks, up to 7 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, up to 2 weeks, up to 1 week, or less than 1 week prior to the surgery.

[0047] In embodiments, the cytokine fusion protein is administered as an adjuvant after a surgery to remove a tumor.

[0048] In embodiments, the cytokine fusion protein is administered up to one week after the surgery, optionally up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 1 day, or less than 1 day after the surgery, or wherein the cytokine fusion protein is administered starting at least two weeks after the surgery.

[0049] In embodiments, the cytokine fusion protein is administered both as a neo-adjuvant prior to a surgery to remove a tumor, and an adjuvant after to a surgery to remove a tumor.

[0050] In embodiments, the cytokine fusion protein is administered up to one week prior to the surgery, optionally up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 1 day, or less than 1 day prior to the surgery, and wherein the cytokine fusion protein is administered up to one week after the surgery, optionally up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 1 day, or less than 1 day after the surgery, or wherein the cytokine fusion protein is administered up to 9 weeks prior to the surgery, optionally up to 8 weeks, up to 7 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, up to 2 weeks, up to 1 week, or less than 1 week prior to the surgery, and wherein the cytokine fusion protein is administered starting at least to two weeks after the surgery.Attorney Docket No. 117802-5021 -WO

[0051] In embodiments, the cancer is a sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, pulmonary cancer, cutaneous, lymphoid, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal-like breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, small cell lung cancer, kidney cancer, gastric cancer, brain cancer, or a CNS tumors.

[0052] In embodiments, provided herein is a fusion protein comprising: an anti-PD-1 antibody; an IL-2 mutein, wherein the anti-PD1 antibody is linked to the IL-2 mutein; an IL-13 superkine linked to the anti-PD- 1 via a first protease sensitive linker (PSL); and an extracellular domain of CD122 linked to the IL-2 mutein via a second PSL. It is contemplated that the IL-13 superkine and the extracellular domain of CD122 act as masking moieties for the fusion protein.

[0053] In embodiments, the anti-PD-1 antibody is a human anti-PD-1 based on nivolumab or pembrolizumab; wherein the anti-PD-1 antibody comprises an lgG1 or lgG4 backbone.

[0054] In embodiments, the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:5, the IL-13 superkine comprises an amino acid sequence of SEQ ID NO:202, the PSL comprises an amino acid sequence of SEQ ID NQ:300, and the extracellular domain of CD122 comprises an amino acid sequence of SEQ ID NO: 350.

[0055] In embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NOs: 83-85 or the amino acid sequence of SEQ ID NOs: 88-91.

[0056] In embodiments, provided herein is a pharmaceutical composition comprising a fusion protein and a pharmaceutically acceptable carrier, the fusion protein comprising: an anti-PD-1 antibody; an IL-2 mutein, wherein the anti-PD1 antibody is linked to the IL-2 mutein; an IL-13 superkine linked to the anti-PD-1 via a first protease sensitive linker (PSL); and an extracellular domain of CD122 linked to the IL-2 mutein via a second PSL.

[0057] In embodiments, provided herein is a nucleic acid composition encoding a fusion protein, the fusion protein comprising: an anti-PD-1 antibody; an IL-2 mutein, wherein the anti-PD1 antibody is linked to the IL- 2 mutein; an IL-13 superkine linked to the anti-PD-1 via a first protease sensitive linker (PSL); and an extracellular domain of CD122 linked to the IL-2 mutein via a second PSL.

[0058] In emodiments, provided herein is an expression vector composition comprising a nucleic acid composition encoding a fusion protein. The fusion protein comprises: an anti-PD-1 antibody; an IL-2Attorney Docket No. 117802-5021 -WOmutein, wherein the anti-PD1 antibody is linked to the IL-2 mutein; an IL-13 superkine linked to the anti-PD-1 via a first protease sensitive linker (PSL); and an extracellular domain of CD122 linked to the IL-2 mu

[0059] tein via a second PSL.

[0060] In embodiments, provided herein is a method of maing a fusion protein comprising culturing a nucleic acid composition encoding the fusion protein or an expression vector composition comprising the fusion protein under conditions where the fusion protein is expressed, and recovering the fusion protein. The fusion protein comprises: an anti-PD-1 antibody; an IL-2 mutein, wherein the anti-PD1 antibody is linked to the IL-2 mutein; an IL-13 superkine linked to the anti-PD-1 via a first protease sensitive linker (PSL); and an extracellular domain of CD122 linked to the IL-2 mutein via a second PSL.BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIGs. 1 A and 1 B depict results of an IL-2R reporter assay of mouse (FIG. 1 A) and human (FIG.1B) MDNA113 masking constructs.

[0062] FIGs. 2A and 2B depict results of a PD-1 / PD-L1 reporter assay of mouse (FIG. 2A) and human (FIG. 2B) MDNA113 masking constructs.

[0063] FIGs. 3A and 3B depict results of an IL-2R reporter assay of mouse (FIG. 3A) and human (FIG.3B) MDNA113 masking constructs upon MMP9 cleavage, releasing the IL-13 mask.

[0064] FIG. 4 shows biodistribution of Cy5-labeled hMDNA113 at 1 mg / kg in whole body mice with MC38 and MC38 / I L13Ra2 tumors on contralateral flanks across timepoints.

[0065] FIG. 5A depicts the results of a tolerability study in in naive C57BI / 6 mice treated with either murine MDNA113 or its unmasked version.

[0066] FIG. 5B shows body weight loss in an MC38IL13Ra2colon carcinoma model (C57BI / 6 mice), treated with either murine MDNA113 or its unmasked version.

[0067] FIG. 6A depicts results of a study in which C57BI / 6 mice carrying the MC38IL13R2colon carcinoma tumor were treated with vehicle, unmasked, MDNA113 and its non-cleavable version (mMDNAI 13NC) and a murine anti-PD-1 antibody. Tumor volume data is presented.

[0068] FIG. 6B depicts results of a study in which C57BI / 6 mice carrying the MC38IL13R2colon carcinoma tumor were rechallenged with MC38IL13R2colon carcinoma tumor following initial treatment, demonstrating that the initial treatment resulted in complete tumor regression. Tumor volume data is presented.Attorney Docket No. 117802-5021 -WO

[0069] FIG. 7 shows results of a study using a B16F10 / IL13Ra2 melanoma model, with tumor volume (left panel) and average tumor growth inhibition (TGI; right panel) data presented.

[0070] FIG. 8 shows quantification of immune cell infiltrates in B16F10IL13R“2melanoma tumors.

[0071] FIG. 9 shows analysis of immune cell infiltrates in 4T 1.2 orthotopic breast tumors.

[0072] FIG. 10A shows results of a Jurkat I L-2RPy bioassay for IL-2 activity of the constructs hMDNA223 / pembrolizumab and hMDNA113B / pembrolizumab (left) and hMDNA223 / nivolumab and hMDNA113B / nivolumab (right).

[0073] FIG. 10B depicts results of a Jurkat IL-2R[3y bioassay for IL-2 activity of the constructs hMDNA223 / pembrolizumab and hMDNAI 13B / pembrolizumab with and without MMP9 cleavage (left) and hMDNA223 / nivolumab and hMDNAI 13B / nivolumab with and without MMP9 cleavage (right).

[0074] FIG. 11 shows results of a PD-1 / PD-L1 reporter assay for the constructs hMDNA223 / pembrolizumab and hMDNAI 13B / pembrolizumab (top) and hMDNA223 / nivolumab and hMDNAI 13B / nivolumab (bottom).

[0075] FIG. 12A shows results of a study in which mice carrying the MC38 colon carcinoma tumor were treated with vehicle, murine MDNA113B or its non-cleavable version (MDNB113NC). Tumor volume data is presented.

[0076] FIG. 12B shows results of a study in which mice carrying the MC38 / IL-13Ra2 colon carcinoma tumor were treated with vehicle, murine MDNA113B or its non-cleavable version (MDNB113NC). Tumor volume data is presented.

[0077] FIG. 13A shows results of a biodistribution study of Cy5.5 labelled MDNA113B in mice implanted with Panc02 / IL-13Rot2<p°s) tumor cells on the right flank and Panc02 / IL-13Roc2(ne9) tumor cells on the right flank. Total flux data in vivo at pre-treatment and indicated time points is presented.

[0078] FIG. 13B depicts results of a biodistribution study of Cy5.5 labelled MDNA113B in mice implanted with PancO2 / IL-13Ra2<p°s) tumor cells on the right flank and Panc02 / IL-13Roc2(ne9) tumor cells on the right flank. Images of ex vivo tumors at 168 hours post-treatment are presented.

[0079] FIG. 14A shows results of analysis of CD8+ tumor infiltrating lymphocytes in a B16F10 / I L-13Ra2 melanoma mouse model treated with MDNA223 or MDNA113B.Attorney Docket No. 117802-5021 -WO

[0080] FIG. 14B shows results of analysis of granzyme B (GrzB)-CD8+ tumor infiltrating lymphocytes in a B16F10 / I L-13Ra2 melanoma mouse model treated with MDNA223 or MDNA113B.

[0081] FIG. 15A shows a schematic of fusion masking constructs MM001, MM002, and MM003, that include anti-PD1, MDNA213, MDNA109FEAA, and MDNA413 components.

[0082] FIG. 15B shows results of a Jurkat I L-2R[3y bioassay for IL-2 activity of the masking constructs of Fig. 15A.

[0083] FIG. 15C shows results of a Jurkat I L-2R[3y bioassay for IL-2 activity of the masking constructs of Fig. 15A, with and without MMP9 cleavage.

[0084] FIG. 16 shows results of a HEK Blue IL-13 assay for IL-13 activity of the masking constructs of Fig.15A, with and without MMP9 cleavage. Fc-MDNA413 was used as a benchmark control.

[0085] FIG. 17A shows a schematic of an anti-PD 1 and anti-VEGF construct (left) and a construct including anti-PD1 and anti-VEGF with masked IL-2 superkine and IL-13Ra moieties.

[0086] FIG. 17B shows results of a Jurkat I L-2R[3y bioassay for IL-2 activity of the constructs of Fig. 17A.

[0087] FIG. 17C shows results of a Jurkat I L-2R[3y bioassay for IL-2 activity of the constructs of Fig. 17A, with and without MMP9 cleavage.

[0088] FIG. 17D shows results of a PD-1 / PD0L1 reporter assay for anti-PD1 activity of the constructs of Fig. 17A.

[0089] FIG. 17E shows results of a VEGF bioassay for anti-VEGF activity of the constructs of Fig. 17A. The bevacizumab biosimilar MAB9947 and recombinant VEGF were used as assay controls.

[0090] FIG. 18A shows a schematic of an IL-18-albumin unmasked fusion construct and an MDNA213-IL-18-albumin masking construct.

[0091] FIG. 18B shows results of a HEK IL-18 bioassay for I L-18 activity of the masking construct of Fig.18A in comparison to the unmasked construct and recombinant IL-18 control.

[0092] FIG. 19A shows a schematic of an anti-CTLA4 scFv-albumin unmasked fusion construct and an MDNA213-anti-CTLA4 scFv-albumin masking construct.

[0093] FIG. 19B shows results of a CTLA4 blockade bioassay for activity of the masking construct of Fig.19A in comparison to the unmasked construct.Attorney Docket No. 117802-5021 -WO

[0094] FIG. 20 shows a schematic of an anti-CD3 scFv-albumin unmasked fusion construct and an MDNA213-anti-CD3 scFv-albumin masking construct.DETAILED DESCRIPTION

[0095] In order for the present disclosure to be more readily understood, certain terms and phrases are defined below as well as throughout the specification.Definitions

[0096] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many terms used in the present disclosure. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.

[0097] As used herein, the term “cytokine” or “cytokine moiety” refers to 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. In embodiments, cytokines include, for example, interleukins (ILs), including species variants, truncated analogs thereof which are capable of stimulating or inducing such cytocidal immune responses. In embodiments, interleukins include, for example, IL-2, IL-12, IL-15, IL-18, IL-4, IL-7, and IL-13. In embodiments, the term “cytokine” is also understood to encompass any variant of a wildtype cytokine that comprises modification and maintains at least a significant portion (such as at least about 50%) of any of its desired function.

[0098] As used herein, “IL-2” means wild-type IL-2, whether native or recombinant. Mature human IL-2 occurs as a 133 amino acid sequence (less the signal peptide, consisting of an additional 20 N-terminal amino acids), as described in Fujita, et. al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of human IL-2 (SEQ ID NO:1; full length) is found in Genbank under accession locator NP_000577.2. The amino acid sequence of mature human IL-2 is depicted in SEQ ID NO:2 (human wild-type mature; position numbering of the substitutions is based on this sequence). The murine (Mus musculus) IL-2 amino acidAttorney Docket No. 117802-5021 -WOsequence is found in Genbank under accession locator (SEQ ID NO:3). The amino acid sequence of mature murine IL-2 is depicted in SEQ ID NO:4.SEQ ID NO:1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATEL KHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS IISTLT SEQ ID NO:2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLA QSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSEQ ID NO:3MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLK LPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTWKLKGSDNTFECQ FDDESATVVDFLRRWIAFCQSIISTSPQSEQ ID NO:4APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKD LQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTWKLKGSDNTFECQFDDESATWDFLRRWIAFCQ SIISTSPQ

[0099] As used herein, “IL-2 mutein” means an IL-2 polypeptide wherein specific substitutions to the interleukin-2 protein have been made. The IL-2 muteins are characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in or at the other residues of the native IL-2 polypeptide chain. In some embodiments, the insertion(s), deletion(s), substitution(s) and / or modification(s) results in an IL-2 mutein that retains the IL-2R binding activity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. The phrase “includes an” can also be used interchangeably with “includes at least one” when referring to for example, the IL-2 muteins of the present disclosure.Attorney Docket No. 117802-5021 -WO

[0100] Muteins also include conservative modifications and substitutions at other positions of IL-2 ( / .e., those that have a minimal effect on the secondary or tertiary structure of the mutein). Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978), and by Argos in EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative changes: Group I: ala, pro, gly, gin, asn, ser, thr; Group II: cys, ser, tyr, thr; Group llkval, ile, leu, met, ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.

[0101] “Numbered in accordance with IL-2” means identifying a chosen amino acid with reference to the position at which that amino acid normally occurs in the mature sequence of wild type IL-2, for example wild-type IL-2 having the sequence of SEQ ID NO:2. For example, in embodiments, R81 refers to the eighty-first amino acid, arginine, that occurs in SEQ ID NO:2; L80 refers to the eightieth amino acid, leucine, that occurs in SEQ ID NO:2; L85 refers to the eighty-fifth amino acid, leucine, that occurs in SEQ ID NO:2; I86 refers to the eighty-sixth amino acid, isoleucine, that occurs in SEQ ID NO:2; I92 refers to the ninety-second amino acid, isoleucine, that occurs in SEQ ID NO:2; F42 refers to the forty-second amino acid, phenylalanine, that occurs in SEQ ID NO:2; and K43 refers to the forty-third amino acid, lysine, that occurs in SEQ ID NO:2.

[0102] As used herein, the abbreviations for the genetically encoded L-enantiomeric amino acids used in the disclosure methods are conventional and are as follows in Table 1.Table 1: Amino acid abbreviationsAttorney Docket No. 117802-5021 -WO

[0103] “Hydrophilic Amino Acid” refers to an amino acid exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophilic amino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gin (Q), Asp (D), Lys (K) and Arg (R).

[0104] The term “cell types having the IL-2RaPy receptor” means the cells known to have this receptor type, i.e., T cells, activated T cells, B cells, activated monocytes, and activated NK cells. The term “cell types having the IL-2R|3y receptor” means the cells known to have that receptor type, i.e., B cells, resting monocytes, resting T cells, and resting NK cells.

[0105] The term “identity,” as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit ( / '.e., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990).Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.Attorney Docket No. 117802-5021 -WO

[0106] The terms “polypeptide,” “protein” or “peptide” refer to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation).

[0107] In the event the mutant IL-2 polypeptides of the disclosure are “substantially pure,” they can be at least about 60% by weight (dry weight) the polypeptide of interest, for example, a polypeptide containing the mutant IL-2 amino acid sequence. For example, the polypeptide can be at least about 75%, about 80%, about 85%, about 90%, about 95% or about 99%, by weight, the polypeptide of interest. Purity can be measured by any appropriate standard method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0108] An “agonist” is a compound that interacts with a target to cause or promote an increase in the activation of the target.

[0109] A “partial agonist” is a compound that interacts with the same target as an agonist but does not produce as great a magnitude of a biochemical and / or physiological effect as the agonist, even by increasing the dosage of the partial agonist.

[0110] A “superagonist” (also referred to as a “superkine”) is a type of agonist that is capable of producing a maximal response greater than the endogenous agonist for the target receptor, and thus has an efficacy of more than 100%.

[0111] “Operably linked” is intended to mean that the nucleotide sequence of interest ( / .e., a sequence encoding an IL-2 mutein) is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). “Regulatory sequences” include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissuespecific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression constructs of the invention can be introduced into host cells to thereby produce the human IL-2 muteins disclosed herein or to produce biologically active variants thereof.Attorney Docket No. 117802-5021 -WO

[0112] As used herein, the term “IL-2 masking moiety” refers to a component of a polypeptide that reduces the activity of an IL-2 moiety. In some embodiments, the IL-2 masking moiety comprises an IL-13 protein, variant, or fragment thereof. In some embodiments, the IL-2 masking moiety further comprises an extracellular domain of CD122, CD132, or CD25. The phrase “includes an” can also be used interchangeably with “includes at least one” when referring to for example, the IL-2 muteins, the IL-2 masking moieties, the IL-13 muteins, or the PSLs of the present disclosure.

[0113] The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell but are still included within the scope of the term as used herein.

[0114] As used herein, the terms “transformation” and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, particle gun, or electroporation.

[0115] As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the compositions.

[0116] As used herein, the term “antibody” or “antibody moiety” 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, trispecific antibodies, etc.), full-length antibodies and antigen binding fragments thereof, so long as they exhibit the desired antigen-binding activity. As used herein, the term “antibody moiety” refers to a full-length antibody or an antigen-binding fragment thereof. As used herein, the term “antigen-binding fragment” 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 notAttorney Docket No. 117802-5021 -WOcomprise a complete antibody structure. In embodiments, 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 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.

[0117] As used herein, the term “anti-PD-1 antibody” refers to any antibody or antigen-binding fragment thereof that binds to PD-1, including inhibitory antibodies. An “anti-PD-1 inhibitor” refers to an inhibitor that binds to and inhibits PD-1. Such anti-PD-1 antibodies and / or inhibitors include but are not limited to pembrolizumab, nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475, among others.

[0118] As used herein, the term “anti-VEGF antibody” refers to any antibody or antigen-binding fragment thereof that binds to VEGF, including inhibitory antibodies. An “anti-VEGF inhibitor” refers to an inhibitor that binds to and inhibits VEGF.

[0119] As used herein, the term “anti-CTLA4 antibody” refers to any antibody or antigen-binding fragment thereof that binds to CTLA4, including inhibitory antibodies. An “anti-CTLA4 inhibitor” refers to an inhibitor that binds to and inhibits CTLA4. Such anti-CTLA-4 antibodies and / or inhibitors include but are not limited to ipilimumab.

[0120] As used herein, the terms “cancer” (or “cancerous”), “hyperproliferative,” “tumor” and / or “neoplastic” to refer to cells having the capacity for autonomous growth ( / .e., an abnormal state or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic disease states may be categorized as pathologic ( / .e., characterizing or constituting a disease state), or they may be categorized as non-pathologic ( / .e., as a deviation from normal but not associated with a disease state). The terms are meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.“Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair. The terms “cancer” or “neoplasm” are used to refer to malignancies of the various organ systems, including those affecting the lung, breast, thyroid, lymph glands and lymphoid tissue, reproductive systems, gastrointestinal organs, and the genitourinary tract, as well as to adenocarcinomas which are generally considered to include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer ofAttorney Docket No. 117802-5021 -WOthe esophagus. Cancers generally can include solid tumors, as well as sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, pulmonary cancer, cutaneous, lymphoid, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal-like breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain cancer, and CNS tumors. CNS tumors include glioma, glioblastoma, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastoma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglia, menangioma, meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, an O6-methylguanine-methyltransferase (MGMT) positive or negative CNS tumor, and furin positive CNS tumor.

[0121] The term “carcinoma” is art-recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0122] As used herein, the term “hematopoietic neoplastic disorders” refers to diseases involving hyperplastic / neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Additional exemplary myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11 : 267-97); lymphoid malignancies include but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia / lymphoma (ATL), cutaneous T cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Sternberg disease.Attorney Docket No. 117802-5021 -WO

[0123] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, / .e., arresting its development; and (c) relieving the disease, / .e., causing regression of the disease. A therapeutically effective amount can be an amount that reduces tumor number, tumor size, and / or increases survival.

[0124] The terms “individual,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0125] The terms “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example in administering any of the fusion proteins as described herein. Thus, such a pharmaceutical composition may be used, for example in administering an IL-2, an IL-2 mutein, an IL-12, an IL-12 mutein, an IL-15, an IL-18, an IL-4, an IL-7, an IL-13, or an IL-13 mutein to a subject. In particular, an IL-2 mutein comprising the substitutions L80F, R81D, L85V, I86V, and I92F is administered in combination with anti-PD-1 to a subject with cancer. In some embodiments, the IL-2 mutein administered further comprises a substitution at position F42A. In some embodiments, the IL-2 administered mutein further comprises a substitution at position K43N.

[0126] The phrase a “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, produces a desired effect (e.g., prophylactic or therapeutic effect). In some embodiments, the therapeutic effect is to reduce tumor number. In some embodiments, theAttorney Docket No. 117802-5021 -WOtherapeutic effect is to reduce tumor size. In some embodiments, the therapeutic effect is to increase survival.

[0127] In some embodiments, unit dosage forms may be within, for example, ampules and vials, including a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dosage forms can be included in multi-dose kits or containers. Compositions disclosed herein, including IL-2 muteins in combination with anti-PD-1 antibodies, as well as compositions comprising any of the fusion proteins as described herein and pharmaceutical compositions thereof can be packaged in a single or multiple unit dosage form for ease of administration and uniformity of dosage.

[0128] A “therapeutically effective amount” will fall in a relatively broad range determinable through experimentation and / or clinical trials. For example, for in vivo injection, e.g., injection directly into the tissue or vasculature of a subject (for example, liver tissue or veins). Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.

[0129] An “effective amount” or “sufficient amount” refers to an amount providing, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a drug), treatments, protocols, or therapeutic regimens agents (including, for example, vaccine regimens), a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured).

[0130] The doses of an “effective amount” or “sufficient amount” for treatment (e.g., to ameliorate or to provide a therapeutic benefit or improvement) typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, disorders, illnesses, pathologies, or complications, for example, caused by or associated with the disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is also a satisfactory outcome. In some embodiments, the effective amount is an amount sufficient to reduce tumor number. In some embodiments, the effective amount is an amount sufficient to reduce tumor size. In some embodiments, the effective amount is an amount sufficient to increase survival.

[0131] “Prophylaxis” and grammatical variations thereof mean a method in which contact, administration or in vivo delivery to a subject is prior to disease. Administration or in vivo delivery to aAttorney Docket No. 117802-5021 -WOsubject can be performed prior to development of an adverse symptom, condition, complication, etc. caused by or associated with the disease. For example, a screen (e.g., genetic) can be used to identify such subjects as candidates for the described methods and uses, but the subject may not manifest the disease. Such subjects therefore include those screened positive for an insufficient amount or a deficiency in a functional gene product (protein), or producing an aberrant, partially functional or non-functional gene product (protein), leading to disease; and subjects screening positive for an aberrant, or defective (mutant) gene product (protein) leading to disease, even though such subjects do not manifest symptoms of the disease.

[0132] Provided herein, e.g., are novel fusion proteins that include (i) an anti-PD1 moiety, and (ii) an IL-2 moiety or an IL-15 moiety attached by a protease sensitive linker (PSL) to (iii) at least one masking and / or targeting moiety comprising an IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof. In embodiments, the IL-13 mutein, IL-13Ra2 binding mutein or IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof is capable of binding to IL-13Ra2 (e.g., an anti-IL-13Ra antibody), and does not bind to IL-13Ro1, and the protease sensitive linker is cleavable by a protease in the tumor microenvironment. Also provided herein are novel fusion proteins that include (i) an anti-PD1 moiety, and (ii) an IL-2 moiety or an IL-15 moiety attached by a protease sensitive linker (PSL) to (iii) at least one masking and / or targeting moiety comprising an IL-13 mutein, an I L-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof. In embodiments, the IL-13 mutein, IL-13Ra2 binding mutein or IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof is capable of binding to IL-13Ra2 (e.g., an anti-l L-13Ra antibody) and binds to IL-13Ra1 , and the protease sensitive linker is cleavable by a protease in the tumor microenvironment. Without being by any particular theory of operation, it is believed that the IL-13 mutein or I L-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof of the fusion protein compositions described herein associates with, obstructs, and / or masks activity of the IL-2 moiety. In addition, the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof allows the fusion protein compositions to bind to IL-13Ra2 expressing tumors. Once localized to the tumor microenvironment, the PSL of the fusion protein undergoes proteolytic cleavage, thereby releasing the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof and “unmasking” the IL-2 moiety. The unmasked IL-2 moiety can then act at the tumor site. Thus, in embodiments, the fusion proteins provided herein advantageously exhibit reduced IL-2 associated toxicity while allowing for targeted activity at IL-Attorney Docket No. 117802-5021 -WO13Ra2 expressing tumors. Such fusion protein compositions are useful for the treatment of IL-13Ra2 expressing cancers.

[0133] Also provided herein are novel IL-2 fusion proteins that include (i) an anti-PD1 moiety attached by a linker to (ii) an IL-2 moiety.

[0134] Aspects of the fusion proteins are further detailed below.A. IL-2 MOIETY

[0135] In embodiments, the fusion proteins provided herein include an IL-2 moiety that comprises a IL-2 or IL-2 mutein. In embodiments, the fusion protein includes a wild-type human IL-2 (SEQ ID NO:2). In some embodiments, the IL-2 moiety includes an IL-2 mutein that includes one or more amino acid substitutions as compared to a wild-type human IL-2 (SEQ ID NO:2). The substituted amino acid residue(s) can be, but are not necessarily, conservative substitutions, which typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In some embodiments, these mutations are at amino acid residues that contact the I L-2R0 and / or the I L-2Ry. In some embodiments, these mutations are at amino acid residues that contact the I L-2Ro.

[0136] More specifically, a mutation (whether conservative or non-conservative, by way of addition(s) or deletion(s)) can be made at one or more positions. In some embodiments, the mutation included in the IL-2 mutein is at one or more of the following amino acid positions as compared to a wild-type human IL-2 (SEQ ID NO:2): I24V, P65H, Q74R, Q74H, Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V, I92F, V93I, and combinations thereof. Exemplary IL-2 muteins that can be included in the fusion proteins are shown in Table 2 below.Table 2: Exemplary IL-2 muteinsAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WO

[0137] In some embodiments, the substitutions in the IL-2 mutein comprises T3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S amino acid substitutions, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2.

[0138] In some embodiments, the IL-2 mutein exhibits increased or enhanced IL-2R0 binding as compared to a wild-type IL-2 (e.g. , human wild-type IL-2, SEQ ID NO:2). In some embodiments, the substitutions in the IL-2 mutein that lead to increased and / or enhanced IL-2R0 binding include L80F, R81D, L85V, I86V, and I92F, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2.

[0139] In some embodiments, the IL-2 mutein having a greater binding affinity for I L-2R as compared to wild-type human IL-2, includes the amino acid substitutions T3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S. In some embodiments, the IL-2 mutein has the amino acid sequence:APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEALKPLEEVLNLA QSKNFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO:5).

[0140] In some embodiments, the IL-2 mutein has increased capabilities to stimulate one or more signaling pathways that are dependent on I L-2R / I L-2Rycheterodimerization. In some embodiments, the IL-Attorney Docket No. 117802-5021 -WO2 mutein has an enhanced capability to stimulate STAT5 phosphorylation in an IL-2R0+ cell as compared to wild-type human IL-2. In some embodiments, the IL-2 mutein stimulates STAT5 phosphorylation in an IL-2Rp+ cell at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the level that wild-type IL-2 stimulates STAT5 phosphorylation in the same cell. In some embodiments, the IL-2 mutein stimulates STAT5 phosphorylation in an IL-2R + cell at a level that is 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or more as compared to the level that wild-type IL-2 stimulates STAT5 phosphorylation in the same cell. In some embodiments, the IL-2R + cell is a T cell. In particular embodiments, the T cell is a CD8+T cell. In some embodiments, the CD8+T cell is a freshly isolated CD8+T cell. In other embodiments, the CD8+T cell T cell is an activated CD8+T cell. In other embodiments, the IL-2R + cell is a natural killer (NK) cell. In some embodiments, the IL-2 mutein comprises substitutions T3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S, as compared to wild-type human IL-2 (SEQ ID NO:2). In some embodiments, the IL-2 mutein comprises SEQ ID NO:5.

[0141] In some embodiments, the mutein has an enhanced capability to stimulate ERK1 / ERK2 signaling in an IL-2R0+ cell as compared to wild-type human IL2. In some embodiments, the IL-2 mutein stimulates pERK1 / ERK2 signaling in an IL-2R + cell at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the level that wildtype IL-2 stimulates pERK1 / ERK2 signaling in the same cell. In some embodiments, the IL-2 mutein stimulates pERK1 / ERK2 phosphorylation in an IL-2R + cell at a level that is 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or more as compared to the level that wild-type IL-2 stimulates pERK 1 / ERK2 phosphorylation in the same cell. In some embodiments, the IL-2R + cell is aT cell. In particular embodiments, the T cell is a CD8+T cell. In some embodiments, the CD8+T cell is a freshly isolated CD8+T cell. In other embodiments, the CD8+T cell T cell is an activated CD8+T cell. In other embodiments, the IL-2R0+ cell is a natural killer (NK) cell. In some embodiments, the IL-2 mutein comprises substitutions T3A, F42A, E62A, L80F, R81 D, L85V, I86V, I92F, and C125S, as compared to wild-type human IL-2 (SEQ ID NO:2). In some embodiments, the IL-2 mutein comprises SEQ ID NO:5.

[0142] STAT5 and ERK1 / 2 signaling can be measured, for example, by phosphorylation of STAT5 and ERK1 / 2 using any suitable method known in the art. For example, STAT5 and ERK1 / 2 phosphorylation can be measured using antibodies specific for the phosphorylated version of these molecules in combination with flow cytometry analysis as described herein. In some embodiments, the mutein has anAttorney Docket No. 117802-5021 -WOenhanced capability to stimulate PI 3-kinase signaling in a I L-2R0+ cell as compared to wild-type human IL2. In some embodiments, the IL-2 mutein stimulates PI 3-kinase signaling in an IL-2R + cell at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less of the level that wild-type IL-2 stimulates PI 3-kinase signaling in the same cell. In some embodiments, the IL-2 mutein stimulates PI 3-kinase signaling in an IL-2R + cell at a level that is 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or more as compared to the level that wild-type IL-2 stimulates PI 3-kinase signaling phosphorylation in the same cell. In some embodiments, the IL-2R + cell is a T cell. In particular embodiments, the T cell is a CD8+T cell. In some embodiments, the CD8+T cell T cell is an activated CD8+T cell. In other embodiments, the I L-2R + cell is a natural killer (NK) cell. PI 3-kinase signaling can be measured using any suitable method known in the art. For example, PI 3-kinase signaling can be measured using antibodies that are specific for phospho-S6 ribosomal protein in conjunction with flow cytometry analysis as described herein. In some embodiments, the IL-2 mutein comprises substitutions T3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S, as compared to wild-type human IL-2 (SEQ ID NO:2). In some embodiments, the IL-2 mutein comprises SEQ ID NO:5.

[0143] In some embodiments the IL-2 mutein is a stimulator of IL-2 and / or IL-15 STAT5 phosphorylation in CD8+T cells. In some embodiments, the mutein is a promoter of IL-2 and / or IL-15 induced proliferation of CD8+T cells. In some embodiments, the mutein is a stimulator of IL-2 dependent, TCRinduced cell proliferation. In some embodiments, the IL-2 mutein comprises substitutions T3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S, as compared to wild-type human IL-2 (SEQ ID NO:2). In some embodiments, the IL-2 mutein comprises SEQ ID NO:5.

[0144] IL-2 promotes Th1, Th9, and Treg T cell differentiation and inhibits Th17 differentiation.Therefore, without being bound by any particular theory of operation, it is believed that IL-2 muteins that function as IL-2 superagonists are capable of promoting Th1, Th9, and / or Treg cell differentiation or inhibiting Th17 cell differentiation. In some embodiments, the IL-2 mutein is a promoter of IL-2 dependent Th1, Th9 and / or Treg differentiation. In some embodiments, the mutein is an inhibitor of Th17 differentiation. In some embodiments, the IL-2 mutein comprises substitutions T3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S, as compared to wild-type human IL-2 (SEQ ID NO:2). In some embodiments, the IL-2 mutein comprises SEQ ID NO:5.Attorney Docket No. 117802-5021 -WO

[0145] In some embodiments, the IL-2 mutein signals less and / or independently of CD25 (for example, has reduced or ablated CD25 binding) as compared to wild-type human IL-2. In some embodiments the reduced and / or independent signaling with regard to CD25 allows for preferential activation of effector T-cells while limiting the stimulation of Tregs. In some embodiments the reduced and / or independent signaling with regard to CD25 allows for reduced toxicity. In some embodiments, the mutein comprises substitutions T3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S, as compared to wild-type human IL-2 (SEQ ID NO:2). In some embodiments, the IL-2 mutein comprises SEQ ID NO:5.

[0146] In some embodiments, the IL-2 mutein is capable of increasing and / or restoring responsiveness to anergic NK cells. In some embodiments, the IL-2 mutein is capable of increasing and / or restoring responsiveness to anergic NK cells in the tumor microenvironment. In some embodiments, the IL-2 mutein comprises substitutions T3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S, as compared to wild-type human IL-2 (SEQ ID NO:2). In some embodiments, the IL-2 mutein comprises SEQ ID NO:5.

[0147] In some embodiments the mutein is an inhibitor an inhibitor of IL-2 dependent activation of natural killer (NK) cells. IL-2 activation of NK cells can be measured by any suitable method known in the art, for example, by measuring IL-2 induced CD69 expression and / or cytotoxicity, as described herein.

[0148] In some embodiments, an increase in I L-2R binding affinity is any binding affinity for I L-2R0 that is greater than the wild-type human IL-2 binding affinity for IL-2R . In some embodiments, the binding affinity is a 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 170-fold, 190-fold, 200-fold, 220-fold, 240-fold or more increase in binding affinity for IL-2Rp as compared to the wild-type human IL-2 binding affinity for I L-2R .

[0149] In some embodiments, an increase in binding capacity for IL-2R is any binding capacity for IL-2Rp that is greater than the wild-type human IL-2 binding capacity for I L-2R . In some embodiments, the binding capacity is a 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 170-fold, 190-fold, 200-fold, 220-fold, 240-fold or more increase in binding capacity for I L-2RP as compared to the wild-type human IL-2 binding capacity for I L-2R .

[0150] In some embodiments, the IL-2 mutein having a greater binding affinity for I L-2RP as compared to wild-type human IL-2 also exhibits reduced binding to CD25 and includes the amino acid substitutionsAttorney Docket No. 117802-5021 -WOT3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S. . In some embodiments, the IL-2 mutein comprises SEQ ID NO:5.

[0151] In some embodiments, a reduction in binding affinity for CD25 is any binding affinity for CD25 that is less than the wild-type human IL-2 binding affinity. In some embodiments, the binding affinity is a 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 170-fold, 190-fold, 200-fold, 220-fold, 240-fold or more decrease in binding affinity for CD25 as compared to the wildtype human IL-2 binding affinity for CD25.

[0152] In some embodiments, the IL-2 mutein having a greater binding affinity for I L-2R and a reduced binding affinity for CD25 as compared to wild-type human IL-2 includes the amino acid substitutions T3A, F42A, E62A, L80F, R81D, L85V, I86V, I92F, and C125S. In some embodiments, the IL-2 mutein comprises SEQ ID NO:5.

[0153] In some embodiments, the IL-2 mutein includes a T3A amino acid substitution to eliminate glycosylation of the IL-2 mutein, which can reduce homogeneity of the IL-2 mutein and IL-2 fusion protein product. In some embodiments, the IL-2 mutein includes a C125S amino acid substitution to eliminate risk of di-sulfide bridge shuffling at this residue to reduce developability risk.

[0154] In some embodiments, the IL-2 mutein of the fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to an IL-2 mutein comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. In some embodiments, the IL-2 mutein of the fusion protein has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions as compared to an IL-2 mutein comprising the amino acid sequence of SEQ ID NO:5, SEQ ID NO: 51, SEQ ID NO: 52, or SEQ ID NO: 53. In embodiments, the variant exhibits greater binding affinity for IL-2R0 as compared to wild-type human IL-2. In embodiments, the variant also exhibits reduced binding to CD25.1. Extended Half-Life IL-2 Moieties

[0155] In some embodiments, the IL-2 moiety includes a polypeptide that increases the in vivo serum halflife of the IL-2 included in the fusion protein (i.e., a “serum half-life enhancing” or “half-life enhancing” polypeptide). In various embodiments, the half-life enhancing polypeptide is a serum albumin (e.g., human serum albumin), a PEG, a PEG-derivative, the Fc region of the IgG subclass of antibodies, or the Fc region of the IgG subclass of antibodies that lacks the IgG heavy chain variable region. Exemplary Fc regions can include a mutation that inhibits complement fixation and Fc receptor binding, or it may be lytic, i.e., able toAttorney Docket No. 117802-5021 -WObind complement or to lyse cells via another mechanism, such as antibody-dependent complement lysis (ADCC; USSN 08 / 355,502 filed Dec. 12, 1994). In some embodiments, half-life can also be extended when the IL-2 moiety is linked to an intact antibody ( / .e., anti-PD 1 antibody, anti-CD3 antibody, or other antibody).

[0156] In some embodiments, the half-life enhancing polypeptide is an Fc region. The “Fc region” can be a naturally occurring or synthetic polypeptide that is homologous to the IgG C-terminal domain produced by digestion of IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. The IL-2 moiety of the fusion protein can include the entire Fc region, or a smaller portion that retains the ability to extend the circulating half-life of the IL-2 moiety of which it is a part. In addition, full-length or fragmented Fc regions can be variants of the wild-type molecule. In some embodiments, the IL-2 moiety of the fusion protein includes an lgG1, lgG2, lgG3, or lgG4 Fc region. In some embodiments, the Fc region is a human lgG1, lgG2, lgG3, or lgG4 Fc region. In some embodiments, the Fc region of the IL-2 moiety includes a monomer that include a CH2-CH3 domain of a human lgG1, lgG2, lgG3 or lgG4 region. In some embodiments, the Fc region of the IL-2 moiety is dimeric and includes two monomers that each include a CH2-CH3 domain of a human lgG1, lgG2, lgG3 or lgG4 region. In some embodiments, the IL-2 moiety includes two monomers, wherein each monomer includes an IL-2 or IL-2 mutein linked to an Fc region monomer that includes a CH2-CH3 domain. In such embodiments, the Fc region monomers dimerize to form a dimeric IL-2 moiety. In some embodiments, the IL-2 moiety includes two monomers, where one monomer includes an IL-2 or IL-2 mutein linked to a first Fc region monomer that includes a CH2-CH3 domain, and the second monomer includes a second Fc region monomer, and does not include an IL-2 or IL-2 mutein. The first and second Fc regions dimerize to form a dimeric Fc region. In embodiments that include a dimeric Fc region, each of the two monomers may include amino acid substitutions that favor the formation of heterodimers (e.g., “knobs-into-holes” or “knobs and holes” amino acid substitutions, see, e.g., US Patent No. 8,216,805, which is incorporated by reference herein for disclosures relating into “knobs and holes” amino acid substitutions). In some embodiments, the Fc region comprises the substitution N297A.

[0157] The Fc region can be “lytic” or “non-lytic,” but is typically non-lytic. A non-lytic Fc region typically lacks a high affinity Fc receptor binding site and a C'1q binding site. The high affinity Fc receptor binding site of murine IgG Fc includes the Leu residue at position 235 of IgG Fc. Thus, the Fc receptor binding site can be destroyed by mutating or deleting Leu 235. For example, substitution of Glu for Leu 235 inhibits the ability of the Fc region to bind the high affinity Fc receptor. The murine C'1q binding site can be functionally destroyed by mutating or deleting the Glu 318, Lys 320, and Lys 322 residues of IgG. For example, substitution of Ala residues for Glu 318, Lys 320, and Lys 322 renders lgG1 Fc unable to direct antibody-Attorney Docket No. 117802-5021 -WOdependent complement lysis. In contrast, a lytic IgG Fc region has a high affinity Fc receptor binding site and a C'1q binding site. The high affinity Fc receptor binding site includes the Leu residue at position 235 of IgG Fc, and the C'1q binding site includes the Glu 318, Lys 320, and Lys 322 residues of lgG1. Lytic IgG Fc has wild-type residues or conservative amino acid substitutions at these sites. Lytic IgG Fc can target cells for antibody dependent cellular cytotoxicity or complement directed cytolysis (CDC). Appropriate mutations for human IgG are also known (see, e.g., Morrison et al., The Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2: 125, 1994).

[0158] In some embodiments, the IL-2 of the IL-2 moiety (e.g., wild-type IL-2 or an IL-2 mutein provided herein) is linked directly or indirectly to the serum half-life enhancing polypeptide. In some embodiments, the half-life enhancing polypeptide is linked to the N-terminus of the IL-2 (e.g., wild-type IL-2 or an IL-2 mutein provided herein). In some embodiments, the half-life enhancing polypeptide is linked to the to the C-terminus of the IL-2 (e.g., wild-type IL-2 or an IL-2 mutein provided herein). In some embodiments, the IL-2 or IL-2 mutein is linked directly to the half-life enhancing polypeptide. In some embodiments, the IL-2 or IL-2 mutein is linked to the half-life enhancing polypeptide via a linker peptide, such as GGGGS. In some embodiments, the linker is (GGGGS)n, wherein n is an integer between 1 and 10. In some embodiments, the linker is GGGGS (SEQ ID NO:401). In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO:402). In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO:403). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NQ:404). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NQ:405).

[0159] In some embodiments, the linker contains one or more protease cleavage sites (e.g., is a protease cleavable linker). Linkers additionally can contain one or more protease cleavage sites or be sensitive to cleavage via oxidation and / or reduction. Peptide linkers that are susceptible to cleavage by enzymes of the complement system, urokinase, tissue plasminogen activator, trypsin, plasmin, caspases, kallikreins, cathepsins, legumain, MMPs, thrombin, urokinase-type plasminogen activator (uPA), matriptase, prostatespecific antigen (PSA), or another enzyme having proteolytic activity may be used in one example.According to another example, a linker may comprise disulfide bonds (for example, the disulfide bonds on a cysteine molecule). According to another example, a linker may comprise a protease-cleavable Pro-Leu-Gly-Leu-Trp-Ala linker (SEQ ID NQ:300), a protease-cleavable Pro-Ala-Gly-Leu-lle-Gly linker (SEQ ID NQ:301 ), a protease-cleavable Val-Cit (VC) linker, a Phe-Arg linker, a Val-Lys linker, a Val-Ala linker, a Val-Arg linker, a Val-Leu-Lys linker, a Gly-Phe-Leu-Gly linker, an Ala-Phe-Lys linker, a pol-L-lysine linker, a beta-Ala-Leu-Ala-Leu linker, an Arg-Arg-Ala-Leu-Ala-Leu linker (SEQ ID NQ:302), a peptidomimetic linker,Attorney Docket No. 117802-5021 -WOa legumain-cleavable Ala-Ala-Asn tripeptide linker, a peptide linker that is cleaved by cathepsin B and other lysosomal proteases, such as Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu, a caspase 3 DEVD sequence, or a self-immolative linker. For example, linkers disclosed in Poreba, M, FEBS J. 287(10): 1936-1969 (2020), incorporated by reference herein, are contemplated by the present disclosure. Since many tumors naturally release high levels of glutathione (a reducing agent) this can reduce the disulfide bonds with subsequent release of the cargo moiety at the site of delivery. In some embodiments, the linkers is a protease-cleavable linkers is a linker cleavable by a matrix metalloprotease (MMP). MMPs are overexpressed in situ at tumors, and linkers cleavable in such contexts are contemplated by the presentation disclosure. For example, linkers disclosed in Hsu, E.J, etal., Nat. Commun. 12(2768): 1-13 (2021), incorporated by reference herein, are contemplated by the present disclosure. In some embodiments, the MMP linker sequence is selected from the group consisting of SGARYRWLTA (SEQ ID NO:406), SGRSYAILTA (SEQ ID NO:407), SRSGRSPAIFTATG (SEQ ID NQ:408), GSSGRSPAIFTAGS (SEQ ID NQ:409), and SGFIANPVTA (SEQ ID NQ:410). In some embodiments, the MMP linker sequence is SGARYRWLTA (SEQ ID NO:411). In some embodiments, the MMP linker sequence is SGRSYAILTA (SEQ ID NO:412). In some embodiments, the MMP linker sequence is SRSGRSPAIFTATG (SEQ ID NO:413). In some embodiments, the MMP linker sequence is GSSGRSPAIFTAGS (SEQ ID NO:414). In some embodiments, the MMP linker sequence is SGFIANPVTA (SEQ ID NO:415). In some embodiments, the MMP linker sequence is PLGLWAPLGLWAPLGLWA (SEQ ID NO:416). In some embodiments, the MMP linker sequence is PLGLWAPLGLWAPLGLWA (SEQ ID NO:417). In some embodiments, the MMP linker sequence is GGSGGTPLGLWAGGSGGT (SEQ ID NO:418). In some embodiments, the MMP linker sequence is GGSGGTPAGLIGGGSGGT (SEQ ID NO:419). In some embodiments, the MMP linker sequence is GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NQ:420). In some embodiments, the PSA linker sequence is GGSGGTHSSKLQGGSGGT (SEQ ID NO:421).

[0160] In some embodiments, the IL-2 moiety includes a polypeptide that functions as an antigenic tag, such as a FLAG sequence. FLAG sequences are recognized by biotinylated, highly specific, anti-FLAG antibodies, as described herein (see also Blanaret al., Science 256:1014, 1992; LeClair etal., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In some embodiments, the IL-2 moiety further comprises a C-terminal c-myc epitope tag.

[0161] In other embodiments, the IL-2 moiety includes a heterologous polypeptide that functions to enhance expression or direct cellular localization of the IL-2 moiety, such as the Aga2p agglutinin subunit (see, e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).Attorney Docket No. 117802-5021 -WO2. I L-2 / Antibody Fusion Moieties

[0162] In other embodiments, the IL-2 moiety of the fusion proteins provided herein includes an antibody or antigen-binding portion thereof (i.e., an “I L-2 / antibody fusion moiety”).

[0163] In some embodiments, the IL-2 / antibody fusion comprises three polypeptides. The first polypeptide comprises an IL-2 or IL-2 mutein attached to a first Fc region. The second polypeptide comprises a heavy chain variable region (VH) and a second Fc region. The third polypeptide comprises a light chain variable region (VL) and a light chain constant region. In this embodiment, the two Fc regions of the first and second polypeptides dimerize, and the VH and VL form an antigen binding domain.

[0164] In some embodiments of the IL-2 / antibody fusion moiety, the IL-2 / antibody fusion moiety includes four polypeptides. The first polypeptide comprises a first antibody heavy chain attached to an IL-2 or IL-2 mutein, wherein the first antibody heavy chain comprises a first heavy chain variable region (VH1) and a first Fc region. The second polypeptide comprises a second antibody heavy chain, wherein the second antibody heavy chain comprises a second heavy chain variable region (VH2) and a second Fc region. The third polypeptide comprises a first variable light chain variable region (VL1) and a light chain constant region. The fourth polypeptide comprises a second variable light chain variable region (VL2) and a light chain constant region. In this embodiment, the two Fc regions of the first and second polypeptides dimerize, and the VH1 and VL1, and VH2 and VL2 each form an antigen binding domain (i.e., a first antigen binding domain and a second antigen binding domain). In some embodiments, the first and second antigen binding domains are the same. In some embodiments, the first and second antigen binding domains are different. In some embodiments the third and fourth polypeptides are the same.

[0165] The antibody or antigen-binding component is attached to the IL-2 or IL-2 mutein using any suitable technique. In some embodiments, the antibody or antigen-binding component is directly linked to the IL-2 or IL-2 mutein. In some embodiments, the antibody or antigen-binding component is linked to the IL-2 or IL-2 mutein using a linker. Any suitable linker, including the linkers described herein can be used to attach the IL-2 or IL-2 mutein to the antibody or antigen-binding component. In embodiments, the linker is a linker peptide having the formula (GGGGS)n, wherein n is an integer between 1 and 10. In some embodiments, the linker is GGGGS (SEQ ID NO:401). In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO:402). In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NQ:403). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NQ:404). In someAttorney Docket No. 117802-5021 -WOembodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:405). In some embodiments, the IL-2 or IL-2 mutein is attached to the C-terminus of an Fc domain of the antibody or antigen binding component.

[0166] The antibody or antigen-binding component of the IL-2 moiety can serve as a targeting moiety. For example, it can be used to localize the chimeric protein to a particular subset of cells or target molecule. Methods of generating cytokine-antibody chimeric polypeptides are described, for example, in U.S. Pat. No.6,617,135.

[0167] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that disrupts the interaction between the PD-1 receptor and its ligand, PD-L1, or PD-L2 and / or is an antibody to a component of the PD-1 / PD-L1 / PD-L2 signaling pathway. Antibodies known in the art which bind to PD-1 and disrupt the interaction between the PD-1 and its ligand, PD-L1 or PD-L2, and stimulate an anti-tumor immune response, are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the antibody or antigen-binding portion thereof binds specifically to PD-1. For example, antibodies that target PD-1 and which can find used in the present invention include, e.g., but are not limited to nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), Pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal Antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (cemiplimab, Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD-1 lgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is from clone: RMP1-14 (rat IgG) - BioXcell cat# BP0146. Other suitable antibodies include anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, herein incorporated by reference. In some embodiments, the antibody or antigen-binding portion thereof binds specifically to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Any antibodies known in the art which bind to PD-L1 and disrupt the interaction between the PD-1 and PD-L1, and stimulates an antitumor immune response, are suitable for use in the chimeric polypeptides disclosed herein. For example, antibodies that target PD-L1 and are in clinical trials, include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genentech). Other suitable antibodies that target PD-LI are disclosed in U.S. Patent No. 7,943,743, herein incorporated by reference. It will be understood by one of ordinary skill that any antibody which binds to PD-1 or PD-L1 or PD-L2, disrupts the PD-1 / PD-L1 / PD-L2 interaction, and stimulates an antitumor immune response, is suitable for use in the IL-2 moiety disclosed herein. In some embodiments, theAttorney Docket No. 117802-5021 -WOIL-2 moiety comprises a fusion to an anti-PD-1 antibody. In some embodiments, the IL-2 moiety comprises a fusion to an anti-PD-L1 antibody. In some embodiments, the IL-2 moiety comprises a fusion to an anti-PD-L2 antibody.

[0168] Exemplary IL-2 / anti-PD-1 antibody fusion moieties that can be included in the fusion proteins are shown in Table 3. In some embodiments, the IL-2 / anti-PD-1 antibody fusion moieties of the fusion protein has the amino acid of one of Table 3. In some embodiments the IL-2 / anti-PD-1 antibody fusion moieties of the fusion protein is a variant of one of the I L-2 / anti-PD-1 antibody fusion moieties in Table 3. In some embodiments, the I L-2 / anti-P D- 1 antibody fusion moieties of the fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to one of the I L-2 / anti-P D- 1 antibody fusion moieties in Table 3. In some embodiments, the IL-2 of the fusion protein has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions as compared to any one of the IL-2 / anti-PD-1 antibody fusion moieties in Table 3. In embodiments, the variant exhibits greater binding affinity for I L-2RP as compared to wild-type human IL-2. In embodiments, the variant also exhibits reduced binding to CD25. In some embodiments, the variant is capable of binding to PD-1 (human PD-1).Table 3: 1 L-2 / anti-PD1 fusion moietiesAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOB. IL-15 MOIETY

[0169] In embodiments, the fusion proteins provided herein include an IL-15 moiety that comprises a IL-15 or IL-15 mutein. In embodiments, the IL-15 fusion protein includes a full length human IL-15 (SEQ ID NO:100). In embodiments, the IL-15 fusion protein includes a mature human IL-15 (SEQ ID NO:101). In some embodiments, the IL-2 moiety includes an IL-2 mutein that includes one or more amino acid substitutions as compared to a full length or a mature human IL-15 (SEQ ID NO:100 or 101). The substituted amino acid residue(s) can be, but are not necessarily, conservative substitutions, which typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0170] Full-length human IL-15:MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYT ESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFL QSFVHIVQMFINTS (SEQ ID NQ:100)

[0171] Mature human IL-15:NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLS SNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:101).Attorney Docket No. 117802-5021 -WOC. IL-13 MOIETY

[0172] In embodiments, an IL-13 mutein acts as a masking moiety. As used herein, a “masking moiety” is a component of a polypeptide or fusion construct that reduces the ability of a ligand, an antibody, or an antigen-binding fragment thereof to specifically bind to its target. In embodiments, the masking moiety reduces the activity of the masked component. In embodiments, the IL-13 mutein masks the activity of an IL-2 moiety. In embodiments, the IL-13 mutein masks the activity of an IL-15 moiety. In embodiments, the IL-13 mutein masks the activity of an IL-12 moiety. In embodiments, the IL-13 mutein masks the binding of an antibody or antigen binding domain of an antibody, and in some embodiments the IL-13 mutein masks the binding of an anti-CD3 antibody and / or antigen binding domains of anti-CD3 antibodies.

[0173] In embodiments, an IL-13 mutein acts as a targeting moiety. As used herein, a “targeting moiety” is a component of a polypeptide or fusion construct that binds to a component associated with a cell. In embodiments, the IL-13 mutein targets the fusion proteins to the tumor microenvironment.

[0174] The fusion proteins provided herein include an IL-13 mutein, an IL-13Ra2 binding mutein, or an IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof, any of which can in some embodiments be linked to the IL-2 moiety or another moiety in the fusion (including for example an anti-PD-1 antibody) by a protease sensitive linker (PSL). When attached to the IL-13 mutein or I L-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof, the IL-2 moiety has reduced IL-2 activity, i.e., the IL-2 moiety is “masked” by the IL-13 mutein, IL-13Ra2 binding mutein, IL-13Ra2 binding mutein or an I L-13Ra2 antibody or antigen-binding fragment, or type II IL-4 receptor antagonist thereof. In some embodiments, the IL-13 mutein, IL-13Ra2 binding mutein, IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof also acts as a targeting moiety, as it is capable of binding to I L-13Ra2 expressing tumors, thereby localizing the fusion protein to the tumor microenvironment. Once localized to the tumor microenvironment, the protease sensitive linker is cleaved by a protease, thereby releasing the IL-13 mutein, I L-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof from the fusion protein. In some embodiments, the “unmasked” IL-2 moiety is then active in the tumor microenvironment, thereby promoting IL-2 anti-tumor activity. In some embodiments, the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof is attached to the IL-2 mutein of the IL-2 moiety by the PSL linker. In some embodiments, the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof is attached to a region of the IL-2 moiety that is not the IL-2 mutein. For example, in some embodiments wherein the IL-2 moiety further includes a half-lifeAttorney Docket No. 117802-5021 -WOenhancing polypeptide (e.g., a serum albumin or an Fc region) or another moiety (e.g., an antibody, e.g., an anti-PD1 antibody), or an additional masking moiety, the IL-13 mutein or I L-13Ra2 antibody or antigenbinding fragment or type II IL-4 receptor antagonist thereof is attached to the half-life enhancing polypeptide, the other moiety, or the additional masking moiety of the IL-2 moiety by the PSL. In some embodiments wherein the IL-2 moiety includes a dimeric Fc region, the IL-2 mutein is attached to one of the Fc region monomers, and the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment or type II IL-4 receptor antagonist thereof is attached to the other Fc region monomer by a PSL.

[0175] In some embodiments, the fusion protein includes two or more IL-13 muteins or IL-13Ra2 antibodies or antigen-binding fragments thereof. In some embodiments, at least one of the IL-13 muteins or IL-13Ra2 antibodies or antigen-binding fragments thereof is attached to the IL-2 mutein of the IL-2 moiety by a PSL. In some embodiments, at least one of the IL-13 muteins or I L-13Ra2 antibodies or antigen-binding fragments or type II IL-4 receptor antagonist thereof is attached to a region of the IL-2 moiety that is not the IL-2 mutein (e.g., a serum albumin, an Fc region, an antibody, e.g., an anti-PD1 antibody, an additional masking moiety).1. IL-13 Mutein

[0176] In embodiments, the IL-13 mutein comprises one or more amino acid substitutions as compared to a wild-type human IL-13:

[0177] PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQR MLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN (SEQ ID NO:200)

[0178] Suitable IL-13 muteins include those that are capable of reducing IL-2 activity of the IL-2 moiety when attached to the fusion protein via the PSL. In some embodiments, the IL-13 mutein is capable of binding to I L-13Ra2 receptor (e.g., an anti-IL-13Ra antibody). In some embodiments, the IL-13 mutein is capable of binding to IL-13Ra1 receptor (e.g., an anti-IL-13Ra antibody). In some embodiments, the IL-13 mutein exhibits enhanced binding to the IL-13Ra2 receptor, and reduced binding to the IL-13Ra1 receptor as compared to wild-type IL-13 (e.g., SEQ ID NQ:200). In exemplary embodiments, the IL-13 mutein is capable of binding to the IL-13Ra2 receptor, and not capable of binding to the IL-13Ra1 receptor.

[0179] In some embodiments an IL-13 mutein of the fusion protein comprises one or more of the amino acids substitutions: (1) L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, K105T (e.g., SEQ ID NQ:201); or (2) L10H, E15R, R86T, D87G, T88R, R108K, Q111 (e.g., SEQ ID NQ:202), which substitutions cause an altered affinity for one or both of IL-13Ra1 and I L-13Ra2.Attorney Docket No. 117802-5021 -WO

[0180] Suitable exemplary IL-13 muteins for use in the fusion proteins described herein are shown in Table 4.Table 4: IL-13 wildtype and muteins

[0181] In some embodiments, the IL-13 mutein of the fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to one of the IL-13 muteins in Table 4. In some embodiments, the IL-13 mutein of the fusion protein has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions as compared to any one of the IL-13 muteins in Table 4 (SEQ ID NO:201 -202). In embodiments, the IL-13 mutein exhibits greater selectivity in binding to I L-13Ro2 versus IL-13Ro1 relative to a wild-type IL-13 sequence (e.g., SEQ ID NQ:200). In embodiments, the IL-13 mutein exhibits increased binding affinity to IL-13Ra1 and decreased binding affinity to IL13Ra2 relative to a wild-type IL-13 sequence (e.g., SEQ ID NQ:200).2. IL-13 M utei n / Anti body Fusion

[0182] In other embodiments, the IL-13 mutein of the fusion proteins provided herein includes an antibody or antigen-binding portion thereof.

[0183] In some embodiments, the IL-13 mutein / antibody fusion comprises three polypeptides. The first polypeptide comprises an IL-13 mutein attached to a first Fc region. The second polypeptide comprises an heavy chain variable region (VH) and a second Fc region. The third polypeptide comprises a light chain variable region (VL) and a light chain constant region. In this embodiment, the two Fc regions of the first and second polypeptides dimerize, and the VH and VL form an antigen binding domain.Attorney Docket No. 117802-5021 -WO

[0184] In some embodiments the IL-13 mutein / antibody fusion includes four polypeptides. The first polypeptide comprises a first antibody heavy chain attached to an IL-13 mutein, wherein the first antibody heavy chain comprises a first heavy chain variable region (VH1) and a first Fc region. The second polypeptide comprises a second antibody heavy chain, wherein the second antibody heavy chain comprises a second heavy chain variable region (VH2) and a second Fc region. The third polypeptide comprises a first variable light chain variable region (VL1) and a light chain constant region. The fourth polypeptide comprises a second variable light chain variable region (VL2) and a light chain constant region. In this embodiment, the two Fc regions of the first and second polypeptides dimerize, and the VH1 and VL1, and VH2 and VL2 each form an antigen binding domain. In some embodiments the third and fourth polypeptides are the same.

[0185] The antibody or antigen-binding component is attached to the IL-13 mutein using any suitable technique. In some embodiments, the antibody or antigen-binding component is directly linked to the IL-13 mutein. In some embodiments, the antibody or antigen-binding component is linked to the IL-13 mutein using a linker. Any suitable linker, including the linkers described herein can be used to attach the IL-13 mutein to the antibody or antigen-binding component. In embodiments, the linker is a linker peptide having the formula (GGGGS)n, wherein n is an integer between 1 and 10. In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO:402). In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO:403). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:404). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NQ:405). In some embodiments, the IL-13 mutein is attached to the C-terminus of an Fc domain of the antibody or antigen binding component. In some embodiments, the IL-13 mutein is attached to N-terminus of the antibody or antigen binding component.

[0186] In some embodiments, the IL-13 mutein comprises a fusion to an antibody or an antigen-binding portion thereof that disrupts the interaction between the PD-1 receptor and its ligand, PD-L1, and / or is an antibody to a component of the PD-1 / PD-L1 signaling pathway.

[0187] In some embodiments, the IL-13 mutein comprises a fusion to an antibody or an antigen-binding portion thereof comprising an anti-CD3 antibody or antigen binding portion thereof and / or an antibody to a component of a CD3 signaling pathway.Attorney Docket No. 117802-5021 -WO

[0188] In some embodiments, the IL-13 mutein comprises a fusion to an IL-12 that disrupts the interaction between the IL-12 receptor, including IL-12R 1 and / or IL-12R02, and its ligand, IL-12.3. Additional Components

[0189] In some embodiments, the masking moieties further comprise a protein binding domain.

[0190] In some embodiments, the protein binding domain is an extracellular domain of CD122, CD132 or CD25.

[0191] In some embodiments, the protein binding domain is an albumin binding domain (ABD), a transferrin binding domain (ABD).

[0192] In some embodiments, the protein binding domain is an IL-13Ra2 domain, an IL-13Ra1 domain, or an IL-4Ra domain.

[0193] In some embodiments, the masking moieties further comprise an extracellular domain of CD122, CD132 or CD25.

[0194] Suitable extracellular domains of CD122, CD132 or CD25 include those that are capable of reducing IL-2 activity of the IL-2 moiety when attached to the fusion protein via the PSL.

[0195] In some embodiments, the extracellular domain of CD122 has the following amino acid sequence:

[0196] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACN LILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHY FERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFR TKPAALGKDT (SEQ ID NO: 350).

[0197] In some embodiments, the extracellular domain of CD132 has the following amino acid sequence:

[0198] LPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQL QKKEIHLYQTFWQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQ YRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKEN(SEQ ID NO: 351).

[0199] In some embodiments, the extracellular domain of CD25 has the following amino acid sequence:Attorney Docket No. 117802-5021 -WO

[0200] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTS SATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQC VQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 352).

[0201] In some embodiments, the masking moieties further comprise an albumin binding domain (ABD). In some embodiments, the ABD polypeptide is a circularly permuted variant of a Protein G-derived albumin binding domain (Uniprot: Q51911) in which the N-terminus corresponds to residue 21 of the parent sequence and the C-terminus precedes residue 21, the termini being joined by a CCSG linker. In some embodiments, the albumin binding domain has the following amino acid sequence:

[0202] AGITSDFYFNAINKAKTVEEVNALKNEILKAHAGGSGTIDQWLLKNAKEDAIAELKK (cp21-ABD, SEQ ID NO: 600).C. PROTEASE SENSITIVE LINKER (PSL)

[0203] The fusion proteins provided herein include a protease sensitive linker (PSL) that attaches the IL-2 moiety and the IL-13 mutein. In some embodiments, the PSL is capable of undergoing cleavage in the tumor microenvironment, thereby “unmasking” the IL-2 moiety. The unmasked IL-2 moiety then promotes antitumor activity in the tumor microenvironment. Exemplary PSLs that can be included in the fusion proteins include PLGLWA (SEQ ID NQ:300), PAGLIG (SEQ ID NQ:301), PLGLVVAPLGLVVAPLGLWA (SEQ ID NO:416), PLGLWAPLGLWAPLGLWA (SEQ ID NO:417) , GGSGGTPLGLWAGGSGGT (SEQ ID NO:418), GGSGGTPAGLIGGGSGGT (SEQ ID NO:419), GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NQ:420), GGSGGTHSSKLQGGSGGT (SEQ ID NO:421), or GGSGGTPLGLWAAEEAHSSKLQGSGG (SEQ ID NO: 422).

[0204] PSLs additionally can contain one or more protease cleavage sites or be sensitive to cleavage via oxidation and / or reduction. Peptide linkers that are susceptible to cleavage by enzymes of the complement system, urokinase, tissue plasminogen activator, trypsin, plasmin, caspases, kallikreins, cathepsins, legumain, MMPs, thrombin, urokinase-type plasminogen activator (uPA), matriptase, PSA, or another enzyme having proteolytic activity may be used in one example. According to another example, a linker may comprise disulfide bonds (for example, the disulfide bonds on a cysteine molecule). According to another example, a PSL may comprise a protease-cleavable Val-Cit (VC) linker, a Phe-Arg linker, a Val-Lys linker, a Val-Ala linker, a Val-Arg linker, a Val-Leu-Lys linker, a Gly-Phe-Leu-Gly linker, an Ala-Phe-Lys linker, a pol-L-lysine linker, a beta-Ala-Leu-Ala-Leu linker, an Arg-Arg-Ala-Leu-Ala-Leu linker (SEQ ID NQ:302), a peptidomimetic linker, a legumain-cleavable Ala-Ala-Asn tripeptide linker, a peptide linker thatAttorney Docket No. 117802-5021 -WOis cleaved by cathepsin B and other lysosomal proteases, such as Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu, a caspase 3 DEVD sequence, or a self-immolative linker. For example, linkers disclosed in Poreba, M, FEBS J. 287(10): 1936-1969 (2020), incorporated by reference herein, are contemplated by the present disclosure. Since many tumors naturally release high levels of glutathione (a reducing agent) this can reduce the disulfide bonds with subsequent release of the cargo moiety at the site of delivery. In some embodiments, the PSL is a protease-cleavable linkers is a linker cleavable by a matrix metalloprotease (MMP). MMPs are overexpressed in situ at tumors, and linkers cleavable in such contexts are contemplated by the presentation disclosure. For example, linkers disclosed in Hsu, E.J., et al., Nat.Commun. 12(2768): 1 -13 (2021), incorporated by reference herein, are contemplated by the present disclosure. In some embodiments, the MMP linker sequence is selected from the group consisting of SGARYRWLTA (SEQ ID NO:406), SGRSYAILTA (SEQ ID NO:407), SRSGRSPAIFTATG (SEQ ID NO:408), GSSGRSPAIFTAGS (SEQ ID NO:409), and SGFIANPVTA (SEQ ID NQ:410). In some embodiments, the MMP linker sequence is SGARYRWLTA (SEQ ID NO:411). In some embodiments, the MMP linker sequence is SGRSYAILTA (SEQ ID NO:412). In some embodiments, the MMP linker sequence is SRSGRSPAIFTATG (SEQ ID NO:413). In some embodiments, the MMP linker sequence is GSSGRSPAIFTAGS (SEQ ID NO:414). In some embodiments, the MMP linker sequence is SGFIANPVTA (SEQ ID NO:415). In some embodiments, the MMP linker sequence is PLGLWAPLGLWAPLGLWA (SEQ ID NO:416). In some embodiments, the MMP linker sequence is PLGLWAPLGLWAPLGLWA (SEQ ID NO:417). In some embodiments, the MMP linker sequence is GGSGGTPLGLWAGGSGGT (SEQ ID NO:418). In some embodiments, the MMP linker sequence is GGSGGTPAGLIGGGSGGT (SEQ ID NO:419). In some embodiments, the MMP linker sequence is GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NQ:420). In some embodiments, the PSA linker sequence is GGSGGTHSSKLQGGSGGT (SEQ ID NO:421). In some embodiments, the linker sequence is GGSGGTPLGLWAAEEAHSSKLQGSGG (SEQ ID NO: 422).D. EXEMPLARY IL-2 FUSION PROTEINS

[0205] In some embodiments, the fusion protein includes an IL-2 moiety that includes an IL-2 mutein according to SEQ ID NO:5 (MDNA109FEAAT3AC125s). In some embodiments, the fusion protein includes an IL-2 moiety that includes an IL-2 mutein according to SEQ ID NO:51 (MDNA109FEAA). In some embodiments, the fusion protein includes an IL-2 moiety that includes an IL-2 mutein according to SEQ ID NO:52 (MDNA109T3AC125s). In some embodiments, the fusion protein includes an IL-2 moiety that includes an IL-2 mutein according to SEQ ID NO:53 (MDNA109).Attorney Docket No. 117802-5021 -WO

[0206] In embodiments, the fusion protein comprises an anti-PD-1 antibody or antigen binding portion thereof.

[0207] In some embodiments, the fusion protein includes an IL-13 having the amino acid sequence of SEQ ID NO:201 (MDNA413-R39 / Q111 , also referred to as MDNA413) or SEQ ID NO:202 (MDNA132.15-L39 / Q111, also referred to as MDNA213). In exemplary embodiments, the PSL has the amino acid sequence of PLGLWA (SEQ ID NQ:300) or PAGLIG (SEQ ID NQ:301).

[0208] In some embodiments, the fusion protein includes: an anti-PD-1 antibody linked to an IL-2 mutein, masked by i) an IL-13 Ra2 selective IL-13 superkine linked to the anti-PD-1 via a first PSL and ii) an extracellular domain of CD122 linked to the IL-2 mutein via a second PSL.

[0209] In some embodiments, the anti-PD-1 antibody is a human anti-PD-1 based on either Nivolumab or Pembrolizumab with an I gG1 or I gG4 backbone.

[0210] In some embodiments, the IL-13 superkine has the amino acid sequence of SEQ ID NO: 202 (MDNA132.15-L39 / Q111, also referred to as MDNA213).

[0211] In some embodiments, the anti-PD-1 antibody is a human anti-PD-1 based on either Nivolumab or Pembrolizumab with an I gG1 or lgG4 backbone, the IL-2 mutein has the amino acid sequence of SEQ ID NO: 5 (MDNA109FEAAT3AC125s), the IL-13 superkine has the amino acid sequence of SEQ ID NO: 202 (MDNA213), the PSL has the amino acid sequence of PLGLWA (SEQ ID NQ:300), and the extracellular domain of CD122 has the amino acid sequence of SEQ ID NO: 350.

[0212] In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NOs: 83-85.

[0213] In some embodiments, the fusion protein has the amino acid sequence of SEQ ID NOs: 88-91.

[0214] Exemplary IL-2 fusion proteins are shown in Table 5.Table 5: Exemplary IL-2 fusion protein>Attorney Docket No. 117802-5021 -WO>>>>Attorney Docket No. 117802-5021 -WO>>>> Attorney Docket No. 117802-5021 -WO>>>>Attorney Docket No. 117802-5021 -WO>>>>> Attorney Docket No. 117802-5021 -WO>>>Attorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WO>>Attorney Docket No. 117802-5021 -WO>>>>>Attorney Docket No. 117802-5021 -WO>>>>Attorney Docket No. 117802-5021 -WO>Attorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOE. EXEMPLARY IL-13 FUSION PROTEINS

[0215] In some embodiments, the fusion protein includes an IL-13 having the amino acid sequence of SEQ ID NO:201 (MDNA413-R39 / Q111 , also referred to as MDNA413) or SEQ ID NO:202 (MDNA132.15-L39 / Q111, also referred to as MDNA213). In exemplary embodiments, the fusion protein includes an IL-13, a cleavage site, and one or more antibodies, antibody fragments, antibody scFv domains, or cytokines. In an embodiment, the antibody, antibody fragment, or antibody scFv domain is an anti-PD-1 antibody, an anti-VEGF antibody, antibody fragment, or antibody scFv domain, an anti-CTLA4 antibody, antibody fragment, or antibody scFv domain, or an anti-CD3 antibody, antibody fragment, or antibody scFv domain. In an embodiment, the cytokine is an IL-18 cytokine. In an embodiment, the fusion protein includes an IL-13 and a hybrid anti-PD-1 and anti-VEGF antibody.

[0216] Exemplary IL-13 fusion proteins and corresponding control constructs are shown in Table 6. Table 6: Exemplary IL-13 fusion proteins>>Attorney Docket No. 117802-5021 -WO>>>>>Attorney Docket No. 117802-5021 -WO>>Attorney Docket No. 117802-5021 -WO> >>>>Attorney Docket No. 117802-5021 -WO>>>>Attorney Docket No. 117802-5021 -WO>Attorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOAttorney Docket No. 117802-5021 -WOF. NUCLEIC ACID MOLECULES ENCODING FUSION PROTEINS

[0217] In some embodiments, the fusion proteins, such as those described above, can be obtained by expression of one or more nucleic acid molecules. For example, in embodiments wherein the fusion is a single polypeptide, the fusion protein is encoded by a single nucleic acid molecule. In other embodiments wherein the fusion protein is made up of two or more polypeptides, each of the two or more polypeptides is encoded by a nucleic acid molecule.

[0218] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded ( / .e., either a sense or an antisense strand).

[0219] The nucleic acid molecules are not limited to sequences that encode polypeptides; some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g., the coding sequence of IL-2, or IL-13) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription.

[0220] Exemplary isolated nucleic acid molecules of the present disclosure can include fragments not found as such in the natural state. Thus, this disclosure encompasses recombinant molecules, such as those in which a nucleic acid sequence (for example, a sequence encoding a mutant IL-2, or IL-13) is incorporated into a vector (e.g., a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location).

[0221] A nucleic acid molecule can contain sequences encoding a “marker” or “reporter.” Examples of marker or reporter genes include p-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neor, G418r), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacz (encoding 0-galactosidase), andAttorney Docket No. 117802-5021 -WOxanthine guanine phosphoribosyltransferase (XGPRT). One of skill in the art will be aware of additional useful reagents, for example, of additional sequences that can serve the function of a marker or reporter.

[0222] The nucleic acids (and the polypeptides they encode) can be those of a mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog, or cat. In one embodiment, the nucleic acid molecules will be those of a human.G. EXPRESSION OF FUSION PROTEINS

[0223] The nucleic acid molecules described above can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transduced with the vector. Accordingly, in addition to the fusion proteins, expression vectors containing one or more nucleic acid molecules encoding a fusion protein and cells transfected with these expression vectors are among the preferred embodiments.

[0224] It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector’s copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, vectors that can be used include those that allow the DNA encoding the fusion protein to be amplified in copy number. Such amplifiable vectors are well known in the art. They include, for example, vectors able to be amplified by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, “Construction of a Modular Dihydrafolate Reductase cDNA: Analysis of Signals Utilized for Efficient Expression”, Mol. Cell. Biol., 2, pp.1304-19 (1982)) or glutamine synthetase (“GS”) amplification (see, e.g., U.S. Pat. No. 5,122,464 and European published application 338,841).

[0225] In some embodiments, the fusion protein will be expressed from vectors, preferably expression vectors. The vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., nonepisomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms ofAttorney Docket No. 117802-5021 -WOexpression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) are included also.

[0226] Exemplary recombinant expression vectors can include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, operably linked to the nucleic acid sequence to be expressed.

[0227] The expression constructs or vectors can be designed for expression of a fusion protein in prokaryotic or eukaryotic host cells.

[0228] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0229] Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters. Strategies to maximize recombinant protein expression in E. coli can be found, for example, in Gottesman (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.), pp. 119-128 and Wada et al. (1992) Nucleic Acids Res. 20:2111-2118. Processes for growing, harvesting, disrupting, or extracting the IL-2 mutein or variant thereof from cells are substantially described in, for example, U.S. Pat. Nos. 4,604,377; 4,738,927;4,656,132; 4,569,790; 4,748,234; 4,530,787; 4,572,798; 4,748,234; and 4,931,543, herein incorporated by reference in their entireties.

[0230] In some embodiments the recombinant fusion protein can also be made in eukaryotes, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in yeast S. cerenvisiae include pYepSed (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corporation, San Diego, Calif.)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cells include Chinese hamster ovary cells (CHO) or COS cells. In mammalian cells, the expression vector’s control functions are often provided by viral regulatory elements. For example,Attorney Docket No. 117802-5021 -WOcommonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0231] The sequences encoding the fusion protein of the present disclosure can be optimized for expression in the host cell of interest. The G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon optimization are well known in the art. Codons within the fusion protein can be optimized to enhance expression in the host cell, such that about 1 %, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0232] Vectors suitable for use include T7-based vectors for use in bacteria (see, for example, Rosenberg et al., Gene 56:125, 1987), the pMSXND expression vector for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors (for example, the expression vector pBacPAK9 from Clontech, Palo Alto, Calif.) for use in insect cells.

[0233] In some embodiments nucleic acid inserts, which encode the fusion protein in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought.

[0234] In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the fusion protein, particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.

[0235] Within these parameters one of skill in the art may select various vector / expression control sequence / host combinations that will express the desired DNA sequences on fermentation or in large scale animal culture, for example, using CHO cells or COS 7 cells.Attorney Docket No. 117802-5021 -WO

[0236] The choice of expression control sequence and expression vector, in some embodiments, will depend upon the choice of host. A wide variety of expression host / vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors with expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including col El, pCRI, pER32z, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Useful expression vectors for yeast cells include the 2 , plasmid and derivatives thereof. Useful vectors for insect cells include pVL 941 and pFastBac™ 1 (GibcoBRL, Gaithersburg, Md.). Cate et al., “Isolation Of The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells”, Cell, 45, pp. 685-98 (1986).

[0237] In addition, any of a wide variety of expression control sequences can be used in these vectors. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage lambda, for example PL, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoA, the promoters of the yeast a-mating system, the polyhedron promoter of Baculovirus, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0238] A T7 promoter can be used in bacteria, a polyhedrin promoter can be used in insect cells, and a cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type within the body. Skilled artisans are well aware of numerous promoters and other regulatory elements which can be used to direct expression of nucleic acids.

[0239] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication, and other genes that encode a selectable marker. For example, the neomycin-resistance (neor) gene imparts G418 resistance to cells in which it is expressed, and thus permitsAttorney Docket No. 117802-5021 -WOphenotypic selection of the transfected cells. Those of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

[0240] Viral vectors that can be used in the invention include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).

[0241] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule that encodes a fusion protein disclosed herein are also features of the invention. A cell of the invention is a transfected cell, i.e., a cell into which a nucleic acid molecule, for example a nucleic acid molecule encoding a fusion protein, has been introduced by means of recombinant DNA techniques. The progeny of such a cell are also considered within the scope of the invention.

[0242] The precise components of the expression system are not critical. For example, a fusion protein can be produced in a prokaryotic host, such as the bacterium E. coli, or in a eukaryotic host, such as an insect cell (e.g, an Sf21 cell), or mammalian cells (e.g, CHO, HEK293, COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993) and Pouwels etal. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0243] The expressed polypeptides can be purified from the expression system using routine biochemical procedures, and can be used, e.g, as therapeutic agents, as described herein.

[0244] In some embodiments, the fusion protein obtained will be glycosylated or unglycosylated depending on the host organism used to produce the mutein. If bacteria are chosen as the host then the fusion protein produced will be unglycosylated. Eukaryotic cells, on the other hand, will glycosylate the IL-2 and IL-13 of the fusion protein, although perhaps notin the same way as native-IL-2 or IL-13 is glycosylated. The fusion protein produced by the transformed host can be purified according to any suitable method. Various methods are known for purifying the fusion protein. See, e.g. Current Protocols in Protein Science, Vol 2. Eds: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T.Wingfield, Unit 6.5 (Copyright 1997, John Wiley and Sons, Inc. The fusion protein can be isolated fromAttorney Docket No. 117802-5021 -WOinclusion bodies generated in E. coli, or from conditioned medium from either mammalian or yeast cultures producing a given mutein using cation exchange, gel filtration, and / or reverse phase liquid chromatography.

[0245] Another exemplary method of constructing a DNA sequence encoding the fusion protein is by chemical synthesis. This includes direct synthesis of a peptide by chemical means of the protein sequence encoding for an IL-2 mutein exhibiting the properties described. This method can incorporate both natural and unnatural amino acids at positions that affect the interactions of IL-2 or IL-13 mutein of the fusion protein with the their corresponding receptors. Alternatively a gene which the fusion protein can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired fusion protein, and preferably selecting those codons that are favored in the host cell in which the recombinant mutein will be produced. In this regard, it is well recognized that the genetic code is degenerate— that an amino acid may be coded for by more than one codon. For example, Phe (F) is coded for by two codons, TIC or TTT, Tyr (Y) is coded for by TAC or TAT and his (H) is coded for by CAC or CAT. Trp (W) is coded for by a single codon, TGG. Accordingly, it will be appreciated that for a given DNA sequence encoding a particular fusion protein, there will be many DNA degenerate sequences that will code for that fusion protein. For example, it will be appreciated that in addition to the preferred DNA sequence for mutein H9, there will be many degenerate DNA sequences that code for the fusion protein shown. These degenerate DNA sequences are considered within the scope of this disclosure. Therefore, “degenerate variants thereof in the context of this invention means all DNA sequences that code for and thereby enable expression of a particular mutein.

[0246] The biological activity of the fusion protein can be assayed by any suitable method known in the art. Such assays include PHA-blast proliferation and NK cell proliferation.H. METHODS OF TREATMENT

[0247] In some embodiments, fusion proteins, and / or nucleic acids expressing them, can be administered to a subject to treat a disorder associated with abnormal apoptosis or a different! ative process (e.g., cellular proliferative disorders or cellular differentiative disorders, such as cancer, by, for example, producing an active or passive immunity). In the treatment of such diseases, the disclosed fusion proteins may possess advantageous properties, such as reduced vascular leak syndrome.

[0248] Examples of cellular proliferative and / or differentiative disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias). A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of sarcoma, carcinoma,Attorney Docket No. 117802-5021 -WOhead and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, pulmonary cancer, cutaneous, lymphoid, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal-like breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain cancer, and CNS tumors. In some embodiments, the cancer for treatment is a solid tumor. In some embodiments, the cancer for treatment includes but is not limited to sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, pulmonary cancer, cutaneous, lymphoid, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal-like breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain cancer, and CNS tumors. CNS tumors include glioma, glioblastoma, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastoma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglia, menangioma, meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, an O6-methylguanine-methyltransferase (MGMT) positive or negative CNS tumor, and furin positive CNS tumor.

[0249] The fusion proteins can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma, or any viral disease, including for example human papillomavirus (HPV) and / or Hepatitis, such as Hepatitis A, Hepatitis B, Hepatitis C, and / or Hepatitis D. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.

[0250] Additional examples of proliferative disorders include hematopoietic neoplastic disorders.

[0251] Alternatively, or in addition to methods of direct administration to patients, in some embodiments, fusion proteins can be used in ex vivo methods. For example, cells e.g., peripheral blood lymphocytes or purified populations of lymphocytes isolated from a patient and placed or maintained in culture) can be cultured in vitro in culture medium and the contacting step can be affected by adding the fusion protein to the culture medium. The culture step can include further steps in which the cells are stimulated or treated with other agents, e.g., to stimulate proliferation, or to expand a population of cells that is reactive to anAttorney Docket No. 117802-5021 -WOantigen of interest (e.g., a cancer antigen or a viral antigen). The cells are then administered to the patient after they have been treated.

[0252] Anti-PD-1 antibodies for use in combination with the fusion proteins disclosed herein for the cancer and / or proliferative disorder treatment methods include but are not limited to nivolumab (OPDIVO®), BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA®), AMP-514 (MEDI0680), Retifanlimab (Zynyz), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872).

[0253] In some embodiments, the fusion protein is used in combination with nivolumab for the treatment of cancer. In some embodiments, the fusion protein is used in combination with pembrolizumab for the treatment of cancer. In some embodiments, the fusion protein is used in combination with BMS-936558 for the treatment of cancer. In some embodiments, the fusion protein is used in combination with MDX-1106 for the treatment of cancer. In some embodiments, the fusion protein is used in combination with ONO-4538 for the treatment of cancer. In some embodiments, the fusion protein is used in combination with AMP224 for the treatment of cancer. In some embodiments, the fusion protein is used in combination with CT-011 for the treatment of cancer. In some embodiments, the fusion protein is used in combination with MK-3475 for the treatment of cancer.

[0254] In some embodiments, the fusion protein is used in combination with an antibody and / or immunotherapy including but not limited to, anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-L1 antagonistic antibodies such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3 such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137 mAbs (anti-4-1 -BB antibodies) such as BMS-663513 urelumab (anti-4-1 BB antibody; see, for example, US Patent Nos. 7,288,638 and 8,962,804, incorporated by reference herein in their entireties); lirilumab (anti-KIR mAB; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors) and PF-05082566 (utomilumab; see, for example, US Patent Nos. 8,821,867; 8,337,850; and 9,468,678, as well as International Patent Application Publication No. WO 2012 / 032433, incorporated by reference herein in their entireties); anti-OX40 mAbs (see, for example, WO 2006 / 029879 or WO 2010 / 096418, incorporated by reference herein in their entireties); anti-GITR mAbs such as TRX518 (see, for example, US Patent No. 7,812,135, incorporated by reference herein in its entirety); anti-CD27 mAbs, such as varlilumab CDX-1127 (see, for example, WO 2016 / 145085 and U.S. Patent PublicationAttorney Docket No. 117802-5021 -WONos. US 2011 / 0274685 and US 2012 / 0213771, incorporated by reference herein in their entireties) anti-ICOS mAbs (for example, MEDI-570, JTX-2011 , and anti-TIM-3 antibodies (see, for example, WO 2013 / 006490 or U.S. Patent Publication No US 2016 / 0257758, incorporated by reference herein in their entireties) for the treatment of cancer.

[0255] In some embodiments, the fusion protein is used in combination with another antibody which can include monoclonal antibodies (mAbs) for the treatment of a solid tumor. In some embodiments, the monoclonal antibody is for the treatment of sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, pulmonary cancer, cutaneous, lymphoid, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal-like breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain cancer, and CNS tumors. CNS tumors include glioma, glioblastoma, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastoma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglia, menangioma, meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, an O6-methylguanine-methyltransferase (MGMT) positive or negative CNS tumor, and furin positive CNS tumor.

[0256] In some embodiments, the fusion protein is used in combination with antibodies for antibodydependent cell-mediated cytotoxicity (ADCC) for the treatment of cancer.

[0257] In some embodiments, the fusion protein as described herein is used in combination with antibodies including dupilumab, nivolumab (OPDIVO®), BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA®), AMP-514 (MEDI0680), Retifanlimab (Zynyz), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872). In some embodiments, the fusion molecule as described herein is used in combination with antibodies including anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-L1 antagonistic antibodies such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3 such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137 mAbs (anti-4-1 -BB antibodies) such as BMS-663513 urelumab (anti-4-1 BB antibody; see, for example, US Patent Nos. 7,288,638 and 8,962,804, incorporated by reference herein in their entireties); lirilumab (anti-KIR mAB; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors) andAttorney Docket No. 117802-5021 -WOPF-05082566 (utomilumab; see, for example, US Patent Nos. 8,821,867; 8,337,850; and 9,468,678, as well as International Patent Application Publication No. WO 2012 / 032433, incorporated by reference herein in their entireties); anti-OX40 mAbs (see, for example, WO 2006 / 029879 or WO 2010 / 096418, incorporated by reference herein in their entireties); anti-GITR mAbs such as TRX518 (see, for example, US Patent No.7,812,135, incorporated by reference herein in its entirety); anti-CD27 mAbs, such as varlilumab CDX-1127 (see, for example, WO 2016 / 145085 and U.S. Patent Publication Nos. US 2011 / 0274685 and US 2012 / 0213771, incorporated by reference herein in their entireties) anti-ICOS mAbs (for example, MEDI-570, JTX-2011 , and anti-TIM-3 antibodies (see, for example, WO 2013 / 006490 or U.S. Patent Publication No US 2016 / 0257758, incorporated by reference herein in their entireties), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-sirs-alpha, and / or anti-CD112R.

[0258] In some embodiments, the fusion protein as described herein is fused with an antibody selected from dupilumab, nivolumab (OPDIVO®), BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA®), AMP-514 (MEDI0680), Retifanlimab (Zynyz), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872). In some embodiments, the fusion protein as described herein is fused with an antibody selected from anti-CTLA4 mAbs, such as ipilimumab, tremelimumab; anti-PD-L1 antagonistic antibodies such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3 such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137 mAbs (anti-4-1-BB antibodies), such as BMS-663513 urelumab (anti-4-1 BB antibody; see, for example, US Patent Nos. 7,288,638 and 8,962,804, incorporated by reference herein in their entireties); lirilumab (anti-KIR mAB; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors) and PF-05082566 (utomilumab; see, for example, US Patent Nos. 8,821,867; 8,337,850; and 9,468,678, as well as International Patent Application Publication No. WO 2012 / 032433, incorporated by reference herein in their entireties), anti-OX40 mAbs (see, for example, WO 2006 / 029879 or WO 2010 / 096418, incorporated by reference herein in their entireties); anti-GITR mAbs such as TRX518 (see, for example, US Patent No. 7,812,135, incorporated by reference herein in its entirety), anti-CD27 mAbs, such as varlilumab CDX-1127 (see, for example, WO 2016 / 145085 and U.S. Patent Publication Nos. US 2011 / 0274685 and US 2012 / 0213771, incorporated by reference herein in their entireties) anti-ICOS mAbs (for example, MEDI-570, JTX-2011, and anti-TIM-3 antibodies (see, for example, WO 2013 / 006490 or U.S. Patent PublicationAttorney Docket No. 117802-5021 -WONo US 2016 / 0257758, incorporated by reference herein in their entireties), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-sirs-alpha, and / or anti-CD112R.I. PHARMACEUTICAL COMPOSITIONS AND METHODS OF ADMINISTRATION

[0259] In some embodiments, fusion proteins can be incorporated into compositions, including pharmaceutical compositions. Such compositions typically include the polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. Such compositions can also comprise anti-PD-1 antibodies. In some embodiments, the composition comprises an IL-2 mutein that is a fusion protein and / or is associated with a CAR-T construct and / or expressed by or associated with an oncolytic virus.

[0260] The anti-PD-1 antibodies and fusion proteins can be administered as a co-composition, simultaneously as two separate compositions, and / or sequentially as two separate compositions. In some embodiments, the anti-PD-1 antibody or inhibitor and fusion protein are administered together as a single co-composition ( / .e., co-formulated). In some embodiments, the anti-PD-1 antibody or inhibitor and fusion protein are administered simultaneously as two separate compositions ( / .e., separate formulations). In some embodiments, the anti-PD-1 antibody or inhibitor and fusion protein are administered sequentially as separate compositions ( / .e., separate formulations). In some embodiments, when the anti-PD-1 antibody or inhibitor and fusion protein are administered sequentially as separate compositions, the anti-PD-1 antibody or inhibitor is administered before the fusion protein. In some embodiments, when the anti-PD-1 antibody or inhibitor and fusion protein are administered sequentially as separate compositions, the fusion protein is administered before the anti-PD-1 antibody or inhibitor. In some embodiments, the anti-PD-1 antibodies include but are not limited to nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475.

[0261] The other immunotherapy agents as described and fusion protein can be administered as a cocomposition, simultaneously as two separate compositions, and / or sequentially as two separate compositions. In some embodiments, the other immunotherapy agents and fusion protein are administered together as a single co-composition ( / .e., co-formulated). In some embodiments, the other immunotherapy agents and fusion protein are administered simultaneously as two separate compositions ( / .e., separate formulations). In some embodiments, the other immunotherapy agents and fusion protein are administered sequentially as separate compositions ( / .e., separate formulations). In some embodiments, when the other immunotherapy agents and fusion protein are administered sequentially as separate compositions, the anti-PD-1 antibody or inhibitor is administered before the fusion protein. In some embodiments, when otherAttorney Docket No. 117802-5021 -WOimmunotherapy agents and fusion protein are administered sequentially as separate compositions, the fusion protein is administered before other immunotherapy agents.

[0262] A pharmaceutical composition is formulated to be compatible with its intended route of administration. The anti-PD-1 antibodies and / or fusion protein may be given orally, but it is more likely that they will be administered through a parenteral route, including for example intravenous administration. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and / or di-basic sodium phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5). The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0263] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate.Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be broughtAttorney Docket No. 117802-5021 -WOabout by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0264] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0265] Oral compositions, if used, generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel™, or corn starch; a lubricant such as magnesium stearate or Sterotes™; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0266] In the event of administration by inhalation, anti-PD-1 antibodies and / or fusion proteins, or the nucleic acids encoding them, are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0267] Systemic administration of the anti-PD-1 antibodies and / or fusion proteins or nucleic acids can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art.Attorney Docket No. 117802-5021 -WO

[0268] In some embodiments, compounds (anti-PD-1 antibodies and / or fusion proteins or nucleic acids) can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0269] In some embodiments, compounds (fusion proteins or nucleic acids) can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20: 1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151-160, 1996, erratum at Am. J. Health Syst. Pharm. 53:325, 1996).

[0270] In one embodiment, the anti-PD-1 antibodies and / or fusion protein or nucleic acids are prepared with carriers that will protect the anti-PD-1 antibodies and / or fusion protein against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0271] Dosage, toxicity and therapeutic efficacy of such anti-PD-1 antibodies, fusion proteins, or nucleic acids compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0272] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initiallyAttorney Docket No. 117802-5021 -WOfrom cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 ( / .e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0273] As defined herein, a therapeutically effective amount of a fusion protein ( / .e., an effective dosage) and / or the anti-PD-1 antibody or inhibitor depends on the polypeptide or antibody selected. In some embodiments, single dose amounts of the fusion protein can be in the range of approximately 0.001 mg / kg to 0.1 mg / kg of patient body weight can be administered. In some embodiments, single dose amounts of the anti-PD-1 antibody or inhibitor can be in the range of approximately 1 mg / kg to 20 mg / kg, or about 5 mg / kg to about 15 mg / kg, or about 10 mg / kg of patient body weight can be administered. In some embodiments, doses of the anti-PD-1 antibody or inhibitor and / or the fusion protein of about 0.005 mg / kg, 0.01 mg / kg, 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg may be administered. In some embodiments, 600,000 lU / kg is administered (IU can be determined by a lymphocyte proliferation bioassay and is expressed in International Units (IU) as established by the World Health Organization 1stInternational Standard for lnterleukin-2 (human)). The dosage may be similar to, but is expected to be less than, that prescribed for PROLEUKIN®. The compositions can be administered one from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the fusion proteins can include a single treatment or, can include a series of treatments. In one embodiment, the compositions are administered every 8 hours for five days, followed by a rest period of 2 to 14 days, e.g., 9 days, followed by an additional five days of administration every 8 hours. In some embodiments, administration is 3 doses administered every 4 days.

[0274] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0275] The examples below are provided to describe certain embodiments of the invention provided herein and are not to be construed to as limiting.Attorney Docket No. 117802-5021 -WOEXAMPLES

[0276] Example 1: Functional Characterization of MDNA113 Constructs in IL-2 and PD-1 Reporter Assays

[0277] The functional characteristics of mouse [SEQ ID NOs: 102-104] and human MDNA113 [SEQ ID NOs:39-41] constructs were assessed by evaluating IL-2 mediated signaling and PD-1 / PD-L1 immune checkpoint blockage.

[0278] For the IL-2 evaluation, a Jurkat IL-2R0Y bioassay was carried out using cells lacking CD25 expression from Promega, and cells were treated with increasing concentration of constructs for 6 h following which the plate was read for luminescence.

[0279] For the PD-1 / PD-L1 evaluation, a co-culture of PD-1 reporter cells and PD-L1 aAPC / CHO-K1 cells was carried out for 6 h followed by addition of luciferase substrate for luminescence, and mouse and human PD-1 / PD-L1 reporter assays from Promega were used.

[0280] As shown in FIGs. 1A-B and 2A-B, both mouse [SEQ ID NOs:102-104] and human MDNA113 [SEQ ID NOs:39-41] constructs exhibit attenuated IL-2R signaling but retain PD-1 / PDL-1 blockade.Furthermore, the human construct (hMDNA113) masks as efficiently as, and is able to block PD-1 / PDL-1 similar to the mouse version (mMDNA113).

[0281] As shown in FIGs. 3A-3B, both mouse [SEQ ID NOs: 102-104] and human MDNA113 [SEQ ID NOs:39-41] constructs were able to be cleaved, removing the masking domain, and upon that cleavage, IL-2 masking was removed and IL-2 function was restored.

[0282] As shown in FIG. 4, Biodistribution of Cy5-labeled hMDNA113 administered via tail vain injection at 1 mg / kg was evaluated in whole body C57BL / 6 mice carrying MC38WTand MC38IL13Ra2tumors on contralateral flanks across timepoints. Samples were embedded for slicing dorsal-to-ventral along the horizontal plane. Images were captured at 35pm pixel resolution using 640nm excitation laser and 680 / 13nm emission filter. Autofluorescence in Gl and Harderian glands. Low marrow and gallbladder signal. The IL-13 masking and targeting domain causes the human MDNA113 [SEQ ID NOs: 45-47] to be preferentially targeted towards IL13Ra2 expressing MC38IL13Ra2tumors and not the MC38WTtumors at all time points.

[0283] As shown in FIG. 5A in vivo tolerability of MDNA113 in naive mice was carried out. C57BI / 6 mice were treated with equimolar doses of anti-mPD1 -IL-2SK[SEQ ID NQs:60-62] and murine MDNA113 [SEQAttorney Docket No. 117802-5021 -WOID NOs: 102-104] on a twice weekly schedule at 4 mg / kg for tolerability analysis. All mice treated with MDNA113 survived until the end of study, while mice treatment with the unmasked version was universally fatal. This indicates the improved tolerability of murine MDNA113.

[0284] As shown in FIG. 5B mice treated with murine MDNA113 [SEQ ID NO: 102-104] in a C57BI / 6, MC38IL13Ra2colon carcinoma model shows no body weight loss compared to mice treated with the unmasked anti-mPD1 -I L-2SK[SEQ ID NOs:60-62] version when dosed at 20mg / kg on a once a week schedule.

[0285] As shown in FIG. 6A C57BI / 6 mice carrying the MC38IL13Ra2colon carcinoma tumor were treated with vehicle, unmasked anti-mPD1-IL-2SK [SEQ ID NOs:60-62], MDNA113 [SEQ ID NOs:102-104] and its non-cleavable version (mMDNA113Nc) [SEQ ID NOs: 105-107] and a murine anti-PD-1 antibody [anti-PD-1 portion of SEQ ID NQ:60-62], Mice were dosed at 1 mg / kg on a once weekly schedule. The left panel of FIG. 6A shows the summary graph, while the right panel of FIG. 6A shows data for individual mice. Data shows that MDNA113 had similar activity compared to its unmasked version with both being able to eliminate the tumor, while anti-PD 1 alone or the masked, non-cleavable MDNA113NC failed to control tumor growth.

[0286] As shown in FIG. 6B mice were rechallenged with MC38IL13Ra2without any additional treatment. Treatment with the murine MDNA113 conferred immunity against rechallenge, thus promoting an adaptive immune system driven memory response, as no tumor regrowth was observed upon rechallenge.

[0287] In vivo efficacy of the murine MDNA113 in a B16F10IL13Ra2melanoma model was carried out. For tumor growth analysis, the average tumor size at time of dosing was 50 mm3, and the mice were dosed once weekly at molar equivalent dose (10 mg / kg; IP). For tumor infiltrates analysis, the average tumor sizeAttorney Docket No. 117802-5021 -WOat time of dosing was 150 mm3, and the mice were dosed once weekly at molar equivalent dose (10 mg / kg; IP). Samples were collected on Day 8 and processed for flow cytometry.

[0288] As shown in FIG. 7, the murine MDNA113 inhibited tumor growth of a B16F10IL13Ra2melanoma model. As shown in FIG. 8, the murine MDNA113 preferentially expanded functionally active CD8+T cells, and did not expand NK cells, and did not expand Tregs in the B16F10 / IL13Ra2 melanoma model.

[0289] Immune cell infiltrates were also analyzed in 4T1.2 orthotopic breast tumors. 4T1.2 tumors in Balb / c mice were treated with equimolar dose (20 mg / kg) of anti-mPD1 -I L-2SKor mMDNA113, and tumors were collected 3 days after for mIF analysis.

[0290] As shown in FIG. 9, murine MDNA113 enhanced tumor infiltration of functionally active CD8+T cells.

[0291] Example 2: In Vitro and In Vivo Characterization of MDNA113B Construct and Variants

[0292] MDNA113B constructs, incorporating the human anti-PD1 antibodies pembrolizumab (“hMDNA113B / pembrolizumab / lgG4” [SEQ ID NOs: 83-85] and (“hMDNA113B / pembrolizumab / lgG1 ” [SEQ ID NOs: 71-73]) or nivolumab (“hMDNA113B / nivolumab / lgG4” [SEQ ID NOs: 89-91]) and(“hMDNA113B / nivolumab / lgG1 ” [SEQ ID NOs: 77-79]) were designed, synthesized, and characterized. Receptor affinity studies were conducted using captured test constructs and receptors as analytes. Results are shown in Table 7. hMDNA113 / pembrolizumab and hMDNA113B / nivolumab had similar binding affinity for PD1 and I L-13Ra2 as respective unmask constructs (hMDNA223 / pembrolizumab and hMDNA223 / nivolumab). Both MDNA113 / pembrolizumab and hMDNA113B / nivolumab had reduced affinity for I L-2Rb compared to respective unmask versions (hMDNA223 / pembrolizumab and hMDNA223 / nivolumab). Unlike human IL-2-Fc, MDNA113 / pembrolizumab, hMDNA113B / nivolumab, hMDNA223 / pembrolizumab and hMDNA223 / nivolumab did not bind IL-2Ra.Table 7: Receptor affinity (KD) of test constructs to human PD-1 , human IL-13Ra2 and human IL-2RAttorney Docket No. 117802-5021 -WO

[0293] The constructs were tested in a Jurkat IL-2R0y bioassay to evaluate for IL-2 activity as described in Example 1 above. As shown in FIG. 10A, the Jurkat IL-2RPy assay, measuring IL-2 downstream signaling, showed reduced potency of hMDNAI 13B / pembrolizumab and hMDNA113B / nivolumab compared to respective unmasked versions (hMDNA223 / pembrolizumab and hMDNA223 / nivolumab). As shown in FIG.10B, the Jurkat IL-2R0Y assay showed that cleavage with MMP9 of hMDNAI 13B / pembrolizumab and hMDNAI 13B / nivolumab restored their IL-2 downstream signaling capacity to similar levels as respective unmasked versions (hMDNA223 / pembrolizumab and hMDNA223 / nivolumab).

[0294] The potency of the test constructs in the Jurkat IL-2R y bioassay of FIG. 10, is shown in Table 8 below.Table 8: Potency of test constructs in Jurkat IL-2R[fy assayAttorney Docket No. 117802-5021 -WO

[0295] Next, the PD-1 / PD-L1 binding of the constructs was evaluated in a PD-1 / PD-L1 reporter assay as described in Example 1 above. As shown in FIG. 11, hMDNA113B / pembrolizumab andhMDNA113B / nivolumab showed a similar capacity to block PD-1 / PD-L1 binding as the respective unmasked constructs (MDNA223 / pembrolizumab and MDNA223 / nivolumab) in the human PD-1 / PD-L1 reporter assay. The potency of the test constructs in the PD-1 / PD-L1 reporter assay is shown below in Table 9.Table 9: Potency of test constructs in human PD-1 / PD-L1 blockade reporter assayAttorney Docket No. 117802-5021 -WO

[0296] An in v7i / o efficacy study of the murine MDNA113B construct [SEQ ID NO: 108-110] was conducted in an MC38 mouse colon tumor model. Tumor bearing mice were treated with same dose of murine MDNA113B or non-cleavable murine MDNA113BNC [SEQ ID NO: 111-113] compared with vehicle control. Animals were treated by intraperitoneal (IP) injection once every week for a total of 3 doses. As shown in FIG. 12A, treatment with MDNA113B resulted in a reduction in tumor volume.

[0297] A second in vivo efficacy study was conducted in an MC38 / IL-13Roc2 mouse colon tumor model. T umor bearing mice treated with same dose of murine MDNA113B or non-cleavable murine MDNA113BNC compared with vehicle control. Animals were treated by intraperitoneal (IP) injection once every week for a total of 2 doses. As shown in FIG. 12B, treatment with MDNA113B resulted in a reduction in tumor volume.

[0298] The biodistribution of Cy5.5-labelled MDNA113B (2 mg / kg by intravenous injection) was investigated in mice implanted with PancO2 / IL-13Ra2(p°s> tumor cells on the right flank and PancO2 / IL-13Rot2<ne9) tumor cells on the right flank. As shown in FIG. 13A, in vivo imaging at pre-treatment and at indicated timepoints following treatment to detect labelled MDNA113B (total flux) at implanted tumor sites showed greater accumulation in PancO2 / IL-13Ra2(p°s> tumors. As shown in FIG. 13B, ex vivo imaging of implanted tumors at 168 hours post-treatment showed greater MDNA113 signal in Panc02 / l L- 13ROC2(P°S) tumors.

[0299] Further, as shown in FIG. 14, an analysis of tumor infiltration lymphocytes in a B16F10 / IL-13Roc2 melanoma model showed increased infiltration of CD8+T cells (FIG. 14A) and CD8+T cells expressing granzyme B (GrzB, FIG. 14B) in tumors from mice treated with MDNA223 [SEQ ID NQ:60-62] or MDNA113B [SEQ ID NO: 108-110]. Tumor-bearing mice were treated with a single dose of the indicated test constructs and tumors were harvested several days thereafter for analysis by flow cytometry.

[0300] The toxicity of the hMDNA113B constructs [SEQ ID NO: 83-85 or SEQ ID NOs: 88-91] is evaluated in a non-human primate model to determine tolerability.

[0301] Example 3: Functional Characterization of Masked Fusion Constructs

[0302] Additional fusion constructs MM001, MM002, and MM003 [SEQ ID NOs: 131-133 , 134-136 , 137-139. ] were synthesized that included a light chain and heavy chain of anti-PD 1 (anti-mouse PD1 , anti-Attorney Docket No. 117802-5021 -WOmPD1), an IL-2 mutein (MDNA109FEAA), IL-13 muteins (MDNA213, MDNA413), and MMP cleavage sites, as shown schematically in FIG. 15A.

[0303] The constructs were tested in a Jurkat IL-2R Y bioassay to evaluate for IL-2 activity as described above. Briefly, Jurkat I L2R y bioassay reporter cells were purchased from Promega, and the kit contained all necessary contents for execution. Cells were thawed and plated as per manufacturer’s protocol. Jurkat IL2F?Py cells were plated into 96 well plates according to the manufacturer’s recommendation in a volume of 50 pL. Test articles were diluted as 3x solutions and then 25 pL were added to each well to achieve the desired concentrations. Cells were treated for 6 hours and then the luciferase substrate was added and incubated for 10 minutes. Luminescence was then measured on the iD5 plate reader.

[0304] As shown in FIG. 15B, MM001 [SEQ ID NO: 131-133), MM002 [SEQ ID NO: 134-136] and MM003 [SEQ ID NO: 137-139] demonstrated 11.9-fold, 11.5-fold and 27.2-fold reduced IL-2 activity respectively compared to non-masked MDNA223 [SEQ ID NO:60-62],

[0305] The constructs were also subjected to mock or cleavage reactions with MMP9 at a scale amendable to downstream analysis. The construct was incubated in a reaction containing 9.5 pg of construct with or without MMP9 in Cleavage Buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCI, 0.05% Brij-35 (w / v), pH 7.5) in a total volume of 160 pL containing 2 pg / mL of MMP9 for 1 hour at 37°C. Following digestion, reactions were placed on ice and diluted in culture media fortesting in the Jurkat I L2R y reporter cell assay.

[0306] As shown in FIG. 15C, in vitro MMP9 cleavage resulted in restoration of the IL-2 activity. The ECsos post-proteolytic cleavage were comparable with non-masked MDNA223.

[0307] Next, the constructs were tested in a HEK-Blue™ IL-13 assay to evaluate for IL-13 activity. Briefly, HEK-Blue™ IL-4 / IL-13 reporter cells (InvivoGen) were plated at 50,000 cells per well in test medium per the manufacturer’s instructions and treated with increasing concentrations of rhlL-13 (R&D Systems 213-ILB-005 / CF) in the presence or absence of 100 nM of constructs for 24 hours. Following incubation for 24 hours, cell supernatant (20 pL) was removed to a new plate and then 180 pL of QUANTI-Blue™ solutionAttorney Docket No. 117802-5021 -WOwas added and incubated for 24 hours at 37°C. Plates were scanned on a conventional plate reader for absorbance at 650 nm. Fc-MDNA413 was used as benchmark control.

[0308] As shown in FIG. 16, Fc-MDNA413 demonstrated 8.6-fold inhibition of the IL-13 induced signaling, as anticipated. MM001, MM002 and MM003 showed attenuation of the IL-13 induced signaling.

[0309] As shown in the schematic of FIG. 17A, the construct MDNA1213 (also termed hMDNA1213 or MDNA1231A) [SEQ ID NOs: 148-151] was designed and synthesized to include light and heavy chain segments of human anti-PD1 (nivolumab) and human anti-VEGF (bevacizumab), in addition to an IL-2 mutein (MDNA109FEAA-T3A-C125S), an IL-13 mutein (MDNA213), and MMP cleavage sites. This construct is disclosed as heterodimeric antibodies that bind anti-PD-1 and anti-VEGF simultaneously. To suppress light-chain mispairing, one arm incorporates a CH-CL crossover (constant-domain swap), yielding an orthogonal HC-LC interface. The molecules include Fc heterodimerization mutations (Knob-into-Hole) to favor HC1-HC2 pairing. A hPDIXhVEGF antibody-only construct, where the anti-PD-1 arm is based on Nivolumab [SEQ ID NOs: 140-143] was also synthesized as a control. The sequence of hPD1 x hVEGF antibody with light and heavy chain segments of human anti-PD 1 (pembrolizumab) and human anti-VEGF (bevacizumab), in addition to an IL-2 mutein (MDNA109FEAA-T3A-C125S), an IL-13 mutein (MDNA213), and MMP cleavage sites, are also provided [SEQ ID NOs: 152-155], alongside with a hPDIXhVEGF antibody-only construct, where anti-PD-1 arm is based on pembrolizumab [SEQ ID NOs: 144-147],

[0310] The constructs were tested in a Jurkat I L-2R y bioassay to evaluate IL-2 activity. Briefly, Jurkat IL2RPy bioassay reporter cells were purchased from Promega, and the kit contained all necessary contents for execution. Cells were thawed and plated as per manufacturer’s protocol. Jurkat I L2R y cells were plated into 96 well plates according to the manufacturer’s recommendation in a volume of 50 pL. Test articles were diluted as 3x solutions and then 25 pL were added to each well to achieve the desired concentrations. Cells were treated for 6 hours and then the luciferase substrate was added and incubated for 10 minutes. Luminescence was then measured on the iD5 plate reader.

[0311] As shown in FIG. 17B, hMDNAI 213 demonstrated 39.2-fold reduced IL-2 activity compared to non-masked MDNA223. MDNA113A [SEQ ID NO: 102-104] was used as control and demonstrated 16.5-fold reduced IL-2 activity compared to non-masked MDNA223.

[0312] The constructs were also subjected to mock or cleavage reactions with MMP9 at a scale amendable to downstream analysis. The construct was incubated in a reaction containing 9.5 g of construct with or without MMP9 in Cleavage Buffer (50 mM Tris, 10 mM CaCL, 150 mM NaCI, 0.05% Brij-Attorney Docket No. 117802-5021 -WO35 (w / v), pH 7.5) in a total volume of 160 pL containing 2 pg / mL of MMP9 for 1 hour at 37°C. Following digestion, reactions were placed on ice and diluted in culture media fortesting in the Jurkat I L2R Y reporter cell assay.

[0313] As shown in FIG. 17C, in vitro MMP9 cleavage resulted in restoration of the IL-2 activity. The EC50 of hMDNA1213 post proteolytic cleavage was comparable with non-masked MDNA223. MDNA113Awas used as a control.

[0314] Next, the constructs were tested in a PD-1 reporter assay to evaluate for anti-PD1 activity. Briefly, human PD-L1 aAPC / CHO-K1 T&U cells were plated into 96 well plates 16 hours prior to assay setup according to the manufacturer’s recommendation in a volume of 100 pL. The following day, test articles were serially diluted as 2x solutions. Media was removed from the pre-plated reporter cells and 40 pL of the 2x test articles and 40 pL of the human PD-1 T&U Effector Cells and were added to each well to achieve desired concentrations. Cells were incubated for 6 hours and then the luciferase substrate was added and incubated for 15 minutes. Luminescence was measured on the iD5 plate reader.

[0315] As shown in FIG. 17D, the hPDIXhVEGF antibody-only construct and hMDNAI 213 demonstrated 59.5-fold and 39.5-fold reduced PD-1 activity respectively compared to non-masked MDNA223 and MDNA113A.

[0316] Further, the constructs were tested in a VEGF bioassay to evaluate for anti-VEGF activity. Briefly, one vial of KDR / NFAT-RE HEK293 Cells (Kit component GA109A) was thawed, resuspended with 4.6 mL of Cell Recovery Medium, mixed and then plated at 25 pL per well. Constructs were diluted directly into assay buffer at 3x concentrated solutions. Bevacizumab biosimilar (MAB9947) was obtained from R&D (MAB9947-100). Recombinant VEGF (rVEGF) was used as an assay control (R&D 11458-VE). Each plate received a VEGF Dose response and inhibition reactions received 1 nM VEGF. For inhibition assays, 25 pL of the concentrated test articles were added to wells according to the plate map and incubated for 15 minutes prior to adding VEGF. Wells lacking test articles were supplemented with 25 pL of Assay Buffer. Following the 15-minute pre-incubation, 25 pL of VEGF was added to each well. Plates were incubated at 37C / 5% CO2 for 6 hours. At the end of the incubation period, Bio-Gio Substrate solution was prepared by resuspending one vial of substrate with the Bio-Gio™ Reagent Buffer (Kit components G7941 ) per the manufacturer's recommendation. Seventy-five (75) microliters of prepared Bio-Gio™ Substrate solutionAttorney Docket No. 117802-5021 -WOwas added to each well and plates were incubated for 5 minutes at room temperature. Plates were analyzed on a Molecular Devices iD5 reader in luminescent mode (integration for 1000 ms).

[0317] As shown in FIG. 17E, the bevacizumab biosimilar, hPDIXhVEGF and hMDNA1213 demonstrated similar IC50s in the assay, demonstrating that anti-VEGF in the fusion construct maintained its activity.

[0318] The / n vitro activity of the MDNA1213B [SEQ ID NOs: 156-159 , 160-163 ] is evaluated in a IL-2R y bioassay, a PD-1 reporter assay, and a VEGF bioassay as described above.

[0319] The in vivo efficacy of the fusion protein MDNA1213B [SEQ ID NOs: 170-173] is evaluated in a MC38 colon cancer mouse model, an MC38 / I L-13Ra2 colon cancer mouse model, and / or a B16-F10 melanoma cancer mouse model to determine tumor growth inhibition, immune cell activation and infiltration, and / or changes in tumor vasculature. The in vivo efficacy of the fusion protein MDNA1213A (comprising mouse anti-PD-1 and anti-VEGF antibody) is evaluated in a MC38 colon cancer mouse model, an MC38 / IL-13Ra2 colon cancer mouse model, and / or a B16-F10 melanoma cancer mouse model to determine tumor growth inhibition, immune cell activation and infiltration, and / or changes in tumor vasculature.

[0320] An IL-13 (MDNA213J-IL-18 fusion construct [SEQ ID NO: 165] was also designed and synthesized, as depicted in the schematic of FIG. 18A. The construct included an IL-18 linked to an albumin and linked to the MDNA213 via a linker including an MMP cleavage site, and the MDNA213 was further modified with a cleavable His tag (via an HRV 3C cleavage site) for purification following synthesis. An IL-18-albumin construct (SEQ ID NO: 164) was also generated.

[0321] The constructs were tested in a HEK IL-18 assay to evaluate for IL-18 activity. Briefly, HEK-Blue IL18 reporter cells were plated at 50,000 cells per well (180 pL per well) in 96 well plates (Corning 3596) in the test medium and treated with the test articles. Test articles were thawed and diluted directly into assay media as 10x concentrated solutions. For agonist assays, 20 pL of the 10x concentrated test articles were added to wells according to the plate map. Plates were incubated at 37C / 5% CO2 for 24 hours. At the end of the incubation period, 20 pL of each cell supernatant was removed to a new plate and then 180 pL of QUANTI-Blue™ solution was added and incubated for 1-2 hours at 37°C. QUANTI-Blue™ solution was prepared by combining QB reagent (kit component, 200 pL per 20 mL) and QB reagent (kit component,Attorney Docket No. 117802-5021 -WO200 pL per 20 mL) in sterile water (Gibco 10977-015). Plates were analyzed on a Molecular Devices iD5 reader in absorbance mode at 650nm. Recombinant IL-18 was used as an assay control.

[0322] As shown in FIG. 18B, the I L-18-albumin (non-masked) showed an ECso of 6.04 pM, whereas MDNA213-IL-18-albumin (masked version) showed an EC50 of 13350 pM, an approximately 2000-fold reduction in IL-18 activity.

[0323] A MDNA213-masked anti-CTLA4 construct (SEQ ID NO: 167) was also designed and synthesized, including an anti-CTLA4 scFv-albumin fusion linked to MDNA213 via a cleavable linker including an MMP cleavage site, as depicted in FIG. 19A. An anti-CTLA4 scFv-albumin construct (SEQ ID NO: 166) was also generated.

[0324] The constructs were tested in a CTLA4 blockade bioassay to evaluate anti-CTLA4 activity. Briefly, the assay was conducted per manufacturer's protocol. For the blockade assay, 25 pL of the concentrated test articles were added to wells. One vial of aAPC / Raji cells (kit component JA1595) was thawed, resuspended with 7.2 mL of Assay Buffer Medium, mixed and then plated at 25 pL per well. The plate was incubated at 37C / 5% CO2 for 6 hours. At the end of the incubation period, Bio-Gio™ Substrate solution was prepared by resuspending one vial of substrate with the Bio-Gio™ Reagent Buffer (Kit components G7941) per the manufacturer's recommendation. Seventy-five (75) microliters of prepared Bio-Gio™ Substrate solution was added to each well and plates were incubated for 5 minutes at room temperature. Plates were analyzed on a Molecular Devices iD5 reader in luminescent mode (integration for 1000 ms). Both the non-masked and masked versions of scFV-anti CTLA4 were tested in the blockade assay.

[0325] As shown in FIG. 19B, the non-masked scFv anti-CTLA4 exhibited an EC50 of 157 nM and masked version (MDNA213-scFv anti CTLA4) showed a 28-fold reduction in the blockade with an EC50 of 4439 nM.

[0326] Similarly to the I L-13-anti-CTLA4 masked construct, an IL-13 (MDNA213)-masked anti-CD3 construct was also designed and synthesized, as depicted in FIG. 20. An in vitro assay is used to assess masking activity of the MDNA213 in the construct.

[0327] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents andAttorney Docket No. 117802-5021 -WOpublications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

[0328] All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, those of skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriate according to the spirit and scope of the invention described herein.

[0329] All references cited herein are hereby incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0330] Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which the claims are entitled.

Claims

Attorney Docket No. 117802-5021 -WOWHAT IS CLAIMED IS:

1. A fusion protein comprising:a) a cytokine or an antibody moiety;b) a protease sensitive linker (PSL); andc) a masking moiety;wherein the PSL attaches the masking moiety to the cytokine or antibody moiety.

2. The fusion protein of claim 1 , wherein the masking moiety comprises an IL-13, an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody, or an antigen-binding fragment thereof, wherein the masking moiety is capable of binding to IL-13Ra2, and does not bind to IL-13Ra1.

3. The fusion protein of claim 1 , wherein the masking moiety comprises an IL-13, an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody, or an antigen-binding fragment thereof, wherein the masking moiety is capable of binding to IL-13Ra2, and does bind to IL-13Ra1.

4. The fusion protein of claim 1 , wherein the masking moiety comprises an IL-13, an IL-13 mutein, or type II IL-4 receptor antagonist thereof, wherein the masking moiety binds with higher affinity to I L-13Ra1 than wild type IL-13 (SEQ ID NO:200), and binds with lower affinity to IL-13Ra2 than wild type IL-13.

5. The fusion protein of any one of claims 1 to 4, wherein the masking moiety comprises an IL-13 mutein that has the amino acid sequence of SEQ ID NO:201 or 202.

6. The fusion protein of any one of claims 1 to 5, further comprising a protein binding domain.

7. The fusion protein of claim 6, wherein the protein binding domain comprises an albumin binding domain (ABD), a transferrin binding domain (TBD), a CD122 domain, a CD25 domain, aCD132 domain, an IL-13Ra2 domain, an IL-13Ra1 domain, or an IL-4Ra domain.

8. The fusion protein of any one of claims 1 to 7, wherein the cytokine or an antibody moiety is an IL-2, an IL-2 mutein, an IL-12, an IL-12 mutein, an IL-15, an IL-18, an IL-4, an IL-7, an IL-13, and IL-13 mutein, an anti-PD-1 antibody, an anti-VEGF antibody, an anti-PDL1 antibody, an anti-PSMA antibody, anAttorney Docket No. 117802-5021 -WOanti-EGFR antibody, an anti-CTLA4 antibody, an anti-CD3 antibody, or a hybrid of two or more of an anti-VEGF antibody, an anti-PSMA antibody, an anti-PD-1 antibody, and anti-PDL1 antibody, an anti-CD3 antibody, and an anti-CTLA4 antibody.

9. The fusion protein of claim 8, wherein the cytokine or an antibody moiety comprises an IL-2 mutein having the amino acid sequence of SEQ ID NO:5, SEQ ID NO 51, SEQ ID NO:52, or SEQ ID NO:53.

10. The fusion protein of any one of claims 1 to 9, wherein the PSL is capable of being cleaved in the tumor microenvironment.

11. The fusion protein of any one of claims 1 to 10, wherein the PSL has the amino acid sequence of PLGLWA (SEQ ID NO:300) or PAGLIG (SEQ ID NQ:301).

12. The fusion protein of any one of claims 1 to 11 , wherein the activity of the cytokine or antibody is reduced by at least two-fold in comparison to an isolated counterpart in the absence of protease cleavage.

13. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 12, and a pharmaceutically acceptable carrier.

14. A nucleic acid composition that encodes the fusion protein of any one of claims 1 to 13.

15. An expression vector composition comprising the nucleic acid composition of claim 14.

16. A method of making a fusion protein of any one of claims 1 to 12, comprising culturing the nucleic acid composition of claim 14 or the expression vector composition of claim 15 under conditions wherein the fusion protein is expressed, and recovering the fusion protein.

17. A method of treating a disease or disorder in a subject in need thereof comprising administering to the subject the fusion protein of any one of claims 1 to 12, or the pharmaceutical composition of claim 13.Attorney Docket No. 117802-5021 -WO18. A cytokine fusion protein comprising:a) an anti-PD1 antibody;b) an IL-2 or an IL-15 moiety comprising an IL-2, an IL-2 mutein, an IL-15, or an IL-15 mutein; c) a protease sensitive linker (PSL); andd) a masking moiety;wherein the PSL attaches the masking moiety to the IL-2 moiety, the IL-15 moiety, or the anti-PD1 antibody.

19. The cytokine fusion protein of claim 18, wherein the masking moiety comprises an IL-13 mutein that comprises the following amino acid substitutions compared to a wild-type human IL-13 (SEQ ID NO:200):a) L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, K105T; orb) L10H, E15R, R86T, D87G, T88R, R108K, Q111.

20. The cytokine fusion protein of claim 18 or 19, wherein the masking moiety comprises an IL-13, an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody, or an antigen-binding fragment thereof, wherein the masking moiety is capable of binding to IL-13Ra2, and does not bind to I L-13Ra1.

21. The cytokine fusion protein of claim 18 or 19, wherein the masking moiety comprises an IL-13, an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody, or an antigen-binding fragment thereof, wherein the masking moiety is capable of binding to IL-13Ra2 and binds to IL-13Ra1.

22. The cytokine fusion protein of any one of claims 18 to 21 , wherein the masking moiety comprises an IL-13 mutein that has the amino acid sequence of SEQ ID NOs: 201 or 202.

23. The cytokine fusion protein of claim 18 or 19, wherein the masking moiety comprises an IL-13, an IL-13 mutein, or type II IL-4 receptor antagonist thereof, wherein the masking moiety binds with higher affinity to IL-13Ra1 than wild type IL-13 (SEQ ID NQ:200), and binds with lower affinity to IL-13Ra2 than wild type IL-13.Attorney Docket No. 117802-5021 -WO24. The cytokine fusion protein of any one of claims 18 to 23, wherein the masking moiety comprises an IL-13 mutein that has the amino acid sequence of SEQ ID NO:201.

25. The cytokine fusion protein of any one of claims 18 to 24, wherein the IL-2 moiety comprises an IL-2 mutein having the amino acid sequence of SEQ ID NO:5, SEQ ID NO 51, SEQ ID NO:52, or SEQ ID NO:53.

26. The cytokine fusion protein of any one of claims 18 to 25, wherein the anti-PD 1 antibody is engineered as “knob-in-hole” (KiH) with mutations in constant region 3 (CH3) in its heavy chains.

27. The cytokine fusion protein of any one of claims 18 to 26, wherein the cytokine fusion protein comprises:a) a first polypeptide comprising an IL-2 or an IL-2 mutein, optionally an IL-2 fusion attached to the second and third constant regions (CH2 and CH3) of the “knob” heavy chain of the anti-PD1 antibody (KiH); b) a second polypeptide comprising the “hole” heavy chain of the anti-PD1 antibody (KiH); andc) a third polypeptide comprising the light chain of the anti-PD1 antibody (KiH).

28. The cytokine fusion protein of any one of claims 18 to 26, wherein the cytokine fusion protein comprises:a) a first polypeptide comprising the “knob” heavy chain of the anti-PD1 antibody (KiH) attached to an IL-2 or an IL-2 mutein, optionally an IL-2 fusion;b) a second polypeptide comprising the “hole” heavy chain of the anti-PD1 antibody (KiH);c) a third polypeptide comprising the light chain of the anti-PD1 antibody (KiH).

29. The cytokine fusion protein of any one of claims 18 to 26, wherein the cytokine fusion protein comprises:a) a first polypeptide comprising the “knob” heavy chain of the anti-PD1 antibody (KiH);b) a second polypeptide comprising the “hole” heavy chain of the anti-PD1 antibody (KiH) attached to an IL-2 or an IL-2 mutein or an IL-2 mutein fusion;c) a third polypeptide comprising the light chain of the anti-PD1 antibody (KiH).Attorney Docket No. 117802-5021 -WO30. The cytokine fusion protein of any one of claims 26 to 29, wherein the masking moiety is attached to the IL-2 or the IL-2 mutein.

31. The cytokine fusion protein of any one of claims 26 to 29, wherein the masking moiety is attached to the antibody heavy chain that is not attached to the IL-2 or the IL-2 mutein.

32. The cytokine fusion protein of any one of claims 26 to 29, wherein the masking moiety is attached to an additional masking moiety.

33. The cytokine fusion protein of any one of claims 18 to 32, wherein the PSL is capable of being cleaved in the tumor microenvironment.

34. The cytokine fusion protein of any one of claims 18 to 33, wherein the PSL has the amino acid sequence of PLGLWA (SEQ ID NO:300) or PAGLIG (SEQ ID NO:301).

35. A cytokine fusion protein comprising sequences selected from the group consisting of:(a) SEQ ID NOs: 6, 7, and 8;(b) SEQ ID NOs: 9, 10, and 11;(c) SEQ ID NOs: 12, 13, and 14;(d) SEQ ID NOs: 15, 16, and 17;(e) SEQ ID NOs: 18, 19, and 20;(f) SEQ ID NOs: 21, 22, and 23;(g) SEQ ID NOs: 24, 25, and 26;(h) SEQ ID NOs: 27, 28, and 29;(i) SEQ ID NOs: 30, 31, and 32;0) SEQ ID NOs: 33, 34, and 35;(k) SEQ ID NOs: 36, 37, and 38;(l) SEQ ID NOs: 39, 40, and 41;(m) SEQ ID NOs: 42, 43, and 44;(n) SEQ ID NOs: 45, 46, and 47;(o) SEQ ID NOs: 48, 49, and 50;Attorney Docket No. 117802-5021 -WO(p) SEQ ID NOs: 71, 72 and 73;(q) SEQ ID NOs: 74, 75 and 76;(r) SEQ ID NOs: 77, 78, and 79;(s) SEQ ID NOs: 80, 81, and 82;(t) SEQ ID NOs: 83, 84, and 85;(u) SEQ ID NOs: 86, 87, and 88;(v) SEQ ID NOs: 89, 90, and 91;(w) SEQ ID NOs: 92, 93, and 94;(x) SEQ ID NOs: 123, 124, and 125;(y) SEQ ID NOs: 128, 129, and 130;(z) SEQ ID NOs: 131, 132, and 133;(aa) SEQ ID NOs: 134, 135, and 136;(ab) SEQ ID NOs: 137, 138, and 139;(ac) SEQ ID NOs: 148, 149, 150, and 151;(ac) SEQ ID NOs: 152, 153, 154, and 155;(ad) SEQ ID NOs: 156, 157, 158, and 159;(ae) SEQ ID NO: 160, 161, 162, and 163;(af) SEQ ID NO: 165;(ag) SEQ ID NO: 167; and(ah) SEQ ID NO: 169.

36. A pharmaceutical composition comprising the cytokine fusion protein of any one of claims 18 to 35, and a pharmaceutically acceptable carrier.

37. A nucleic acid composition that encodes the cytokine fusion protein of any one of claims 18 to 35.

38. An expression vector composition comprising the nucleic acid composition of claim 37.

39. A method of making a cytokine fusion protein of any one of claims 18 to 35, comprising culturing the nucleic acid composition of claim 37 or the expression vector composition of claim 38 under conditions wherein the cytokine fusion protein is expressed, and recovering the cytokine fusion protein.Attorney Docket No. 117802-5021 -WO40. A method of treating an I L-13Ra2 expressing cancer in a subject in need thereof comprising administering to the subject the cytokine fusion protein of any one of claims 18 to 35, or the pharmaceutical composition of claim 36.

41. The method of claim 40, wherein the cytokine fusion protein comprises one or more of SEQ ID NOs:6-50, 71-94, 108-116, or 121-124.

42. The method of claim 40 or 41 , wherein the cytokine fusion protein is administered as a neoadjuvant prior to a surgery to remove a tumor.

43. The method of claim 42, wherein the cytokine fusion protein is administered up to one week prior to the surgery, optionally up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 1 day, or less than 1 day prior to the surgery, or wherein the cytokine fusion protein is administered up to 9 weeks prior to the surgery, optionally up to 8 weeks, up to 7 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, up to 2 weeks, up to 1 week, or less than 1 week prior to the surgery.

44. The method of any one of claims 40 to 41 , wherein the cytokine fusion protein is administered as an adjuvant after a surgery to remove a tumor.

45. The method of claim 44, wherein the cytokine fusion protein is administered up to one week after the surgery, optionally up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 1 day, or less than 1 day after the surgery, or wherein the cytokine fusion protein is administered starting at least two weeks after the surgery.

46. The method of any one of claims 40 to 41 , wherein the cytokine fusion protein is administered both as a neo-adjuvant prior to a surgery to remove a tumor, and an adjuvant after to a surgery to remove a tumor.

47. The method of claim 46, wherein the cytokine fusion protein is administered up to one week prior to the surgery, optionally up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, upAttorney Docket No. 117802-5021 -WOto 1 day, or less than 1 day prior to the surgery, and wherein the cytokine fusion protein is administered up to one week after the surgery, optionally up to 7 days, up to 6 days, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 1 day, or less than 1 day after the surgery, or wherein the cytokine fusion protein is administered up to 9 weeks prior to the surgery, optionally up to 8 weeks, up to 7 weeks, up to 6 weeks, up to 5 weeks, up to 4 weeks, up to 3 weeks, up to 2 weeks, up to 1 week, or less than 1 week prior to the surgery, and wherein the cytokine fusion protein is administered starting at least to two weeks after the surgery.

48. The method of any one of claims 40 to 47, wherein the cancer is a sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, pulmonary cancer, cutaneous, lymphoid, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal-like breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, small cell lung cancer, kidney cancer, gastric cancer, brain cancer, or a CNS tumors.

49. A fusion protein comprising:a) an anti-PD-1 antibody;b) an IL-2 mutein, wherein the anti-PD1 antibody is linked to the IL-2 mutein;b) an IL-13 superkine linked to the anti-PD-1 via a first protease sensitive linker (PSL); and c) an extracellular domain of CD122 linked to the IL-2 mutein via a second PSL.

50. The fusion protein of claim 49, wherein the anti-PD-1 antibody is a human anti-PD-1 based on nivolumab or pembrolizumab and wherein the anti-PD-1 antibody comprises an lgG1 or lgG4 backbone.

51. The fusion protein of claim 49 or 50, wherein the the IL-2 mutein comprises an amino acid sequence of SEQ ID NO:51, the IL-13 superkine comprises an amino acid sequence of SEQ ID NO:202, the PSL comprises an amino acid sequence of SEQ ID NQ:300, and the extracellular domain of CD122 comprises an amino acid sequence of SEQ ID NO: 350.

52. The fusion protein of any one of claims 49 to 51, wherein the fusion protein comprises the amino acid sequence of SEQ ID NOs: 83-85 or the amino acid sequence of SEQ ID NOs: 88-91.Attorney Docket No. 117802-5021 -WO53. A pharmaceutical composition comprising the fusion protein of any one of claims 49 to 52, and a pharmaceutically acceptable carrier.

54. A nucleic acid composition that encodes the fusion protein of any one of claims 49 to 52.

55. An expression vector composition comprising the nucleic acid composition of claim 54.

56. A method of making a fusion protein of any one of claims 49 to 52, comprising culturing the nucleic acid composition of claim 54 or the expression vector composition of claim 55 under conditions wherein the fusion protein is expressed, and recovering the fusion protein.

57. A method of treating a disease or disorder in a subject in need thereof comprising administering to the subject the fusion protein of any one of claims 49 to 52, or the pharmaceutical composition of claim 53.

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