Il-2 fusion proteins

Abstract

Provided herein are novel IL-2 fusion proteins that include an IL-2 moiety attached to an IL-2 masking moiety that includes an IL-13 mutein, an IL-13Ra2 binding mutein, an IL-13Ra2 antibody or antigen-binding fragment thereof, an extracellular domain of CD122, an extracellular domain of CD132, or an extracellular domain of CD25 by a protease sensitive linker (PSL). In some embodiments, the IL-2 fusion proteins include an IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment thereof that is capable of binding to IL- 13Ra2, and does not bind to IL-13Ro1, and the protease sensitive linker is cleavable by a protease in the tumor microenvironment. Such IL-2 fusion proteins are useful, for example, for the treatment of IL-13Ro2 expressing cancers.

Description

IL-2 FUSION PROTEINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 to U.S. Patent Application Nos. 63 / 510,580, filed on June 27, 2023, 63 / 594,694, filed on October 31 , 2023, 63 / 595,044, filed on November 1 , 2023, 63 / 595,271 , filed on November 1, 2023, and 63 / 575,960, filed on April 8, 2024, all of which are expressly incorporated herein 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-2RP; CD122), and the interleukin 2 receptor gamma (IL-2Ry;CD132; common gamma chain). The first receptor to be identified was the IL-2Ra, which is a 55 kD polypeptide (p55) that appears upon T cell activation and was originally called Tac (for T activation) antigen. The IL-2Ra binds IL-2 with a Kd of approximately 10'8M and is also known as the “high affinity” IL-2 receptor. Binding of IL-2 to cells expressing only the IL-2Ra does not lead to any detectable biologic response. In most circumstances, IL-2 works through three different receptors: the IL-2Rct, the IL-2Rp, and the IL-2Ry. Most cells, such as resting T cells, are not responsive to IL-2 since they only express the IL-2RJ3, and the IL-2Ry, which have low affinity for IL-2. Upon stimulation, resting T cells express the relatively high affinity IL-2 receptor IL-2Ra.Binding of IL-2 to the IL-2Ra causes this receptor to sequentially engage the IL-2Rp, and the IL-2Ry, bringing about T cell activation. IL-2 “superkines” with augmented action due to enhanced binding affinity for IL-2R0 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 are novel IL-2 fusion proteins that include an IL-2 moiety attached to at least one IL-2 masking moiety comprising an IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigenbinding fragment thereof by a protease sensitive linker (PSL). In embodiments, the IL-2 fusion protein includes at least one IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment thereof that is capable of binding to IL-13Ra2, and does not bind to IL-13Ro1 , and the protease sensitive linker is cleavable by a protease in the tumor microenvironment. When the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment thereof is attached to the IL-2 by the PSL, the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment thereof of the IL-2 fusion proteins described herein binds to and masks the activity of the IL-2 moiety. In addition, the IL-13 mutein or I L-13Ra2 antibody or antigen-binding fragment thereof allows the subject IL-2 fusion protein to bind to IL-13Ra2 expressing tumors. Once localized to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing the IL- 13 mutein or IL-13Ra2 antibody or antigen-binding fragment 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 subject IL-2 fusion proteins provided herein advantageously exhibit reduced IL-2 associated toxicity while allowing for targeted activity at IL-13Ra2 expressing tumors. Such IL-2 fusion proteins are particularly useful for the treatment of IL-13Ra2 expressing cancers.

[0007] In one aspect, provided herein is an IL-2 cytokine fusion protein comprising: a) an IL-2 moiety comprising an IL-2 or an IL-2 mutein, optionally an IL-2 mutein fusion; b) at least one protease sensitive linker (PSL); and c) at least one IL-2 masking moiety comprising an IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment thereof, wherein the PSL attaches the IL-2 masking moiety to the IL-2 moiety, optionally wherein the IL-2 masking moiety is capable of binding to IL-13 Ra2 , and does not bind to IL-13Ra1 .

[0008] In some embodiments, the masking moiety comprises an IL-13 mutein that has the amino acid sequence of any one of SEQ ID N0s:200-241 . In some embodiments, the masking moiety comprises at least one IL-13 mutein that comprises the following amino acid substitutions compared to a wild-type human IL-13 (SEQ ID NQ:200): a) L10H, E15R, R86T, D87G, T88R, R108K, Q111 , b) L10H, E15R, R86T, D87G, T88R, R108K, R111, c) L10H, R86T, D87G, T88R, R108K, Q111 , or d) L10H, R86T, D87G, T88R, R108K, R111. In some embodiments, the masking moiety comprises at least one IL-13 mutein that has the amino acid sequence of SEQ ID NO:216 or SEQ ID NO:228.

[0009] In some embodiments, provided herein are novel IL-2 fusion proteins that include an IL-2 moiety attached to at least one IL-2 masking moiety comprising an IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment thereof by a protease sensitive linker (PSL). In embodiments, the IL-2 fusion protein includes at least one IL-13 mutein, IL-13Ra2 binding mutein or IL-13Ra2 antibody or antigen-binding fragment thereof that is capable of binding to IL-13Ra2 and does not bind 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 thereof is attached to the IL-2 by the PSL, the IL-13 mutein or I L-13Ra2 antibody or antigen-binding fragment thereof of the IL-2 fusion proteins described herein binds to and masks the activity of the IL-2 moiety. In addition, the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment thereof allows the subject IL-2 fusion protein to bind to I L-13Ra2 expressing tumors. Once localized to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment 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 subject IL-2 fusion proteins provided herein advantageously exhibit reduced IL-2 associated toxicity while allowing for targeted activity at IL-13Ra2 expressing tumors. Such IL-2 fusion proteins are particularly useful for the treatment of IL-13Ra2 expressing cancers.

[0010] In one aspect, provided herein is an IL-2 cytokine fusion protein comprising: a) an IL-2 moiety comprising an IL-2 or an IL-2 mutein, optionally an IL-2 mutein fusion; b) at least one protease sensitive linker (PSL); and c) at least one IL-2 masking moiety comprising an IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment thereof, wherein the PSL attaches the IL-2 masking moiety to the IL-2 moiety, optionally wherein the IL-2 masking moiety is capable of binding to IL-13 Ra2 , and does not bind to IL-13Ra1 .

[0011] In some embodiments, the masking moiety comprises an IL-13 mutein that has the amino acid sequence of any one of SEQ ID NQs:200-241 . In some embodiments, the masking moiety comprises at least one IL-13 mutein that comprises the following amino acid substitutions compared to a wild-type human IL-13(SEQ ID NQ:200): a) L10H, E15R, R86T, D87G, T88R, R108K, Q111 , b) L10H, E15R, R86T, D87G, T88R, R108K, R111 , c) L10H, R86T, D87G, T88R, R108K, Q111 , or d) L10H, R86T, D87G, T88R, R108K, R111. In some embodiments, the masking moiety comprises at least one IL-13 mutein that has the amino acid sequence of SEQ ID NO:216 or SEQ ID NO:228.

[0012] In some embodiments, the masking moiety further comprises an extracellular domain of CD122, an extracellular domain of CD132, or an extracellular domain of CD25.

[0013] In some embodiments, the IL-2 moiety comprises an IL-2 mutein having the amino acid sequence of any one of SEQ ID NOs:5-24 and 105. In some embodiments, the IL-2 mutein has the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:9. In some embodiments, the IL-2 mutein further comprises a T3A and a C125S amino acid substitution.

[0014] In some embodiments, the IL-2 moiety further comprises an albumin or an Fc domain or an antibody attached to the IL-2 or IL-2 mutein. In certain embodiments, the albumin is a human albumin, optionally wherein the human albumin is recombinant human albumin. In certain embodiments, the antibody is engineered as “knob-in-hole” (KiH) with mutations in constant region 3 (CH3) of the antibody heavy chains.

[0015] In some embodiments, the IL-2 moiety comprises an IL-2 x anti-PD1 fusion protein comprising: a) a first polypeptide comprising 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 domain; b) a second polypeptide comprising a second antibody heavy chain, wherein the second antibody heavy chain comprises a second heavy chain variable region (VH2) and a second Fc domain; c) a third polypeptide comprising a first variable light chain variable region (VL1) and a light chain constant region; and d) a fourth polypeptide comprising a second variable light chain variable region (VL2) and a light chain constant region, wherein VH1 and VL1 form a first PD-1 binding domain, and VH2 and VL2 form a second PD-1 binding domain.

[0016] In some embodiments, the first PD-1 binding domain and second PD-1 binding domain have the same amino acid sequence. In certain embodiments, the first PD-1 binding domain and second PD-1 binding domain have different amino acid sequences. In some embodiments, the IL-2 masking moiety is attached to the IL-2 or the IL-2 mutein. In some embodiments, the IL-2 masking moiety is attached to the second Fc domain, and the IL-2 or the IL-2 mutein is attached to the first Fc domain.

[0017] In some embodiments, the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising an IL-2 or an IL-2 mutein or an IL-2 fusion attached to the second and third constant regions (CH2 and CH3) of the “knob” heavy chain of the antibody (KiH); b) a second polypeptidecomprising the “hole” heavy chain of the antibody (KiH); and c) a third polypeptide comprising the light chain of the antibody (KiH).

[0018] In some embodiments, the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising the “knob” heavy chain of the antibody (KiH) attached to an IL-2 or an IL-2 mutein or an IL-2 fusion; b) a second polypeptide comprising the “hole” heavy chain of the antibody (KiH); and c) a third polypeptide comprising the light chain of the antibody (KiH).

[0019] In some embodiments, the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising the “knob” heavy chain of the antibody (KiH); b) a second polypeptide comprising the “hole” heavy chain of the antibody (KiH) attached to an IL-2 or an IL-2 mutein or an IL-2 mutein fusion; and c) a third polypeptide comprising the light chain of the antibody (KiH).

[0020] In some embodiments, the IL-2 masking moiety is attached to the IL-2 or IL-2 mutein or IL-2 mutein fusion in the IL-2 moiety. In some embodiments, the IL-2 masking moiety is attached to the antibody (KiH) heavy chain that is not attached to an IL-2 or an IL-2 mutein or an IL-2 mutein fusion.

[0021] In some embodiments, the antibody (KiH) is anti-PD1 (KiH).

[0022] In some embodiments, the IL-2 mutein has an amino acid sequence of any one of SEQ ID NOs: 5-24 and 105. In some embodiments, the IL-2 mutein further comprises a T3A and a C125S amino acid substitution

[0023] In some embodiments, the PSL is capable of being cleaved in the tumor microenvironment. In certain embodiments, the PSL has the amino acid sequence of PLGLVVAPLGLVVAPLGLWA, PLGLWAPLGLWAPLGLWA , GGSGGTPLGLWAGGSGGT, GGSGGTPAGLIGGGSGGT, GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT, or GGSGGTHSSKLQGGSGGT.

[0024] In another aspect, provided herein is an IL-2 cytokine fusion protein comprising sequences selected from the group consisting of (a) SEQ ID NOs: 302, 303, and 304; (b) SEQ ID NOs: 299, 300, and 301 ; (c) SEQ ID NOs: 305, 306, and 307; (d) SEQ ID NOs: 308, 309, and 310; (e) SEQ ID NOs: 311 , 312, and 313; (f) SEQ ID NOs: 314, 315, and 316; (g) SEQ ID NOs: 317, 318, and 319; (h) SEQ ID NOs: 320, 321 , and 322; (I) SEQ ID NO: 323; (j) SEQ ID NOs:329, 330, and 331; (k) SEQ ID NOs:332, 333, and 334; (I) SEQ ID NOs:335, 336, and 337; (m) SEQ ID NOs:338, 339, and 340; (n) SEQ ID NOs:353, 354, and 355; (o) SEQ ID NOs:359, 360, and 361 ; (p) SEQ ID NOs:362, 363, and 364; (q) SEQ ID NOs: 365, 366, and 367; (r) SEQ ID NOs: 325, 326, and 327; (s) SEQ ID NOs: 353, 354, and 355; and (t) SEQ ID NOs: 324.

[0025] In another aspect, provided herein is a pharmaceutical composition comprising one of the IL-2 cytokine fusion protein disclosed herein, and a pharmaceutically acceptable carrier.

[0026] Also provided herein are nucleic acid compositions and expression vector compositions comprising nucleic acids that encode the subject IL-2 cytokine fusion proteins, host cells that include the nucleic acid composition or expression vector composition, and methods of making the IL-2 cytokine fusion proteins using the host cells.

[0027] In another aspect, provided herein is a method of treating an IL-13Ra2 expressing cancer in a subject in need thereof comprising administering to the subject the IL-2 cytokine fusion protein.

[0028] In another aspect, provided herein is a method of treating a cancer in a subject in need thereof comprising administering to the subject an IL-2 cytokine fusion protein comprising: a) an IL-2 mutein; and b) an albumin or an Fc domain or an antibody; wherein the IL-2 mutein has an amino acid sequence of any one of SEQ ID NOs: 5-24 and 105 and optionally further comprises a T3A and / or a C125S amino acid substitution.

[0029] In another aspect, the IL-2 cytokine fusion protein comprises one or more of SEQ ID NOs:1-367.

[0030] In another aspect, the IL-2 cytokine fusion protein has the amino acid sequence of SEQ ID NO:53.

[0031] In another aspect, the IL-2 cytokine fusion protein is administered as a neo-adjuvant prior to a surgery to remove a tumor.

[0032] In another aspect, the IL-2 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 IL-2 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.

[0033] In another aspect, the IL-2 cytokine fusion protein is administered as an adjuvant after a surgery to remove a tumor.

[0034] In another aspect, the IL-2 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 IL-2 cytokine fusion protein is administered starting at least two weeks after the surgery.

[0035] In another aspect, the IL-2 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.

[0036] In another aspect, the IL-2 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 IL-2 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 IL-2 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 IL-2 cytokine fusion protein is administered starting at least to two weeks after the surgery.

[0037] In another aspect, 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A provides graphs, summarizing a Jurkat I L2 Rpy reporter analysis of constructs MDNA109- Alb (SEQ ID NO: 56) and MDNA132.15-PSL-MDNA109-Alb (SEQ ID NO: 298) (also referred to as “MDNA213-PSL-MDNA109-Albumin"). The assay was performed independently on two plates. Plots and curve fits are presented for each construct on the respective plate.

[0039] Figure 1B provides graphs of the Jurkat IL2R(3y reporter analysis of constructs MDNA11 (SEQ ID NO: 53) and MDNA132-PSL-MDNA11T3 / C125(SEQ ID NO: 294). The assay was performed independently on two plates. Plots and curve fits are presented for each construct on the respective plate.

[0040] Figure 2 provides sensograms for BLI conducted on constructs as indicated, human CD122 as ligand and constructs (MDNA109-albumin (SEQ ID NO: 56) and MDNA132.15-PSL-MDNA109-albumin (SEQ ID NO: 298)) as analytes.

[0041] Figure 3 provides sensograms for BLI conducted on indicated constructs with receptor, human IL13Ra2 as ligand and constructs (Fc-MDNA132.15 (SEQ ID NO: 270) and MDNA132.15-PSL-MDNA109- albumin (SEQ ID NO: 298)) as analytes.

[0042] Figure 4 provides the summary of a study showing that proteolytic activation by MMP9 restores IL-2 activity of MDNA213-PSL-MDNA109-Albumin (SEQ ID NO: 298).

[0043] Figure 5 provides the summary of a study showing that MDNA223T3AC125S-fPSL2f-MDNA213 (SEQ ID NOs: 299-301) exhibits reduced IL-2 activity and no change in PD-1 / PDL-1 blockade.

[0044] Figure 6 provides the summary of a study showing that proteolytic activation by MMP9 restores IL-2 activity of MDNA223T3AC125S-fPSL2f-MDNA213 (SEQ ID NOs: 299-301).

[0045] Figure 7 provides the summary of a study showing that MDNA223T3AC125S-fPSAf-MDNA213: Anti- mPD1 (H) (SEQ ID NOs: 302-304) exhibits 12-fold reduced potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade

[0046] Figure 8 provides the summary of a study showing that MDNA223T3AC125S-fPSL3f-MDNA213: Anti- mPD1(H) (SEQ ID NOs: 305-307) exhibits 11-fold reduced potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade.

[0047] Figure 9 provides the summary of a study showing that MDNA223T3AC125S-fPSL2fPS L3f- MDNA213:Anti-mPD1(H) (SEQ ID NOs: 308-310) exhibits 10-fold reduced potency (i.e., masked activity) in IL- 2 mediated signaling and no change in PD-1 / PDL-1 blockade.

[0048] Figure 10 provides the summary of a study showing that MDNA223T3AC125S-GS-MDNA213: anti- mPD1 (H) (SEQ ID NOs: 311-313) exhibits 11-fold reduced potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade

[0049] Figure 11 provides the summary of a study showing that MDNA223T3AC125S: Anti-mPDI (H)- fPSL2f- MDNA213 (SEQ ID NOs: 314-316) exhibits 3-fold reduced potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade.

[0050] Figure 12 provides the summary of a study showing that anti-mPD1 (K)-fPSL2f-MDNA213: MDNA223T3AC125S(SEQ ID NOs: 317-319) Exhibits 2-fold reduced potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade.

[0051] Figure 13 provides the summary of a study showing that MDNA223T3AC125S-fPSL2f-MDNA213: Anti- mPD1 (H)-fPSL2f-MDNA213 (SEQ ID NOs: 320-322) exhibits 39-fold reduced potency (i.e., masked activity) in IL-2 mediated Signaling and no change in PD-1 / PDL-1 blockade.

[0052] Figure 14 provides the summary of a study showing that MDNA223T3AC125S: Anti-mPD1 (H)-fPSL2f- MDNA213 Anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NOs: 320-322) is sensitive to MMP9 cleavage in vitro.

[0053] Figure 15 provides the summary of a study showing that MDNA223T3AC125S: Anti-mPD1 (H)-fPSL2f- MDNA213 Anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NOs: 320-322) is activated upon MMP9 cleavage as observed in the IL-2R reporter assay.

[0054] Figure 16 provides the summary of a study showing that MDNA213-fPSL2f- MDNA11T3AC125S(SEQ ID NO: 323) Exhibits ~10-fold reduced potency (i.e., masked activity) in IL-2 mediated signaling.

[0055] Figure 17 provides the summary of a study showing that MDNA213-fPSL2f- MDNA11T3AC125S(SEQ ID NO: 323) is sensitive to MMP9 cleavage in vitro.

[0056] Figure 18 provides the summary of a study showing that MDNA213-fPSL2f- MDNA11T3AC125S(SEQ ID NO: 323) is activated upon MMP9 cleavage as observed in the IL-2 reporter assay.

[0057] Figure 19 shows SDS-PAGE Analysis. Image of the reducing SDS-PAGE gel for various BiSKITs. Positions of molecular weight markers are shown on the left.

[0058] Figure 20 shows SDS-PAGE Analysis. Image of the reducing SDS-PAGE gel for various BiSKITs. Positions of molecular weight markers are shown on the left.

[0059] Figures 21 A and 21 B show graphs of the Jurkat IL2Rpy Reporter analysis. Plots and curve fits are presented for each construct on each plate. Data is presented as mean + SEM.

[0060] Figure 22 shows graphs of the CTLL-2 proliferation analysis of constructs as indicated. Plots and curve fits are presented for each construct on each plate. Data is presented as mean + SEM.

[0061] Figure 23 shows results of a human PBMC Proliferation assay: 3 PBMC donors were stimulated with different concentrations of indicated constructs for 48 h. The proliferation was analyzed by BrDU incorporation. Data presented as mean + SEM.

[0062] Figure 24 shows results of a human pSTAT5 assay: 3 PBMC donors were stimulated with different concentrations of indicated constructs for 15 min. pSTAT5 induction in naive CD8+T cells, NK cells, and Tregs are shown.

[0063] Figure 25 shows a compilation of EC50 data for masked and non-masked MDNA223 tested across the PBMC samples from three human donors for naive CD8+T cells and Tregs.

[0064] Figure 26 shows the frequency of cells, CD8+T cells, NK cells and Tregs expressing pSTAT5 in the 3 donors for various constructs. Human PBMCs were stimulated with masked and non-masked MDNA11 indicated for 15 min and analyzed for pSTAT5 by flow cytometry. Data is presented as singlicate.

[0065] Figure 27 shows a compilation of EC50 data for masked and non-masked MDNA11 tested across the PBMC samples from three human donors for naive CD8+T cells and Tregs.

[0066] Figure 28 shows EC50 ratios of naive CD8+T cells to TregS. EC50 ratios of naive CD8 T-cells to Tregs is an average of individual ratios of different PMBC samples used.

[0067] Figure 29 shows cell receptor (IL13Ra2) binding kinetics with Fc-MDNA213 (SEQ ID NOs: 270-271) and MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParH(SEQ ID NOs: 320-322). IL-13Ra2 expressing EMT6 / IL13Ra2 cells were subjected to increasing concentrations of indicated constructs and binding detected by anti-Fc antibody.

[0068] Figure 30 shows CBC Analysis: Complete blood count on day 3 post treatment with MDNA223A3 / S125masked and non-masked versions. Mice were treated (IP) with constructs as indicated. Blood was collected for CBC analysis 72 h post treatment. Data is presented as mean + SEM.

[0069] Figure 31 shows CBC Analysis: Complete blood count on day 3 post treatment with MDNA11 A3 / S125 masked and non-masked versions. Mice were treated (IP) with constructs as indicated. Blood was collected for CBC analysis 72 h post treatment. Data is presented as mean + SEM.

[0070] Figure 32 shows a study using a MC-38 Colon Carcinoma Model: C57BI / 6 mice were IT treated with indicated constructs at 15 g / tumor on twice weekly dosing regimen, 4 doses in total. Data presented as average tumor measurements on different days and as mean + SEM

[0071] Figure 33 shows a study using an EMT6 / IL13Ra2 Breast Tumor Model: Balb / c mice were IP treated 4 days post cell implantation with indicated constructs at equimolar doses on once weekly dosing regimen, 2 doses in total. Data presented as average tumor measurements on different days and as mean + SEM.

[0072] Figure 34 shows a study using a MC-38 Colon Carcinoma Model: C57BI / 6 mice were IP treated with indicated constructs at equimolar doses on once weekly dosing regimen, 2 doses in total. Data presented as average body weight (left) and tumor measurements (right) on different days and as mean + SEM.

[0073] Figure 35 shows Body weight of animals in once weekly dosing regimen. The average body weight measurement of each group is indicated. Data presented as mean + SD.

[0074] Figure 36 shows body weight of animals in twice weekly dosing regimen. The average body weight measurement of each group is indicated. Data presented as mean + SD.

[0075] Figure 37 shows a Survival Curve for animals in the MTD study with MDNA1 13AA3 / S125. Mice were treated at the indicated dose and dosing schedule. Kaplan-Meier plots show overall survival for each group. Mice that were alive at the end of the study were censored at Day 18. N=3 mice per group.

[0076] Figure 38 shows graphs of the Jurkat IL2Rpy Reporter analysis. Plots and curve fits are presented for each construct on each plate. Data is presented as mean+SEM

[0077] Figure 39 shows graphs of HEK Blue IL-2 Reporter analysis. OD650nm data was plotted as a function of the cytokine concentration (nM) on a semi-log graph. A four-parameter logistic curve fit is presented as a solid line. Error bars represent the standard error of the means of the replicate wells.

[0078] Figure 40 shows graphs of the Mouse PD-1 / PD-L1 Blockade Assay. Plots and curve fits are presented for each construct on each plate.

[0079] Figure 41 shows SDS-PAGE Analysis. Image of the reducing SDS-PAGE gel for various MASK-ITs. Positions of molecular weight markers are shown on the left.

[0080] Figure 42A shows graphs of the Jurkat I L2 Rpy or HEK Blue IL-2 Reporter analysis. Plots and curve fits are presented for each construct on each plate. Data is presented as mean + SEM.

[0081] Figures 42B and 42C show graphs of the Jurkat IL2Rpy or HEK Blue IL-2 Reporter analysis Plots and curve fits are presented for each construct on each plate. Data is presented as mean + SEM.

[0082] Figure 43 shows representative sensorgrams of unlabelled and labelled MDNA223 and MDNA113 showing binding to mouse CD122.

[0083] Figure 44 shows in vivo imaging data at the indicated time points. A549 and A375 tumors are on the left and right flank respectively as viewed.

[0084] Figure 45 shows results of a MC-38 TGI Study: Mice were treated as indicated. Data represented as mean + SEM.

[0085] Figure 46 shows results of a MC-38 TGI Study: Mice were treated as indicated. Data represented as mean + SEM.

[0086] Figure 47 shows PD response. Mice were treated with constructs as indicated. Samples were collected on day 3 for CBC analysis. Data represented as mean + SEM.

[0087] Figure 48 shows Body weight: Animals in different groups were treated as indicated on either a once weekly or twice weekly dosing regimen. Body weight measurements were taken twice weekly during the study.

[0088] Figure 49 shows Jurkat IL-2 reporter assay. Data presented as mean + SEM.

[0089] Figure 50 shows SDS-PAGE for detection of cleavage by MMP9

[0090] Figure 51 shows IL-13Ra2 binding by flow cytometry. The constructs as indicated were titrated on A375 cells. Fc-MDNA213 was titrated against both A375 and A549 cells.

[0091] Figure 52 shows results of a MC38 Tumor growth inhibition study: C57BI / 6 mice were treated IP as indicated. Data presented as mean + SEM.

[0092] Figure 53 shows results of a MC38 Tumor growth inhibition study: C57BI / 6 mice were treated IP as indicated. Data presented as mean + SEM.

[0093] Figure 54 shows results of a MC38 Tumor growth inhibition study: C57BI / 6 mice were treated IP as indicated. Data presented as mean + SEM.

[0094] Figure 55 shows a Survival Curve for neo-adjuvant effect of MDNA113, MDNA223 or anti-mPD1 in 4T 1 .2 breast tumor model.

[0095] Figure 56 shows a Survival Curve for adjuvant effect of MDNA223 or anti-mPD1 in 4T 1 .2 breast tumor model.

[0096] Figure 57 provides sensograms for BLI conducted on indicated constructs with human CD122 (IL- 2Rp) as ligand and constructs MDNA223A3 / 3125(SEQ |D NOS:107-109), MDNA113AA3 / S125(SEQ ID NOs: 320- 322) and MDNA113BA3 / S125(SEQ ID NOs: 335-337) as analytes.

[0097] Figure 58 provides the summary of a study showing that MDNA113BT3 / C125is activated upon MMP9 cleavage as observed in the IL-2R reporter assay.

[0098] Figure 59 shows a Kaplan-Meier survival plot of number of mice alive out of total (n / 8) over 65 study days. Mice were implanted with 4T1 .2 tumor cells orthotopically and treated with MDNA11 (5 mg / kg) as a neoadjuvant and adjuvant with surgical tumor resection (Day 16). ‘Comparison to control (surgery; no treatment). Mantel-Cox test. N = 8 per group.

[0099] Figure 60 shows individual tumor volumes in mice after initial tumor implantation and subsequent rechallenges with 4T 1 .2 tumors. N = 5 in control group. N=7 and 3 in the neoadjuvant and adjuvant groups respectively.

[0100] Figure 61 shows multiplex immunofluorescence images of 4T 1 .2 tumors excised from control mice and mice treated with MDNA11 (5 mg / kg) demonstrating immune cell infiltration. N = 4 per group.

[0101] Figure 62 shows individual quantification of immune cell infiltrates in total tumor area through Multiplex Immunofluorescence images of 4T1.2 tumors excised from control mice and mice treated with MDNA11 (5 mg / kg) . N = 4 per group.DETAILED DESCRIPTION

[0102] 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

[0103] 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.

[0104] 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 NOH ; 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 acid sequence is found in Genbank under accession locator (SEQ ID NOG). The amino acid sequence of mature murine IL-2 is depicted in SEQ ID NO:4.SEQ ID NO:1MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKH LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTL TSEQ ID NO:2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQS KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSEQ ID NO:3MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLP RMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDE SATWDFLRRWIAFCQSIISTSPQSEQ ID N0:4APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQ CLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIIST SPQ

[0105] 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-2Rp 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.

[0106] 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 II I :val, lie, leu, met, ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.

[0107] 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 wildtype 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.

[0108] 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 abbreviations

[0109] "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).

[0110] 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 typeshaving the I L-2RPy receptor" means the cells known to have that receptor type, i.e., B cells, resting monocytes, and resting NK cells.

[0111] 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 (i.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.

[0112] 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).

[0113] 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

[0114] An “agonist" is a compound that interacts with a target to cause or promote an increase in the activation of the target.

[0115] 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.

[0116] 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%.

[0117] “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 thenucleotide 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., tissue-specific 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] As used herein, the term “anti-PD-1 antibody” refers to any antibody 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 nivolumab, BMS-936558, MDX-1106, ONO- 4538, AMP224, CT-011 , and MK-3475, among others.

[0123] 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, nonsmall cell carcinoma of the lung, cancer of the small intestine and cancer of the 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.

[0124] 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.

[0125] 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.

[0126] 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, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. A therapeutically effective amount can be an amount that reduces tumor number, tumor size, and / or increases survival.

[0127] 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.).

[0128] 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 an IL-2 mutein to a subject. In particular, an IL-2 mutein comprising the substitutions L80F, R81 D, L85V, I86V, and I92F is administered incombination 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.

[0129] 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, the therapeutic effect is to reduce tumor size. In some embodiments, the therapeutic effect is to increase survival.

[0130] 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. IL-2 muteins in combination with anti-PD-1 antibodies, and pharmaceutical compositions thereof can be packaged in a single or multiple unit dosage form for ease of administration and uniformity of dosage.

[0131] 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.

[0132] 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).

[0133] 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 anamount 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.

[0134] “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 a subject 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.I. DETAILED DESCRIPTION

[0135] Provided herein are novel IL-2 fusion protein compositions that include an IL-2 moiety attached to an IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment thereof by a protease sensitive linker (PSL). In embodiments, the IL-2 fusion protein compositions include an IL-13 mutein, an IL- 13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment thereof that is capable of binding to IL-13Ro2, and does not bind 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 IL-13Ra2 antibody or antigen-binding fragment thereof of the IL-2 fusion protein compositions described herein binds to and masks the activity of the IL-2 moiety. In addition, the IL-13 mutein or I L-13Ra2 antibody or antigen-binding fragment thereof allows the subject IL-2 fusion protein compositions to bind to I L-13Ro2 expressing tumors. Once localized to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment thereof and “unmasking” the IL-2 moiety. The unmasked IL-2 moiety can then act at the tumor site. Thus, in embodiments, the subject IL-2 fusion proteins provided herein advantageously exhibit reduced IL-2 associated toxicity while allowing for targeted activity at IL-13Ra2 expressing tumors. Such IL-2 fusion protein compositions are useful for the treatment of IL-13Ra2 expressing cancers. Aspects of the subject IL-2 fusion protein are further detailed below.A. IL-2 MOIETY

[0136] In embodiments, the subject IL-2 fusion proteins provided herein include an IL-2 moiety that comprises a IL-2 or IL-2 mutein. In embodiments, the IL-2 fusion protein includes a wild-type human IL-2(SEQ ID N0:2). 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 IL-2R|3 and / or the IL-2Ry.

[0137] More specifically, a mutation (whether conservative or non-conservative, by way of addition(s) or deletion(s)) can be made at one or more of 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 toa wild-type human IL-2 (SEQ ID NO:2): I24V, P65H, Q74R, Q74H, Q74N, Q74S, L80F, L80V, R811, R81T, R81 D, L85V, I86V, I89V, I92F, V93I, and combinations thereof. Exemplary IL-2 muteins that can be included in the subject IL-2 fusion proteins are shown in Table 2 below.Table 2: Exemplary IL-2 muteins

[0138] In some embodiments, the substitutions in the IL-2 mutein comprise L80F, R81 D, L85V, I86V, and I92F, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises F42A substitution, wherein numbering is in accordance with the wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises Y45A substitution, wherein numbering is in accordance with the wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises E62A substitution, wherein numbering is in accordance with the wild-type human IL- 2 of SEQ ID NO:2. In some embodiments, the substitutions in the IL-2 mutein comprise F42A, L80F, R81 D, L85V, I86V, and I92F, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the substitutions in the IL-2 mutein comprise F42A, Y45A, L80F, R81 D, L85V, I86V, and I92F, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the substitutions in the IL-2 mutein comprise F42A, E62A, L80F, R81 D, L85V, I86V, and I92F, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the substitutions in the IL-2 mutein comprise F42A, Y45A, E62A, L80F, R81D, L85V, I86V, and I92F, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the substitutions in the IL-2 mutein comprise E62A, L80F, R81 D, L85V, I86V, and I92F, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the substitutions in the IL-2 mutein comprise Y45A, E62A, L80F, R81 D, L85V, I86V, and I92F, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the substitutions in the IL-2 mutein comprise Y45A and E62A , numbered in accordance with wild-type human IL-2 of SEQ ID NO:2.

[0139] In some embodiments, the IL-2 mutein exhibits increased or enhanced IL-2R|3 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-2Rp binding include L80F, R81 D, L85V, I86V, and I92F, numbered in accordance with wild-type human IL-2 of SEQ ID NO:2. In some embodiments, an IL-2 mutein for use in the IL-2 fusion proteins described herein comprises L80F, R81 D, L85V, I86V, and I92F and exhibits increased IL-2Rp binding. In some embodiments, an IL-2 mutein for use in the invention further comprises a substitution at position F42A. In some embodiments, the IL-2 mutein for use in the invention further comprises a substitution at position K43N . In some embodiments, the mutein comprises substitutions L80F, R81 D, L85V, I86V, and I92F, and one or more substitutions selected from the group consisting of F42A, Y45A, and E62A, all as compared to wild-type human IL-2 (SEQ ID NO:2).

[0140] In some embodiments, the amino acid substitutions increasing IL-2R|3 binding affinity include: L80F, R81 D, L85V, I86V, and I92F. In some embodiments, the amino acid substitutions that increase IL-2 R|3 binding affinity include: L80F, R81 D, L85V, I86V, and I92F.

[0141] In some embodiments, the subject IL-2 mutein having a greater binding affinity for I L-2R|3 as compared to wild-type human IL-2, includes the amino acid substitutions L80F, R81 D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the amino acid sequence:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID N0:5).

[0142] In some embodiments, the IL-2 mutein has increased capabilities to stimulate one or more signaling pathways that are dependent on I L-2Rp / IL-2Rycheterodimerization. In some embodiments, the subject IL-2 mutein has an enhanced capability to stimulate STAT5 phosphorylation in an IL-2Rp+ 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-2Rp+ 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-2RP+ 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-2Rp+ cell is a natural killer (NK) cell. In some embodiments, the IL-2 mutein comprises substitutions L80F, R81 D, L85V, I86V, and I92F, as compared to wild-type human IL-2 (SEQ ID NO:2).

[0143] In some embodiments, the mutein has an enhanced capability to stimulate ERK1 / ERK2 signaling in an IL-2Rf>+ cell as compared to wild-type human IL-2. In some embodiments, the IL-2 mutein stimulates pERK1 / ERK2 signaling 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 pERK1 / ERK2 signaling in the same cell. In some embodiments, the IL-2 mutein stimulates pERK1 / ERK2 phosphorylation in an IL-2Rp+ 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 pERK1 / ERK2 phosphorylation in the same cell. In some embodiments, the IL-2Rp+ 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-2Rp+ cell is a natural killer (NK) cell. In some embodiments, the IL-2 mutein comprises substitutions L80F, R81 D, L85V, I86V, and I92F, as compared to wild-type human IL-2 (SEQ ID NO:2).

[0144] 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 flowcytometry analysis as described herein. In some embodiments, the mutein has an enhanced capability to stimulate PI 3-kinase signaling in a IL-2RP+ cell as compared to wild-type human IL-2. In some embodiments, the IL-2 mutein stimulates PI 3-kinase signaling 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 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-2RJ3+ 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|3+ 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 IL-2Rp+ cell is a natural killer (NK) cell. In some embodiments, the IL-2 mutein comprises substitutions L80F, R81 D, L85V, I86V, and I92F, as compared to wild-type human IL-2 (SEQ ID NO:2). PI3-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.

[0145] 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, TCR-induced cell proliferation. In some embodiments, the IL-2 mutein comprises substitutions L80F, R81 D, L85V, I86V, and I92F, as compared to wild-type human IL-2 (SEQ ID NO:2).

[0146] 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 Th 17 differentiation. In some embodiments, the IL-2 mutein comprises substitutions L80F, R81 D, L85V, I86V, and I92F, as compared to wild-type human IL-2 (SEQ ID NO:2).

[0147] 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 T regs. In some embodiments the reduced and / or independent signaling with regard to CD25 allows for reduced toxicity. In some embodiments, the mutein comprises substitutions L80F,R81 D, L85V, I86V, and I92F, and one or more substitutions selected from the group consisting of F42A, Y45A, and E62A, all as compared to wild-type human IL-2 (SEQ ID NO:2). In some embodiments, the IL-2 mutein comprises SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NQ:10.

[0148] 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 L80F, R81 D, L85V, I86V, and I92F, as compared to wild-type human IL-2 (SEQ ID NO:2).

[0149] 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.

[0150] In some embodiments, an increase in IL-2R(3 binding affinity is any binding affinity for IL-2Rp that is greater than the wild-type human IL-2 binding affinity for IL-2Rp. 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-2R|3 as compared to the wild-type human IL-2 binding affinity for IL-2Rp.

[0151] In some embodiments, an increase in binding capacity for IL-2R|3 is any binding capacity for IL- 2Rp that is greater than the wild-type human IL-2 binding capacity for IL-2Rp. 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-2R|3 as compared to the wild-type human IL-2 binding capacity for IL-2R|3.

[0152] In some embodiments, the subject 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 substitutions F42A, L80F, R81 D, L85V, I86V, and I92F. In some embodiments, the reduce binding affinity is about 220-fold, i.e., from about Kd of 6.6 nM for wild-type human IL-2 to about 1.4 M for the mutein comprising F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the amino acid sequence:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:6; also referred to as H9-F42A).

[0153] In some embodiments, the subject 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 substitutions K43N, L80F, R81 D, L85V, I86V, and I92F. In some embodiments, the reduce binding affinity is due to allowing for glycosylation at position 43 with the K43N substitution. By substituting lysine for asparagine (K43N), CD25 binding is reduced and / or ablated in the IL-2 mutein comprising the amino acid substitutions K43N, L80F, R81 D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the amino acid sequence:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFNFYMPKKATELKHLQCLEEELKPLEEVLNLAQS KNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NOY; also referred to as H9-K43N).

[0154] 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 wild-type human IL-2 binding affinity for CD25.

[0155] In some embodiments, the subject IL-2 mutein having a greater binding affinity for IL-2R|3 and a reduced binding affinity for CD25 as compared to wild-type human IL-2 includes the amino acid substitutions F42A, Y45A L80F, R81 D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the amino acid sequence:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNLAQ SKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:8; H9- F42A / Y45A; H9-FYAA).

[0156] In some embodiments, the subject IL-2 mutein having a greater binding affinity for IL-2R|3 and a reduced binding affinity for CD25 as compared to wild-type human IL-2 includes the amino acid substitutions F42A, E62A L80F, R81 D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the amino acid sequence:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEALKPLEEVLNLAQ SKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:9; H9- F42A / E62A; H9-FEAA).

[0157] In some embodiments, the subject IL-2 mutein having a greater binding affinity for I L-2R|3 and a reduced binding affinity for CD25 as compared to wild-type human IL-2 includes the amino acid substitutionsF42A, Y45A, E62A, L80F, R81 D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the amino acid sequence:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEALKPLEEVLNLAQ SKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:10; H9- F42A / Y45A / E62A; H9-FYEAAA).

[0158] 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. In some embodiments, the IL-2 mutein is a variant of MDNA109 (SEQ ID NO:5) or MDN109FEAA (SEQ ID NO:9) that further includes a T3A and / or C125S substitution.

[0159] In some embodiments, the IL-2 mutein of the IL-2 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-2 muteins in Table 2. In some embodiments, the IL-2 mutein of the IL-2 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 muteins in Table 2 (SEQ ID NO:5-24). In embodiments, the variant exhibits greater binding affinity for I L-2R|3 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

[0160] In some embodiments, the IL-2 moiety includes a polypeptide that increases the in vivo serum half-life of the IL-2 included in the IL-2 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, 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 to bind complement or to lyse cells via another mechanism, such as antibody-dependent complement lysis (ADCC; USSN 08 / 355,502 filed Dec. 12, 1994).

[0161] 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 subject IL-2 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 Fcregions can be variants of the wild-type molecule. In some embodiments, the IL-2 moiety of the subject IL-2 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, Ig G3 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, Ig G3 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.

[0162] 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'1 q 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-dependent 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'1 q 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).

[0163] In some embodiments, the half-life enhancing polypeptide is a serum albumin. In exemplary embodiments, the serum albumin is a human serum albumin or variant thereof that is capable of extending theserum half-life of the attached IL-2 or IL-2 mutein. In some embodiments, the human serum albumin has the following amino acid sequence:DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDK LCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARR HPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVA RLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIA EVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKC CKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFH ADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAAL GL (SEQ ID NQ:400).

[0164] 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).

[0165] 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 Val-Cit (VC) linker, a Phe-Arg linker, a Val-Lys linker, a Vai-Ala linker, a Val-Arg linker, a Val-Leu-Lys linker, a Gly-Phe-Leu-Glylinker, an Ala-Phe-Lys linker, a pol-L-lysine linker, a beta-Ala-Leu-Ala-Leu linker, an Arg -Arg -Al a- Leu -Al a- Leu linker, a peptidomimetic linker, a 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 metal loprotease (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 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 PLGLWAPLGLVVAPLGLWA (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).

[0166] 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 Blanar et al., Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In some embodiments, the IL-2 moiety further comprises a C-terminal c-myc epitope tag.

[0167] 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).

[0168] Exemplary IL-2 moieties that include a half-life enhancing polypeptide for use in the subject IL-2 fusion polypeptides described herein are shown in Table 3. In some embodiments, the IL-2 moiety of the IL-2 fusion protein has the amino acid of one of SEQ ID NOs:25-73. In some embodiments the IL-2 moiety of the IL-2 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-2 moieties in Table 3 (SEQ ID NOs:25-67). In some embodiments, the IL-2 of the IL-2 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 moieties in Table 3 (SEQ ID NOs:25-67). In embodiments, the IL-2 exhibits greater binding affinity for IL-2R|3 as compared to wild-type human IL-2. In embodiments, the variant also exhibits reduced binding to CD25.Table 3: Exemplary IL-2 Moieties with Enhanced Serum Half-Life2. IL-2 / Antibody Fusion Moieties

[0169] In other embodiments, the IL-2 moiety of the IL-2 fusion proteins provided herein includes an antibody or antigen-binding portion thereof (i.e., an "IL-2 / antibody fusion moiety”).

[0170] 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 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.

[0171] In some embodiments of the I L-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 thirdpolypeptide 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.

[0172] 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 NO:403) In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:404). In some embodiments, 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. In some embodiments, the IL-2 or IL-2 mutein is attached to N-terminus of the antibody or antigen binding component.

[0173] 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.

[0174] 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 , and / or is an antibody to a component of the PD-1 / PD-L1 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 , 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 antigenbinding 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 anti-tumor 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- Ll 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 , disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response, is suitable for use in the IL-2 moiety disclosed herein. In some embodiments, the IL-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.

[0175] Exemplary IL-2 / anti-PD-1 antibody fusion moieties that can be included in the subject IL-2 fusion proteins are shown in Table 4. In some embodiments, the IL-2 moiety of the IL-2 fusion protein has the amino acid of one of Table 4. In some embodiments the IL-2 moiety of the IL-2 fusion protein is a variant of one of the IL-2 moieties in Table 4. In some embodiments, the IL-2 moiety of the IL-2 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-2 moieties in Table 4. In some embodiments, the IL-2 of the IL-2 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 moieties in Table 4. In embodiments, the variant exhibits greater binding affinity for IL-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 4: IL-2 / anti-PD1 fusion moieties

[0176] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets CTLA-4 and disrupts its interaction with CD80 and CD86. Exemplary antibodies that target CTLA-4 include ipilimumab (MDX-010, MDX-101 , Bristol-Myers Squibb), which is FDA approved, and tremelimumab (ticilimumab, CP-675, 206, Pfizer), currently undergoing human trials. Other suitable antibodies that target CTLA-4 are disclosed in WO 2012 / 120125, U.S. Patents No. 6,984720, No. 6,682,7368, and U.S. Patent Applications 2002 / 0039581 , 2002 / 0086014, and 2005 / 0201994, herein incorporated by reference. It will be understood by one of ordinary skill that any antibody which binds to CTLA-4, disrupts its interaction with CD80 and CD86, and stimulates an anti-tumor immune response, is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti- CTLA-4 antibody.

[0177] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen- binding portion thereof that targets LAG-3 and disrupts its interaction with MHC class II molecules. An exemplary antibody that targets LAG-3 is IMP321 (Immutep), currently undergoing human trials. Other suitableantibodies that target LAG-3 are disclosed in U.S. Patent Application 2011 / 0150892, herein incorporated by reference. It will be understood by one of ordinary skill that any antibody which binds to LAG-3, disrupts its interaction with MHC class II molecules, and stimulates an anti-tumor immune response, is suitable for use in the the IL-2 moiety disclosed herein. In some embodiments, the the IL-2 moiety comprises a fusion to an anti- LAG-3 antibody.

[0178] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets TIGIT and disrupts its interaction with CD155 (PVR) and / or CD112 (PVRL2, nectin- 2). It will be understood by one of ordinary skill that any antibody which binds to TIGIT, disrupts its interaction with CD155 (PVR) and / or CD112 (PVRL2, nectin-2), and stimulates an anti-tumor immune response or an immune stimulatory response that results in anti-tumor activity overall, is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti-TIGIT antibody.

[0179] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets CD112R (also known as PVRIG) and disrupts its interaction with CD112 and / or PVRL2 / nectin-2. It will be understood by one of ordinary skill that any antibody which binds to CD112R, disrupts its interaction with CD112 and / or PVRL2 / nectin-2, and stimulates an anti-tumor immune response or an immune stimulatory response that results in anti-tumor activity overall, is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti-CD112R antibody.

[0180] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen- binding portion thereof that targets B7-H3 or B7-H4. The B7 family does not have any defined receptors but these ligands are upregulated on tumor cells or tumor-infiltrating cells. An exemplary antibody that targets B7-H3 is MGA271 (Macrogenics) is currently undergoing human trials. Other suitable antibodies that target B7 family members are disclosed in U.S. Patent Application 2013 / 0149236, herein incorporated by reference. It will be understood by one of ordinary skill that any antibody which binds to B7-H3 or H4, and stimulates an anti-tumor immune response, is suitable for use in the IL-2 moieties disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti- B7-H3 or B7-H4 antibody.

[0181] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets TIM-3 and disrupts its interaction with galectin 9. Suitable antibodies that target TIM-3 are disclosed in U.S. Patent Application 2013 / 0022623, herein incorporated by reference. It will be understood by one of ordinary skill that any antibody which binds to TIM-3, disrupts its interaction with galectin9, and stimulates an anti-tumor immune response, is suitable for use in the IL-2 moieties disclosed herein. In some embodiments, IL-2 moiety comprises a fusion to an anti- TIM-3 antibody.

[0182] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets 4-1 BB / CD137 and disrupts its interaction with CD137L. It will be understood by one of ordinary skill that any antibody which binds to 4-1 BB / CD137, disrupts its interaction with CD137L or another ligand, and stimulates an anti-tumor immune response or an immune stimulatory response that results in anti-tumor activity overall, is suitable for use in the IL-2 moieties disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti-4-1 BB / CD137 antibody.

[0183] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets GITR and disrupts its interaction with its interaction with its ligand. It will be understood by one of ordinary skill that any antibody which binds to GITR, disrupts its interaction with GITRL or another ligand, and stimulates an anti-tumor immune response or an immune stimulatory response that results in anti-tumor activity overall, is suitable for use in the IL-2 moieties disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti-GITR antibody.

[0184] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets 0X40 and disrupts its interaction with its ligand. It will be understood by one of ordinary skill that any antibody which binds to 0X40, disrupts its interaction with OX40L or another ligand, and stimulates an anti-tumor immune response or an immune stimulatory response that results in anti-tumor activity overall, is suitable for use in the IL-2 moieties disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti-OX40 antibody.

[0185] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets CD40 and disrupts its interaction with its ligand. It will be understood by one of ordinary skill that any antibody which binds to CD40, disrupts its interaction with its ligand, and stimulates an anti-tumor immune response or an immune stimulatory response that results in anti-tumor activity overall, is suitable for use in the IL-2 moieties disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti-CD40 antibody

[0186] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets ICOS and disrupts its interaction with its ligand. It will be understood by one of ordinary skill that any antibody which binds to ICOS, disrupts its interaction with its ligand, and stimulates an anti-tumor immune response or an immune stimulatory response that results in anti-tumor activity overall, issuitable for use in the IL-2 moieties disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti-ICOS antibody.

[0187] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets CD28 and disrupts its interaction with its ligand. It will be understood by one of ordinary skill that any antibody which binds to CD28, disrupts its interaction with its ligand, and stimulates an anti-tumor immune response or an immune stimulatory response that results in anti-tumor activity overall, is suitable for use in the IL-2 moieties disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti-CD28 antibody. In some embodiments, the IL-2 moiety comprises a fusion to an anti-CD3 antibody, including a T-cell engager antiCD3 antibody.

[0188] In some embodiments, the IL-2 moiety comprises a fusion to an antibody or an antigen-binding portion thereof that targets IFNct and disrupts its interaction with its ligand. It will be understood by one of ordinary skill that any antibody which binds to IFNct, disrupts its interaction with its ligand, and stimulates an anti-tumor immune response or an immune stimulatory response that results in anti-tumor activity overall, is suitable for use in the IL-2 moieties disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion to an anti-l FNa antibody.3. Additional IL-2 Fusion Moieties

[0189] In some embodiments, the IL-2 moiety comprises a fusion to a tumor antigen or polypeptide targeting a tumor antigen. Generally, tumor antigens allow for distinguishing the tumor cells from their normal cellular counterparts and can include, for example, tumor-specific antigens (TSA) as well as tumor-associated antigens (TAA). In some embodiments, a tumor antigen is a protooncogene and / or a tumor suppressor, as well as overexpressed or aberrantly expressed cellular proteins, tumor antigens produced by oncogenic viruses, oncofetal antigens, altered cell surface glycolipids and glycoproteins, and / or cell type-specific differentiation antigens. Such tumor antigens can include melanoma antigens, cancer-testis antigens, epithelial tumor antigens, cell cycle regulatory proteins, prostate specific antigens (including prostate carcinoma antigens, such as for example those disclosed in U.S. Pat. No. 5,538,866) lymphoma (U.S. Pat. Nos. 4,816,249; 5,068,177; and 5,227,159). Tumor antigens can include for example, but are not limited to, HMW mucins bound by 2G3 and 369F10, c-erbB-2 related tumor antigen (an approximately 42 kD or 55 kD glycoprotein), the approximately 40, 60, 100 and 200 kD antigens bound by 113F1 , 9-O-acetyl GD3, p97, alphafetoprotein (AFP) (for example, for germ cell tumors and / or hepatocellular carcinoma), carcinoembryonic antigen (CEA) (for example, for bowel cancers occasional lung or breast cancer), CA-125 (for example, for ovarian cancer), MUC-1 (for example, for breast cancer), epithelial tumor antigen (ETA) (for example, forbreast cancer), tyrosinase (for example, for malignant melanoma), melanoma-associated antigen (MAGE) (for example, for malignant melanoma), cancer / testis antigen 1 (CTAG1B), melanoma-associated antigen 1 (MAGEA1), abnormal Ras products, abnormal p53 products, overexpression of cyclins (including, for example, cyclin B1), mutation in fibronectin, posttranslational alteration in the MUC1 glycoprotein, secreted tumor antigens (including, for example, gangliosides).

[0190] Other fusions can include fusions with pro-apoptotic payloads. Such exemplary sequences are provided in the table below. In some embodiments, and IL-2 moiety as described herein is fused to a pro- apoptotic payload, for example a BAD, BAX, BAK, BIK, and / or BIDsequence. In some embodiments, the pro- apoptotic payload is a Bcl-2 domain containing peptide and / or a susequence of a BAD, BAX, BAK, BIK, and / or BID sequence. Exemplary pro-apoptotic fusions are provided below, in Table 5.Table 5: List of Selected Pro-Apoptotic Fusion Partners

[0191] Other fusions can include fusions with anti-apoptotic payloads for use in prolonging activation of CD8 cells, NK cells and anergic NK cells as well, and such exemplary sequences are provided in the table below. Such prolonged activation of T-cells can prove beneficial in cancer therapy treatment methods.Table 6: List of Exemplary IL-2 Anti-Apoptotic Fusion Amino Acid SequencesB. IL-2 MASKING MOIETY

[0192] The subject IL-2 fusion proteins provided herein include an IL-13 mutein, an IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment thereof that is linked to the IL-2 moiety by a protease sensitive linker (PSL). When attached to the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment 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 IL-13Ra2 antibody or antigen-binding fragment 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 thereof is capable of binding to I L-13Ro2 expressing tumors, thereby localizing the IL-2 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, IL-13Ra2 binding mutein or an IL-13Ra2 antibody or antigen-binding fragment thereof from the IL-2 fusion protein. In some embodiments, the “unmasked” IL-2 moiety is then active in the tumor microenvironment, thereby promoting IL-2 anti-tumor activity.

[0193] In some embodiments, the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment 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 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-life enhancing polypeptide (e.g., a serum albumin or an Fc region), the IL-13 mutein or IL-13Ra2 antibody or antigen-binding fragment thereof is attached to the half-life enhancing polypeptide 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 thereof is attached to the other Fc region monomer by a PSL.

[0194] In some embodiments, the IL-2 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 aPSL. In some embodiments, at least one of the IL-13 muteins or IL-13Ra2 antibodies or antigen-binding fragments thereof is attached to a region of the IL-2 moiety that is not the IL-2 mutein (e.g., a serum albumin or an Fc region).1. IL-13 Mutein

[0195] In embodiments, the IL-13 mutein comprises one or more amino acid substitutions as compared to a wild-type human IL-13:

[0196] PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRML SGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN (SEQ ID NO:200)

[0197] Suitable IL-13 muteins include those that are capable of reducing IL-2 activity of the IL-2 moiety when attached to the IL-2 fusion protein via the PSL. In some embodiments, the IL-13 mutein is capable of binding to IL-13Ra2 receptor. 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-13Rct2 receptor, and not capable of binding to the IL-13Ra1 receptor.

[0198] In some embodiments an IL-13 mutein of the subject IL-2 fusion protein comprises one or more of the amino acids substitutions: (1) L10F, L10I, L10V, L10A, L10D, L10T, L10H; (2) R11S, R11 N, R11 H, R11 L, R11 I; (3) I14L, I14F, 114V, I14M; (4) V18L, V18F, V18I; (5) E12A, (6) R65D, (7) R86K, R86T, R86M; (8) D87E, D87K, D87R, D87G, D87S; (9) T88I, T88K, T88R; (10) K89R, K89T, K89M; (11) L101 F, L101 I, L101Y, L101H, L101 N; (12) K104R, K104T, K104M; (13) K105T, K105A, K105R, K105E; (14) F107L, F107I, F107V, F107M; (15) R108K, R108T, R108M; and (16) E15R, which substitutions cause an altered affinity for one or both of IL- 13Ra 1 and IL-13Ra2. In other embodiments, modified residues are at two or more, three or more, four or more, five or more, and not more than 14 amino acids within the combined set of contact residues defined above. As described in International Patent Publication WO 2013 / 112871 , the disclosure of which is incorporated by reference herein in its entirety.

[0199] Sets of modifications may include the following specific changes: (1) L10H; L10A; (2) R11 L; (4) V18I; (7) R86M; R86K; R86T; (8) D87K; D87G; (9) T88R, T88S; T88K; (10) K89R; (11) L101 N; (12) K104R; (13) K105A; K105E; (14) R108K; (15) E15R as compared to a wild-type human IL-13 (SEQ ID NQ:200). In some embodiments, the modification includes any one of the recited specific changes. In some embodiments, the modification includes L10H. In some embodiments, the modification includes L10A. In some embodiments, the modification includes R11 L. In some embodiments, the modification includes E15R. In some embodiments, the modification includes V18I. In some embodiments, the modification includes R86M.In some embodiments, the modification includes R86K. In some embodiments, the modification includes R86T. In some embodiments, the modification includes D87K. In some embodiments, the modification includes D87G. In some embodiments, the modification includes T88R. In some embodiments, the modification includes T88S. In some embodiments, the modification includes T88K. In some embodiments, the modification includes K89R. In some embodiments, the modification includes L101 N. In some embodiments, the modification includes K104R. In some embodiments, the modification includes K105A. In some embodiments, the modification includes K105E. In some embodiments, the modification includes R108K.

[0200] In some embodiments, the IL-13 mutein of the IL-2 fusion protein has greater selectivity in in binding to IL-13Ra2 versus IL-13Ra1 relative to a wild-type IL-13 sequence (SEQ ID NG:200). Specific sets of modifications that provide for greater selectivity in binding to IL-13Ra2 versus IL-13Ra1 relative to a wild-type IL-13 sequence may include, without limitation:• [L10H, E15R, R86T, D87G, T88R, R108K] (MDNA132 + E15R or MDNA132.15, e.g. SEQ ID NO:228)• [L10H, R86T, D87G, T88R, R108K] (C11 e.g. SEQ ID NO:229)• [L10D, R111, V181, R86K, D87K, K89R, R108K] (for example, 02, e.g. SEQ ID NO:224)• [L10A, R86T, D87G, T88K, K89R, L101 N, K104R, K105A, R108K] (for example, 03, e.g. SEQ ID NO:225)• [L10V, K89R, L101 N, K105E, R108T] (for example, 04, e.g. SEQ ID NO:211• [R11S, I14M, T88S, L101 N, K105A, R108K] (for example, C7, e.g. SEQ ID NO:212)• [L10H, R11 L, V18I, R86K, D87E, K89R, L101 N, K105T, R108K] (C9, e.g. SEQ ID NO:226)• [L10A, V18F, R86K, D87K, K89R, L101 I, K104R, R108K] (D7, e.g. SEQ ID NO:231)• [L10T / D; R111; V18I; R86K; D87K / G; T88S; K89R; L101Y; K104R; K105T; R108K]• [L10A / V; R86T; D87G; T88K; K89R; L101N; K104R; K105A / E; R108K / T]

[0201] In some embodiments, the set of modifications comprises L1 OH, R86T, D87G, T88R, and R108K (C11 e g. SEQ ID NO:229). In some embodiments, the set of modifications comprises L10V, K89R, L101 N, K105E, R108T (C4, e.g., SEQ ID NO:211). In some embodiments, the set of modifications comprises R11 S, I14M, T88S, L101N, K105A, and R108K (C7, e.g. SEQ ID NO:212). In some embodiments, the set of modifications comprises L10H, R11 L, V18I, R86K, D87E, K89R, L101 N, K105T, and R108K (C9, e.g. SEQ ID NO:226). In some embodiments, the set of modifications comprises L10H, E15R, R86T, D87G, T88R, and R108K (MDNA132 + E15R, e.g. SEQ ID NO:228). In some embodiments, the set of modifications comprises L10A, V18F, R86K, D87K, K89R, L101 I, K104R, and R108K (D7, e.g. SEQ ID NO:231). In some embodiments, the set of modifications comprises L10T / D, R111, V18I, R86K, D87K / G, T88S, K89R, L101Y,K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10T, R111, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10T, R11 I, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10D, R111, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10D, R111, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, R108K. In some embodiments, the set of modifications comprises L10A / V, R86T, D87G, T88K, K89R, L101 N, K104R, K105A / E, and R108K / T. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101 N, K104R, K105A, and R108K. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108K. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101 N, K104R, K105A, and R108T. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101 N, K104R, K105E, and R108T. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101 N, K104R, K105A, and R108K. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101 N, K104R, K105E, and R108K. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105A, an dR108T. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101 N, K104R, K105E, and R108T.

[0202] In some embodiments, the IL-13 mutein of the IL-2 fusion protein has greater selectivity in binding to IL-13Ra1 versus IL-13Ra2 relative to a wild-type IL-13 sequence (e.g., SEQ ID NO:200). Specific sets of modifications that provide for greater selectivity in binding to IL-13Ra1 v IL-13Ra2 relative to a wild-type IL-13 sequence may include, without limitation:• [L10V, V18I, D87S, D88S, L101 F, K104R, K105T]• [R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T]• [L10V, V18I, D87S, T88S, L101 F, K104R, K105T]• [L10V / I; D87S; T88S; K89R; L101 H / F; K104R; K105T]• [L10I; V18I; R86T; D87G; T88S; K89R; L101Y / H; K104R; K105A]• [L10V; V18I; D87S; T88S; L101 F; K104R; K105T]• [V18I, R86T, D87G, T88S, L101Y, K104R, K105A]• [R11 I, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M] which substitutions are optionally combined with the substitutions [E12A / G / S, R65D / E],

[0203] In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101 F, K104R, and K105T. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S,K89M, L101Y, K104R, and K105T. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, and K105T. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, and K105T. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, and K105A. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, and K105T. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, and K105A. In some embodiments, the set of modifications comprises R11 I, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, and F107M. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises R111, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101 F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M , L101Y, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D / E. In some embodiments, the set of modifications comprises R111, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101 F, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M , L101Y, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T,D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D / E. In some embodiments, the set of modifications comprises R111, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101 F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65D / E. In some embodiments, the set of modifications comprises R111, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101 F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101 Y, K104R, K105A, E12A, and R65D. In some embodiments, the set of modifications comprises R11 I, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M, E12A, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101 F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S,L101Y, K104R, K105A, E12G, and R65D. In some embodiments, the set of modifications comprises R11 I, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M, E12G, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101 Y, K104R, K105A, E12S, and R65D. In some embodiments, the set of modifications comprises R11 I, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M, E12S, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101 F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65E. In some embodiments, the set of modifications comprises R11 I, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M, E12A, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101 F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65E In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65E. In some embodiments, the set of modifications comprises R111, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M, E12G, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101 F, K104R, K105T, E12S, and R65E. In someembodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101 H / F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101 F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65E. In some embodiments, the set of modifications comprises R11 I, V18I, R86K, D87G, T88S, L101 H, K104R, K105A, F107M, E12S, and R65E. In some embodiments, the set of modifications comprises L10V, E12A, V18I, R65D, D87S, T88S, L101 F, K104R, and K105T (see, for example, IL-13dn; SEQ ID NO:232).

[0204] Suitable exemplary IL-13 muteins for use in the subject IL-2 fusion proteins described herein are shown in Table 7.Table 7: IL-13 wildtype and muteins

[0205] In some embodiments, the IL-13 mutein of the IL-2 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 7. In some embodiments, the IL-13 mutein of the IL-2 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 muteins in Table 7 (SEQ ID N0:200-241 ). In embodiments, the IL-13 mutein exhibits greater selectivity in binding to IL-13Ra2 versus IL-13Ro1 relative to a wild-type IL- 13 sequence (e.g , SEQ ID NQ:200).2. Extended Half-Life IL-13 Muteins

[0206] In some embodiments, the IL-13 mutein of the subject IL-2 fusion proteins includes a polypeptide that increases the in vivo serum half-life of the IL-2 fusion protein (i.e., a "serum half-life enhancing” or "half-life enhancing” polypeptide). The half-life enhancing polypeptide can be any of the half-life enhancing polypeptides described herein. In various embodiments, the half-life enhancing polypeptide is a serum albumin (e.g., human serum albumin), a PEG, a PEG-derivative, or the Pc region of the IgG subclass of antibodies that lacks the IgG heavy chain variable region.

[0207] In some embodiments, the half-life enhancing polypeptide is an Fc region. The Fc region can include the entire Fc region, or a smaller portion that retains the ability to extend the circulating half-life of the IL-2 fusion protein 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-13 mutein is attached to an lgG1 , lgG2, lgG3, or lgG4 Fc region. In some embodiments, the Fc region is a human IgG 1 , lgG2, lgG3, or Ig G4 Fc region. In some embodiments, the Fc region includes a monomer that include a CH2-CH3 domain of a human lgG1 , lgG2, lgG3 or Ig G4 region. In some embodiments, the Fc region 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 Fc region includes two monomers, wherein each monomer includes an IL-13 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 Fc region that is attached to two IL-13 muteins. 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.

[0208] In some embodiments, the half-life enhancing polypeptide is a serum albumin. In exemplary embodiments, the serum albumin is a human serum albumin or variant thereof that is capable of extending the serum half-life of the attached IL-13 mutein. In some embodiments, the human serum albumin has the following amino acid sequence:DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDK LCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARR HPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVA RLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIA EVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKC CKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFH ADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAAL GL (SEQ ID NG:400).

[0209] In some embodiments, the IL-13 mutein 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-13 mutein. In some embodiments, the half-life enhancing polypeptide is linked to the to the C-terminus of the IL- 13 mutein. In some embodiments, the IL-13 mutein is linked directly to the half-life enhancing polypeptide. In some embodiments, the IL-13 mutein is linked to the half-life enhancing polypeptide via a linker peptide. Any suitable linker can be used to attach the IL-13 mutein to the half-life enhancing polypeptide, include linkers described herein (e.g., gly-ser linkers described herein).

[0210] Exemplary IL-13 muteins that include a half-life enhancing polypeptide for use in the subject IL-2 fusion polypeptides described herein are shown in Table 8. In some embodiments, the IL-13 mutein is attached to a half-life enhancing polypeptide and has the amino acid of one of SEQ ID NOs:242-281 . In some embodiments, the IL-13 mutein is attached to a half-life enhancing polypeptide and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to one of sequences in Table 8 (SEQ ID NOs:242-281 ). In some embodiments, the IL-13 mutein is attached to a half-life enhancing polypeptide and has 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions as compared to any one of the sequences in Table 8 (SEQ ID NOs: 242-281 ) In embodiments, the IL-13 mutein is attached to a half-life enhancing polypeptide, exhibits greater selectivity in binding to IL-13Rct2 versus IL- 13Ra1 relative to a wild-type IL-13 sequence (e.g., SEQ ID NQ:200)Table 8: IL-13 Extended Half-Life Fusions3. IL-13 Mutein / Antibody Fusion

[0211] In other embodiments, the IL-13 mutein of the IL-2 fusion proteins provided herein includes an antibody or antigen-binding portion thereof.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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. Exemplary IL-13 mutein / anti-PD1 fusions are depicted below in Table 9.Table 9: IL-13 anti-PD1 fusions4. IL-13Ra2 Antibodies

[0216] In some embodiments, the subject IL-2 masking moieties comprise an IL-13Ra2 antibody or antigen-binding fragment thereof. Suitable IL-13Ra2 antibody or antigen-binding fragment thereof include, but are not limited to, SAB1406031 (Sigma), HPA067363 (Sigma), HPA067363 (Sigma), AV53557 (Sigma), WH0003598M1 (Sigma), AP1145 (Sigma), E7U7B (Cell Signaling), 2H25L68 (ThermoFisher), 018 (ThermoFisher), PA5-96045v (ThermoFisher), PA5-106798 (ThermoFisher), PA5-46976 (ThermoFisher), PA5-44066 (ThermoFisher), PA5-47732 (ThermoFisher), PA5-143269 (ThermoFisher), PA5-86898 (ThermoFisher), PA5-87205 (ThermoFisher), BS-2461 R (ThermoFisher), 2E10 (ThermoFisher), and those disclosed in W02008146911 and WO2014072888, incorporated herein by reference in their entireties. As used herein, “antigen-binding fragment thereof' refers to antigen-binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.5. Additional Components

[0217] In some embodiments, the subject IL-2 masking moieties further comprise an extracellular domain of CD122, CD132 or CD25.

[0218] Suitable extracellular domain of CD122, CD132 or CD25 include those that are capable of reducingIL-2 activity of the IL-2 moiety when attached to the IL-2 fusion protein via the PSL

[0219] In some embodiments, the extracellular domain of CD122 has the following amino acid sequence:

[0220] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLIL GAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQWHVETHRCNISWEISQASHYFER HLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAA LGKDT (SEQ ID NO: 350).

[0221] In some embodiments, the extracellular domain of CD132 has the following amino acid sequence:

[0222] LPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQK KEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTD WDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKEN (SEQ ID NO: 351).

[0223] In some embodiments, the extracellular domain of CD25 has the following amino acid sequence:

[0224] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSA TRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFWGQMVYYQCVQGY RALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 352).C. PROTEASE SENSITIVE LINKER (PSL)

[0225] The IL-2 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 subject IL-2 fusion proteins include PLGLVVAPLGLVVAPLGLVVA (SEQ ID NO:416), PLGLWAPLGLWAPLGLWA(SEQ ID NO:417) , GGSGGTPLGLWAGGSGGT (SEQ ID NO:418), GGSGGTPAGLIGGGSGGT (SEQ ID NO:419), GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NO:420), or GGSGGTHSSKLQGGSGGT (SEQ ID NO:421).

[0226] 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 comprisedisulfide 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, a peptidomimetic linker, a 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 PSL is a protease-cleavable linkers is a linker cleavable by a matrix metal loprotease (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 PLGLVVAPLGLVVAPLGLVVA (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).D. EXEMPLARY IL-2 FUSION PROTEINS

[0227] In some embodiments, the IL-2 fusion protein includes an IL-2 moiety that includes an IL-2 mutein according to SEQ ID NO:5 (MDNA109) or SEQ ID NO:9 (MDNA109FEAA). In some embodiments, the IL-2 mutein is a variant of MDNA109 (SEQ ID NO:5) or MDN109FEAA (SEQ ID NO:9) that further includes a T3A and / or C125S substitution.

[0228] In some embodiments, the IL-2 moiety includes a human albumin polypeptide, optionally wherein the human albumin is recombinant human albumin. In exemplary embodiments, the IL-2 moiety includes an anti-PD-1 antibody or antigen binding portion thereof. In some the IL-2 moiety is attached to a half-life enhancing polypeptide such as a human serum albumin polypeptide or an Fc region.

[0229] In some embodiments, the IL-2 fusion protein includes an IL-13 having the amino acid sequence of MDNA132 (SEQ ID NO:216) or MDNA132.15 (SEQ ID NO:228). In exemplary embodiments, the PSL has the amino acid sequence of PLGLWAPLGLWAPLGLWA (SEQ ID NO: 417), or GGSGGTPLGLWAGGSGGT (SEQ ID NO: 418), or GGSGGTPAGLIGGGSGGT (SEQ ID NO: 419), or GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NO: 420), or GGSGGTHSSKLQGGSGGT (SEQ ID NO: 421).

[0230] Exemplary subject IL-2 fusion proteins are shown in Tables 10a and 10b.Table 10a: Exemplary subject IL-2 fusion proteinTable 10b: Description and rationale of constructs designed for testing masking efficiencyIllE. NUCLEIC ACID MOLECULES ENCODING IL-2 FUSION PROTEINS

[0231] In some embodiments, the subject IL-2 fusion protein, such as those described above, can be obtained by expression of one or more nucleic acid molecules. For example, in embodiments wherein the IL-2 fusion is a single polypeptide, the IL-2 fusion protein is encoded by a single nucleic acid molecule. In other embodiments wherein the IL-2 fusion protein is made up of two or more polypeptides, each of the two or more polypeptides is encoded by a nucleic acid molecule.

[0232] 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, orsynthetic 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 doublestranded or single-stranded ( / . e. , either a sense or an antisense strand).

[0233] 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.

[0234] 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).

[0235] A subject 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-hosphotransferase (HPH), thymidine kinase (TK), lacz (encoding p-galactosidase), and xanthine 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.

[0236] The subject 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.F. EXPRESSION OF MUTANT IL-2, IL-4, OR IL-13 GENE PRODUCTS

[0237] 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 subject IL-2 fusion proteins, expression vectors containing one or more nucleic acid molecules encoding a subject IL-2 fusion protein and cells transfected with these expression vectors are among the preferred embodiments.

[0238] 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 IL-2 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).

[0239] In some embodiments, the subject IL-2 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 of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) are included also.

[0240] 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.

[0241] The expression constructs or vectors can be designed for expression of an IL-2 fusion protein in prokaryotic or eukaryotic host cells.

[0242] 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.

[0243] 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.

[0244] In some embodiments the recombinant IL-2 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 54:1 13-123), 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, commonly 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.).

[0245] The sequences encoding the IL-2 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 IL-2 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.

[0246] 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.

[0247] In some embodiments nucleic acid inserts, which encode the subject IL-2 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.

[0248] 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 subject IL-2 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.

[0249] 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.

[0250] 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 p 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).

[0251] 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 TAG or TRC system, the major operator and promoter regions of phage lambda, for example PL, the controlregions 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.

[0252] 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.

[0253] 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 permits phenotypic 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.

[0254] 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.).

[0255] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule that encodes a subject IL-2 mutein 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 IL-2, IL-4, or IL-13 mutein or bifunctional molecule, has been introduced by means of recombinant DNA techniques. The progeny of such a cell are also considered within the scope of the invention.

[0256] The precise components of the expression system are not critical. For example, a subject IL-2 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 inMolecular Biology, John Wiley and Sons, New York, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0257] 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.

[0258] In some embodiments, the IL-2 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 IL-2 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 not in the same way as native-IL-2 or IL-13 is glycosylated. The IL-2 fusion protein produced by the transformed host can be purified according to any suitable method. Various methods are known for purifying the IL-2 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 IL-2 fusion protein can be isolated from inclusion 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.

[0259] Another exemplary method of constructing a DNA sequence encoding the subject IL-2 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 IL-2 fusion protein with the their corresponding receptors. Alternatively a gene which the IL-2 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 IL-2 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 IL-2 fusion protein, there will be many DNA degenerate sequences that will code for that IL-2 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 IL-2 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.

[0260] The biological activity of the IL-2 fusion protein can be assayed by any suitable method known in the art. Such assays include PHA-blast proliferation and NK cell proliferation.G. METHODS OF TREATMENT

[0261] In some embodiments, subject IL-2 fusion proteins, and / or nucleic acids expressing them, can be administered to a subject to treat a disorder associated with abnormal apoptosis or a differentiative 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 IL-2 fusion proteins may possess advantageous properties, such as reduced vascular leak syndrome.

[0262] 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, 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. 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.

[0263] The subject IL-2 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.

[0264] Additional examples of proliferative disorders include hematopoietic neoplastic disorders.

[0265] Alternatively, or in addition to methods of direct administration to patients, in some embodiments, subject IL-2 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 subject IL-2 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 an antigen of interest (e.g., a cancer antigen or a viral antigen). The cells are then administered to the patient after they have been treated.

[0266] Anti-PD-1 antibodies for use in combination with the subject IL-2 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®), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872).

[0267] In some embodiments, the subject IL-2 fusion protein is used in combination with nivolumab for the treatment of cancer. In some embodiments, the IL subject IL-2 fusion protein is used in combination with pembrolizumab for the treatment of cancer. In some embodiments, the subject IL-2 fusion protein is used in combination BMS-936558 for the treatment of cancer. In some embodiments, the subject IL-2 fusion protein is used in combination MDX-1106 for the treatment of cancer. In some embodiments, the subject IL-2 fusion protein is used in combination ONO-4538 for the treatment of cancer. In some embodiments, the subject IL-2 fusion protein is used in combination AMP224 for the treatment of cancer. In some embodiments, the subject IL-2 fusion protein is used in combination CT-011 for the treatment of cancer. In some embodiments, the subject IL-2 fusion protein is used in combination MK-3475 for the treatment of cancer.

[0268] In some embodiments, the subject IL-2 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 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) for the treatment of cancer.

[0269] In some embodiments, the subject IL-2 fusion protein is used in combination with another antibody which can include monoclonal antibodies 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.

[0270] In some embodiments, the subject IL-2 fusion protein is used in combination with antibodies for antibody-dependent cell-mediated cytotoxicity (ADCC) for the treatment of cancer.

[0271] In some embodiments, the subject IL-2 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®), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872). In some embodiments, the IL-2, IL-4, or IL-13 mutein and / or bifunctional 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) 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- 0X40 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, M EDI-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.

[0272] In some embodiments, the subject IL-2 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®), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872). In some embodiments, the subject IL-2 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, M EDI-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 theirentireties), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-sirs-alpha, and / or anti-CD112R.H. PHARMACEUTICAL COMPOSITIONS AND METHODS OF ADMINISTRATION

[0273] In some embodiments, subject IL-2 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.

[0274] The anti-PD-1 antibodies and subject IL-2 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 subject IL-2 fusion protein are administered together as a single co-composition ( / .e. , co-formulated). In some embodiments, the anti-PD-1 antibody or inhibitor and subject IL-2 fusion protein are administered simultaneously as two separate compositions ( / .e., separate formulations). In some embodiments, the anti-PD-1 antibody or inhibitor and subject IL-2 fusion protein are administered sequentially as separate compositions ( / .e., separate formulations). In some embodiments, when the anti-PD-1 antibody or inhibitor and IL-2 fusion protein are administered sequentially as separate compositions, the anti-PD-1 antibody or inhibitor is administered before the IL-2 fusion protein. In some embodiments, when the anti-PD-1 antibody or inhibitor and IL-2 fusion protein are administered sequentially as separate compositions, the IL-2 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.

[0275] The other immunotherapy agents as described and IL-2 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 IL-2 fusion protein are administered together as a single co-composition ( / .e., co-formulated). In some embodiments, the other immunotherapy agents and IL-2 fusion protein are administered simultaneously as two separate compositions ( / .e., separate formulations). In some embodiments, the other immunotherapy agents and IL-2 fusion protein are administered sequentially as separate compositions ( / .e., separate formulations). In some embodiments, when the other immunotherapy agents and IL-2 fusion protein are administered sequentially as separate compositions, the anti-PD-1 antibody or inhibitor is administered before the IL-2 fusion protein. In some embodiments, when other immunotherapy agents and IL-2 fusion protein are administered sequentially as separate compositions, the IL-2 fusion protein is administered before other immunotherapy agents.

[0276] A pharmaceutical composition is formulated to be compatible with its intended route of administration. The anti-PD-1 antibodies and / or IL-2 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.

[0277] 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 brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0278] 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 compoundinto 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.

[0279] 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

[0280] In the event of administration by inhalation, anti-PD-1 antibodies and / or IL-2 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.

[0281] Systemic administration of the anti-PD-1 antibodies and / or IL-2 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, or creams as generally known in the art.

[0282] In some embodiments, compounds (anti-PD-1 antibodies and / or IL-2 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.

[0283] In some embodiments, compounds (subject IL-2 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).

[0284] In one embodiment, the anti-PD-1 antibodies and / or IL-2 fusion protein or nucleic acids are prepared with carriers that will protect the anti-PD-1 antibodies and / or IL-2 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.

[0285] Dosage, toxicity and therapeutic efficacy of such anti-PD-1 antibodies, IL-2 fusion proteis, 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.

[0286] 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 initially from 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.

[0287] As defined herein, a therapeutically effective amount of a subject IL-2 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 IL-2 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 IL-2 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 subject IL-2 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.

[0288] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0289] The examples below are provided to describe certain embodiments of the invention provided herein and are not to be construed to as limiting.ADDITIONAL EMBODIMENTS

[0290] In embodiments, provided herein is an IL-2 cytokine fusion protein comprising: (a) an IL-2 moiety comprising an IL-2 or an IL-2 mutein, optionally an IL-2 mutein fusion; (b) a protease sensitive linker (PSL); and (c) an IL-2 masking moiety; wherein the PSL attaches the IL-2 masking moiety to the IL-2 moiety.

[0291] In embodiments, provided herein is an IL-2 cytokine fusion protein comprising: (a) an IL-2 moiety comprising an IL-2 or an IL-2 mutein, optionally an IL-2 mutein fusion; (b) at least one one protease sensitive linker (PSL); and (c) at least one IL-2 masking moiety; wherein the at least one PSL attaches the IL-2 masking moiety to the at least one IL-2 moiety.

[0292] 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 IL-2 masking moiety is capable of binding to IL-13Ra2, and does not bind to IL-13Ra1.

[0293] In embodiments, the masking moiety comprises an IL-13 mutein that has the amino acid sequence of any one of SEQ ID NQs:200-241 .

[0294] 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 NQ:200): ( a) L10H, E15R, R86T, D87G, T88R, R108K, Q111 , (b) L10H, E15R, R86T, D87G, T88R, R108K, R111 , (c) L10H, R86T, D87G, T88R, and R108K, Q111 , or (d) L10H, R86T, D87G, T88R, and R108K, R111.

[0295] In embodiments, the masking moiety comprises an IL-13 mutein that has the amino acid sequence of SEQ ID NO:216 or SEQ ID NO:228.

[0296] In embodiments, the masking moiety further comprises an extracellular domain of CD122, an extracellular domain of CD132, or an extracellular domain of CD25.

[0297] In embodiments, the IL-2 moiety comprises an IL-2 mutein having the amino acid sequence of any one of SEQ ID NOs:5-24 and 105.

[0298] In embodiments, the IL-2 mutein has the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:9.

[0299] In embodiments, the IL-2 mutein further comprises a T3A and a C125S amino acid substitution.

[0300] In embodiments, the IL-2 moiety further comprises an albumin, an Fc domain, or an antibody attached to the IL-2 or IL-2 mutein.

[0301] In embodiments, the albumin is a human albumin, optionally wherein the human albumin is recombinant human albumin.

[0302] In embodiments, the IL-2 moiety comprises an IL-2 x anti-PD1 fusion protein comprising: (a) a first polypeptide comprising an IL-2 or IL-2 mutein attached to a first Fc domain; (b) a second polypeptide comprising an heavy chain variable region (VH) and a second Fc domain; and (c) a third polypeptide comprising a light chain variable region (VL) and a light chain constant region, wherein the VH and VL form a PD-1 binding domain.

[0303] In embodiments, the second polypeptide is an antibody heavy chain.

[0304] In embodiments, the IL-2 moiety comprises an IL-2 x anti-PD1 fusion protein comprising: (a) a first polypeptide comprising a first antibody heavy chain attached to an IL-2 or IL-2 mutein, wherein the firstantibody heavy chain comprises a first heavy chain variable region (VH1) and a first Fc domain; (b) a second polypeptide comprising a second antibody heavy chain, wherein the second antibody heavy chain comprises a second heavy chain variable region (VH2) and a second Fc domain; (c) a third polypeptide comprising a first variable light chain variable region (VL1) and a light chain constant region; and (d) a fourth polypeptide comprising a second variable light chain variable region (VL2) and a light chain constant region, wherein VH1 and VL1 form a first PD-1 binding domain, and VH2 and VL2 form a second PD-1 binding domain.

[0305] In embodiments, the first PD-1 binding domain and second PD-1 binding domain have the same amino acid sequence.

[0306] In embodiments, the first PD-1 binding domain and second PD-1 binding domain have different amino acid sequences.

[0307] In embodiments, the antibody is engineered as “knob-in-hole” (KiH) with mutations in constant region 3 (CH3) in its heavy chains.

[0308] In embodiments, the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: (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 antibody (KiH); (b) a second polypeptide comprising the “hole” heavy chain of the antibody (KiH); and (c) a third polypeptide comprising the light chain of the antibody (KiH).

[0309] In embodiments, the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: (a) a first polypeptide comprising the “knob” heavy chain of the 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 antibody (KiH); (c) a third polypeptide comprising the light chain of the antibody (KiH).

[0310] In embodiments, the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: (a) a first polypeptide comprising the “knob” heavy chain of the antibody (KiH); (b) a second polypeptide comprising the “hole” heavy chain of the 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 antibody (KiH).

[0311] In embodiments, the IL-2 masking moiety is attached to the IL-2 or IL-2 mutein.

[0312] In embodiments, the IL-2 masking moiety is attached to the antibody heavy chain that is not attached to the IL-2 or IL-2 mutein.

[0313] In embodiments, the antibody (KiH) is anti-PD1 (KiH).

[0314] In embodiments, the IL-2 mutein has an amino acid sequence of any one of SEQ ID NOs: 5-24 and 105.

[0315] In embodiments, the IL-2 mutein further comprises a T3A and a C125S amino acid substitution.

[0316] In embodiments, the PSL is capable of being cleaved in the tumor microenvironment.

[0317] In embodiments, the PSL has the amino acid sequence of PLGLWAPLGLVVAPLGLWA,PLGLWAPLGLWAPLGLWA, GGSGGTPLGLWAGGSGGT, GGSGGTPAGLIGGGSGGT, GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT, or GGSGGTHSSKLQGGSGGT.

[0318] In embodiments, provided herein is an IL-2 cytokine fusion protein comprising sequences selected from the group consisting of: (a) SEQ ID NOs: 302, 303, and 304; (b) SEQ ID NOs: 299, 300, and 301 ; (c) SEQ ID NOs: 305, 306, and 307; (d) SEQ ID NOs: 308, 309, and 310; (e) SEQ ID NOs: 311 , 312, and 313; (f) SEQ ID NOs: 314, 315, and 316; (g) SEQ ID NOs: 317, 318, and 319; (h) SEQ ID NOs: 320, 321 , and 322; (I) SEQ ID NO: 323; (j) SEQ ID NOs:329, 330, and 331 ; (k) SEQ ID NOs:332, 333, and 334; (I) SEQ ID NOs:335, 336, and 337; (m) SEQ ID NOs:338, 339, and 340; (n) SEQ ID NOs:353, 354, and 355; (o) SEQ ID NOs:359, 360, and 361 ; (p) SEQ ID NOs:362, 363, and 364; (q) SEQ ID NOs:365, 366, and 367; (r) SEQ ID NOs:325, 326, and 327; (s) SEQ ID NOs:353, 354, and 355 and (t) SEQ ID NO: 324.

[0319] In embodiments, provided herein is a pharmaceutical composition comprising an IL-2 cytokine fusion protein described herein, and a pharmaceutically acceptable carrier.

[0320] In embodiments, provided herein is a nucleic acid composition that encodes an IL-2 cytokine fusion protein described herein.

[0321] In embodiments, provided herein is an expression vector composition comprising a nucleic acid composition described herein.

[0322] In embodiments, provided herein is a method of making an IL-2 cytokine fusion protein described herein, comprising culturing a nucleic acid composition described herein or an expression vector composition described herein under conditions wherein the IL-2 cytokine fusion protein is expressed, and recovering the IL-2 cytokine fusion protein.

[0323] 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 an IL-2 cytokine fusion protein described herein, or a pharmaceutical composition described herein.

[0324] In embodiments, the IL-2 cytokine fusion protein comprises one or more of SEQ ID NOs:1-367.

[0325] In embodiments, provided herein is a method of treating a cancer in a subject in need thereof comprising administering to the subject an IL-2 cytokine fusion protein comprising: (a) an IL-2 mutein; and (b) an albumin or an Fc domain or an antibody; wherein the IL-2 mutein has an amino acid sequence of any one of SEQ ID NOs: 5-24 and 105 and optionally further comprises a T3A and / or a C125S amino acid substitution.

[0326] In embodiments, the IL-2 cytokine fusion protein comprises one or more of SEQ ID NOs:1-367.

[0327] In embodiments, the IL-2 cytokine fusion protein has the amino acid sequence of SEQ ID NO:53.

[0328] In embodiments, the IL-2 cytokine fusion protein is administered as a neo-adjuvant prior to a surgery to remove a tumor.

[0329] In embodiments, the IL-2 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. In embodiments, the IL-2 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.

[0330] In embodiments, the IL-2 cytokine fusion protein is administered as an adjuvant after a surgery to remove a tumor.

[0331] In embodiments, the IL-2 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. In embodiments, the IL-2 cytokine fusion protein is administered starting at least two weeks after the surgery.

[0332] In embodiments, the IL-2 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.

[0333] In embodiments, the IL-2 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 IL-2 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. In embodiments, the IL-2 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 IL-2 cytokine fusion protein is administered starting at least to two weeks after the surgery.

[0334] 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.EXAMPLESExample 1: Assessing IL-13 Superkine Masking of IL-2 Super Agonist Activity in an In Vitro IL-2 Reporter AssayA. Introduction

[0335] MDNA132 is an IL-13 superkine that selectively binds to IL-13 decoy receptor (IL-13Ra2) with no binding to IL-13 functional receptor (IL-13Ra1).

[0336] MDNA132.15-PSL-MDNA109-Alb (SEQ ID NO:298; also referred to herein as MDNA213-PSL- MDNA109-albT3 / C125) was produced with the rationale to mask the IL-2 “beta only” super-agonist MDNA109- Alb with MDNA132.15 (also referred to herein as “MDNA213”) using the protease sensitive linker (PSL) which is hypothesized to get cleaved once MDNA132.15 docks M DNA109-Alb to IL-13 decoy receptor overexpressing tumors. The masking will help peripheral activation of T cells thereby improving tolerability.MDNA109-Alb was used as a comparator for this construct on the plate. MDNA132-PSL-MDNA11T3 / C125(SEQ ID NO: 294) was also expressed and purified in CHO and tested for IL-2 activity in the Jurkat I L-2R(3y bioassay. Additional constructs, MDNA213-PSL-MDNA109-albT3 / C125(SEQ ID NO: 298) and MDNA223A3 / S125- fPSL2f-MDNA213Lin(SEQ ID NOs: 299-301) were designed and produced to further validate the proof of concept. MDNA213-PSL-MDNA109-albT3 / C125had 3 copies of PSL not flanked by 'GS' residues while a single copy of PSL was flanked by ‘GS’ residues in MDNA223A3 / s125-fPSL2f-MDNA213Li".B. Methods

[0337] JURKAT I L2R(3y BIOASSAY - The Jurkat IL2R£y bioassay from Promega is designed for intended use with novel IL-2 and IL-15 molecules that are engineered for reduced CD25 binding since the IL2Rpy bioassay Jurkat cells lack CD25 expression. It is a simple and homogenous assay that is sensitive, reproducible and can be completed in a day. Since the cells lack CD25 receptor, the engineered IL-2 molecule will bind to the CD122 receptor expressed on the cells and trigger signaling through pSTAT5 which is detected through luminescence in the Jurkat reporter cells.

[0338] Jurkat IL-2Rpy cells were plated into 96 well plates according to the manufacturer’s recommendation in a volume of 50 piL. The constructs were diluted as 3x solutions and then 25 piL were added to each well (Table 11). 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. Two plates were run for the assay, with each plate containing serial dilutions of the test constructs in quadruplicate wells.Table 11 : Constructs used in the Jurkat IL-2Rpy Reporter AssayC. Results

[0339] MDNA109-Alb and MDNA132.15-PSL-MDNA109-Alb both resulted in a clear dose response (Figure 1A). ECsos for these dose response curves differed by 18.8-fold with MDNA109-Alb having the higher potency (lower EC50). Similar results were obtained with MDNA223A3 / s125-fPSL2f-MDNA213Lin(masked version) exhibiting a reduced potency in the Jurkat I L2Rpy reporter assay compared to MDNA223 (nonmasked version). The EC50 of DNA223A3'si25-fPSL2f-MDNA213^ (1624 pM) was 12.3-fold lower than that of MDNA223 (131.9 pM). This is consistent with the intended masking of MDNA109FEAA moiety of MDNA223 by MDNA213.

[0340] The EC50S are summarized in Table 12.Table 12: Summarized EC50s for MDNA132.15-PSL-MDNA109-Alb and fold change from MDNA109-Alb EC50 in the Jurkat IL-2Rpy assay

[0341] MDNA11 (SEQ ID NO: 53) and MDNA132-PSL-MDNA11 T3 / C125 (SEQ ID NO: 294) both resulted in a clear dose response (Figure 1 B). ECsos for the two dose response curves differed by ~5.6-fold with MDNA11exhibiting a higher potency (lower ECso at 509 pM) and MDNA132-PSL-MDNA11T3 / C125demonstrating an EC50 of 2835 pM.D. Conclusion

[0342] MDNA132.15-PSL-MDNA109-Alb exhibited a lower potency by 18.8-fold demonstrating that MDNA132.15 can mask the activity of MDNA109-Alb.Example 2: Assessing IL-13 Superkine Masking of IL-2 Super Agonist Binding to CD122A. Introduction

[0343] The binding affinity experiment presented in this example is a confirmation of Example 1 to validate the masking effect pf MDNA132.15 IL-13 superkine The observation in the previous experiment indicated masking of MDNA109-Albumin by MDNA132.15 through a decrease in potency of IL-2 induced pSTAT5.B. Methods

[0344] Ni-NTA biosensors (Sartorius cat. #18-5101 ) were hydrated for each experiment in a 96-well plate (Greiner Part #655209) as required by the Octet RH16 Biolayer Inferometer (BLI) instrument manufacturer (Sartorius). The ligand (200 nMHis-CD122) was immobilized to the Ni-NTA biosensors and then dipped into a titration of MDNA109-Alb (Construct #7741-044, 82.8 KDa) or MDNA132.15-PSL-MDNA109-Alb (Construct #7741 _046, 97.05 KDa) analytes. All dilutions were performed in PBS buffer (11.9 mM phosphate pH 7.4, 137 mM sodium chloride, 2.7 mM potassium chloride) containing kinetics reagent (Sartorius cat. #18-1105).Assays occurred in a 384-well tilted well assay plate (Sartorius cat. #18-5080) and the volume per well was 50 pL at a temperature of 25°C. The assay baseline drift was subtracted out using the protein-bound biosensor dipped into buffer instead of the respective analyte. Negative control experiments were included where the ligand was not loaded onto the biosensors and a mid-range concentration of analyte was tested in order to determine "bare” biosensor binding profiles. There was no significant non-specific binding observed in all experiments. A blocking step was added using Superblock (ThermoFisher cat. #37515).

[0345] The general BLI step conditions used were as follows:• Biosensors check in assay buffer (30 sec)• His-CD122 (ligand) immobilization (12 min)• Blocking with Superblock (2 min)• Baseline in assay buffer (3 min)• Analyte titration association (5 min)• Dissociation back into baseline buffer (15 min)C. Results

[0346] The construct MDNA132.15-PSL-MDNA109-albumin was tested for binding affinity to human CD122 with MDNA109-albumin as experimental control. The data, as sensorgrams, is presented in Figure 2.

[0347] As observed, both MDNA109-albumin and MDNA132.15-PSL-MDNA109-albumin showed affinity to human CD122 albeit with distinctive konvalues generating a 6.9-fold difference in binding affinity constant KD with MDNA109-albumin and MDNA132.15-PSL-MDNA109-albumin exhibiting a KD of 4.6 nM and 31.6 nM respectively. The shift in the binding affinity constant is indicative of a masking effect on binding of MDNA109- albumin by MDNA132.15 fused together via PSL (protease sensitive linker).D. Conclusion

[0348] The binding affinity data clearly established the masking effect observed by 6.9-fold difference in KD constant between the constructs, MDNA109-albumin and MDNA132.15-PSL-MDNA109-albumin. The data will be further validated by cleaving the construct using MMP9 and testing the recuperation of MDNA109-albumin binding.Example 3: Characterization of a tumor-targeting and activatable T-MASK platform to enhance tumor accumulation and tolerability of potent immune modulators

[0349] The novel T-MASK (Targeted Metallo / protease Activated SuperKine) platform involves fusion of a dual tumor-targeting / masking domain to potent immune modulator(s) via a metal lo-protease (MMP) sensitive linker (PSL) to achieve the following objectives: (1) reduce / fine-tune the potency of the immune modulator by steric hindrance to increase systemic tolerability and (2) promote retention in the tumor microenvironment (TME) to maximize MMP cleavage and restore full potency at the intended target site. As proof of concept, we selected as the tumor-targeting / masking domain an IL-13 superkine (MDNA213, also referred to herein as, "MDNA132 15”) with high selectivity and affinity for the IL-13 decoy receptor IL-13Ra2, a tumor associated antigen expressed in many aggressive solid tumors.Example 3a: Assessing IL-2 Super Agonist Masking of IL-13 Superkine Binding to IL-13Ra2A. Introduction

[0350] MDNA132 is an IL-13 superkine that selectively binds to IL-13 decoy receptor (IL-13Ra2) with no binding to IL-13 functional receptor (IL-13Ra1 ).

[0351] M DNA132.15-PSL-M DNA109-Alb was produced with the rationale to mask the MDNA109-Alb with MDNA132.15 using the protease sensitive linker (PSL) which is hypothesized to get cleaved once MDNA132.15 docks MDNA109-Alb to IL-13 decoy receptor over-expressing tumors.B. Methods

[0352] Ni-NTA biosensors (Sartorius cat. #18-5101) were hydrated for each experiment in a 96-well plate (Greiner Part #655209) as required by the Octet RH16 Biolayer Inferometer (BLI) instrument manufacturer (Sartorius). The ligand (200 nMHis-CD122) was immobilized to the Ni-NTA biosensors and then dipped into a titration of MDNA109-Alb (Construct #7741-044, 82.8 KDa) or MDNA132.15-PSL-MDNA109-Alb (Construct #7741 046, 97.05 KDa) analytes. All dilutions were performed in PBS buffer (11.9 mM phosphate pH 7.4, 137 mM sodium chloride, 2.7 mM potassium chloride) containing kinetics reagent (Sartorius cat. #18-1105).Assays occurred in a 384-well tilted well assay plate (Sartorius cat. #18-5080) and the volume per well was 50 pL at a temperature of 25°C. The assay baseline drift was subtracted out using the protein-bound biosensor dipped into buffer instead of the respective analyte. Negative control experiments were included where the ligand was not loaded onto the biosensors and a mid-range concentration of analyte was tested in order to determine "bare” biosensor binding profiles. There was no significant non-specific binding observed in all experiments. A blocking step was added using Superblock (ThermoFisher cat. #37515).

[0353] The general BLI step conditions used were as follows:• Biosensors check in assay buffer (30 sec)• His-CD122 (ligand) immobilization (12 min)• Blocking with Superblock (2 min)• Baseline in assay buffer (3 min)• Analyte titration association (5 min)• Dissociation back into baseline buffer (15 min)C. Results

[0354] The construct MDNA132.15-PSL-MDNA109-albumin was tested for binding affinity to human IL- 13Ra2 with Fc-MDNA132.15 (KIH) as experimental control. The data, as sensorgrams, is presented in Figure 3.

[0355] As observed, both Fc-MDNA132.15 (KIH) and MDNA132.15-PSL-MDNA109-albumin showed affinity to human IL-13Ra2 though with distinctive konand kOff values generating a 11.5-fold difference in binding affinity constant Ko with Fc-MDNA132.15 (KIH) and MDNA132.15-PSL-MDNA109-albumin exhibiting a KD of0.32 nM and 3.7 nM respectively. The shift in the binding affinity constant is indicative of a masking effect on binding of MDNA132.15 by MDNA109-albumin when fused together via PSL (protease sensitive linker).D. Conclusion

[0356] The binding affinity data clearly established the masking effect observed by 11 ,5-fold difference in KD constant between the constructs, Fc-MDNA132.15 and MDNA132.15-PSL-MDNA109-albumin.Example 3b: Proteolytic Activation by MMP9 Restores IL-2 Activity of MDNA132.15-PSL- MDNA109-Albumin

[0357] The ability of proteolytic cleavage of the MDNA132.15-PSL-MDNA109-albumin (also referred to as “MDNA213-PSL-MDNA109-Alb”) to restore the IL-2 activity of MDNA109 was assessed.

[0358] Pre-activated MMP9 was purchased from Sigma Millipore (SAE0078). The constructs were incubated in a reaction containing 9.5 g of protein with or without 2 pg / mL of MMP9 in Cleavage Buffer (50 mM Tris, 10 mM CaCE, 150 mM NaCI, 0.05% Brij-35 (w / v), pH 7.5) in a total volume of 160 pL for 1 hour at 37°C.Following digestion, reactions were placed on ice and diluted in culture media for testing in the Jurkat I L2RPy reporter cell assay. Aliquots were also taken and inactivated by the addition of SDS-PAGE loading buffer and samples were stored at -80°C until analysis. Samples were analyzed by SDS-PAGE using 4-12% Bis-Tris Bolt polyacrylamide gels (Invitrogen) under reducing conditions. A molecular weight protein marker (Novex™ Sharp Pre-stained Protein Standard #LC5800) was also run on the gel. After electrophoresis, the gel was stained with Simply Blue Safe Stain (Invitrogen).

[0359] The MMP9 cleavage data for MDNA213-PSL-MDNA109-albT3 / C125is shown in Figure 4, left.MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-albT3 / C125) without MMP9 (Lane 2) resolved as a single band at 109 kDa. Presence of MMP9 (Lane 3) resulted in cleavage producing an additional band at 83 kDa corresponding to MDNA109-alb (non-masked version, Lane 1). However, there was evidence of incomplete cleavage of MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-albT3 / C125) with nearly 50% of it remaining uncleaved.

[0360] Subsequently, the reaction mix post MMP9 cleavage was tested in the Jurkat IL-2 assay (Figure 4, right). The activity of MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-alb™ci25)waspartially restored to the level of MDNA109-albumin. The potency of the MMP9 cleavage product (EC50 = 1144 pM) was higher than that of mock cleaved reaction (EC50 = 5108 pM) but remained ~3-fold lower in potency compared to MDNA109-albumin (EC50 = 337 pM). This is consistent with the incomplete cleavage of MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-albT3 / ci25).Example 3c: Functional Characterization of MDNA223-PSL-MDNA132.15 in IL-2 and PD-1 Reporter AssaysA. Introduction

[0361] MDNA132 is an IL-13 superkine that selectively binds the IL-13 decoy receptor (IL-13Ra2) but not the functional IL-13Ra1 . IL-13Roc2 is over-expressed in a broad range of tumors including glioblastomas, pancreas, bladder, and colon cancers but not in normal tissues with the exception of the testes, suggesting that it is a tumor associated antigen (TAA). Therefore, there is a potential to leverage MDNA132 to selectively deliver a therapeutic payload to IL-13Ra2 over-expressing tumors without eliciting undesired response in normal tissues (i.e., reduced risk of toxicity).

[0362] MDNA223 (aka anti-mPD1-MDNA109FEAA KIH) is a Bifunctional Superkine Immuno Therapy (BiSKIT) involving fusion of anti-PD1 with MDNA109FEAA. The latter is an IL-2 superkine with enhanced affinity for I L-2Rp and no binding to IL-2Ra, resulting in selective activation of immune effector cells (CD8+ T and NK cells) with diminished capacity to stimulate immune suppressive Tregs. Lack of binding to IL-2Ra can also reduce risk of toxicity. MDNA223 is design to enable cis-binding to PD-1 and IL-2R expressed on CD8+ T cells to elicite immune checkpoint blockade (via binding to PD-1) and functional stimulation (via binding to IL- 2R) on the same cells. In vivo studies in mouse syngeneic tumor models have shown that MDNA223 has superior anti-cancer activity over the combination anti-PD1 + MDNA19 (i.e. MDNA109FEAA-Fc), thus identifying this BiSKIT as a potential candidate for further development.

[0363] To target MDNA223 to the tumor microenvironment (TME) to potentially enhance efficacy and reduce risk of systemic toxicity (i.e. broaden therapeutic index), a mouse (m)MDNA223*-PSL-MDNA132.15 construct was produced based on the following rationale:• T3A and C125S mutations were added to the MDNA109FEAA moiety (i.e IL-2 portion of MDNA11 . T3A mutation is used in other IL-2 drugs including Proleukin to eliminate glycysolation, which can reduce homogeneity of the drug product. C125S mutation is used to eliminate risk of di-sulfide bridge shuffling at this residue to reduce developability risk. These mutation are denoted as in the name of this construct.Fusion of MDNA132.15 to MDNA223 to enable targeting of IL-13Rcc2 expressing tumors. Direct fusion of MDNA132.15 to the MDNA109FEAA portion of the BiSKIT by a protease-sensitive linker (PSL) can potentially reduce activity of MDNA109FEAA as a result of steric hindrance. The fusion isnot expected to affect PD-1 blockade. Cleavage of the PSL within the TME by therein proteases relases MDNA223* from MDNA132.15, thereby relieving steric hindrance and restoring full activity to MDNA223*. In effect, this enables MDNA223 to be targeted to the TME while attenuating its activity when in circulation to reduce risk of peripheral toxicity.B. MethodsJurkat IL2Rpy Assay

[0364] Cells were plated into 96 well plates according to the manufacturer’s recommendation in a volume of 50 pi L. Test articles were serial diluted and added (25 pi L) to each well to achieve the final test concentrations listed in Table 13. Cells were incubated for 6 hours and then the luciferase substrate was added and incubated for 10 minutes. Luminescence was measured on the ID5 plate reader.Table 13: Constructs tested in the Jurkat I L2Rpy Reporter Assay.PD-1 Reporter Assay

[0365] In the PD-1 / PD-L1 Blockade Bioassay, PD-L1 aAPC / CHO-K1 cells are used to engage PD-1 effector cells through the T-cell receptor (TCR) or the PD-1 receptor. When PD-L1 engages PD-1 , TCR signaling, and the downstream luciferase reporter driven by an NEAT response element are inhibited. PD-1 blocking antibodies prevent the interaction of PD-1 and PD-L1 , allowing TCR signaling and subsequent NFAT-reporter luminescence.

[0366] Mouse 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 piL. The following day, test articles were serially diluted as 2x solutions. Media was removed from the pre-plated reporter cells and 40 piL of the 2x test articles and 40 piL of the Mouse PD-1 T&U Effector Cells and were added to each well to achieve the concentrations listed in Table 14. 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.Table 14: Constructs tested in the PD-1 Reporter Assay.C. ResultsPotency in the Jurkat I L2Rpy Reporter Assay to Measure IL-2R Signaling

[0367] As shown in Figure 5, mMDNA223-PSL-MDNA132.15 (SEQ ID NOs:299-301 , masked version) exhibited reduced potency in the Jurkat I L2Rpy reporter assay compared to MDNA223 (unmasked version). The EC50of mMDNA223-PSL-MDNA132.15 (1624 pM) was 12.3-fold lower than that of mMDNA223 (131.9 pM). The reduced potency of mMDNA223-PSL-MDNA132.15 on inducing IL-2R signaling is consistent with the masking effect of MDNA132.15 fused to the MDNA109FEAA portion of the construct.Potency of PD-1 / PD-L1 Reporter Assay to Assess Immune Checkpoint Blockade:

[0368] In the PD-1 reporter assay (Figure 5) both mMDNA223 (EC50 = 3 .7 nM) and mMDNA223-PSL- MDNA132.15 (EC50 = 3.9 nM) showed similar potency at blockade of PD-1 / PD-L1 immune checkpoint. Potency of both constructs were highly comparable to the parental anti-mPD1 antibody (EC50 = 3.4 nM). These data showed that fusion of MDNA132.15 to the MDNA109FEAA portion of the BiSKIT had no effect on the functionality of the anti-PD1 portion.D. Conclusion

[0369] mMDNA223*-PSL-MDNA132.15 (EC50 = 1624 pM) showed reduced potency compared to mMDNA223 (EC50 = 131 .9 pM) in IL-2R signaling. Both mMDNA223*-PSL-MDNA132.15 (EC50 = 3.9 nM) and mMDNA223 (EC50 = 3.7 nM) maintained similar potency at PD-1 / PD-L1 immune checkpoint blockade. Therefore, fusion of MDNA132.15 to mMDNA223 attenuated (i.e. masked) the activity of MDNA109FEAA (IL- 2R signaling) but had no effect on anti-PD1 (immune checkpoint blockade), consistent with the intent of the construct design.Example 3d: Proteolytic Activation by MMP9 Restores IL-2 Activity of MDNA223-PSL- MDNA132.15

[0370] The ability of proteolytic cleavage of "MDNA223-fPSLf-MDNA213” or “MDNA223A3 / S125. fPSL2f-MDNA213Lin" (SEQ ID NOs:299-301) to restore the IL-2 activity of MDNA109 was assessed.

[0371] Results from the MMP9 cleavage of MDNA223A3 / s125-fPSL2f-MDNA213Linare presented in Figure 6, left. MDNA223 (anti-mPD1-MDNA109FEAA KIH, Lane 1) resolved under reducing condition as 3 bands corresponding to heavy chain 1 (HC1 ; fused to MDNA109FEAA; ~65 KDa, heavy chain 2 (HC2, ~52 kDa) and light chain (LC, ~25 kDa). Lane 2 shows the mock cleavage of MDNA223-fPSLf-MDNA213 KIH where the size of HC1 (~90 kDa) is increased by the addition of fPSLf-MDNA213. Incubation at 37°C for 1 hour (Lane 3) resulted in near complete cleavage of MDNA223A3 / s125-fPSL2f-MDNA213Linwhen MMP9 was used at a concentration of 2 g / mL. These results demonstrated that MDNA223A3 / s125-fPSL2f-MDNA213L'ncan be efficiently cleaved by MMP9 to release the MDNA213 masking domain without affecting the integrity of remaining construct as judged by size.

[0372] The cleaved MDNA223A3 / s125-fPSL2f-MDNA213Linconstruct further exhibited IL-2 activity (Figure 6, right).

[0373] Following MMP9 cleavage of MDNA223A3 / s125-fPSL2f-MDNA213Linto remove the MDNA213 domain, the IL-2 activity was restored to similar potency of MDNA223 evident from similar EC50 of MDNA223 (607 pM) and cleaved MDNA223A3 / s125-fPSL2f-MDNA213Lin(419 pM). Therefore, MMP9 cleavage relieved MDNA223- fPSLf-MDNA213 of the masking effect of MDNA213.Example 3e: Additional Functional Characterization of MDNA223-PSL-MDNA132.15 in IL-2 and PD-1 Reporter Assays

[0374] The functional characteristics of additional MDNA223-PSL-MDNA132.15 (also referred to as "MDNA223-PSL-MDNA213”) constructs were assessed using different PSL linkers, as shown in Table 15 below. For each of these constructs IL-2 mediated signaling and PD-1 / PD-L1 immune checkpoint blockage were assessed.Table 15: Exemplary PSL Linkers

[0375] As shown in Figures 7-10, each of these MDNA223-PSL-MDNA132.15 constructs exhibited reduced IL-2 mediated signaling potency as compared to MDNA223, and no change in PD-1 / PD-L1 blockade.Example 3f: Functional Characterization of MDNA223T3AC125S: Anti-mPD1(H)- fPSL2f-MDNA213 and Anti-mPD1(K)-fPSL2f-MDNA213: MDNA223T3AC125Sin IL-2 and PD-1 Reporter Assays

[0376] The functional characteristics of MDNA223T3ACI25S:anti-mPD1 (H)- fPSL2f-MDNA213 (SEQ ID NOs:314-316) and anti-mPD1 (K)-fPSL2f-MDNA213: MDNA223T3AC125S (SEQ ID NOs:317-319) were assessed. In the MDNA223T3Aci25s:anti-mPDI(H)- fPSL2f-MDNA213 construct, MDNA213 is attached to the C terminal of the “hole” Fc domain homodimer by the PSL. In the anti-mPD1(K)-fPSL2f-MDNA213: MDNA223T3ACI25S construct, MDNA213 is attached to the C terminal of the "knob” homodimer. For each of these constructs, IL-2 mediated signaling and PD-1 / PD-L1 immune checkpoint blockage were assessed.

[0377] As shown in Figures 11 and 12, the IL-2 mediated signaling potency of the MDNA223T3Aci25s: antj_ mPD1(H)- fPSL2f-MDNA213 and anti-mPD1 (K)-fPSL2f-MDNA213: MDNA223T3AC125S constructs were reduced 3- and 2- fold, respectively as compared to MDNA223. Further, no change in PD-1 / PD-L1 blockade was observed for either of these constructs.Example 3g: Functional Characterization of MDNA223T3AC125S-fPSL2f-MDNA213: Anti-mPDI(H)- fPSL2f-MDNA213 in IL-2 and PD-1 Reporter Assays

[0378] The functional characteristics of MDNA223T3Aci25s.fPSL2f-MDNA213: Anti-mPD1(H)-fPSL2f- MDNA213 (SEQ ID NQ:320-322) was assessed. In the MDNA223T3Aci25s.fPSL2f-MDNA213: Anti-mPDI(H)- fPSL2f-MDNA213 construct, a first MDNA213 is attached to the C terminal of the “hole” Fc domain homodimer by the PSL, and a second MDNA213 is attached to the MDNA109FEAA223T3ACI25S |[__2 mediated signaling and PD-1 / PD-L1 immune checkpoint blockage were assessed.

[0379] As shown in Figure 13, the IL-2 mediated signaling potency of the MDNA223T3AC125S-fPSL2f- MDNA213: Anti-mPD1 (H)-fPSL2f-MDNA213 construct was reduced 39-fold, as compared to MDNA223, and there was no change in PD-1 / PDL-1 blockade. Further, as shown in Figure 14, the MDNA223T3AC125S-fPSL2f- MDNA213: Anti-mPD1 (H)-fPSL2f-MDNA213 construct is sensitive to MMP9 cleavage in vitro, and shows activity upon MMP9 cleavage in an IL-2R reporter assay (Figure 15).Example 3h: Functional Characterization of MDNA213-fPSL2f- MDNA11T3AC125SIL-2 and PD-1 Reporter Assays

[0380] The functional characteristics of MDNA213-fPSL2f- MDNA11 TSACI 25s (SEQ |DNO:323) was assessed. In the MDNA213-fPSL2f- MDNA11T3AC125Sconstruct, an MDNA213 is attached to the MDNA109FEAA223T3AC125Sof the MDNA11 123T3AC125Sconstruct by a CSL. IL-2 mediated signaling and PD- 1 / PD-L1 immune checkpoint blockage were assessed.

[0381] As shown in Figure 16, the IL-2 mediated signaling potency of the MDNA213-fPSL2f- MDNA11T3AC125Sconstruct was reduced 21-fold, as compared to MDNA223. Further, as shown in Figure 17, the MDNA213-fPSL2f- MDNA11T3AC125Sconstruct is sensitive to MMP9 cleavage in vitro, and shows activity upon MMP9 cleavage in an IL-2R reporter assay (Figure 18).Example 4: Characterization of a tumor-targeting and activatable T-MASK platform to enhance tumor accumulation and tolerability of potent immune modulatorsA. Background

[0382] The novel T-MASK (Targeted Metallo / protease Activated SuperKine) platform involves fusion of a dual tumor-targeting / masking domain to potent immune modulator(s) via a metal lo-protease (MMP) sensitive linker (PSL) to achieve the following objectives: (1) reduce / fine-tune the potency of the immune modulator by steric hindrance to increase systemic tolerability and (2) promote retention in the tumor microenvironment (TME) to maximize MMP cleavage and restore full potency at the intended target site. As proof of concept, we selected as the tumor-targeting / masking domain an IL-13 superkine (MDNA213) with high selectivity and affinity for the IL-13 decoy receptor IL-13Rcc2, a tumor associated antigen expressed in many aggressive solid tumors. MDNA213 is fused via PSL to MDNA1 1 and MDNA223, both containing a not-alpha, beta-enhanced IL-2 fused with albumin or anti-PD1 antibody respectively. We present preliminary results on characterization of both T-MASK constructs demonstrating conditional fine-tuning of IL-2R agonism.B. Methods

[0383] T -MASK optimization included assessment of PSL linker and orientation of the tumor- targeting / masking domain. In vitro IL-2 and PD-1 / PDL-1 reporter assays were performed to evaluate IL-2R stimulation and anti-PD1 blockade respectively. In vitro MMP assay was used to validate cleavability of T- MASK constructs and restoration of full potency.C. Results

[0384] T-MASK constructs showed approximately 10-40 folds reduction in potency compared to respective non-masked versions in IL-2R induced p-STAT5 reporter cell assay. Extent of fine-tuning can be adjusted by length and composition of PSL and orientation of the domains, providing versatility to the T-MASK platform and potential to engage the complete repertoire of peripherally circulating immune cells. In the case of MDNA223 (anti-PDI-IL^Wne), fusion of MDNA213 to generate the MDNA223-CSL-MDNA213 T-MASK construct resulted in reduced IL-2R agonism but had no effect on potency of PD1 / PDL-1 blockade as expected. Unmasking of MDNA223-PSL-MDNA213 constructs (also referred to as "MDNA113" constructs), such as for example, by MMP-mediated cleavage fully restored its IL-2R signaling activity to the same level as the non-masked MDNA223 construct. Similar data were obtained with the masked version of MDNA11 , demonstrating robustness of the T-MASK platform. In vivo studies are ongoing to evaluate the effect of T- MASK constructs on peripheral immune cell expansion (systemic response), tumor retention (maximize activation) and tumor growth inhibition (targeted response).Example 5: Restoration of IL-2R Signaling Activity following Proteolytic Cleavage in vitroA. Methods

[0385] MMP9 Cleavage: 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 CaCI2, 150 mM NaCI, 0.05% Brij-35 (w / v), pH 7.5) in a total volume of 160 pL. Four distinctive conditions were tested to observe difference in cleavage efficiency, if any. Following digestion, reactions were placed on ice and diluted in culture media for testing in the Jurkat IL2Rpy reporter cell assay. Aliquots were also taken and flash frozen in liquid nitrogen prior to storage at - 80°C for subsequent SDS-PAGE analysis.

[0386] Jurkat I L2R(3y Reporter Assay: Cells were plated into 96 well plates in a volume of 50 pL according to the manufacturer’s recommendation. Test articles were serially diluted and added (25 pL) to each well. Cells were incubated for 6 hours and then the luciferase substrate was added and incubated for additional 10 minutes. Luminescence was measured on the i D5 plate reader.B. Results

[0387] The results are shown in Figures 19 and 20. With the exception of MASK A4, all BiSKITs were digested by MMP9 to varying extent (60-70%) under the conditions described in methodology. MASK A4 was designed without a cleavage site to demonstrate selectivity of MMP proteolysis.

[0388] MDNA223 (aka anti-mPD1-MDNA109FEAA KIH, resolved under reducing conditions as 3 bands corresponding to heavy chain 1 (HC1 ; fused to MDNA109FEAA; ~65 KDa, heavy chain 2 (HC2, ~52 kDa) and light chain (LC, ~25 kDa).

[0389] The mock cleavage of constructs MASK A1 to MASK A4 resolved as 3 bands where the size of HC1 (~90 kDa) is increased by the addition of fPSLf-MDNA213. Incubation with MMP9 resulted in cleavage of constructs wherein the bands corresponded to MDNA223 as anticipated except for MASK A4 since it had no PSL i.e., MMP9 cleavage site.

[0390] Mock and MMP9 cleavage of MASK A5 and MASK A6 resolved as 3 bands where the size of HC1 (-70 kDa) is increased by the addition of fPSLf-MDNA213. Incubation with MMP9 resulted in cleavage of constructs wherein the bands corresponded to MDNA223 as anticipated.

[0391] Mock cleavage of MASK A7 resolved as 3 bands where the size of HC1 (-90 kDa) and HC2 (-70 kDa) is increased by the addition of fPSLf-MDNA213. Incubation with MMP9 resulted in cleavage of constructs wherein the bands corresponded to MDNA223 as anticipated. Mock cleavage of of MASK B1 resolved as a single band of -110 kDa whereas MMP9 digested resolved as 2 bands, -84 kDa and -12 kDa corresponding to MDNA11 and MDNA213 respectively.C. Restoration of IL-2 Activity

[0392] MASK-IT constructs A7 and MASK B1 were reported to exhibit -40 and 20-fold reduction respectively in IL-2 activity in the Jurkat I L2Rf>y Reporter Cell Assay and sensitivity to MMP9 digestion as described above. MASK A7 and MASK B1 were further tested for restoration of IL-2 activity post MMP9 cleavage.

[0393] In the Jurkat IL- 1 L2RPy cell reporter assay (Figures 21 A and 21 B), both MASK A7 and MASK B1 exhibited reduced IL-2 activity in absence of MMP9 (mock digestion). However, the activity was restored to similar potency as MDNA223 or MDNA11 in presence of MMP9 under all the conditions tested. Hence, MMP9 cleavage relieved both constructs from the masking effect of MDNA213, consistent with the design of this MASK-IT construct. The EC50 for MDNA223 and MDNA11 was reported at 0.5 nM. MASK A7 and MASK B1 exhibited reduced potency with an EC50 of 2.9 nM for both. The potency was reinstated upon MMP9 cleavage of both MASK A7 and MASK B1 with EC50s between 0.5 - 0.6 nM for different cleavage conditions.D. IL-2 Function of MDNA1 13 is Masked in Murine CTLL-2 and Human PBMC Proliferation Assay

[0394] CTLL2 cells were plated into 96 well plates at 30,000 cells per well in media containing the T-STIM proliferation supplement. Following plating, cells were treated with increasing concentrations of the test or control sample for 48 hours. After treatment, Cell Titer Blue viability reagent (Promega G8080) was added to each well and the plates were scanned at 560Ex / 590Em after development of the fluorescent viability signal.

[0395] As shown in Figure 22, a rightward shift of the curve with MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParH(MASK A7; SEQ ID NOs:320, 321 , and 322) with respect to MDNA223 was indicative of its lower potency. The EC50of MDNA113 (4.3 nM) was 14-fold lower than MDNA223 (0.3 nM). Similarly, MDNA213-fPSL2f- MDNA11A3 / S125(SEQ ID NO:323) showed 113-fold lower activity at (14 nM) than MDNA11 (0.12 nM).

[0396] Data from the human PBMC proliferation assay are presented in Figure 23. Three human PBMC donors were stimulated with masked and non-masked versions of MDNA223 for 48 h and subjected to analysis of BrDU incorporation as readout for proliferation. Both MDNA223 (non-masked) and MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParH(masked) induced PBMC proliferation with the latter showing reduced potency. While EC50 values were implicated based on logistic curve fitting, failure to achieve clear upper threshold within the dose-range tested rendered these values less reliable for comparisons.Comparison of stimulation index of MDNA223 and MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHclearly showed an attenuation of latter at all concentrations tested. Similar observations were made with masked and nonmasked MDNA11.E. Attenuation of Th1 Response by MDNA113 in IL-2 Mediated pSTAT5 Induction in Human PBMCs

[0397] Human PBMCs were subjected to treatment with MDNA223 (non-masked) and MDNA223A3 / S125- fPSL2f-MDNA213Lin / ParH(masked) for 15 min and harvested for flow cytometric analysis of immune subsets. The results are shown in Figures 24-28.

[0398] MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHattenuated the IL-2 mediated pSTAT5 induction in human PBMCs in both naive CD8+T cells and Tregs. The EC50S revealed a ~7-fold reduction in pSTAT5 induction in naive CD8+T cells with MDNA223 and MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHexhibiting an EC50 of 776.9 pM and 5402 pM respectively (average from 3 independent donors,). A diminution in pSTAT5 induction in Tregs was observed to be ~10-fold with an average EC50 of 8504 pM for MDNA223A3 / s125-fPSL2f- MDNA213Lin / ParHcompared to 829.4 pM for MDNA223.

[0399] ECso ratios of naive CD8+T-cells to Tregs is considered a useful metric to evaluate the therapeutic potential of a construct since it reflects the balance between anti-tumor and pro-tumor immune response. Acomparable ratio between non-masked and masked MDNA223 demonstrated the therapeutic potential of MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHis not compromised even though the IL-2 activity is masked.

[0400] Similar observations were made with masked and non-masked MDNA11 . The EC50S revealed a ~7-fold reduction in pSTAT5 induction in naive CD8+T cells with MDNA11 and MDNA213 -fPSL2f- MDNA11A3 / S125exhibiting an EC50 of 512.8 pM and 3674.3 pM respectively (average from 3 independent donors,). A diminution in pSTAT5 induction in Tregs was observed to be ~5-fold with an average EC50 of 2825 pM for MDNA213 -fPSL2f-MDNA11A3 / S125compared to 611.6 pM for MDNA11.F. Receptor Binding Affinity

[0401] EMT6 (WT) and EMT6 / IL-13Ra2 cells were thawed and cultured in T75 flasks with 15% FBS in Waymouths MB752 / 1 (w / L-Glu). At 80% confluency, cells were passaged using 0.05% Trypsin EDTA. Cells were counted on hemocytometer and seeded at 1 :10 subculture ratio into 6-well TC-treated plates. At 80% confluency, cells were removed by 20min incubation with cell-stripper (non-enzymatic detachment solution). Cells were transferred to 96 U-bottom and treated for 30 min with constructs (5-fold 6-point curve). Fc- MDNA213 was used as control. After incubation, cells were stained with anti-Fc antibody for detection of cell receptor binding. Cell receptor binding of constructs and anti-Fc was determined by flow cytometry. Affinity (EC50) was measured by Graph Pad Prism.

[0402] The data is presented in Figure 29. Fc-MDNA213 (positive control) binds to the cell receptor at the two concentrations tested. MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHbinds albeit with lower affinity compared to Fc-MDNA213 to the EMT6 / IL-13Ra2 cells in vitro. The binding was observed to be dose dependent. The decrease in binding compared to Fc-MDNA213 may be explained by the steric hinderance from MDNA223 fusion using a protease sensitive linker.G. Pharmacodynamic assessment of MDNA113

[0403] Pharmacodynamic response to MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHwas studied in comparison to the non-masked MDNA223 for a reduction in peripheral lymphocyte expansion in vivo in mice when administered intra-peritoneally (IP).

[0404] The peripheral expansion of lymphocytes measured by CBC analysis, 72 h post treatment with masked and non-masked MDNA223A3 / S125and MDNA1 1A3 / S125is shown in Figures 30 and 31. The peripheral lymphocyte expansion at equivalent doses of non-masked and masked MDNA223A3 / S125or MDNA11A3 / S125demonstrated a significant reduction with respective masked versions. These data indicate that MDNA223A3 / S125and MDNA11A3 / S125were shielded efficiently by masking domain MDNA213 in circulation and successfully reduced the peripheral lymphocyte expansion. However, increasing the dose of MDNA223A3 / S125-fPSL2f-MDNA213Lin / ParHand MDNA213-fPSL2f-MDNA1 1A3 / S125(from 2 mg / kg to 5, 7.5 and 10 mg / kg) resulted in peripheral lymphocyte expansion that was analogous to vehicle treated animals.

[0405] The other blood cells including neutrophils (PMNs), monocytes, basophils and eosinophils, as expected, were unaffected upon treatment with masked or non-masked MDNA223A3 / S125and MDNA11A3 / S125.H. In Vivo Tumor Growth Inhibition in MC-38 Colon Carcinoma Model

[0406] As shown in Figure 32, the masked (MDNA223A3 / s125-fPSL2f-MDNA213Lin / PsrH) and non-masked versions of MDNA223 showed comparable tumor growth inhibition providing strong evidence that MDNA113 was cleaved by proteases in the TME resulting in release of MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHfrom masking domain (MDNA213) thereby restoring its full potency and therapeutic potential. MDNA223A3 / S125- fNCLf-MDNA213Lin / ParHexhibited moderate tumor growth inhibition supporting the sheltering of MDNA223 in vivo when masked by MDNA213 via a non-cleavable linker.I. In Vivo Tumor Growth Inhibition in EMT6 / IL13Ra2 Breast Tumor Model

[0407] As shown in Figure 33, data indicated that tumor growth inhibition on treatment with equimolar doses of MDNA23A3 / s125-fNCLf-MDNA213Lin / ParHwas considerably weaker compared to MDNA223A3 / S125- fPSL2f-MDNA213Lin / ParHand MDNA223. The therapeutic potency of MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHand MDNA223 was comparable. In addition, the efficacy of MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHand MDNA223 was significantly superior to MDNA1 1 (IL-2 super-agonist) alone or its co-administration with anti- mPD1 .J. In Vivo Tumor Growth Inhibition in MC-38 Colon Carcinoma Model

[0408] As shown in Figure 34, systemic administration of MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHexhibited tumor growth inhibition in the MC-38 model without body weight loss compared to MDNA223 wherein the animals showed a significant reduction in body weight post administration of first dose. Dosing was initiated at an average tumor size of 40 mm3. MDNA223 and MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParHwere used at equimolar doses of 7.5 mg / kg and 8.8 mg / kg respectively.Conclusion:

[0409] MDNA223-PSL-MDNA213 is a novel T-MASK construct designed to increase tolerability while leveraging the synergy between PD1 / PDL-1 blockade and IL-2R agonism for immunotherapy. Ongoing studies investigate alternative tumor-targeting / masking domains and immune modulators, including other cytokines and potent therapeutic agents, to potentially broaden the utility of the T-MASK platform.Example 6: MTD Study of MDNA113 in comparison to non-masked MDNA223A. Nomenclature

[0410] The following constructs are referred to in the Examples:• M DNA1 13A3 / si25 or MDNA113AA3 / S125= MDNA223A3si25_fpsL2f-MDNA213L^rH• MDNA1 13B = MDNA223A3 / s125-fPSL2f-ecCD122: MDNA213• MDNA223A3 / S125= MDNA223• MDNA113NC= MDNA223-2x(GS-MDNA213) [SEQ ID NOs: 325 -327]• MDNA113BNC= Anti-mPD1 (K)-MDNA109FEAA-T3AC125S-fPSL2f-ecCD122: Anti-mPD1(H)-GS- MDNA213 [SEQ ID NOs: 353 - 355]B. Objective

[0411] The objective of the study was to evaluate the maximum tolerated dose (MTD) of MDNA113A3 / S125in one of the commonly used mice strains, C57BI / 6 to see whether MDNA1 13A3 / S125would be tolerated better than MDNA223A3 / S125.C. Methods

[0412] Female C57BI / 6 mice aged 8 weeks at the start of dosing were used for this study. The study design is outlined in Table 16 below.Table 16: Dosing for each group in the MTD study with MDNA113A3 / S125and MDNA223A3 / S125*Molar equivalent doses between MDNA223A3 / S125and MDN A113A3 / S125.D. Results

[0413] Body weights of all animals are presented in Figure 35 and Figure 36 for once weekly and twice weekly dosing regimens respectively.• Group 1 animals (MDNA223A3 / S12520 mg / kg, once weekly) showed transient weight loss post all three doses administered while Group 5 animals (MDNA1 13A3 / S12523 mg / kg, once weekly) exhibited a mild transient weight loss after the first two doses.• Animals in both Group 2 (MDNA223A3 / S12510 mg / kg, once weekly) and Group 6 (MDNA1 13A3 / S12512 mg / kg, once weekly) showed a transient weight loss post first and third dose which was regained as the study progressed.• Animals in both Group 3 (MDNA223A3 / S1257 mg / kg, once weekly) and Group 7 (MDNA1 13A3 / S1258 mg / kg, once weekly) showed a mild transient weight loss post first and third dose that recovered on subsequent days of the study.• All animals in Group 4 (MDNA223A3 / S1257 mg / kg, twice weekly) succumbed after the second dose. While two of three animals in Group 8 (MDNA113A3 / S1258 mg / kg, twice weekly) were found dead after the second dose, one animal survived all the four doses and gained weight remainder of the study period.• All animals in Group 9 (MDNA223A3 / S1254 mg / kg, twice weekly) succumbed after the second dose whilst all animals in Group 10 (MDNA223A3 / S1254 mg / kg, twice weekly) survived to the end of study. The animals in Group 10 exhibited a mild transient weight loss post second dose that recuperated and was subsequently consistent through the study period.

[0414] A survival curve is shown in Figure 37.• All animals in Group 1 to 3 (MDNA223A3 / S125once weekly groups) and Group 5 to 7 survived till the end of study ((MDNA113A3 / S125once weekly groups).• All animals in Group 4 (MDNA223A3 / S1257 mg / kg twice weekly) and 2 / 3 animals in Group 8 (MDNA1 13A3 / S1258 mg / kg twice weekly) were found dead post the second dose. The remaining animal in Group 8 survived till end of study. Thus, a survival percentage of 33% was observed in Group 8.• All animals in Group 9 (MDNA223A3 / S1254 mg / kg twice weekly) succumbed post second dose while all animals in Group 10 (MDNA113A3 / S1254.7 mg / kg twice weekly) survived to the end of study(100% survival).E. Conclusion

[0415] Both MDNA113A3 / S125and the non-masked version MDNA223A3 / S125were tolerated well at all doses when administered IP in a once weekly dosing schedule. Hence, MTD for either MDNA223A3 / S125or MDNA113A3 / S125was not reached.

[0416] The non-masked version MDNA223A3 / S125was not tolerated well when administered IP on a twice weekly dosing schedule at either of the doses tested, 7 mg / kg and 4 mg / kg. All animals died post the second dose within the week of dosing cycle. MDNA113A3 / S125on the other hand was tolerated better with one of 3 animals surviving the molar equivalent 8 mg / kg and all animals surviving the molar equivalent 4.7 mg / kg twice weekly dosing. Hence, the MTD of MDNA1 13A3 / S125was established at 4.7 mg / kg twice weekly dosing regimen . The data suggests MTD of MDNA223A3 / S125to be lower than 4 mg / kg at twice weekly dosing regimen .Example 7: In Vitro Assessment of MASK-ITs to establish reduced IL-2 activity and retained PD-1 blockadeA. Objective

[0417] To augment the masking effect on MDNA223 or other IL-2 BiSKITs in periphery, a number of new constructs were designed with an additional masking domain i.e., extracellular domain of human CD122 or CD25. The constructs were assessed for reduced IL-2 activity and retention of PD-1 blockade in comparison to non-masked MDNA223.B. MethodsThe constructs were tested in the Jurkat IL2Rpy reporter assay (cells that lack the CD25 receptor) and HEK Blue IL-2 assay (cells that express the trimeric receptor, CD25 / CD122 / CD132) .The constructs were tested in the PD-1 / PDL-1 blockade reporter assay for validating the potency of anti-PD1 .

[0418] The constructs were tested for restoration of IL-2R agonism post MMP9 cleavage.C. Results

[0419] Non-masked MDNA223A3 / S125and MDNA113A3 / S125were used as benchmark and experimental control respectively. Since Anti-mPD1-MDNA109A3 / s125-fPSL2f-ecCD25:MDNA213 and Anti-mPD1 -H9TA3 / S125- fPSL2f-ecCD25: MDNA213 could not be purified as monomeric fraction for the formation of oligomers during the purification process, both P1 & P2 fractions of the constructs were tested in the assay. While P1 fraction of Anti-mPD1-MDNA109A3 / s125-fPSL2f-ecCD25:MDNA213 exhibited complete loss of IL-2 activity, the P2 fraction showed ~41 -fold reduction in IL-2 signaling compared to non-masked MDNA223A3 / S125. It also showed ~5-fold better masking efficiency than MDNA1 13A3 / S125wherein the masking domain comprised of only MDNA213 as opposed to MDNA213 and extracellular domain of CD25 in Anti-mPD1 -MDNA109A3 / s125-fPSL2f-ecCD25: MDNA213 (Figure 38A).

[0420] MDNA223A3 / s125-fPSL2f-ecCD122:MDNA213 demonstrated 453-fold attenuation in IL-2 signaling compared to non-masked MDNA223A3 / S125and ~47-fold improved masking than MDNA113A3 / S125(Figure 38B).

[0421] The EC50S are summarized in Table 17.Table 17: ECso determinations (in pM) for the various test samples tested in Jurkat IL2R Y bioassay.

[0422] Constructs including the ones harboring the IL-2 mutein with CD25 binding retained were tested in the HEK Blue IL-2 reporter assay for the presence of trimeric receptor complex (CD25 / CD122 / CD132).

[0423] Fc-MDNA109T3 / C125and anti-hPD1 -MDNA109A3 / S125were used as experimental controls and demonstrated similar potency. Rh IL-2 used as benchmark control was —3.5 fold less potent consistent with fact that MDNA109 is a beta-enhanced IL-2 mutein. The two fractions, P1 and P2, of MASK-IT Anti- mPD1 MDNA109A3 / s125-fPSL2f-ecCD25:MDNA213 exhibited an attenuated potency with a reduction of ~1 1 and ~8-fold respectively (Figure 39A).

[0424] To compare the potency of MASK-IT, Anti-mPD1 -H9TA3 / s12MPSL2f-ecCD25:MDNA213, H9TT3 / C125- albumin and Anti-hPD1-H9TA3 / S125were used as experimental controls for non-availability of anti-mPD1 - H9TA3 / S125 Compared to rh IL-2 used as benchmark, H9TT3 / c125-albumin and Anti-hPD1 -H9TA3 / S125showed lower potency. The IL-2 signaling potency of Anti-mPD1-H9TA3 / s125-fPSL2f-ecCD25:MDNA213 was however, masked -124 and ~43-fold respectively for P1 and P2 fractions (Figure 39B).

[0425] Non-masked MDNA223A3 / S125and Anti-hPD1-H9TFEAAA3 / S125were used as experimental controls to compare the respective reduction in IL-2 potency of MDNA223A3 / s125-fPSL2f-ecCD122:MDNA213 and Anti- hPD1-H9TFEAAA3 / s125-fPSL2f-ecCD122:MDNA213. An attenuation of ~65-fold and ~688-fold was observed respectively. Of note, Anti-hPD1 -H9TFEAAA3 / S125exhibited a ~7-fold lower potency than MDNA223A3 / S125in the assay suggesting that H9TFEAA is a weaker IL-2 agonist than MDNA109FEAA (Figure 39C).

[0426] The EC50 values for all are summarized in Table 18.Table 18: EC50 determinations (in pM) for the various test samples tested in HEK Blue IL-2 assay.

[0427] MASK-ITs tested for PD-1 / PDL-1 blockade demonstrated similar potency as non-masked MDNA223A3 / S125and benchmark control, anti-mPD1 antibody (Figure 40).

[0428] MASK-ITs were digested using MMP9 and analyzed by SDS-PAGE. MDNA223 (aka anti-mPD1- MDNA109FEAA KIH) resolved under reducing conditions as 3 bands corresponding to heavy chain 1 (HC1 ; fused to MDNA109FEAA; ~65 KDa), heavy chain 2 (HC2, ~52 kDa) and light chain (LC, ~25 kDa).

[0429] All constructs tested (i.e., MASK-ITs of MDNA223, anti-mPD1-H9TFEAA, anti-mPD1-MDNA109 fractions 1 and 2) showed near complete cleavage in MMP9 reactions. In absence of rMMP9, the constructs resolved as 3 bands where HC1 (~90 kDa) and HC2 (~70 kDa) were increased in size by fusion fPSL2f- ecCD122:MDNA213. Cleavage by MMP9 released the fPSL2f-ecCD122:MDNA213 masking domain as indicated by presence of proteins bands of similar size as those of MDNA223 (Figure 41).

[0430] Restoration of IL-2 Activity post MMP9 Cleavage

[0431] MASK-ITs were tested for IL-2 activity post MMP9 cleavage either in the Jurkat IL2RBv or HEK Blue IL-2 reporter assay. These studies showed that IL-2 activity was restored to similar potency as their respectivenon-masked versions following cleavage by MMP9 (Figures 42A and 42B; Table 19) with the exception of Anti-mPD1-H9TA3 / s125-fPSL2f-ecCD25:MDNA213 (P2 fraction).Table 19: EC50 determinations (in pM) for the various constructs tested in IL-2 Reporter assay in presence or absence of MMP9D. Conclusion

[0432] The designed constructs when tested in the Jurkat IL2Rpy reporter assay or HEK Blue IL-2 assay exhibited reduced IL-2 activity compared to non-masked controls and MDNA113A3 / S125(utilizing only MDNA213, IL-13 superkine as masking domain). The data is summarized below with masking domain of each construct underlined for inference:• MDNA223A3 / s125-fPSL2f-ecCD122:MDNA213 showed 453-fold reduced IL-2 activity in comparison to non-masked MDNA223A3 / S125and ~47-fold improved masking than MDNA113A3 / S125(MDNA223A3 / S125- fPSL2f-MDNA213Lin / ParH) in the Jurkat IL2Rpy assay• The P1 fraction of Anti-mPD1 -MDNA109A3 / s125-fPSL2f-ecCD25:MDNA213 showed abrogation whilst the P2 fraction showed ~41-fold reduction in IL-2 signaling compared to non-masked MDNA223A3 / S125. The masking was ~5-fold improved than MDNA1 13A3 / S125(MDNA223A3 / s125-fPSL2f-MDNA213Lin / ParH) in the Jurkat IL2Rpy assay.• The IL-2 signaling potency of Anti-mPD1-H9TA3 / s125-fPSL2f-ecCD25:MDNA213 was attenuated -124 and ~43-fold respectively for the P1 and P2 fractions compared to non-masked Anti-hPD1-H9TA3 / S125in the HEK Blue IL-2 assay.• A 688-fold reduction in IL-2 activity of Anti-hPD1 -H9TFEAAA3 / si25-fPSL2f-ecCD122:MDNA213 was observed in HEK Blue IL-2 assay in comparison to Anti-hPD1 -H9TFEAAA3 / S125.

[0433] The MASK-ITs were observed to be sensitive to proteolytic cleavage in vitro and restored the attenuated IL-2 function post cleavage by MMP9.Example 8: In Vivo Imaging of labelled MDNA113 and non-masked MDNA223 to validate accumulation in IL-13Ra2 expressing tumorsA. Objective

[0434] In Vivo Imaging System (MS) was used to visualize labeled protein or other analytes in mice to understand distribution and accumulation. Multiple images were acquired over time to provide a longitudinal view of the kinetics of drug biodistribution in live animals. This study investigated the potential for durable and selective tumor accumulation of MDNA113 in mice bearing I L-13Rcc2 expressing tumors resulting in a superior anti-tumor response. The study will validate the IL-13Ra2 targeting role of MDNA213 in MDNA1 13.B. Methods

[0435] Labelling of masked and non-masked MDNA223: A VivoTag800 IN VIVO NIR fluorochrome (Perkin Elmer, cat. #NEV11107) was used to label MDNA113 and MDNA223 according to the manufacturer’s protocol.

[0436] Binding analysis by Biolayer Interferometry: To establish that the labeling did not alter the receptor binding properties, MDNA113 and MDNA223 were tested for binding to one of the cognate receptors, mouse CD122, by Biolayer Interferometry (BLI)ZOctet.

[0437] BLI step conditions used were as follows: Biosensor check in PBS Kinetics buffer (0.5 minutes), ligand protein immobilization (10 minutes), blocking with Superblock (2 minutes), baseline in PBS Kineticsbuffer (2 minutes), analyte titration association (5 minutes) and analyte dissociation back into baseline buffer (15 minutes).

[0438] In Vivo Imaging: Several human cell lines are known to express IL-13Ra2 including melanoma cell line, A375 (human melanoma) xenograft model, which expresses IL-13Ra2, was used to assess MDNA113 and MDNA223 targeting and accumulation in athymic nude mice. A549, a human lung tumor with low / no IL- 13Ra2 expression, was used as a control on the contra-lateral flank of mice.

[0439] Animal Information (Vendor, Species, Strain, Sex, Age or Weight, and Number):• Vendor: Charles River Laboratories• Species: Mouse• Strain: Athymic Nude (strain code 490)• Sex: Female• Age / Weight: 5-6 weeks• Number: 12 (9+ 3 extra) Animals were be purpose-bred, specific pathogen-free, and experimentally naive at the start of the study. Age / Weight refers to the Age / Weight at the time of delivery from the vendor.

[0440] The study design is shown in Table 20.Table 20: Study Design for in vivo imaging with labeled MDNA113 and MDNA223

[0441] Epifluorescence IVIS imaging used excitation=785 nm and emission=810 nm with mice in dorsal position at pre-dose (baseline image) and 4h, 24h, 72h, 120h and 168 h post-injection of labelled constructs. Following the last imaging timepoint (168hr post-injection), mice were euthanized, and tumors were collected for ex-vivo imaging.C. Results

[0442] VivoTag800 Labeling: A total of 1 .5-1 .8 mg of each of construct was subjected to the labelling reaction. Table 21 below summarizes the labelling results. The calculations do not take into account the added molecular weight of the VivoTag800 fluorochrome. The protein: vi votag800 ratio higher than 1 is indicative of successful labeling.Table 21 : Protein labeling and absorbance data for MDNA223 and MDNA113

[0443] Labelled constructs were tested for binding to mouse CD122 receptor by Octet before in vivo imaging study.

[0444] Receptor Binding Analysis by BLI / Octet: Binding affinity by BLI / Octet was performed to confirm that the receptor binding profile of MDNA113 and MDNA223 were not impacted by labelling with vivot800.Unlabelled and labelled versions of both constructs showed similar binding properties to mouse CD122, indicating no change in binding affinity following labeling with vivotag800.

[0445] The binding affinity constant (KD) calculated from steady state kinetics was 320 nM and 240 nM respectively for unlabeled and labeled MDNA223. Similarly, MDNA113 exhibited KD of 1 .8 uM and 4.0 uM respectively for unlabeled and labeled versions (Figure 43).

[0446] Of note, the Ko of MDNA113 was ~10-fold higher than MDNA223 consistent with a reduction in binding affinity of MDNA113 to mouse CD122 due to fusion of the masking / targeting domain MDNA213 causing steric hinderance.

[0447] In Vivo Imaging: A375 (IL-13Ra2 positive; right flank) and A549 (IL-13Ra2 negative; left flank) cells were engrafted in flanks of athymic nude mice and allowed to grow into established tumors. Mice with established tumors were IV injected with labelled MDNA223 or MDNA113 and IVIS images were acquired at pre-dose, 4h, 24h, 72h, 120h, and 168h post-treatment and ex-vivo post euthanasia. Compiled images are shown in Figure 44.

[0448] Accumulation of labelled MDNA113 was observed in A375 tumors (right flank) as compared to A549 tumors (left flank) in some of the mice. At earlier time-points (i.e., up to 24 hours), MDNA113 was distributed throughout the body of mice. As MDNA113 is cleared from systemic distribution at later timepoints (72 to 168 hours), accumulation in IL-13Ra2 expressing tumors (A375) was evident in at least one of three mice. MDNA113 localization to A375 tumors is consistent with the hypothesis of MDNA213 mediated targeting of MDNA113 to IL-13Roc2 expressing tumors. Following in vivo imaging at the 168-hour time-point, mice were euthanized, and their intact tumors were resected for ex-vivo imaging. Ex-vivo images confirmed selective accumulation of MDNA113 in IL-13Ra2 expressing tumors (A375).

[0449] Accumulation of labelled MDNA223 (experimental control) was observed to transiently occur in vivo as evident from the 72h data image. Additionally, the ex-vivo images post termination showed low accumulation in both A549 (IL-13Ra2 negative) and A375 cells (IL-13Ra2 positive) tumors suggesting lack of specific targeting in absence of the MDNA213 domain.D. Conclusion

[0450] BLI / Octet studies showed that labeling of MDNA223 and MDNA113 did not alter their binding profiles.

[0451] In vivo and ex vivo imaging showed durable and targeted accumulation of MDNA113 in IL-13Roc2 expressing A375 tumors in mice for ~10 days. Accumulation of MDNA223 in vivo was observed to be transient, however, ex-vivo images showed non-targeted accumulation in both A549 (IL-13Ra2 negative) and A375 cells (IL-13Ra2 positive) tumors.Example 9: MC-38 tumor growth inhibition (TGI) study to evaluate the efficacy of MDNA113 andMDNA223-fPSL2f-ecCD122:MDNA213 (MDNA113B)A. Objective

[0452] The objective of the study was to investigate the therapeutic efficacy of MDNA113 and MDNA223- fPSL2f-ecCD122:MDNA213 (MDNA113B).B. Methods

[0453] The study design is shown in Table 22.Table 22: Study design for MC38 TGI studyC. Results

[0454] MDNA113 exhibited superior tumor growth inhibition compared to non-masked MDNA223 when treated once weekly. This may be because of conditional activation of MDNA113 at tumor site / TME uponproteolytic cleavage by proteases. Four of 7 mice showed complete regression of tumor when treated with MDNA113 compared to one of seven complete regressions when treated with MDNA223 (Figure 45).

[0455] MDNA223-fPSL2f-ecCD122:MDNA213 was however, less potent than non-masked MDNA223 which may be attributed to incomplete proteolytic activation at tumor site. Two of 7 mice showed complete regression of tumor when treated with MDNA223-fPSL2f-ecCD122:MDNA213.

[0456] On a twice weekly dosing schedule, MDNA113 showed significant tumor growth inhibition compared to non-cleavable version of MDNA113. Two of 7 mice showed complete regression of tumor when treated with MDNA113 (Figure 46).D. Conclusion

[0457] Both MDNA113 and MDNA223-fPSL2f-ecCD122:MDNA213 showed significant tumor growth inhibition which was comparable with non-masked MDNA223.Example 10: PD (pharmacodynamic) Study to evaluate peripheral lymphocyte expansion in miceA. Objective

[0458] The objective of the study was to investigate the peripheral lymphocyte expansion in response to non-masked MDNA223, and masked MDNA223 i.e., MDNA113A and MDNA113B .B. Methods

[0459] Balb / c were used for the study. The animals were dosed per Table 23.Table 23: Study design for PD study in mice• Animals were acclimated for 3 days prior to start of the study.• After acclimation, animals were closed per table above.• Samples were collected and sent for CBC analysis.C. Results

[0460] MASK-ITs MDNA113A and MDNA113B demonstrated attenuated peripheral lymphocyte expansion compared to non-masked MDNA223. The masked response could however be rescued on increasing the dose of the construct. While MDNA113A exhibited a similar response to non-masked MDNA223 at a 5-fold higher dose i.e., 11.7 mg / kg, MDNA113B displayed at a 10-fold higher dose which is 24 mg / kg. However, even at 24 mg / kg, the lymphocyte expansion was significantly (p=0.02) lower than non-masked MDNA223 (Figure 47).D. Conclusion

[0461] MASK-ITs show attenuated lymphocyte expansion in periphery compared to non-masked MDNA223 providing strong evidence for reduced systemic toxicity. The data was consistent with intent of construct design.Example 11 : MTD Study to measure maximum tolerated dose for MDNA113A and MDNA113BA. Objective

[0462] The study is being conducted to investigate the tolerability of MDNA113B compared to MDNA113A and non-masked version, MDNA223.B. Methods

[0463] C57BI / 6 mice were used for the study. The study design is shown in Table 24.Table 24: MTD study design of MDNA113A and MDNA113BC. Results

[0464] All doses were well tolerated by mice. The body weight measurements are presented in Figure 48. A steep body weight loss is observed with MDNA223 (30 mg / kg once weekly) group and with MDNA113A (8 mg / kg twice weekly) group. However, the mice recovered in the following days of the study. No body weight loss was observed in any of the MDNA113B groups.D. Conclusion

[0465] All tested doses were well tolerated, and no deaths were observed in any groups. Hence, MTD was not reached.Example 12: IL-2 Activity pre-and post MMP-9 Digestion (Non-cleavable MASK-ITs)A. Objective

[0466] The objective of the study was to investigate the restoration of IL-2 activity, if any, on proteolytic activation of MASK-ITs, cleavable and non-cleavable versions.B. Methods

[0467] The constructs were subjected to mock (absence of MMP9) or MMP9 digestion at 5 ug / mL for 1 hr at 37°C and processed for assessment of IL-2 activity in the Jurkat I L-2by reporter assay.C. Results

[0468] The data presented in Figure 49 shows that the cleavable version of MASK-IT, MDNA113 restores its IL-2 activity post proteolytic activation in presence of MMP9. The non-cleavable versions of both MDNA113A and MDNA113B do not show the restoration of IL-2 activity, as anticipated. The non-cleavable versions lack the PSL site for cleavage and hence, unmasking of MDNA223.

[0469] The EC50S are summarized in Table 25 and the evidence of presence or absence of cleavage is shown in Figure 50.Table 25: EC50 of MASK-ITs in presence or absence of MMP9D. Conclusion

[0470] The non-cleavable versions were not activated post proteolytic cleavage by MMP9.Example 13: Cell receptor binding assay to validate the affinity of MDNA113 to IL-13Ra2 expressing cells in vitroA. Objective

[0471] The study was conducted with A375 human melanoma cells (positive for IL-13Ra2) and A549 human lung epithelial cells (negative for IL-13Ra2) to evaluate the binding of MDNA113 to IL-13Rcc2 expressing cells for the presence of MDNA213 (IL-13 superkine).B. Methods

[0472] Constructs:1 . Control (tested on A549 cells only): Fc-MDNA213 At 1800nM and 600nM only2. Test articles (tested on A375 cells):(i) MDNA223(ii) Fc-MDNA213(iii) MDNA113A(iv) MDNA113BAll titrated with 3-fold, 8-pt curve starting at 1800nM.• Cells at 70-80% confluency will be washed with 2 mL of DPBS• 1 mL of corning cell stripper will be added.• Cells will be incubated at 37°C for 20 minutes to detach.• Cells will be gently reverse pipetted, and well washed with 2 mL of culture media.• Cells will be transferred to 50mL tube and centrifuged at 400G for 10 minutes• Cells will be resuspended in 4mL culture media, and an aliquot will be taken for counting on hemocytometer.• Cells will be seeded at 100K cells (in 1 OOp L) into a 96-well u-bottom plate.• Cells will be centrifuged at 400G for 3 minutes, supernatant to be aspirated.• Cells will be resuspended in 100 pL of constructs (in culture media).• Cells will be incubated at 4°C for 30 minutes.

[0473] Staining• Cells will be centrifuged at 400G for 3 minutes, supernatant to be aspirated.• Cells will be resuspended in 200pL of PBS• Cells will be centrifuged at 400G for 3 minutes, supernatant to be aspirated.• Cells will be washed 2X more times with 200pL of PBS.• Cells will be resuspended in 10Op L of anti-Fc PE (Abeam #AB98596) staining solution.• Cells will be incubated at 4°C for 30 minutes.• 100 pL MACS buffer will be added to each well.• Plate to be centrifuged at -400 x g for 3 minutes.• Cells to be washed 2X more times with 200pL MACS buffer.• Cells will be resuspended in 200 pL 0.5%PFA in MACS / PBS buffer.• Unstained cells will be used for negative population.• Cells to be acquired on Flow Cytometer.

[0474] Flow Analysis• An anti-Fc PE histogram to be made and Mean Fluorescent Intensity (MFI) to be observed for each sample.

[0475] Relative Mean Fluorescent Intensity to be calculated for each sample relative to untreated (no construct) control.C. Results

[0476] The data from the study is shown in Figure 51. Fc-MDNA213 binds strongly to the IL-13Ra2 expressed on A375 cells while no binding is observed on A549. MASK-ITs, MDNA113A and MDNA113B tested on A375 cells showed similar binding pattern as MDNA213 albeit slightly reduced. MDNA223 shows non-specific binding at the highest tested dose.D. Conclusion

[0477] MDNA113A and MDNA113B show dose dependent binding to IL-13Ra2 expressed on A375 cells that was moderately reduced compared to Fc-MDNA213.Example 14: MC-38 Tumor Growth inhibition study to establish efficacy of MDNA113A and MDNA113BA. Objective

[0478] The objective of the study was to establish in vivo efficacy of MDNA113A and MDNA113B.B. Methods

[0479] Eighty-eight C57BI / 6 mice were used for the study. The study design is shown in Table 26.Table 26: Study design for MC38 TGI study (IP administration)C. Results

[0480] The data in Figure 52 shows that MDNA223 treatment is superior to monotherapy with MDNA11 or Anti-mPD1 and their co-administration. The data is consistent with our previous observation in CT26 colon model and B16F10 melanoma model.

[0481] The data for groups treated with cleavable and non-cleavable MDNA113A is shown in Figure 53. At a dose of 18 mg / kg, MDNA113A showed superior efficacy compared to DNA113ANC.

[0482] The data for groups treated with cleavable and non-cleavable MDNA113B is shown in Figure 54. At a dose of 37 mg / kg, MDNA113B showed superior efficacy compared to DNA113ANC. This provides strong evidence that MDNA113B is activated at tumor site releasing MDNA223 from masking by ecCD122 and MDNA213.D. Conclusion

[0483] Both MDNA113A and MDNA113B are proteolytically activated at the tumor site and exhibit efficacy similar to non-masked MDNA223.Example 15: Adjuvant effect of MDNA223 and Neo-adjuvant Effect of MDNA113 and non-maskedMDNA223 in 4T1.2 orthotopic breast tumor modelA. Objective

[0484] The objective of the study was to investigate the neo-adjuvant or adjuvant effect of MDNA223 and MDNA113 in the 4T1 .2 breast orthotopic tumor model.B. Methods

[0485] Balb / c mice were used for the study. The study design is shown in Table 27.Table 27: Study design for adjuvant / neo-adjuvant effect of MDNA113 in 4T1.2 orthotopic breast tumor model.C. Results

[0486] Both MDNA223 and MDNA113 provided significant survival benefit when used as neo-adjuvant as shown in Figure 55. The deaths observed in no treatment control and anti-mPD1 neo-adjuvant treatment were due to distant metastasis which was absent with MDNA113 or MDNA223 neo-adjuvant treatment.

[0487] The survival benefit with adjuvant treatment was however less pronounced for MDNA223 albeit being superior to anti-mPD1 alone treatment (Figure 56).

[0488] MDNA113 cannot be used as adjuvant because it requires proteolytic activation at tumor site.D. Conclusion

[0489] MDNA223 has a profound neo-adjuvant effect compared to when used as an adjuvant treatment and provides significant survival benefit when used as neo-adjuvant in 4T1.2 orthotopic breast tumor model.

[0490] MDNA113 also prolongs survival when used as neo-adjuvant adjuvant in 4T1 .2 orthotopic breast tumor model escaping distant metastasis observed in no treatment control and anti-mPD1 treated mice.Example 16: Binding Affinity assessment of MDNA113 to CD122 (IL-2R )A. Nomenclature

[0491] The following constructs are referred to in the Examples:• MDNA113^25or MDNA113A^25= MDNA223A3 / s125-fPSL2f-MDNA213Un / ParH[SEQ ID NOs: 320 -322]• MDNA113B = MDNA223A3 / s125-fPSL2f-ecCD122: MDNA213 [SEQ ID NOs: 338 -340]• MDNA223A3 / S125= MDNA223 [SEQ ID NOs: 107 -109]B. Objective

[0492] The objective of the study was to evaluate the binding affinity to CD122 (IL-2Rp) of masked (MDNA113) compared to non-masked MDNA223.C. Method

[0493] Binding affinity assessment was performed on BLI Octet The general BLI step conditions used were as follows:• Biosensor check in assay buffer (30 sec).• His-CD25 / His-CD122 (ligand) immobilization (12 min).• Blocking with Superblock (2 min).• Baseline in assay buffer (3 min).• Test construct variant (analyte) titration association (5 min).• Dissociation back into baseline buffer (15 min)D. Results

[0494] Both MD113A and MDNA113B exhibited a reduced binding to CD122 compared to non-masked MDNA223 albeit at varying affinities. MDNA113A showed ~1.6-fold attenuated affinity while MDNA113B demonstrated 163-fold attenuated affinity compared to non-masked MDNA223 (Figure 57).E. Conclusion

[0495] MDNA113B shows significantly attenuated binding affinity in comparison to non-masked MDNA223.Example 17: In Vitro Assessment of MASK-ITs (with MDNA223T3 / C125) to establish reduced IL-2 activityA. Nomenclature

[0496] The following constructs are referred to in the Examples:• MDNA223T3 / C125[SEQ ID NOs: 74-76]• MDNA 113BT3 / C125: [SEQ ID: 365-367]B. Objective

[0497] The objective of the study was to validate the reduced IL-2 activity of MDNA113BT3 / C125 compared to non-masked MDNA223T3 / C125 that is reversible by proteolytic cleavage.C. Method

[0498] The constructs were tested in the Jurkat I L2R(E>y reporter assay (cells that lack the CD25 receptor) in the presence and absence of MMP9.D. Results

[0499] MDNA113BT3 / C125 showed attenuated IL-2 activity compared to the non-masked MDNA223T3 / C125 by ~630-fold . The IL-2 activity was restored upon in vitro MMP9 cleavage (Figure 58).E. Conclusion

[0500] MDNA113BT3 / C125 was activated on proteolytic cleavage.Example 18: Neo-adjuvant administration of MD A11A. Background

[0501] MDNA11 is an albumin-fused ‘beta-enhanced not-alpha' IL-2 superkine that preferentially expands and activates immune effector cells over immune suppressive Tregs. MDNA11 effectively inhibited subcutaneously implanted syngeneic tumors with complete tumor regression and a potent memory response. Mice treated with MDNA11 demonstrated increased tumor infiltration of CD8+ T and NK cells and elevated circulating memory and antigen-specific CD8+ T cells. Given these results, it was investigated whether preconditioning of immune cells against cancer cells by MDNA11 prior to tumor resection could prevent metastasis and prolong survival. This study compared the efficacy of MDNA11 in neo-adjuvant and adjuvant settings in an aggressive orthotopic breast cancer model prone to distant metastasis following tumor removal.B. Objective

[0502] To compare the effect of neoadjuvant and adjuvant treatment with MDNA11 in a syngeneic 4T 1 .2 orthotopic breast cancer model with surgery to completely resect the tumors, and to assess the effect of neoadjuvant and adjuvant MDNA11 treatment in conferring long-term protection from subsequent tumor challenges.C. Method

[0503] Female Bal b / c mice were implanted with 4T 1 .2 tumor cells in the mammary fat pad and tumors were allowed to grow to a palpable size. Mice were either untreated (control), treated with surgery only to remove tumors, or given a single dose of MDNA11 intraperitoneally either as a neo-adjuvant 4 days prior to surgery or as an adjuvant 2 days following surgery. Surviving mice from all treatment cohorts were rechallenged with 4T 1 .2 cells in the right hind leg at study day 65 and left hind at study day 98. Throughout the rechallenge phase of the study, survival, tumor volumes and signs of metastasis at necropsy were measured.

[0504] Data collected included survival rate, necropsy at endpoint to record signs of metastasis, and multiplex immunofluorescence (MIF) analysis for immune cell infiltration on excised tumors.D. Results

[0505] As shown in Table 28 and Figures 59-62, all control mice that had tumors resected but received no treatment died by study day 54 with gross metastasis in multiple organs. In the adjuvant group where mice received a single dose of MDNA11 following tumor resection, 3 of 8 (37.5%) survived to study end (study day 134). Of the 5 mice in the adjuvant group that died while on study, 4 had distant metastasis. In contrast, of the 8 mice that were administered a single dose of MDNA11 prior to tumor resection, 7 (87.5%) survived to study day 134 with no observable clinical symptoms. The single mouse that was found dead had no metastasis. Remaining mice in the neo-adjuvant (n = 7) and adjuvant (n = 3) were re-challenged at day 65 by implanting 4T 1 .2 cells subcutaneously in their flanks. A second re-challenge was performed on study day 98. Mice did not receive any additional treatment and on both re-challenge occasions there was no tumor growth ormetastasis whereas control naive mice that were also implanted with 4T 1 .2 cancer cells showed robust tumor growth at the implanted site.Table 28: Summary of Necropsy FindingsE. Conclusion

[0506] A single dose of MDNA11 in a neo-adjuvant setting conferred immediate and long-term protection from metastatic disease in the 4T 1 .2 orthotopic breast cancer model.***

[0507] 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 and publications 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.

[0508] 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.

[0509] 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.

[0510] 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

WHAT IS CLAIMED IS:1 . An IL-2 cytokine fusion protein comprising: a) an IL-2 moiety comprising an IL-2 or an IL-2 mutein, optionally an IL-2 mutein fusion; b) a protease sensitive linker (PSL); and c) an IL-2 masking moiety wherein the PSL attaches the IL-2 masking moiety to the IL-2 moiety.

2. The IL-2 cytokine 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 IL-2 masking moiety is capable of binding to IL-13Ra2, and does not bind to IL-13Ra1 .

3. The IL-2 cytokine fusion protein of claim 1 or 2, wherein the masking moiety comprises an IL-13 mutein that has the amino acid sequence of any one of SEQ ID N0s:200-241 .

4. The IL-2 cytokine fusion protein of any one of claims 1 to 3, 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 NQ:200): a) L10H, E15R, R86T, D87G, T88R, R108K, Q111 , b) L10H, E15R, R86T, D87G, T88R, R108K, R111 , c) L10H, R86T, D87G, T88R, and R108K, Q111, or d) L10H, R86T, D87G, T88R, and R108K, R111.

5. The IL-2 cytokine 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:216 or SEQ ID NO:228.

6. The IL-2 cytokine fusion protein of any one of claims 1 to 5, wherein the masking moiety further comprises an extracellular domain of CD122, an extracellular domain of CD132, or an extracellular domain of CD25.

7. The IL-2 cytokine fusion protein of any one of claims 1 to 6, wherein the IL-2 moiety comprises an IL-2 mutein having the amino acid sequence of any one of SEQ ID NOs:5-24 and 105.

8. The IL-2 cytokine fusion protein of claim 7, wherein the IL-2 mutein has the amino acid sequence of SEQ ID N0:5 or SEQ ID N0:9.

9. The IL-2 cytokine fusion protein of claim 8, wherein the IL-2 mutein further comprises a T3A and a C125S amino acid substitution.

10. The IL-2 cytokine fusion protein of any one of claims 1 to 9, wherein the IL-2 moiety further comprises an albumin, an Fc domain, or an antibody attached to the IL-2 or IL-2 mutein.11 . The IL-2 cytokine fusion protein of claim 10, wherein the albumin is a human albumin, optionally wherein the human albumin is recombinant human albumin.

12. The IL-2 cytokine fusion protein of claim 10, wherein the antibody is engineered as “knob-in-hole” (KiH) with mutations in constant region 3 (CH3) in its heavy chains.

13. The IL-2 cytokine fusion protein of claim 12, wherein the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: 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 antibody (KiH); b) a second polypeptide comprising the “hole” heavy chain of the antibody (KiH); and c) a third polypeptide comprising the light chain of the antibody (KiH).

14. The IL-2 cytokine fusion protein of claim 12, wherein the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising the “knob” heavy chain of the 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 antibody (KiH); c) a third polypeptide comprising the light chain of the antibody (KiH).

15. The IL-2 cytokine fusion protein of claim 12, wherein the IL-2 moiety comprises an IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising the “knob” heavy chain of the antibody (KiH); b) a second polypeptide comprising the “hole” heavy chain of the 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 antibody (KiH).16 The IL-2 cytokine fusion protein of any one of claims 12 to 15, wherein the IL-2 masking moiety is attached to the IL-2 or the IL-2 mutein.17 The IL-2 cytokine fusion protein of any one of claims 12 to 16, wherein the IL-2 masking moiety is attached to the antibody heavy chain that is not attached to the IL-2 or the IL-2 mutein.

18. The IL-2 cytokine fusion protein of any one of claims 12 to 17, wherein the antibody (KiH) is anti-PD1 (KiH).

19. The IL-2 cytokine fusion protein of any one of claims 12 to 18, wherein the IL-2 mutein has an amino acid sequence of any one of SEQ ID NOs: 5-24 and 105.

20. The IL-2 cytokine fusion protein of claim 19, wherein the IL-2 mutein further comprises a T3A and a C125S amino acid substitution.21 . The IL-2 cytokine fusion protein of any one of claims 1 to 20, wherein the PSL is capable of being cleaved in the tumor microenvironment.

22. The IL-2 cytokine fusion protein of any one of claims 1 to 21 , wherein the PSL has the amino acid sequence of PLGLVVAPLGLVVAPLGLVVA, PLGLWAPLGLWAPLGLWA, GGSGGTPLGLWAGGSGGT, GGSGGTPAGLIGGGSGGT, GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT, or GGSGGTHSSKLQGGSGGT.

23. An IL-2 cytokine fusion protein comprising sequences selected from the group consisting of:(a) SEQ ID NOs: 302, 303, and 304;(b) SEQ ID NOs: 299, 300, and 301 ;(c) SEQ ID NOs: 305, 306, and 307;(d) SEQ ID NOs: 308, 309, and 310;(e) SEQ ID NOs: 311 , 312, and 313;(f) SEQ ID NOs: 314, 315, and 316;(g) SEQ ID NOs: 317, 318, and 319;(h) SEQ ID NOs: 320, 321 , and 322;(i) SEQ ID NO: 323;(j) SEQ ID NOs:329, 330, and 331 ;(k) SEQ ID NOs:332, 333, and 334;(l) SEQ ID NOs:335, 336, and 337;(m) SEQ ID NOs:338, 339, and 340;(n) SEQ ID NOs:353, 354, and 355;(o) SEQ ID NOs:359, 360, and 361 ;(p) SEQ ID NOs:362, 363, and 364;(q) SEQ ID NOs:365, 366, and 367;(r) SEQ ID NOs:325, 326, and 327;(s) SEQ ID NOs:353, 354, and 355 and(t) SEQ ID NO: 324.

24. A pharmaceutical composition comprising the IL-2 cytokine fusion protein of any one of claims 1 to 23, and a pharmaceutically acceptable carrier.

25. A nucleic acid composition that encodes the IL-2 cytokine fusion protein of any one of claims 1 to 23.

26. An expression vector composition comprising the nucleic acid composition of claim 25.

26. A method of making an IL-2 cytokine fusion protein of any one of claims 1 to 23, comprising culturing the nucleic acid composition of claim 25 or the expression vector composition of claim 26 under conditions wherein the IL-2 cytokine fusion protein is expressed, and recovering the IL-2 cytokine fusion protein.

27. A method of treating an I L-13Ra2 expressing cancer in a subject in need thereof comprising administering to the subject the IL-2 cytokine fusion protein of any one of claims 1 to 23, or the pharmaceutical composition of claim 24.

28. The method of claim 27, wherein the IL-2 cytokine fusion protein comprises one or more of SEQ ID NOs:1-367.

29. A method of treating a cancer in a subject in need thereof comprising administering to the subject an IL-2 cytokine fusion protein comprising: a) an IL-2 mutein; and b) an albumin or an Fc domain or an antibody; wherein the IL-2 mutein has an amino acid sequence of any one of SEQ ID NOs: 5-24 and 105 and optionally further comprises a T3A and / or a C125S amino acid substitution.

30. The method of claim 29, wherein the IL-2 cytokine fusion protein comprises one or more of SEQ ID NOs:1-367.31 . The method of claim 29 or 30, wherein the IL-2 cytokine fusion protein has the amino acid sequence of SEQ ID NO:53.

32. The method of any one of claims 27-31 , wherein the IL-2 cytokine fusion protein is administered as a neo-adjuvant prior to a surgery to remove a tumor.

33. The method of claim 32, wherein the IL-2 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 IL-2 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.

34. The method of any one of claims 27-31 , wherein the IL-2 cytokine fusion protein is administered as an adjuvant after a surgery to remove a tumor.

35. The method of claim 34, wherein the IL-2 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 IL-2 cytokine fusion protein is administered starting at least two weeks after the surgery.

36. The method of any one of claims 32-35, wherein the IL-2 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.

37. The method of claim 36, wherein the IL-2 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 IL-2 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 IL-2 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 IL-2 cytokine fusion protein is administered starting at least to two weeks after the surgery.

38. The method of any one of claims 27-37, 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.

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