Il-2 fusion proteins

Abstract

Provided herein are novel IL-2 fusion proteins comprising an IL-2 moiety linked to an IL-2 masking moiety by a protease sensitive linker (PSL), the IL-2 masking moiety comprising 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. In some embodiments, the IL-2 fusion protein comprises an IL-13 mutein or an IL-13Ra2 antibody or an antigen binding fragment thereof, the IL-13 mutein or the IL-13Ra2 antibody or the antigen binding fragment thereof is capable of binding to IL-13R [alpha] 2, but not to IL-13R [alpha] 1, and the protease-sensitive linker can be cleaved by a protease in the tumor microenvironment. Such IL-2 fusion proteins are useful, for example, in the treatment of cancers that express IL-13R [alpha] 2.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Patent Application No. 63 / 510,580, filed June 27, 2023; U.S. Patent Application No. 63 / 594,694, filed October 31, 2023; U.S. Patent Application No. 63 / 595,044, filed November 1, 2023; U.S. Patent Application No. 63 / 595,271, filed November 1, 2023; and U.S. Patent Application No. 63 / 575,960, filed April 8, 2024, all of which are expressly incorporated herein by reference in their entirety. Background Technology

[0003] Interleukin-2 (IL-2) is a pluripotent cytokine primarily produced by activated CD4+ T cells and plays a crucial role in generating normal immune responses. IL-2 promotes the proliferation and expansion of activated T lymphocytes, promotes B cell growth, and activates monocytes and natural killer cells. It is due to these activities that IL-2 has been tested and is used as an approved cancer treatment (aldesleukin, Proleukin®). In eukaryotic cells, human IL-2 is synthesized as a 153-amino acid precursor polypeptide, from which 20 amino acids are removed to produce mature, secreted IL-2 (Taniguchi 1983). Recombinant human IL-2 has been produced in *E. coli* (Rosenberg 1984), insect cells (Smith 1985), and mammalian COS cells (Taniguchi 1983).

[0004] Interleukin-2 (IL-2) is a type I cytokine with a four-alpha helix bundle, initially identified as a T-cell growth factor (Morgan et al., Science). Science IL-2 promotes the differentiation of CD4+ T helper cells (Zhu et al., Annual Review of Immunology 193: 1007 (1976)), but it was subsequently shown to have a wide range of effects. IL-2 promotes the differentiation of CD4+ T helper cells (Zhu et al., Annual Review of Immunology 193: 1007 (1976)). Annual review of immunology )》28: 445 (2010); Liao et al., Nature Immunology ( Nat Immunol )》 9: 1288 (2008); and Liao et al., Nature Immunology 12: 551 (2011)) and regulation of T (Treg) cell development (Cheng et al., Immunological Review) ... Immunol Rev(2011)》 241: 63 (Liao et al., Immunology) , inducing natural killer cells and cytotoxic CD8+ T cells (Liao et al., Immunology) Immunity ( )》 38: 13 (2013)), and mediates activation-induced cell death (AICD) (Lenardo et al., Nature ( ) 38: 13 (2013)). Nature )》 353: 858 (1991)).

[0005] IL-2 exerts its function by interacting with three different receptors: interleukin-2 receptor α (IL-2Rα; CD25), interleukin-2 receptor β (IL-2Rβ; CD122), and interleukin-2 receptor γ (IL-2Rγ; CD132; shared γ chain). The first receptor to be identified is IL-2R. It is a 55 kDa polypeptide (p55) that appears during T cell activation and was initially called Tac (representing T activation) antigen. IL-2R With approximately 10 -8 M of K d It binds to IL-2 and is also known as the "high-affinity" IL-2 receptor. IL-2 binds to IL-2 receptors that only express IL-2R. Binding of IL-2 to cells does not elicit any detectable biological response. In most cases, IL-2 functions through three distinct receptors: IL-2Rα, IL-2Rβ, and IL-2Rγ. Most cells, such as resting T cells, are unresponsive to IL-2 because they express only IL-2Rβ and IL-2Rγ, which have low affinity for IL-2. Upon stimulation, resting T cells express the relatively high-affinity IL-2 receptor IL-2Rα. Binding of IL-2 to IL-2Rα leads to this receptor sequentially binding to IL-2Rβ and IL-2Rγ, thereby activating T cells. An IL-2 “superkine” has previously been developed, which enhances its effects due to its increased binding affinity to IL-2Rβ (Levin et al., Nature 484: 529 (2012)).

[0006] Despite the immense potential of IL-2 in cancer therapy, its clinical application remains relatively limited, partly due to the severe toxicities associated with high doses. Because of IL-2's short serum half-life of several minutes, high doses are typically required to achieve optimal immunomodulatory effects. However, such high doses inevitably lead to severe toxicities, including vascular leakage syndrome (VLS), pulmonary edema, hypotension, and cardiotoxicity. Therefore, novel and effective IL-2 cancer therapies are still needed to minimize IL-2-related toxicities. Summary of the Invention

[0007] This article provides a novel IL-2 fusion protein comprising an IL-2 moiety linked to at least one IL-2 masking portion via a protease-sensitive linker (PSL), said at least one IL-2 masking portion comprising an IL-13 mutant protein, an IL-13Ra2-binding mutant protein, or an IL-13Ra2 antibody or an antigen-binding fragment thereof. In embodiments, the IL-2 fusion protein comprises at least one IL-13 mutant protein, an IL-13Ra2-binding mutant protein, or an IL-13Ra2 antibody or an antigen-binding fragment thereof, said at least one IL-13 mutant protein, IL-13Ra2-binding mutant protein, or IL-13Ra2 antibody or an antigen-binding fragment thereof capable of binding to IL-13Rα2 but not to IL-13Rα1, and said protease-sensitive linker can be cleaved by proteases in the tumor microenvironment. When the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment is linked to IL-2 via PSL, the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment of the IL-2 fusion protein described herein binds to the IL-2 moiety and masks its activity. Furthermore, the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment allows the subject IL-2 fusion protein to bind to tumors expressing IL-13Rα2. Once localized to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment and “demasking” the IL-2 moiety. The demasked IL-2 moiety can then provide antitumor activity at the localized tumor site. Therefore, in the embodiments, the subject IL-2 fusion protein provided herein advantageously exhibits reduced IL-2-related toxicity while allowing targeting activity against tumors expressing IL-13Rα2. These IL-2 fusion proteins can be used specifically to treat cancers that express IL-13Rα2.

[0008] On one hand, this article provides an IL-2 cytokine fusion protein comprising: a) an IL-2 moiety containing IL-2 or an IL-2 mutant protein, optionally an IL-2 mutant protein fusion; b) at least one protease-sensitive linker (PSL); and c) at least one IL-2 masking moiety containing an IL-13 mutant protein, an IL-13Ra2-binding mutant protein, or an IL-13Ra2 antibody or an antigen-binding fragment thereof, wherein the PSL links the IL-2 masking moiety to the IL-2 moiety, optionally wherein the IL-2 masking moiety is capable of binding to IL-13Ra2 but not to IL-13Ra1.

[0009] In some embodiments, the masking portion comprises an IL-13 mutant protein having the amino acid sequence of any of SEQ ID NO: 200-241. In some embodiments, the masking portion comprises at least one IL-13 mutant protein having the following amino acid substitutions compared to wild-type human IL-13 (SEQ ID NO: 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 portion comprises at least one IL-13 mutant protein having the amino acid sequence of SEQ ID NO: 216 or SEQ ID NO: 228.

[0010] In some embodiments, this document provides a novel IL-2 fusion protein comprising an IL-2 moiety linked to at least one IL-2 masking portion via a protease-sensitive linker (PSL), said at least one IL-2 masking portion comprising an IL-13 mutant protein, an IL-13Ra2-binding mutant protein, or an IL-13Ra2 antibody or an antigen-binding fragment thereof. In embodiments, the IL-2 fusion protein comprises at least one IL-13 mutant protein, an IL-13Ra2-binding mutant protein, or an IL-13Ra2 antibody or an antigen-binding fragment thereof, said at least one IL-13 mutant protein, IL-13Ra2-binding mutant protein, or IL-13Ra2 antibody or an antigen-binding fragment thereof capable of binding to IL-13Rα2 but not to IL-13Rα1, and said protease-sensitive linker can be cleaved by proteases in the tumor microenvironment. When the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment is linked to IL-2 via PSL, the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment of the IL-2 fusion protein described herein binds to the IL-2 moiety and masks its activity. Furthermore, the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment allows the subject IL-2 fusion protein to bind to tumors expressing IL-13Rα2. Once localized to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment and “demasking” the IL-2 moiety. The demasked IL-2 moiety can then provide antitumor activity at the localized tumor site. Therefore, in the embodiments, the subject IL-2 fusion protein provided herein advantageously exhibits reduced IL-2-related toxicity while allowing targeting activity against tumors expressing IL-13Rα2. These IL-2 fusion proteins can be used specifically to treat cancers that express IL-13Rα2.

[0011] On one hand, this article provides an IL-2 cytokine fusion protein comprising: a) an IL-2 moiety containing IL-2 or an IL-2 mutant protein, optionally an IL-2 mutant protein fusion; b) at least one protease-sensitive linker (PSL); and c) at least one IL-2 masking moiety containing an IL-13 mutant protein, an IL-13Ra2-binding mutant protein, or an IL-13Ra2 antibody or an antigen-binding fragment thereof, wherein the PSL links the IL-2 masking moiety to the IL-2 moiety, optionally wherein the IL-2 masking moiety is capable of binding to IL-13Ra2 but not to IL-13Ra1.

[0012] In some embodiments, the masking portion comprises an IL-13 mutant protein having the amino acid sequence of any of SEQ ID NO: 200-241. In some embodiments, the masking portion comprises at least one IL-13 mutant protein having the following amino acid substitutions compared to wild-type human IL-13 (SEQ ID NO: 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 portion comprises at least one IL-13 mutant protein having the amino acid sequence of SEQ ID NO: 216 or SEQ ID NO: 228.

[0013] In some embodiments, the masking portion further includes an extracellular domain of CD122, an extracellular domain of CD132, or an extracellular domain of CD25.

[0014] In some embodiments, the IL-2 portion comprises an IL-2 mutant protein having the amino acid sequence of any one of SEQ ID NO: 5-24 and 105. In some embodiments, the IL-2 mutant protein has the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9. In some embodiments, the IL-2 mutant protein further comprises T3A and C125S amino acid substitutions.

[0015] In some embodiments, the IL-2 portion further comprises an albumin or Fc domain or antibody linked to the IL-2 or an IL-2 mutant protein. In some embodiments, the albumin is human albumin, optionally wherein the human albumin is recombinant human albumin. In some embodiments, the antibody is engineered to have a mutated "knob-in-hole" (KiH) in constant region 3 (CH3) of its heavy chain.

[0016] In some embodiments, the IL-2 portion comprises an IL-2 x anti-PD1 fusion protein, the IL-2 x anti-PD1 fusion protein comprising: a) a first polypeptide comprising a first antibody heavy chain linked to IL-2 or an IL-2 mutant protein, 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.

[0017] In some embodiments, the first PD-1 binding domain and the second PD-1 binding domain have the same amino acid sequence. In some embodiments, the first PD-1 binding domain and the second PD-1 binding domain have different amino acid sequences. In some embodiments, the IL-2 masking portion is linked to the IL-2 or the IL-2 mutant protein. In some embodiments, the IL-2 masking portion is linked to the second Fc domain, and the IL-2 or the IL-2 mutant protein is linked to the first Fc domain.

[0018] In some embodiments, the IL-2 portion comprises an IL-2 x antibody (KiH) fusion protein, the IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising IL-2 or an IL-2 mutant protein or IL-2 fusion protein linked to a second constant region and a third constant region (CH2 and CH3) of the "palm" heavy chain of the antibody (KiH); b) a second polypeptide comprising the "mortar" 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 portion comprises an IL-2 x antibody (KiH) fusion protein, the IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising a "pestle" heavy chain of the antibody (KiH) linked to IL-2 or an IL-2 mutant protein or an IL-2 fusion body; b) a second polypeptide comprising a "mortar" heavy chain of the antibody (KiH); and c) a third polypeptide comprising a light chain of the antibody (KiH).

[0020] In some embodiments, the IL-2 portion comprises an IL-2 x antibody (KiH) fusion protein, the IL-2 x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising a "pestle" heavy chain of the antibody (KiH); b) a second polypeptide comprising a "mortar" heavy chain of the antibody (KiH) linked to IL-2 or an IL-2 mutant protein or an IL-2 mutant protein fusion; and c) a third polypeptide comprising a light chain of the antibody (KiH).

[0021] In some embodiments, the IL-2 masking portion is linked to the IL-2 or the IL-2 mutant protein or IL-2 mutant protein fusion in the IL-2 portion. In some embodiments, the IL-2 masking portion is linked to the antibody (KiH) heavy chain that is not linked to IL-2 or the IL-2 mutant protein or IL-2 mutant protein fusion.

[0022] In some embodiments, the antibody (KiH) is anti-PD1 (KiH).

[0023] In some embodiments, the IL-2 mutant protein has the amino acid sequence of any one of SEQ ID NO: 5-24 and 105. In some embodiments, the IL-2 mutant protein further comprises T3A and C125S amino acid substitutions.

[0024] In some embodiments, the PSL can be cleaved within the tumor microenvironment. In some embodiments, the PSL has the amino acid sequence PLGLVVAPLGLVVAPLGLVVA, PLGLWAPLGLWAPLGLWA, GGSGGTPLGLWAGGSGGT, GGSGGTPAGLIGGGSGGT, GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT, or GGSGGTHSSK LQGGSGGT.

[0025] On the other hand, this article provides an IL-2 cytokine fusion protein comprising sequences selected from the group consisting of: (a) SEQ ID NO: 302, 303, and 304; (b) SEQ ID NO: 299, 300, and 301; (c) SEQ ID NO: 305, 306, and 307; (d) SEQ ID NO: 308, 309, and 310; (e) SEQ ID NO: 311, 312, and 313; (f) SEQ ID NO: 314, 315, and 316; (g) SEQ ID NO: 317, 318, and 319; (h) SEQ ID NO: 320, 321, and 322; (i) SEQ ID NO: 323; (j) SEQ ID NO: 329, 330, and 331; (k) SEQ ID NO: 332, 333, and 334; (l) SEQ ID NO: 335, 336 and 337; (m) SEQ ID NO: 338, 339 and 340; (n) SEQ ID NO: 353, 354 and 355; (o) SEQ ID NO: 359, 360 and 361; (p) SEQ ID NO: 362, 363 and 364; (q) SEQ ID NO: 365, 366 and 367; (r) SEQ ID NO: 325, 326 and 327; (s) SEQ ID NO: 353, 354 and 355; and (t) SEQ ID NO: 324.

[0026] On the other hand, this article provides a pharmaceutical composition comprising one of the IL-2 cytokine fusion proteins disclosed herein and a pharmaceutically acceptable carrier.

[0027] This document also provides a nucleic acid composition and an expression vector composition comprising a nucleic acid encoding a subject IL-2 cytokine fusion protein; a host cell comprising the nucleic acid composition or the expression vector composition; and a method for preparing the IL-2 cytokine fusion protein using the host cell.

[0028] On the other hand, this article provides a method for treating cancers expressing IL-13Ra2 in subjects of need, the method comprising administering the IL-2 cytokine fusion protein to the subject.

[0029] On the other hand, this article provides a method for treating cancer in a subject in need, the method comprising administering an IL-2 cytokine fusion protein to the subject, the IL-2 cytokine fusion protein comprising:

[0030] a) IL-2 mutant protein; and

[0031] b) Albumin or Fc domain or antibody;

[0032] The IL-2 mutant protein described herein has the amino acid sequence of any one of SEQ ID NO: 5-24 and 105 and optionally further comprises T3A and / or C125S amino acid substitutions.

[0033] On the other hand, the IL-2 cytokine fusion protein comprises one or more of SEQ ID NO: 1-367.

[0034] On the other hand, the IL-2 cytokine fusion protein has the amino acid sequence of SEQ ID NO: 53.

[0035] On the other hand, the IL-2 cytokine fusion protein is administered as a novel adjuvant prior to surgical procedures used to remove tumors.

[0036] On the other hand, the IL-2 cytokine fusion protein is administered up to one week before 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 before the surgery, or the IL-2 cytokine fusion protein is administered up to 9 weeks before 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 before the surgery.

[0037] On the other hand, the IL-2 cytokine fusion protein is administered as an adjuvant after surgery to remove tumors.

[0038] On the other hand, the IL-2 cytokine fusion protein is administered at most one week after the surgery, optionally at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 1 day or less after the surgery, or the IL-2 cytokine fusion protein is administered at least two weeks after the surgery.

[0039] On the other hand, the IL-2 cytokine fusion protein is administered as a novel adjuvant before and after surgery for tumor removal.

[0040] On the other hand, the IL-2 cytokine fusion protein is administered at most one week before the surgery, optionally at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 1 day, or less than 1 day before the surgery, and wherein the IL-2 cytokine fusion protein is administered at most one week after the surgery, optionally at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 1 day, or less than 1 day after the surgery, or wherein the IL-2 cytokine fusion protein is administered at most 9 weeks before the surgery, optionally at most 8 weeks, at most 7 weeks, at most 6 weeks, at most 5 weeks, at most 4 weeks, at most 3 weeks, at most 2 weeks, at most 1 week, or less than 1 week before the surgery, and wherein the IL-2 cytokine fusion protein is administered starting at least two weeks after the surgery.

[0041] On the other hand, the cancers mentioned are sarcomas, carcinomas, head and neck cancers, glioblastomas, bladder cancers, oral cancers, mesotheliomas, pancreatic cancers, liver cancers, colorectal cancers, lung cancers, skin cancers, lymphomas, gastrointestinal cancers, prostate cancers, ovarian cancers, breast cancers, basaloid breast tumors, endometrial cancers, multiple myelomas, melanomas, lymphomas, lung cancers, small cell lung cancers, kidney cancers, stomach cancers, brain cancers, or CNS tumors. Attached Figure Description

[0042] Figure 1A A graph summarizing the JurkatIL2Rβγ reporter gene analysis of the 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") is provided. The assays were performed independently on two plates. Plots and curve fittings for each construct are presented on the respective plates.

[0043] Figure 1B The constructs MDNA11 (SEQ ID NO: 53) and MDNA132-PSL-MDNA11 are provided. T3 / C125 A graph of the Jurkat IL2Rβγ reporter gene analysis (SEQ ID NO: 294). The assays were performed independently on two plates. Plots and curve fittings for each construct are presented on the respective plates.

[0044] Figure 2 Sensing maps of BLI performed on the indicated constructs are provided, with human CD122 as the ligand and the constructs (MDNA109-albumin (SEQ ID NO: 56) and MDNA132.15-PSL-MDNA109-albumin (SEQ ID NO: 298)) as the analytes.

[0045] Figure 3 Sensing maps of BLI performed on indicated constructs with receptors are provided, with human IL13Ra2 as the ligand and constructs (Fc-MDNA132.15 (SEQ ID NO: 270) and MDNA132.15-PSL-MDNA109-albumin (SEQ ID NO: 298)) as the analytes.

[0046] Figure 4 A research summary is provided, which shows that proteolytic activation of MMP9 restores IL-2 activity of MDNA213-PSL-MDNA109-albumin (SEQ ID NO: 298).

[0047] Figure 5 A research summary is provided, which shows that mDNA223 T3AC125S -fPSL2f- MDNA213 (SEQ ID NO:299-301) exhibited reduced IL-2 activity and no change in PD-1 / PDL-1 blockade.

[0048] Figure 6 A research summary is provided, showing that proteolytic activation of MMP9 restores mDNA223. T3AC125S IL-2 activity of -fPSL2f-MDNA213 (SEQ ID NO: 299-301).

[0049] Figure 7 A research summary is provided, which shows that mDNA223 T3AC125S -fPSAf- MDNA213: anti-mPD1(H) (SEQ ID NO: 302-304) exhibited a 12-fold reduction in IL-2-mediated signal transduction efficacy (i.e., masking activity) and no change in PD-1 / PDL-1 blockade.

[0050] Figure 8 A research summary is provided, which shows that mDNA223 T3AC125S -fPSL3f- MDNA213: anti-mPD1(H) (SEQ ID NO: 305-307) exhibited an 11-fold reduction in IL-2-mediated signal transduction efficacy (i.e., masking activity) and no change in PD-1 / PDL-1 blockade.

[0051] Figure 9 A research summary is provided, which shows that mDNA223 T3AC125S-fPSL2fPSL3f-MDNA213: anti-mPD1(H) (SEQ ID NO: 308-310) exhibited a 10-fold reduction in IL-2-mediated signal transduction efficacy (i.e., masking activity) and no change in PD-1 / PDL-1 blockade.

[0052] Figure 10 A research summary is provided, which shows that mDNA223 T3AC125S -GS- MDNA213: anti-mPD1(H) (SEQ ID NO: 311-313) exhibited an 11-fold reduction in IL-2-mediated signal transduction efficacy (i.e., masking activity) and no change in PD-1 / PDL-1 blockade.

[0053] Figure 11 A research summary is provided, which shows that mDNA223 T3AC125S Anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NO: 314-316) exhibited a 3-fold reduction in IL-2-mediated signal transduction efficacy (i.e., masking activity) and no change in PD-1 / PDL-1 blockade.

[0054] Figure 12 A research summary is provided, which shows anti-mPD1(K)-fPSL2f-MDNA213:MDNA223 T3AC125S (SEQ ID NO: 317-319) exhibited a 2-fold reduction in IL-2-mediated signal transduction efficacy (i.e., masking activity) and no change in PD-1 / PDL-1 blockade.

[0055] Figure 13 A research summary is provided, which shows that mDNA223 T3AC125S -fPSL2f-MDNA213: Anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NO: 320-322) exhibited a 39-fold reduction in IL-2-mediated signal transduction efficacy (i.e., masking activity) and no change in PD-1 / PDL-1 blockade.

[0056] Figure 14 A research summary is provided, which shows that mDNA223 T3AC125S Anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NO: 320-322) is sensitive to MMP9 cleavage in vitro.

[0057] Figure 15 A research summary is provided, which shows that mDNA223T3AC125S Anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NO: 320-322) is activated upon MMP9 cleavage, as observed in IL-2R reporter gene assays.

[0058] Figure 16 A research summary is provided, which shows that mDNA213-fPSL2f-mDNA11 T3AC125S (SEQ ID NO:323) exhibits approximately 10-fold reduced IL-2-mediated signal transduction efficacy (i.e., masking activity).

[0059] Figure 17 A research summary is provided, which shows that mDNA213-fPSL2f-mDNA11 T3AC125S (SEQ ID NO:323) showed sensitivity to MMP9 cleavage in vitro.

[0060] Figure 18 A research summary is provided, which shows that mDNA213-fPSL2f-mDNA11 T3AC125S (SEQ ID NO:323) is activated upon MMP9 cleavage, as observed in IL-2 reporter gene assays.

[0061] Figure 19 SDS-PAGE analysis is shown. Images of reduced SDS-PAGE gels of various BiSKITs are presented. The positions of molecular weight markers are shown on the left.

[0062] Figure 20 SDS-PAGE analysis is shown. Images of reduced SDS-PAGE gels of various BiSKITs are presented. The positions of molecular weight markers are shown on the left.

[0063] Figure 21A and 21B A graph of the Jurkat IL2Rβγ reporter gene analysis is shown. Graphs and curve fits for each construct are presented on each plate. Data are presented as averages. + SEM presentation.

[0064] Figure 22 Graphs of CTLL-2 proliferation analysis of the constructs as indicated are shown. Graphs and curve fits for each construct are presented on each plate. Data are presented as mean + SEM.

[0065] Figure 23Human PBMC proliferation assays are shown: results of stimulating three PBMC donors with different concentrations of the indicated constructs for 48 hours. Proliferation was analyzed by BrDU incorporation. Data are presented as mean + SEM.

[0066] Figure 24 Human pSTAT5 assays are shown: results of stimulating three PBMC donors with different concentrations of the indicated constructs for 15 minutes. pSTAT5 induction in primordial CD8+ T cells, NK cells, and Treg cells is also shown.

[0067] Figure 25 The EC50 results for masked and unmasked mDNA223 were shown in PBMC samples from three human donors for primordial CD8+ T cells and Tregs. 50 Data assembly.

[0068] Figure 26 The frequencies of pSTAT5 expression in cells, CD8+ T cells, NK cells, and Tregs from three donors are shown for various constructs. Human PBMCs were stimulated with masked and unmasked mDNA11 for 15 minutes, and pSTAT5 was analyzed by flow cytometry. Data are presented in single-sample format.

[0069] Figure 27 The EC50 of masked and unmasked mDNA11 was shown in PBMC samples from three human donors for primordial CD8+ T cells and Tregs. 50 Data assembly.

[0070] Figure 28 This shows the primary CD8+ T cells and T cells. reg EC 50 Ratio. Primary CD8 T cells to T cells reg EC 50 The ratio is the average of the individual ratios of the different PMBC samples used.

[0071] Figure 29 The cell receptor (IL13Ra2) is shown in relation to Fc-MDNA213 (SEQ ID NO: 270-271) and MDNA223. A3 / S125 -fPSL2f-MDNA213 Lin / ParH Binding kinetics of (SEQ ID NO: 320-322). EMT6 / IL13Ra2 cells expressing IL-13Ra2 underwent increased concentrations of all constructs and binding was detected by anti-Fc antibody.

[0072] Figure 30 The use of mDNA223 was shown. A3 / S125CBC analysis on day 3 post-treatment with both masked and unmasked versions: complete blood count. Mice were treated with the construct (IP) as instructed. Blood was collected 72 hours post-treatment for CBC analysis. Data are presented as mean values. + SEM presentation.

[0073] Figure 31 The use of MDNA11 was shown A3 / S125 CBC analysis on day 3 post-treatment with both masked and unmasked versions: complete blood count. Mice were treated with the construct (IP) as instructed. Blood was collected 72 hours post-treatment for CBC analysis. Data are presented as mean values. + SEM presentation.

[0074] Figure 32 This study demonstrates the use of the MC-38 colon cancer model: C57Bl / 6 mice were treated with IT at 15 µg / tumor of the indicated construct twice weekly for a total of four doses. Data are presented as mean and average tumor measurements on different days. + SEM presentation.

[0075] Figure 33 This study demonstrates the use of an EMT6 / IL13Rα2 breast tumor model: Balb / c mice were treated with the indicated construct at an equimolar dose on day 4 post-cell implantation, following a once-weekly dosing regimen, for a total of two administrations. Data are presented as mean and average tumor measurements over different days. + SEM presentation.

[0076] Figure 34 This study demonstrates the use of the MC-38 colon cancer model: C57Bl / 6 mice were treated with the indicated construct at an equimolar dose on a once-weekly dosing regimen for a total of two administrations. Data are presented as mean body weight (left) and tumor measurements (right) on different days, along with the mean. + SEM presentation.

[0077] Figure 35 The body weights of the animals in the once-weekly dosing regimen are shown. The mean body weight measurement for each group is indicated. Data are presented in... + SD rendering.

[0078] Figure 36 The body weights of the animals in the twice-weekly dosing regimen are shown. The mean body weight measurement for each group is indicated. Data are presented in... + SD rendering.

[0079] Figure 37 This demonstrates the use of MDNA113A A3 / S125Survival curves of animals in the MTD study. Mice were treated according to the indicated dose and dosing schedule. The Kaplan-Meier graph shows the overall survival rate for each group. Mice that survived at the end of the study were examined on day 18. N = 3 mice / group.

[0080] Figure 38 shows a graph of the Jurkat IL2Rβγ reporter gene analysis. Graphs and curve fits for each construct are presented on each plate. Data are presented as averages. + SEM presentation.

[0081] Figure 39 shows a plot of the HEK Blue IL-2 reporter gene analysis. OD650nm data are plotted as a function of cytokine concentration (nM) on a semi-logarithmic plot. The four-parameter logistic curve fit is presented as a solid line. Error bars represent the standard error of the mean values ​​of the replicate wells.

[0082] Figure 40 The graphs of mouse PD-1 / PD-L1 blockade assays are shown. Graphs and curve fittings for each construct are presented on each plate.

[0083] Figure 41 The SDS-PAGE analysis is shown. Images of the reduced SDS-PAGE gels for each mask-it are displayed. The positions of molecular weight markers are shown on the left.

[0084] Figure 42A Graphs of Jurkat IL2Rβγ or HEK Blue IL-2 reporter gene analyses are shown. Graphs and curve fits for each construct are presented on each plate. Data are presented as averages. + SEM presentation.

[0085] Figure 42B and 42C Graphs of Jurkat IL2Rβγ or HEK Blue IL-2 reporter gene analyses are shown. Graphs and curve fits for each construct are presented on each plate. Data are presented as averages. + SEM presentation.

[0086] Figure 43 Representative sensor maps showing unlabeled and labeled MDNA223 and MDNA113 binding to mouse CD122 are shown.

[0087] Figure 44 In vivo imaging data at the indicated time points are shown. As shown in the figure, tumors A549 and A375 are located in the left and right flanks, respectively.

[0088] Figure 45Results of the MC-38 TGI study are shown: mice were treated as instructed. Data are presented as mean values. + SEM.

[0089] Figure 46 Results of the MC-38 TGI study are shown: mice were treated as instructed. Data are presented as mean values. + SEM.

[0090] Figure 47 PD response is shown. Mice were treated with the construct as indicated. Samples were collected on day 3 for CBC analysis. Data are presented as mean. + SEM.

[0091] Figure 48 Weights are shown: Animals in different groups were treated as indicated with once-weekly or twice-weekly dosing regimens. Weight was measured twice weekly during the study period.

[0092] Figure 49 The Jurkat IL-2 reporter gene assay is shown. Data are presented as averages. + SEM presentation.

[0093] Figure 50 An SDS-PAGE for detecting MMP9 cuts is shown.

[0094] Figure 51 IL-13Ra2 binding by flow cytometry is shown. The construct as indicated was titrated on A375 cells. Fc-MDNA213 was titrated on A375 and A549 cells.

[0095] Figure 52 Results of the MC38 tumor growth inhibition study are shown: C57Bl / 6 mice were treated with IP as instructed. Data are presented as mean values. + SEM presentation.

[0096] Figure 53 Results of the MC38 tumor growth inhibition study are shown: C57Bl / 6 mice were treated with IP as instructed. Data are presented as mean values. + SEM presentation.

[0097] Figure 54 Results of the MC38 tumor growth inhibition study are shown: C57Bl / 6 mice were treated with IP as instructed. Data are presented as mean values. + SEM presentation.

[0098] Figure 55Survival curves of novel adjuvant effects of MDNA113, MDNA223, or anti-mPD1 in the 4T1.2 breast tumor model are shown.

[0099] Figure 56 Survival curves of adjuvant effects of mDNA223 or anti-mPD1 in the 4T1.2 breast tumor model are shown.

[0100] Figure 57 Sensing maps of BLI performed on the indicated constructs are provided, with human CD122 (IL-2Rβ) as the ligand and construct mDNA223. A3 / S125 (SEQ ID NO: 107-109), MDNA113A A3 / S125 (SEQ ID NO: 320-322) and mDNA113B A3 / S125 (SEQ ID NO: 335-337) was used as the analyte.

[0101] Figure 58 A research summary was provided, which showed that mDNA113B T3 / C125 It is activated upon MMP9 cleavage, as observed in IL-2R reporter gene assays.

[0102] Figure 59 The Kaplan-Meyer survival plot shows the number of mice (n / 8) that survived out of the total number of mice in the 65-day study. 4T1.2 tumor cells were orthotopically implanted into mice and treated with mDNA11 (5 mg / kg) as a novel adjuvant, as well as adjuvant plus surgical tumor resection (day 16). Compared with the control group (surgical, no treatment). Mantel-Cox test. N = 8 animals / group.

[0103] Figure 60 The volume of a single tumor in mice after initial tumor implantation and subsequent tumor re-stimulation with 4T1.2 is shown. N = 5 in the control group. N = 7 and 3 in the novel adjuvant group and the adjuvant group, respectively.

[0104] Figure 61 Multiplex immunofluorescence images of 4T1.2 tumors excised from control mice and mice treated with mDNA11 (5 mg / kg) are shown, revealing immune cell infiltration. N = 4 mice / group.

[0105] Figure 62 The quantification of immune cell infiltration in individual total tumor regions is shown by multiplex immunofluorescence images of 4T1.2 tumors excised from control mice and mice treated with mDNA11 (5 mg / kg). N = 4 mice / group. Detailed Implementation

[0106] To make this disclosure easier to understand, certain terms and phrases are defined below and throughout the specification.

[0107] definition

[0108] All references cited herein are incorporated herein by reference in their entirety as if fully explained. Unless otherwise defined, 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 pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology ( Dictionary of Microbiology and Molecular Biology ) 3rd Edition John Wiley & Sons (New York, NY, 2001); March, *Advanced Organic Chemistry: Reactions, Mechanisms, and Structures* Advanced Organic Chemistry Reactions, Mechanisms and Structure ) 5th Edition John Willie & Son Publishing (NY 2001); and Sambrook and Russell, *Molecular Cloning: A Laboratory Manual* Molecular Cloning: A Laboratory Manual ) 3rd Edition Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provides a general guide to many of the terms used in this disclosure for those skilled in the art. Where appropriate, unless otherwise indicated, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and / or parameters.

[0109] As used herein, “IL-2” refers to wild-type IL-2, whether natural or recombinant. Mature human IL-2 appears as a 133-amino acid sequence (minus the signal peptide, consisting of an additional 20 N-terminal amino acids), as described by Fujita et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS USA), 80, 7437-7441 (1983). The amino acid sequence of human IL-2 (SEQ ID NO:1; full length) is found in Genbank with the accession NP_000577.2. The amino acid sequence of mature human IL-2 (human wild-type mature; substitution position numbering based on this sequence) is depicted in SEQ ID NO:2. Mouse (house mouse ( Mus musculus The amino acid sequence of IL-2 (SEQ ID NO: 3) was found in Genbank by the accession locator. The amino acid sequence of mature mouse IL-2 is depicted in SEQ ID NO: 4.

[0110] SEQ ID NO: 1

[0111] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0112] SEQ ID NO: 2

[0113] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0114] SEQ ID NO: 3

[0115] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0116] SEQ ID NO: 4

[0117] APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0118] As used herein, "IL-2 mutant protein" refers to an IL-2 polypeptide in which a specific substitution has been made to the interleukin-2 protein. IL-2 mutant proteins are characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or other residues in the native IL-2 polypeptide chain. In some embodiments, the insertions, deletions, substitutions, and / or modifications result in an IL-2 mutant protein that retains IL-2Rβ binding activity. Exemplary mutant proteins may include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. When referring to, for example, the IL-2 mutant protein of this disclosure, the phrase "comprising one / a" may also be used interchangeably with "comprising at least one / a."

[0119] Mutant proteins also include conserved modifications and substitutions at other sites on IL-2 (i.e., those conserved modifications and substitutions that have minimal impact on the secondary or tertiary structure of the mutant protein). Such conserved substitutions include those described by Dayhoff in *The Atlas of Protein Sequence and Structure*. The Atlas of Protein Sequence and Structure ) 5 (1978) and Argos in the Journal of the European Society for Molecular Biology ( EMBO J Those conserved substitutions described in .)》, 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conserved changes: Group I: ala, pro, gly, gln, asn, ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met, ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.

[0120] "Based on IL-2 number" means that the selected amino acid is identified by referencing the normal position of the amino acid in the mature sequence of wild-type IL-2 (e.g., wild-type IL-2 having the sequence of SEQ ID NO: 2). For example, in the embodiments, R81 refers to the eighty-first amino acid in SEQ ID NO: 2, arginine; L80 refers to the eightieth amino acid in SEQ ID NO: 2, leucine; L85 refers to the eighty-fifth amino acid in SEQ ID NO: 2, leucine; I86 refers to the eighty-sixth amino acid in SEQ ID NO: 2, isoleucine; I92 refers to the ninety-second amino acid in SEQ ID NO: 2, isoleucine; F42 refers to the forty-second amino acid in SEQ ID NO: 2, phenylalanine; and K43 refers to the forty-third amino acid in SEQ ID NO: 2, lysine.

[0121] As used herein, the abbreviations for the genetically encoded L-enantiomer amino acids used in the methods of this disclosure are conventional and are shown in Table 1 below.

[0122] Table 1: Amino Acid Abbreviations

[0123]

[0124]

[0125] "Hydrophilic amino acids" refer to amino acids exhibiting hydrophobicity less than zero, according to the normalized common hydrophobicity class defined by Eisenberg et al., 1984, *Journal of Molecular Biology* 179: 125-142. Genetically encoded hydrophilic amino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K), and Arg (R).

[0126] The term "having IL-2R" "Cell types of γ-receptors" refers to cells known to possess this receptor type, namely T cells, activated T cells, B cells, activated monocytes, and activated NK cells. The term "having IL-2 receptors" is used in conjunction with this. "Cell types of γ receptors" refers to cells known to have the receptor type, namely B cells, resting monocytes, and resting NK cells.

[0127] As used herein, the term "identity," when referring to a polypeptide or DNA sequence, refers to the subunit sequence identity between two molecules. Molecules are considered identical at said position when the subunit position in two molecules is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide). The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. Generally, sequences are aligned to obtain the highest-order match. If necessary, identity can be calculated using publicly available techniques and widely available computer programs, such as the GCS software package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and FASTA (Atschul et al., Journal of Molecular Biology 215:403, 1990). Sequence identity can be measured using sequence analysis software, such as the sequence analysis package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wisconsin 53705), using its default parameters.

[0128] The terms “polypeptide,” “protein,” or “peptide” refer to any chain of amino acid residues, regardless of its length or post-translational modifications (e.g., glycosylation or phosphorylation).

[0129] If the mutant IL-2 peptides of this disclosure are "substantially pure," they can be at least about 60% by weight (dry weight) of the peptide of interest, such as peptides containing the amino acid sequence of mutant IL-2. For example, the peptide can be at least about 75% by weight, about 80% by weight, about 85% by weight, about 90% by weight, about 95% by weight, or about 99% by weight of the peptide of interest. Purity can be measured by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0130] An "agonist" is a compound that interacts with a target to cause or promote an increase in the activation of the target.

[0131] A "partial agonist" is a compound that interacts with an agonist on the same target, but the biochemical and / or physiological effects produced by a partial agonist are less potent than those of an agonist, even with increased dosage.

[0132] "Super agonists" (also known as "super factors") are a type of agonist that can produce a greater maximum response to the target receptor than endogenous agonists, and therefore have more than 100% efficacy.

[0133] "Operationally ligated" means that the nucleotide sequence of interest (i.e., the sequence encoding the IL-2 mutant protein) is ligated to a regulatory sequence in a manner that allows for the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in a host cell when a 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), *Gene Expression Technology: Methods in Enzymology*, 185 (Academic Press, San Diego, California). Regulatory sequences include those that guide constitutive expression of nucleotide sequences in many types of host cells and those that guide expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of expression vectors can depend on factors such as the choice of host cells to be transformed, the desired protein expression level, etc. The expression constructs of the present invention can be introduced into host cells to produce the human IL-2 mutant protein disclosed herein or its biologically active variants.

[0134] As used herein, the term "IL-2 masking moiety" refers to a polypeptide component that reduces the activity of an IL-2 moiety. In some embodiments, the IL-2 masking moiety comprises an IL-13 protein, a variant thereof, or a fragment thereof. In some embodiments, the IL-2 masking moiety further comprises an extracellular domain of CD122, CD132, or CD25. When referring to, for example, the IL-2 mutant protein, IL-2 masking moiety, IL-13 mutant protein, or PSL of this disclosure, the phrase "comprising one" may also be used interchangeably with "comprising at least one".

[0135] The terms “host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to the specific subject cell but also to the offspring or potential offspring of such cells. Such offspring may differ in fact from the parent cell due to mutations or environmental influences that may result in certain modifications in the offspring, but are still included within the scope of the terminology used herein.

[0136] As used herein, the terms “conversion” and “transfection” refer to a variety of recognized techniques used to introduce foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipid transfection, particle gun or electroporation.

[0137] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Complementary active compounds (e.g., antibiotics) may also be incorporated into the composition.

[0138] As used herein, the term "anti-PD-1 antibody" refers to any antibody that binds to PD-1, including inhibitory antibodies. "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.

[0139] As used herein, the terms “cancer” (or “cancerous”), “hyperproliferative,” “tumor,” and / or “hyperplastic” refer to cells possessing autonomous growth capacity (i.e., an abnormal state or condition characterized by rapid proliferative cell growth). Hyperproliferative and hyperplastic disease states can be classified as pathological (i.e., characterizing or constituting a disease state) or nonpathological (i.e., deviating from normal but not associated with a disease state). The terms are intended to include cells, tissues, or organs encompassing all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignant transformation, regardless of histopathological type or stage of invasiveness. “Pathological hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of nonpathological hyperproliferative cells include cell proliferation associated with wound healing. The term "cancer" or "hyperplasia" is used to refer to malignant diseases of various organ systems, including those affecting the lungs, breasts, thyroid gland, lymph nodes and lymphatic tissue, reproductive system, gastrointestinal organs, and genitourinary tract, as well as adenocarcinomas generally considered to include malignant tumors such as most colon cancers, renal cell carcinomas, prostate cancer and / or testicular tumors, non-small cell lung cancer, small bowel cancer, and esophageal cancer. Cancer can generally include solid tumors as well as sarcomas, carcinomas, head and neck cancers, glioblastomas, bladder cancer, oral cancers, mesotheliomas, pancreatic cancers, liver cancers, colorectal cancers, lung cancers, skin cancers, lymphomas, gastrointestinal cancers, prostate cancers, ovarian cancers, breast cancers, basaloid breast tumors, endometrial cancers, multiple myelomas, melanomas, lymphomas, lung cancers (including small cell lung cancer), kidney cancers, stomach cancers, brain cancers, and CNS tumors. CNS tumors include gliomas, glioblastomas, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, meningiomas, neuroblastomas, retinoblastomas, medulloblastomas, adult pituitary adenomas, O6-methylguanine-methyltransferase (MGMT) positive or negative CNS tumors, and furin-positive CNS tumors.

[0140] The term "cancer" is recognized in this field as a malignant disease of epithelial or endocrine tissue, including respiratory cancers, gastrointestinal cancers, genitourinary cancers, testicular cancers, breast cancers, prostate cancers, endocrine cancers, and melanomas. "Adenocarcinoma" refers to cancer originating from glandular tissue or where tumor cells form identifiable glandular structures.

[0141] As used herein, the term "hematopoietic neoplastic disease" refers to a disease involving proliferative / necrotrophic cells of hematopoietic origin, such as those derived from bone marrow, lymphocytes, or erythrocyte lineages or their precursor cells. Preferably, the disease originates from poorly differentiated acute leukemia (e.g., erythrocytic leukemia and acute megakaryocytic leukemia). Other exemplary myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) (as reviewed in Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11:267-97); and lymphoid malignancies including, but not limited to, acute lymphoblastic leukemia (ALL) (including B-cell ALL and T-cell ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Other forms of malignant lymphoma include, but are not limited to, non-Hodgkin lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphoblastic leukemia (LGF), Hodgkin's disease, and Reed-Stemberg disease.

[0142] As used herein, the terms “treatment” and “treating” refer to achieving a desired pharmacological and / or physiological effect. The effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects attributable to the disease. As used herein, the term “treatment” encompasses any treatment of a disease in mammals, particularly humans, and includes: (a) preventing the development of the disease in subjects who are susceptible to or at risk of developing the disease but have not yet been diagnosed with it; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing its remission. A therapeutically effective amount may be an amount that reduces the number of tumors, decreases the size of tumors, and / or increases survival.

[0143] The terms “individual,” “subject,” and “patient” are used interchangeably in this document and refer to mammals including, but not limited to: human and non-human primates, including apes and humans; mammalian locomotion animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0144] The terms “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gas, 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 biologically or otherwise undesirable, for example, that can be administered to a subject without causing substantially undesirable biological effects. Therefore, such pharmaceutical compositions can be used, for example, to administer an IL-2 mutant protein to a subject. Specifically, an IL-2 mutant protein comprising substitutions for L80F, R81D, L85V, I86V, and I92F is combined with an anti-PD-1 agent and administered to a subject with cancer. In some embodiments, the administered IL-2 mutant protein further comprises a substitution at position F42A. In some embodiments, the administered IL-2 mutant protein further comprises a substitution at position K43N.

[0145] As used herein, the phrase "unit dosage form" refers to a physically discrete unit suitable for use in a single dose to administer treatment to a subject; each unit contains a predetermined amount of a drug optionally associated with a drug carrier (excipient, diluent, mediator, or filler) that, when administered in one or more doses, produces a desired effect (e.g., a preventative or therapeutic effect). In some embodiments, the therapeutic effect is a reduction in the number of tumors. In some embodiments, the therapeutic effect is a reduction in the size of the tumor. In some embodiments, the therapeutic effect is an increase in survival.

[0146] In some embodiments, unit dosage forms may be in, for example, ampoules and vials, comprising liquid compositions or compositions in a lyophilized or freeze-dried state; for example, a sterile liquid carrier may be added prior to in vivo administration or delivery. Individual unit dosage forms may be included in multi-dose kits or containers. IL-2 mutant proteins and their pharmaceutical compositions in combination with anti-PD-1 antibodies may be packaged in single or multiple unit dosage forms to facilitate administration and dosage consistency.

[0147] The "therapeutic effective dose" will fall within a relatively wide range that can be determined experimentally and / or through clinical trials. For example, for in vivo injection, such as direct injection into the subject's tissues or vascular system (e.g., liver tissue or veins). Other effective doses can be readily determined by those skilled in the art through routine trials that establish dose-response curves.

[0148] "Effective amount" or "sufficient amount" means the amount of a subject, administered alone or in combination with one or more other combinations (such as therapeutic agents, drugs, etc.), treatments, regimens, or medical regimens (including, for example, vaccine regimens), to provide a detectable response of any duration (long-term or short-term), any measurable or detectable degree, or any expected result or benefit of any duration (e.g., minutes, hours, days, months, years, or cure) to a subject in a single or multiple doses.

[0149] An "effective" or "sufficient" dose used for treatment (e.g., to improve or provide therapeutic benefit or improvement) typically provides a response, to a measurable extent, to one, many, or all adverse symptoms, consequences, or complications of the disease, such as one or more adverse symptoms, symptoms, diseases, pathologies, or complications caused by or associated with the disease, but mitigation, reduction, inhibition, containment, limitation, or control of the progression or worsening of the disease is also a satisfactory result. In some embodiments, an effective dose is an amount sufficient to reduce the number of tumors. In some embodiments, an effective dose is an amount sufficient to reduce the size of the tumor. In some embodiments, an effective dose is an amount sufficient to increase survival.

[0150] "Prevention" and its grammatical variations refer to methods of contacting, administering, or delivering to a subject before the onset of disease. Administration or delivery to a subject may be performed before adverse symptoms, signs, complications, etc., caused by or associated with the disease occur. For example, screening (e.g., genetic screening) may be used to identify subjects as candidates for the methods and uses, even if such subjects do not exhibit the disease. Therefore, such subjects include those who are screened positive for insufficient or absent functional gene products (proteins), or for producing disease-causing aberrations, partially functional, or non-functional gene products (proteins); and those who are screened positive for disease-causing aberrations or defects (mutant) gene products (proteins), even if such subjects do not exhibit symptoms of the disease.

[0151] I. Detailed Description

[0152] This document provides novel IL-2 fusion protein compositions comprising an IL-2 moiety linked to an IL-13 mutant protein, an IL-13Ra2-binding mutant protein, or an IL-13Ra2 antibody or an antigen-binding fragment thereof via a protease-sensitive linker (PSL). In embodiments, the IL-2 fusion protein composition comprises an IL-13 mutant protein, an IL-13Ra2-binding mutant protein, or an IL-13Ra2 antibody or an antigen-binding fragment thereof, wherein the IL-13 mutant protein, IL-13Ra2-binding mutant protein, or IL-13Ra2 antibody or an antigen-binding fragment thereof is capable of binding to IL-13Rα2 but not to IL-13Rα1, and the protease-sensitive linker is cleavable by proteases in the tumor microenvironment. Without being bound by any particular operational theory, it is assumed that the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment in the IL-2 fusion protein compositions described herein binds to the IL-2 moiety and masks its activity. Furthermore, the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment allows the subject IL-2 fusion protein composition to bind to tumors expressing IL-13Rα2. Once localized to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment and “demasking” the IL-2 moiety. The demasked IL-2 moiety can then act on the tumor site. Thus, in the embodiments, the subject IL-2 fusion protein provided herein advantageously exhibits reduced IL-2-related toxicity while allowing targeting activity against tumors expressing IL-13Rα2. Such IL-2 fusion protein compositions can be used for the treatment of cancers expressing IL-13Rα2. Aspects of the subject IL-2 fusion protein are further detailed below.

[0153] A. IL-2 section

[0154] In embodiments, the subject matter IL-2 fusion protein provided herein comprises an IL-2 moiety containing IL-2 or an IL-2 mutant protein. In embodiments, the IL-2 fusion protein comprises wild-type human IL-2 (SEQ ID NO: 2). In some embodiments, the IL-2 moiety comprises an IL-2 mutant protein containing one or more amino acid substitutions compared to wild-type human IL-2 (SEQ ID NO: 2). The substituted amino acid residues may be, but are not necessarily, conserved substitutions, which generally include substitutions from the group consisting of: 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 contacting IL-2Rβ and / or IL-2Rγ.

[0155] More specifically, mutations can be made at one or more sites (whether conserved or non-conserved, by addition or deletion). In some embodiments, the mutations included in the IL-2 mutant protein compared to wild-type human IL-2 (SEQ ID NO: 2) are located at one or more of the following amino acid positions: I24V, P65H, Q74R, Q74H, Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V, I92F, V93I, and combinations thereof. Exemplary IL-2 mutant proteins that may be included in the subject IL-2 fusion protein are shown in Table 2 below.

[0156] Table 2: Exemplary IL-2 mutant proteins

[0157]

[0158]

[0159]

[0160] In some embodiments, the substitutions in the IL-2 mutant protein comprise L80F, R81D, L85V, I86V, and I92F, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the IL-2 mutant protein further comprises an F42A substitution, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the IL-2 mutant protein further comprises a Y45A substitution, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the IL-2 mutant protein further comprises an E62A substitution, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutant protein comprise F42A, L80F, R81D, L85V, I86V, and I92F, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutant protein comprise F42A, Y45A, L80F, R81D, L85V, I86V, and I92F, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutant protein comprise F42A, E62A, L80F, R81D, L85V, I86V, and I92F, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutant protein comprise F42A, Y45A, E62A, L80F, R81D, L85V, I86V, and I92F, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutant protein comprise E62A, L80F, R81D, L85V, I86V, and I92F, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutant protein include Y45A, E62A, L80F, R81D, L85V, I86V, and I92F, according to the wild-type human IL-2 number of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutant protein include Y45A and E62A, according to the wild-type human IL-2 number of SEQ ID NO: 2.

[0161] In some embodiments, the IL-2 mutant protein exhibits increased or enhanced IL-2Rβ binding compared to wild-type IL-2 (e.g., human wild-type IL-2, SEQ ID NO: 2). In some embodiments, substitutions in the IL-2 mutant protein that result in increased and / or enhanced IL-2Rβ binding include L80F, R81D, L85V, I86V, and I92F, according to the wild-type human IL-2 number in SEQ ID NO: 2. In some embodiments, the IL-2 mutant protein used for the IL-2 fusion protein described herein comprises L80F, R81D, L85V, I86V, and I92F and exhibits enhanced IL-2Rβ binding. In some embodiments, the IL-2 mutant protein used in the present invention further comprises a substitution at position F42A. In some embodiments, the IL-2 mutant protein used in the present invention further comprises a substitution at position K43N. In some embodiments, the mutant protein comprises substitutions for L80F, R81D, L85V, I86V, and I92F, and one or more substitutions selected from the group consisting of F42A, Y45A, and E62A, all of which are compared to wild-type human IL-2 (SEQ ID NO: 2).

[0162] In some embodiments, amino acid substitutions that increase IL-2Rβ binding affinity include: L80F, R81D, L85V, I86V, and I92F.

[0163] In some embodiments, the subject IL-2 mutant protein having a higher binding affinity for IL-2Rβ compared to wild-type human IL-2 includes amino acid substitutions of L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutant protein has the following amino acid sequence: (SEQ ID NO: 5).

[0164] In some embodiments, the IL-2 mutant protein has an increased stimulatory effect depending on one or more IL-2Rβ / IL-2Rγ ratios. cThe ability of heterodimerized signaling pathways. In some embodiments, the subject IL-2 mutant protein has an enhanced ability to stimulate STAT5 phosphorylation in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the level of STAT5 phosphorylation in IL-2Rβ+ cells stimulated by the IL-2 mutant protein 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 of STAT5 phosphorylation in the same cells stimulated by wild-type IL-2. In some embodiments, the level of STAT5 phosphorylation in the same cells stimulated by the IL-2 mutant protein is 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or more compared to the level of STAT5 phosphorylation in IL-2Rβ+ cells stimulated by wild-type IL-2. In some embodiments, the IL-2Rβ+ cells are T cells. In a specific embodiment, the T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. In other embodiments, the CD8+ T cells are activated CD8+ T cells. In other embodiments, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutant protein comprises substitutions of L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0165] In some embodiments, the mutant protein has an enhanced ability to stimulate ERK1 / ERK2 signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the level of IL-2 mutant protein stimulating pERK1 / ERK2 signaling in IL-2Rβ+ cells 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 of pERK1 / ERK2 signaling stimulated by wild-type IL-2 in the same cells. In some embodiments, the phosphorylation levels of pERK1 / ERK2 in the same cells stimulated by the IL-2 mutant protein are 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or more compared to the level of pERK1 / ERK2 phosphorylation in wild-type IL-2-stimulated IL-2Rβ+ cells. In some embodiments, IL-2Rβ+ cells are T cells. In a specific embodiment, the T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. In other embodiments, the CD8+ T cells are activated CD8+ T cells. In other embodiments, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutant protein comprises substitutions of L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0166] 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 instance, STAT5 and ERK1 / 2 phosphorylation can be measured using a combination of antibodies specific to the phosphorylated versions of these molecules and flow cytometry analysis as described herein. In some embodiments, the mutant protein has an enhanced ability to stimulate PI3-kinase signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the level of PI3-kinase signaling in IL-2Rβ+ cells stimulated by the IL-2 mutant protein 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 of PI3-kinase signaling in the same cells stimulated by wild-type IL-2. In some embodiments, the level of phosphorylation of PI3-kinase signaling in the same cells stimulated by the IL-2 mutant protein is 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or more compared to the level of PI3-kinase signaling in wild-type IL-2-stimulated IL-2Rβ+ cells. In some embodiments, IL-2Rβ+ cells are T cells. In a specific embodiment, the T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are activated CD8+ T cells. In other embodiments, IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutant protein comprises substitutions for L80F, R81D, L85V, I86V, and I92F 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, PI3-kinase signaling can be measured using flow cytometry analysis as described herein, using antibodies specific to phosphoryl-S6 ribosomal protein.

[0167] In some embodiments, the IL-2 mutant protein is a stimulator of IL-2 and / or IL-15 STAT5 phosphorylation in CD8+ T cells. In some embodiments, the mutant protein is a promoter of IL-2 and / or IL-15-induced CD8+ T cell proliferation. In some embodiments, the mutant protein is a stimulator of IL-2-dependent TCR-induced cell proliferation. In some embodiments, the IL-2 mutant protein comprises substitutions for L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0168] IL-2 promotes Th1, Th9, and Treg T cell differentiation and inhibits Th17 differentiation. Therefore, without being bound by any particular operational theory, it is considered that IL-2 mutant proteins acting as IL-2 superagonists can promote Th1, Th9, and / or Treg cell differentiation or inhibit Th17 cell differentiation. In some embodiments, the IL-2 mutant protein is a promoter of IL-2-dependent Th1, Th9, and / or Treg differentiation. In some embodiments, the mutant protein is an inhibitor of Th17 differentiation. In some embodiments, the IL-2 mutant protein comprises substitutions for L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0169] In some embodiments, the IL-2 mutant protein emits less and / or CD25-independent signaling (e.g., reduced or eliminated CD25 binding) compared to wild-type human IL-2. In some embodiments, reduced and / or independent CD25-related signaling allows preferential activation of effector T cells while limiting Treg stimulation. In some embodiments, reduced and / or independent CD25-related signaling results in reduced toxicity. In some embodiments, the mutant protein comprises substitutions for L80F, R81D, L85V, I86V, and I92F, and one or more substitutions selected from the group consisting of F42A, Y45A, and E62A, all compared to wild-type human IL-2 (SEQ ID NO: 2). In some embodiments, the IL-2 mutant protein comprises SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0170] In some embodiments, the IL-2 mutant protein can increase and / or restore responsiveness to unresponsive NK cells. In some embodiments, the IL-2 mutant protein can increase and / or restore responsiveness to unresponsive NK cells in the tumor microenvironment. In some embodiments, the IL-2 mutant protein comprises substitutions for L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0171] In some embodiments, the mutant protein is an inhibitor, an inhibitor of IL-2-dependent natural killer (NK) cell activation. As described herein, IL-2 activation of NK cells can be measured by any suitable method known in the art, such as measuring IL-2-induced CD69 expression and / or cytotoxicity.

[0172] In some embodiments, the increase in IL-2Rβ binding affinity is that any binding affinity for IL-2Rβ is greater than the binding affinity of wild-type human IL-2 for IL-2Rβ. In some embodiments, the binding affinity is an increase of 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 190, 200, 220, 240, or more times compared to the binding affinity of wild-type human IL-2 for IL-2Rβ.

[0173] In some embodiments, the increase in IL-2Rβ binding affinity is any binding affinity to IL-2Rβ greater than that of wild-type human IL-2 to IL-2Rβ. In some embodiments, the binding affinity is an increase of 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 190, 200, 220, 240, or more times compared to the binding affinity of wild-type human IL-2 to IL-2Rβ.

[0174] In some embodiments, the subject IL-2 mutant protein, which has a higher binding affinity for IL-2Rβ compared to wild-type human IL-2, also exhibits reduced binding to CD25 and includes amino acid substitutions of F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the binding affinity is reduced by approximately 220-fold, from approximately 6.6 nM Kd in wild-type human IL-2 to approximately 1.4 µM in the mutant protein, which comprises F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0175] (SEQ ID NO: 6; also known as H9-F42A).

[0176] In some embodiments, the subject IL-2 mutant protein, exhibiting a higher binding affinity for IL-2Rβ compared to wild-type human IL-2, also shows reduced binding to CD25 and includes amino acid substitutions of K43N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the reduced binding affinity is due to allowing glycosylation with a K43N substitution at position 43. By replacing asparagine (K43N) with lysine, CD25 binding is reduced and / or eliminated in IL-2 mutant proteins containing amino acid substitutions of K43N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0177] (SEQ ID NO: 7; also known as H9-K43N).

[0178] In some embodiments, reduced binding affinity for CD25 means that any binding affinity for CD25 is lower than that for wild-type human IL-2. In some embodiments, the binding affinity is a reduction of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 190, 200, 220, 240, or more times compared to the binding affinity of wild-type human IL-2 for CD25.

[0179] In some embodiments, the subject IL-2 mutant protein, exhibiting higher binding affinity for IL-2Rβ and decreased binding affinity for CD25 compared to wild-type human IL-2, includes amino acid substitutions of F42A, Y45A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0180] (SEQ ID NO: 8; H9-F42A / Y45A; H9-FYAA).

[0181] In some embodiments, the subject IL-2 mutant protein having a higher binding affinity for IL-2Rβ and a lower binding affinity for CD25 compared to wild-type human IL-2 includes amino acid substitutions of F42A, E62A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0182] (SEQ ID NO: 9; H9-F42A / E62A; H9-FEAA).

[0183] In some embodiments, the subject IL-2 mutant protein having a higher binding affinity for IL-2Rβ and a lower binding affinity for CD25 compared to wild-type human IL-2 includes amino acid substitutions of F42A, Y45A, E62A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0184] (SEQ ID NO: 10; H9–F42A / Y45A / E62A; H9-FYEAAA).

[0185] In some embodiments, the IL-2 mutant protein includes a T3A amino acid substitution to eliminate glycosylation of the IL-2 mutant protein, which can reduce the homogeneity of the IL-2 mutant protein and the IL-2 fusion protein product. In some embodiments, the IL-2 mutant protein includes a C125S amino acid substitution to eliminate the risk of disulfide bridge rearrangement at this residue, thereby reducing the risk of spreadability issues. In some embodiments, the IL-2 mutant protein is a further variant of mDNA109 (SEQ ID NO: 5) or MDN109FEAA (SEQ ID NO: 9) including T3A and / or C125S substitutions.

[0186] In some embodiments, the IL-2 mutant protein 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 with one of the IL-2 mutant proteins in Table 2. In some embodiments, the IL-2 mutant protein of the IL-2 fusion protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to any of the IL-2 mutant proteins in Table 2 (SEQ ID NO: 5-24). In embodiments, the variant exhibits a greater binding affinity for IL-2Rβ compared to wild-type human IL-2. In embodiments, the variant also exhibits reduced binding to CD25.

[0187] 1. The portion of IL-2 with an extended half-life

[0188] In some embodiments, the IL-2 portion includes a polypeptide that increases the in vivo serum half-life of IL-2 included in the IL-2 fusion protein (i.e., a "serum half-life enhancer" or "half-life enhancing" polypeptide). In various embodiments, the half-life enhancing polypeptide is the Fc region of serum albumin (e.g., human serum albumin), PEG, a PEG derivative, or an IgG subclass lacking the variable region of the IgG heavy chain. Exemplary Fc regions may include mutations that inhibit complement fixation and Fc receptor binding, or they may be cleaved, i.e., capable of binding to complement or lysed cells via another mechanism (such as antibody-dependent complement cleavage; USSN 08 / 355,502, filed December 1994).

[0189] In some embodiments, the half-life-enhancing peptide is an Fc region. An “Fc region” can be a naturally occurring or synthetic peptide homologous to the C-terminal domain of IgG produced by digesting IgG with papain. The molecular weight of an IgG Fc is approximately 50 kDa. The IL-2 moiety of the subject IL-2 fusion protein may include the entire Fc region or a smaller portion that retains the ability to extend the circulating half-life of the IL-2 moiety in which it resides. Furthermore, the full-length or fragmented Fc region may be a variant of the wild-type molecule. In some embodiments, the IL-2 moiety of the subject IL-2 fusion protein includes an IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region of the IL-2 moiety includes a monomer comprising a CH2-CH3 domain of a human IgG1, IgG2, IgG3, or IgG4 region. In some embodiments, the Fc region of the IL-2 moiety is a dimer and comprises two monomers, each of which includes a CH2-CH3 domain of a human IgG1, IgG2, IgG3, or IgG4 region. In some embodiments, the IL-2 portion comprises two monomers, each comprising IL-2 or an IL-2 mutant protein linked to an Fc region monomer comprising a CH2-CH3 domain. In such embodiments, the Fc region monomer dimerizes to form a dimerized IL-2 portion. In some embodiments, the IL-2 portion comprises two monomers, one of which comprises IL-2 or an IL-2 mutant protein linked to a first Fc region monomer comprising a CH2-CH3 domain, and the second monomer comprises a second Fc region monomer but does not comprise IL-2 or an IL-2 mutant protein. The first and second Fc regions dimerize to form a dimerized Fc region. In embodiments comprising a dimerized Fc region, each of the two monomers may include an amino acid substitution that favors the formation of a heterodimer (e.g., a "mortar and pestle" or "mortar and pestle" amino acid substitution, see, for example, U.S. Patent No. 8,216,805, which is incorporated herein by reference for the disclosure of "mortar and pestle" amino acid substitutions). In some embodiments, the Fc region comprises a substituted N297A.

[0190] The Fc region can be either "cleavable" or "non-cleavable," but is typically non-cleavable. Non-cleavable Fc regions generally lack both high-affinity Fc receptor binding sites and C'1q binding sites. The high-affinity Fc receptor binding site of mouse IgG Fc includes the Leu residue at position 235 of the IgG Fc. Therefore, the Fc receptor binding site can be disrupted by mutation or deletion of Leu 235. For example, replacing Leu 235 with Glu inhibits the ability of the Fc region to bind to the high-affinity Fc receptor. The mouse C'1q binding site can be functionally disrupted by mutation or deletion of Glu 318, Lys 320, and Lys 322 residues of IgG. For example, replacing Glu 318, Lys 320, and Lys 322 with Ala residues prevents IgG1 Fc from undergoing directed antibody-dependent complement cleavage. Conversely, cleavable IgG Fc regions possess both high-affinity Fc receptor binding sites and C'1q binding sites. The high-affinity Fc receptor binding site at position 235 of IgG Fc includes the Leu residue, while the C'1q binding site includes Glu 318, Lys 320, and Lys 322 residues of IgG1. Cleavage IgG Fc has wild-type residues or conserved amino acid substitutions at these sites. Cleavage IgG Fc can target cells to produce antibody-dependent cytotoxicity or complement-directed cell lysis (CDC). Appropriate mutations in human IgG are also known (see, for example, Morrison et al., The Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2:125, 1994).

[0191] In some embodiments, the half-life-enhancing polypeptide is serum albumin. In an exemplary embodiment, the serum albumin is human serum albumin or a variant thereof capable of extending the serum half-life of linked IL-2 or IL-2 mutant proteins. In some embodiments, human serum albumin has the following amino acid sequence:

[0192] (SEQ ID NO:400).

[0193] In some embodiments, the IL-2 portion of the IL-2 moiety (e.g., wild-type IL-2 or the IL-2 mutant protein provided herein) is directly or indirectly linked to a serum half-life enhancing peptide. In some embodiments, the half-life enhancing peptide is linked to the N-terminus of IL-2 (e.g., wild-type IL-2 or the IL-2 mutant protein provided herein). In some embodiments, the half-life enhancing peptide is linked to the C-terminus of IL-2 (e.g., wild-type IL-2 or the IL-2 mutant protein provided herein). In some embodiments, IL-2 or the IL-2 mutant protein is directly linked to the half-life enhancing peptide. In some embodiments, IL-2 or the IL-2 mutant protein is linked to the half-life enhancing peptide via a linker peptide such as GGGGS. In some embodiments, the linker is (GGGGS). n ,in nIt is an integer between 1 and 10. In some embodiments, the connector is GGGGS (SEQ ID NO: 401). In some embodiments, the connector is GGGGSGGGGS (SEQ ID NO: 402). In some embodiments, the connector is GGGGSGGGGSGGGGS (SEQ ID NO: 403). In some embodiments, the connector is GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 404). In some embodiments, the connector is GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 405).

[0194] In some embodiments, the linker contains one or more protease cleavage sites (e.g., it is a protease-cleavable linker). Alternatively, the linker may contain one or more protease cleavage sites or be sensitive to cleavage by oxidation and / or reduction. In one example, a peptide linker readily cleaved by enzymes of the complement system, urokinase, tissue plasminogen activator, trypsin, plasmin, caspase, kallikrein, cathepsin, podocyte oleanolic acid, MMP, thrombin, urokinase-type plasminogen activator (uPA), matriptase, prostate-specific antigen (PSA), or another proteolytically active enzyme may be used. According to another example, the linker may contain disulfide bonds (e.g., disulfide bonds on a cysteine ​​molecule). According to another example, the adapter may comprise a protease-cleavable Val-Cit (VC) adapter, a Phe-Arg adapter, a Val-Lys adapter, a Val-Ala adapter, a Val-Arg adapter, a Val-Leu-Lys adapter, a Gly-Phe-Leu-Gly adapter, an Ala-Phe-Lys adapter, a pol-L-lysine adapter, a β-Ala-Leu-Ala-Leu adapter, an Arg-Arg-Ala-Leu-Ala-Leu adapter, a peptide mimic adapter, a pod protein-cleavable Ala–Ala–Asn tripeptide adapter, a peptide adapter cleaved by cathepsin B and other lysosomal proteases (such as Gly–Phe–Leu–Gly and Ala–Leu–Ala–Leu, caspase 3 DEVD sequence), or a self-canceling adapter. This disclosure envisions, for example, Poreba, M, *Journal of the Federation of European Biochemical Societies* (…). FEBS JThe linker disclosed in Nature Communications 287(10):1936-1969 (2020), which is incorporated herein by reference. Many tumors naturally release high levels of glutathione (a reducing agent), which can reduce disulfide bonds and subsequently release cargo portions at the delivery site. In some embodiments, the linker is a protease-cleavable linker, a linker that can be cleaved by matrix metalloproteinases (MMPs). MMPs are overexpressed in situ at tumor sites, and this disclosure contemplates linkers that are cleavable in such cases. For example, Hsu, EJ et al., Nature Communications (2020) Nat.Commun. The connector disclosed in 12(2768):1-13 (2021), which is incorporated herein by reference. In some embodiments, the MMP connector 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 NO: 410). In some embodiments, the MMP connector sequence is SGARYRWLTA (SEQ ID NO: 411). In some embodiments, the MMP connector sequence is SGRSYAILTA (SEQ ID NO: 412). In some embodiments, the MMP connector sequence is SRSGRSPAIFTATG (SEQ ID NO: 413). In some embodiments, the MMP connector sequence is GSSGRSPAIFTAGS (SEQ ID NO: 414). In some embodiments, the MMP connector sequence is SGFIANPVTA (SEQ ID NO: 415). In some embodiments, the MMP connector sequence is PLGLVVAPLGLVVAPLGLVVA (SEQ ID NO: 416). In some embodiments, the MMP connector sequence is PLGLWAPLGLWAPLGLWA (SEQ ID NO: 417). In some embodiments, the MMP connector sequence is GGSGGTPLGLWAGGSGGT (SEQ ID NO: 418). In some embodiments, the MMP connector sequence is GGSGGTPAGLIGGGSGGT (SEQ ID NO: 419). In some embodiments, the MMP connector sequence is GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NO: 420). In some embodiments, the PSA connector sequence is GGSGGTHSSKLQGGSGGT (SEQ ID NO: 421).

[0195] In some embodiments, the IL-2 moiety includes a polypeptide that functions as an antigen tag, such as the FLAG sequence. As described herein, the FLAG sequence is recognized by biotinylated, highly specific anti-FLAG antibodies (see also Blanar et al., Science 256:1014, 1992; LeClair et al., Proceedings of the National Academy of Sciences of the United States of America 89:8145, 1992). In some embodiments, the IL-2 moiety further includes a C-terminal c-myc epitope tag.

[0196] In other embodiments, the IL-2 moiety includes a heterologous polypeptide, such as the Aga2p lectin subunit, which has the function of enhancing the expression of the IL-2 moiety or guiding cell localization (see, for example, Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0197] Table 3 shows exemplary IL-2 moieties comprising half-life-enhancing peptides used in the subject matter IL-2 fusion peptides described herein. In some embodiments, the IL-2 moieties of the IL-2 fusion protein have amino acids from one of SEQ ID NO: 25-73. In some embodiments, the IL-2 moieties of the IL-2 fusion protein have at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with one of the IL-2 moieties in Table 3 (SEQ ID NO: 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 compared to any of the IL-2 moieties in Table 3 (SEQ ID NO: 25-67). In embodiments, the IL-2 exhibits a greater binding affinity for IL-2Rβ compared to wild-type human IL-2. In embodiments, the variants also exhibit reduced binding to CD25.

[0198] Table 3: Exemplary IL-2 fractions with enhanced serum half-life

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215] 2. IL-2 / antibody fusion portion

[0216] In other embodiments, the IL-2 portion of the IL-2 fusion protein provided herein includes an antibody or its antigen-binding portion (i.e., the "IL-2 / antibody fusion portion").

[0217] In some embodiments, the IL-2 / antibody fusion comprises three polypeptides. The first polypeptide comprises IL-2 or an IL-2 mutant protein linked to a first Fc region. The second polypeptide comprises a heavy chain variable region (VH) and a second Fc region. The third polypeptide comprises a light chain variable region (VL) and a light chain constant region. In this embodiment, the two Fc regions of the first and second polypeptides dimerize, and the VH and VL form an antigen-binding domain.

[0218] In some embodiments of the IL-2 / antibody fusion portion, the IL-2 / antibody fusion portion comprises four peptides. A first peptide comprises a first antibody heavy chain linked to IL-2 or an IL-2 mutant protein, wherein the first antibody heavy chain includes a first heavy chain variable region (VH1) and a first Fc region. A second peptide comprises a second antibody heavy chain, wherein the second antibody heavy chain includes a second heavy chain variable region (VH2) and a second Fc region. A third peptide comprises a first variable light chain variable region (VL1) and a light chain constant region. A fourth peptide 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 peptides dimerize, and VH1 and VL1, as well as 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 antigen-binding domain and the second antigen-binding domain are identical. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are different.

[0219] An antibody or antigen-binding component is linked to IL-2 or an IL-2 mutant protein using any suitable technique. In some embodiments, the antibody or antigen-binding component is directly linked to IL-2 or an IL-2 mutant protein. In some embodiments, a connector is used to link the antibody or antigen-binding component to IL-2 or an IL-2 mutant protein. Any suitable connector, including those described herein, can be used to link IL-2 or an IL-2 mutant protein to an antibody or antigen-binding component. In embodiments, the connector is of formula (GGGGS). n The linker peptide, of which n It is an integer between 1 and 10. In some embodiments, the connector is GGGGS (SEQ ID NO: 401). In some embodiments, the connector is GGGGSGGGGS (SEQ ID NO: 402). In some embodiments, the connector is GGGGSGGGGSGGGGS (SEQ ID NO: 403). In some embodiments, the connector is GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 404). In some embodiments, the connector is GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 405). In some embodiments, IL-2 or the IL-2 mutant protein is linked to the C-terminus of the Fc domain of the antibody or antigen-binding component. In some embodiments, IL-2 or the IL-2 mutant protein is linked to the N-terminus of the antibody or antigen-binding component.

[0220] The antibody or antigen-binding component of the IL-2 moiety can act as a targeting moiety. For example, it can be used to localize the chimeric protein to a specific subset of cells or target molecules. Methods for generating cytokine-antibody chimeric peptides are described, for example, in U.S. Patent No. 6,617,135.

[0221] In some embodiments, the IL-2 portion comprises a fusion with an antibody or its antigen-binding portion thereof, the fusion disrupting the interaction between the PD-1 receptor and its ligand PD-L1, and / or an antibody targeting a component of the PD-1 / PD-L1 signaling pathway. Antibodies known in the art that bind to PD-1 and disrupt the interaction between PD-1 and its ligand PD-L1 and stimulate an antitumor immune response are suitable for the chimeric peptides disclosed herein. In some embodiments, the antibody or its antigen-binding portion specifically binds to PD-1. For example, antibodies targeting PD-1 and found to be used in this invention include, 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.), pitilizumab (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 Medicine Co., Ltd.). HengRui), human monoclonal antibody REGN2810 (cimiprimab, Regeneron Pharmaceuticals), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is derived from clone: ​​RMP1-14 (rat IgG) - BioXcell catalog number BP0146. Other suitable antibodies include the anti-PD-1 antibody disclosed in U.S. Patent No. 8,008,449, which is incorporated herein by reference. In some embodiments, the antibody or its antigen-binding moiety specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Any antibody known in the art that binds to PD-L1 and disrupts the interaction between PD-1 and PD-L1 and stimulates an antitumor immune response is suitable for the chimeric peptides disclosed herein. For example, antibodies targeting PD-L1 and currently in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genentech).Other suitable antibodies targeting PD-L1 are disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference. Those skilled in the art will understand that any antibody that binds to PD-1 or PD-L1, disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for the IL-2 portion disclosed herein. In some embodiments, the IL-2 portion comprises a fusion with an anti-PD-1 antibody. In some embodiments, the IL-2 portion comprises a fusion with an anti-PD-L1 antibody.

[0222] Table 4 shows exemplary IL-2 / anti-PD-1 antibody fusion moieties that may be included in the subject IL-2 fusion protein. In some embodiments, the IL-2 moieties of the IL-2 fusion protein have one of the amino acids listed in Table 4. In some embodiments, the IL-2 moieties of the IL-2 fusion protein are variants of one of the IL-2 moieties in Table 4. In some embodiments, the IL-2 moieties of the IL-2 fusion protein have at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with 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 compared to any of the IL-2 moieties in Table 4. In embodiments, the variants exhibit a greater binding affinity for IL-2Rβ compared to wild-type human IL-2. In embodiments, the variants also exhibit reduced binding to CD25. In some embodiments, the variants are capable of binding to PD-1 (human PD-1).

[0223] Table 4: IL-2 / Anti-PD1 Fusion Portion

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237] In some embodiments, the IL-2 portion comprises a fusion of an antibody or its antigen-binding portion that targets CTLA-4 and disrupts its interaction with CD80 and CD86. Exemplary antibodies targeting CTLA-4 include FDA-approved ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb) and tremelimumab (ticilimumab, CP-675, 206, Pfizer), both currently in human trials. Other suitable antibodies targeting CTLA-4 are disclosed in WO 2012 / 120125, U.S. Patent Nos. 6,984,720, 6,682,7368, and U.S. Patent Applications 2002 / 0039581, 2002 / 0086014, and 2005 / 0201994, all of which are incorporated herein by reference. Those skilled in the art will understand that any antibody that binds to CTLA-4, disrupts its interaction with CD80 and CD86, and stimulates an anti-tumor immune response is suitable for the chimeric peptides disclosed herein. In some embodiments, the IL-2 portion comprises a fusion with an anti-CTLA-4 antibody.

[0238] In some embodiments, the IL-2 portion comprises a fusion of an antibody or its antigen-binding portion that targets LAG-3 and disrupts its interaction with MHC class II molecules. An exemplary antibody targeting LAG-3 is IMP321 (Immutep, Inc.), which is currently undergoing human trials. Other suitable antibodies targeting LAG-3 are disclosed in U.S. Patent Application 2011 / 0150892, which is incorporated herein by reference. Those skilled in the art will understand that any antibody that binds to LAG-3, disrupts its interaction with MHC class II molecules, and stimulates an antitumor immune response is suitable for the IL-2 portion disclosed herein. In some embodiments, the IL-2 portion comprises a fusion of an anti-LAG-3 antibody.

[0239] In some embodiments, the IL-2 portion comprises a fusion with an antibody or its antigen-binding moiety that targets TIGIT and disrupts its interaction with CD155 (PVR) and / or CD112 (PVRL2, cohesin-2). Those skilled in the art will understand that any antibody that binds to TIGIT, disrupts its interaction with CD155 (PVR) and / or CD112 (PVRL2, cohesin-2), and stimulates an antitumor immune response or immunostimulatory response with overall antitumor activity is suitable for the chimeric peptides disclosed herein. In some embodiments, the IL-2 portion comprises a fusion with an anti-TIGIT antibody.

[0240] In some embodiments, the IL-2 portion comprises a fusion with an antibody or its antigen-binding moiety that targets CD112R (also known as PVRIG) and disrupts its interaction with CD112 and / or PVRL2 / adhesionin-2. Those skilled in the art will understand that any antibody that binds to CD112R, disrupts its interaction with CD112 and / or PVRL2 / adhesionin-2, and stimulates an antitumor immune response or immunostimulatory response with overall antitumor activity is suitable for the chimeric peptides disclosed herein. In some embodiments, the IL-2 portion comprises a fusion with an anti-CD112R antibody.

[0241] In some embodiments, the IL-2 moiety comprises a fusion with an antibody or antigen-binding portion thereof targeting B7-H3 or B7-H4. The B7 family has no defined receptors, but these ligands are upregulated on tumor cells or tumor-infiltrating cells. An exemplary antibody targeting B7-H3 is MGA271 (Macrogenics), which is currently undergoing human trials. Other suitable antibodies targeting members of the B7 family are disclosed in U.S. Patent Application 2013 / 0149236, which is incorporated herein by reference. Those skilled in the art will understand that any antibody that binds to B7-H3 or H4 and stimulates an anti-tumor immune response is suitable for the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion with an anti-B7-H3 or B7-H4 antibody.

[0242] In some embodiments, the IL-2 portion comprises a fusion of an antibody or its antigen-binding portion that targets TIM-3 and disrupts its interaction with galectin 9. Suitable antibodies targeting TIM-3 are disclosed in U.S. Patent Application 2013 / 0022623, which is incorporated herein by reference. Those skilled in the art will understand that any antibody that binds to TIM-3, disrupts its interaction with galectin 9, and stimulates an antitumor immune response is suitable for the IL-2 portion disclosed herein. In some embodiments, the IL-2 portion comprises a fusion of an anti-TIM-3 antibody.

[0243] In some embodiments, the IL-2 moiety comprises a fusion with an antibody or its antigen-binding moiety that targets 4-1BB / CD137 and disrupts its interaction with CD137L. Those skilled in the art will understand that any antibody that binds to 4-1BB / CD137, disrupts its interaction with CD137L or another ligand, and stimulates an antitumor immune response or immunostimulatory response with overall antitumor activity is suitable for the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion with an anti-4-1BB / CD137 antibody.

[0244] In some embodiments, the IL-2 moiety comprises a fusion of an antibody or its antigen-binding moiety that targets GITR and disrupts its interaction with its ligand. Those skilled in the art will understand that any antibody that binds to GITR, disrupts its interaction with GITRL or another ligand, and stimulates an antitumor immune response or immunostimulatory response with overall antitumor activity is suitable for the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion of an anti-GITR antibody.

[0245] In some embodiments, the IL-2 moiety comprises a fusion with an antibody or its antigen-binding portion that targets OX40 and disrupts its interaction with its ligand. Those skilled in the art will understand that any antibody that binds to OX40, disrupts its interaction with OX40L or another ligand, and stimulates an antitumor immune response or immunostimulatory response with overall antitumor activity is suitable for the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion with an anti-OX40 antibody.

[0246] In some embodiments, the IL-2 portion comprises a fusion with an antibody or its antigen-binding portion that targets CD40 and disrupts its interaction with its ligand. Those skilled in the art will understand that any antibody that binds to CD40, disrupts its interaction with its ligand, and stimulates an antitumor immune response or immunostimulatory response with overall antitumor activity is suitable for the IL-2 portion disclosed herein. In some embodiments, the IL-2 portion comprises a fusion with an anti-CD40 antibody.

[0247] In some embodiments, the IL-2 portion comprises a fusion with an antibody or its antigen-binding portion that targets ICOS and disrupts its interaction with its ligands. Those skilled in the art will understand that any antibody that binds to ICOS, disrupts its interaction with its ligands, and stimulates an antitumor immune response or immunostimulatory response with overall antitumor activity is suitable for the IL-2 portion disclosed herein. In some embodiments, the IL-2 portion comprises a fusion with an anti-ICOS antibody.

[0248] In some embodiments, the IL-2 portion comprises a fusion with an antibody or its antigen-binding portion that targets CD28 and disrupts its interaction with its ligand. Those skilled in the art will understand that any antibody that binds to CD28, disrupts its interaction with its ligand, and stimulates an antitumor immune response or immunostimulatory response with overall antitumor activity is suitable for the IL-2 portion disclosed herein. In some embodiments, the IL-2 portion comprises a fusion with an anti-CD28 antibody. In some embodiments, the IL-2 portion comprises a fusion with an anti-CD3 antibody, including a T-cell conjugate anti-CD3 antibody.

[0249] In some embodiments, the IL-2 moiety comprises a fusion with an antibody or its antigen-binding moiety that targets IFNα and disrupts its interaction with its ligands. Those skilled in the art will understand that any antibody that binds to IFNα, disrupts its interaction with its ligands, and stimulates an antitumor immune response or immunostimulatory response with overall antitumor activity is suitable for the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety comprises a fusion with an anti-IFNα antibody.

[0250] 3. Additional IL-2 fusion components

[0251] In some embodiments, the IL-2 portion comprises a fusion with a tumor antigen or a peptide targeting a tumor antigen. Typically, tumor antigens enable the differentiation of tumor cells from their normal cell counterparts and may include, for example, tumor-specific antigens (TSA) and tumor-associated antigens (TAA). In some embodiments, tumor antigens are proto-oncogenes and / or tumor suppressors, as well as overexpressed or aberrantly expressed cellular proteins, tumor antigens generated by oncogenic viruses, meconium antigens, altered cell surface glycolipids and glycoproteins, and / or cell type-specific differentiation antigens. Such tumor antigens may include melanoma antigens, cancer-testis antigens, epithelial tumor antigens, cell cycle regulatory proteins, prostate-specific antigens (including prostate cancer antigens, such as the prostate cancer antigen disclosed in U.S. Patent No. 5,538,866), and lymphoma antigens (U.S. Patent Nos. 4,816,249; 5,068,177; and 5,227,159). Tumor antigens may include, for example, but not limited to, HMW mucins that bind to 2G3 and 369F10, c-erbB-2-associated tumor antigens (glycoproteins of approximately 42 kD or 55 kD), and tumor antigens of approximately 40 kD, 60 kD, 100 kD, and 200 kD. Antigens that bind kD to 113F1, 9-O-acetylGD3, p97, alpha-fetoprotein (AFP) (e.g., for germ cell tumors and / or hepatocellular carcinoma), carcinoembryonic antigen (CEA) (e.g., for colorectal cancer, incidental lung cancer, or breast cancer), CA-125 (e.g., for ovarian cancer), MUC-1 (e.g., for breast cancer), epithelial tumor antigen (ETA) (e.g., for breast cancer), tyrosinase (e.g., for malignant melanoma), melanoma-associated antigen (MAGE) (e.g., for malignant melanoma), cancer / testis antigen 1 (CTAG1B), melanoma-associated antigen 1 (MAGEA1), aberrant Ras products, aberrant p53 products, overexpression of cyclins (including, for example, cyclin B1), mutations in fibronectin, post-translational alterations of MUC1 glycoprotein, and secreted tumor antigens (including, for example, gangliosides).

[0252] Other fusions may include fusions with pro-apoptotic payloads. Exemplary sequences of this type are provided in the table below. In some embodiments, the IL-2 moiety as described herein is fused to a pro-apoptotic payload (e.g., BAD, BAX, BAK, BIK, and / or BID sequences). In some embodiments, the pro-apoptotic payload is a peptide containing a Bcl-2 domain and / or sequences of BAD, BAX, BAK, BIK, and / or BID. Exemplary pro-apoptotic fusions are provided in Table 5 below.

[0253] Table 5: List of selected pro-apoptotic fusion couples

[0254]

[0255]

[0256] Other fusions may include fusions with an anti-apoptotic payload for prolonging the activation of CD8 cells, NK cells, and unresponsive NK cells, and such exemplary sequences are provided in the table below. Such prolonged activation of T cells may prove beneficial in cancer therapeutic approaches.

[0257] Table 6: List of exemplary IL-2 anti-apoptotic fusion amino acid sequences

[0258]

[0259]

[0260]

[0261] B.IL-2 shielding section

[0262] The IL-2 fusion proteins described herein include IL-13 mutant proteins, IL-13Ra2-binding mutant proteins, or IL-13Ra2 antibodies or their antigen-binding fragments linked to the IL-2 moiety via a protease-sensitive linker (PSL). When linked to an IL-13 mutant protein or an IL-13Ra2 antibody or its antigen-binding fragment, the IL-2 moiety exhibits reduced IL-2 activity; that is, the IL-2 moiety is "masked" by the IL-13 mutant protein, IL-13Ra2-binding mutant protein, IL-13Ra2 antibody or its antigen-binding fragment. In some embodiments, the IL-13 mutant protein, IL-13Ra2-binding mutant protein, IL-13Ra2 antibody or its antigen-binding fragment can bind to tumors expressing IL-13Rα2, thereby localizing the IL-2 fusion protein to the tumor microenvironment. Once located in the tumor microenvironment, the protease-sensitive linker is cleaved by the protease, thereby releasing the IL-13 mutant protein, the IL-13Ra2-binding mutant protein, or the IL-13Ra2 antibody or its antigen-binding fragment from the IL-2 fusion protein. In some embodiments, the IL-2 moiety is then “demasked” to be active in the tumor microenvironment, thereby promoting the antitumor activity of IL-2.

[0263] In some embodiments, the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment is linked to the IL-2 mutant protein of the IL-2 moiety via a PSL linker. In some embodiments, the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment is linked to a region of the IL-2 moiety that is not the IL-2 mutant protein. For example, in some embodiments, the IL-2 moiety further includes a half-life-enhancing peptide (e.g., serum albumin or an Fc region), and the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment is linked to the half-life-enhancing peptide of the IL-2 moiety via PSL. In some embodiments, the IL-2 moiety includes a dimerized Fc region, the IL-2 mutant protein is linked to one of the Fc region monomers, and the IL-13 mutant protein or IL-13Ra2 antibody or its antigen-binding fragment is linked to another Fc region monomer via PSL.

[0264] In some embodiments, the IL-2 fusion protein comprises two or more IL-13 mutant proteins or IL-13Ra2 antibodies or antigen-binding fragments thereof. In some embodiments, at least one of the IL-13 mutant proteins or IL-13Ra2 antibodies or antigen-binding fragments thereof is linked to the IL-2 mutant protein of the IL-2 moiety via PSL. In some embodiments, at least one of the IL-13 mutant proteins or IL-13Ra2 antibodies or antigen-binding fragments thereof is linked to a region of the IL-2 moiety that is not an IL-2 mutant protein (e.g., serum albumin or the Fc region).

[0265] 1. IL-13 mutant protein

[0266] In the embodiment, compared with wild-type human IL-13:

[0267] Compared to PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN (SEQ ID NO: 200), the IL-13 mutant protein contains one or more amino acid substitutions.

[0268] Suitable IL-13 mutant proteins include those that reduce the activity of the IL-2 moiety when linked to an IL-2 fusion protein via PSL. In some embodiments, the IL-13 mutant protein is capable of binding to the IL-13Rα2 receptor. In some embodiments, compared to wild-type IL-13 (e.g., SEQ ID NO: 200), the IL-13 mutant protein exhibits enhanced binding to the IL-13Rα2 receptor and decreased binding to the IL-13Rα1 receptor. In an exemplary embodiment, the IL-13 mutant protein is capable of binding to the IL-13Rα2 receptor but not to the IL-13Rα1 receptor.

[0269] In some embodiments, the IL-13 mutant protein of the subject IL-2 fusion protein comprises one or more of the following amino acid substitutions: (1) L10F, L10I, L10V, L10A, L10D, L10T, L10H; (2) R11S, R11N, R11H, R11L, R11I; (3) I14L, I14F, I14V, 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, L101I, L101Y, L101H, L101N; (12) K104R, K104T, K104M; (13) K105T, K105A, K105R, K105E; (14) F107L, F107I, F107V, F107M; (15) R108K, R108T, R108M; and (16) E15R, wherein the substitutions result in a change of affinity for one or both of IL-13Rα1 and IL-13Rα2. In other embodiments, the modified residues are located at two or more, three or more, four or more, five or more, and no more than 14 amino acids within the set of contact residues defined above. As described in International Patent Publication WO 2013 / 112871, the disclosure of which is incorporated herein by reference in its entirety.

[0270] Compared to wild-type human IL-13 (SEQ ID NO: 200), the modification set may include the following specific variations: (1) L10H; L10A; (2) R11L; (4) V18I; (7) R86M; R86K; R86T; (8) D87K; D87G; (9) T88R, T88S; T88K; (10) K89R; (11) L101N; (12) K104R; (13) K105A; K105E; (14) R108K; (15) E15R. In some embodiments, the modification includes any one of the listed specific variations. In some embodiments, the modification includes L10H. In some embodiments, the modification includes L10A. In some embodiments, the modification includes R11L. 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 L101N. 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.

[0271] In some embodiments, the IL-13 mutant protein of the IL-2 fusion protein exhibits greater selectivity for binding to IL-13Rα2 compared to IL-13Rα1, relative to the wild-type IL-13 sequence (SEQ ID NO: 200). The specific set of modifications that provide greater selectivity for binding to IL-13Rα2 than IL-13Rα1 relative to the wild-type IL-13 sequence may include, but is not limited to:

[0272] [L10H, E15R, R86T, D87G, T88R, R108K] (mDNA132 + E15R or mDNA132.15, e.g., SEQ ID NO: 228)

[0273] [L10H, R86T, D87G, T88R, R108K] (C11, e.g., SEQ ID NO: 229)

[0274] [L10D, R11I, V18I, R86K, D87K, K89R, R108K] (e.g., C2, e.g., SEQ ID NO: 224)

[0275] [L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, R108K] (e.g., C3, e.g., SEQ ID NO: 225)

[0276] [L10V, K89R, L101N, K105E, R108T] (e.g., C4, e.g., SEQ ID NO: 211)

[0277] [R11S, I14M, T88S, L101N, K105A, R108K] (e.g., C7, e.g., SEQ ID NO: 212)

[0278] [L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, R108K] (C9, e.g., SEQ ID NO:226)

[0279] [L10A, V18F, R86K, D87K, K89R, L101I, K104R, R108K] (D7, e.g., SEQ ID NO: 231)

[0280] [L10T / D; R11I; V18I; R86K; D87K / G; T88S; K89R; L101Y; K104R; K105T; R108K]

[0281] [L10A / V; R86T; D87G; T88K; K89R; L101N; K104R; K105A / E; R108K / T]

[0282] In some embodiments, the modification set includes L10H, R86T, D87G, T88R, and R108K (C11, e.g., SEQ ID NO: 229). In some embodiments, the modification set includes L10V, K89R, L101N, K105E, and R108T (C4, e.g., SEQ ID NO: 211). In some embodiments, the modification set includes R11S, I14M, T88S, L101N, K105A, and R108K (C7, e.g., SEQ ID NO: 212). In some embodiments, the modification set includes L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, and R108K (C9, e.g., SEQ ID NO: 226). In some embodiments, the modification set includes L10H, E15R, R86T, D87G, T88R, and R108K (mDNA132 + E15R, e.g., SEQ ID NO: 228). In some embodiments, the modification set includes L10A, V18F, R86K, D87K, K89R, L101I, K104R, and R108K (D7, e.g., SEQ ID NO: 231). In some embodiments, the modification set includes L10T / D, R11I, V18I, R86K, D87K / G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the modification set includes L10T, R11I, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the modification set includes L10T, R11I, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the modification set includes L10D, R11I, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the modification set includes L10D, R11I, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the modification set includes L10A / V, R86T, D87G, T88K, K89R, L101N, K104R, K105A / E, and R108K / T. In some embodiments, the modification set includes L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108K. In some embodiments, the modification set includes L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108K.In some embodiments, the modification set includes L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108T. In some embodiments, the modification set includes L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108T. In some embodiments, the modification set includes L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108K. In some embodiments, the modification set includes L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108K. In some embodiments, the modification set includes L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and dR108T. In some embodiments, the modification set includes L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108T.

[0283] In some embodiments, the IL-13 mutant protein of the IL-2 fusion protein exhibits greater selectivity for binding to IL-13Rα1 than IL-13Rα2 relative to the wild-type IL-13 sequence (e.g., SEQ ID NO: 200). The specific set of modifications that provide greater selectivity for binding to IL-13Rα1 than IL-13Rα2 relative to the wild-type IL-13 sequence may include, but is not limited to:

[0284] [L10V, V18I, D87S, D88S, L101F, K104R, K105T]

[0285] [R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T]

[0286] [L10V, V18I, D87S, T88S, L101F, K104R, K105T]

[0287] [L10V / I; D87S; T88S; K89R; L101H / F; K104R; K105T]

[0288] [L10I; V18I; R86T; D87G; T88S; K89R; L101Y / H; K104R; K105A]

[0289] [L10V; V18I; D87S; T88S; L101F; K104R; K105T]

[0290] [V18I, R86T, D87G, T88S, L101Y, K104R, K105A]

[0291] [R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M]

[0292] The substitution is optionally combined with the substitutions [E12A / G / S, R65D / E].

[0293] In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, and K105T. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, and K105T. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, and K105T. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, and K105A. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, and K105A. In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, and F107M. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A / G / S, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A / G / S, and R65D / E.In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A / G / S, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65D / E. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D / E. In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65D / E. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65D / E. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D / E.In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65D / E. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D / E. In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D / E. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65D / E. In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D / E. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D.In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65D. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D. In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65D. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65D. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65D. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65D. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D. In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D.In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12S, and R65D. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65D. In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65E. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65E.In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65E. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65E. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65E. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65E. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65E. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65E. In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65E. In some embodiments, the modification set includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the modification set includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65E. In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the modification set includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65E. In some embodiments, the modification set includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65E.In some embodiments, the modification set includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the modification set includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65E. In some embodiments, the modification set includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65E. In some embodiments, the modification set includes L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T (see, for example, IL-13dn; SEQ ID NO:232).

[0294] Table 7 shows suitable exemplary IL-13 mutant proteins applicable to the IL-2 fusion proteins described herein.

[0295] Table 7: Wild-type and mutant IL-13 proteins

[0296]

[0297]

[0298]

[0299]

[0300]

[0301] In some embodiments, the IL-13 mutant protein 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 with one of the IL-13 mutant proteins in Table 7. In some embodiments, the IL-13 mutant protein of the IL-2 fusion protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to any of the IL-2 mutant proteins in Table 7 (SEQ ID NO: 200-241). In embodiments, the IL-13 mutant protein exhibits greater selectivity for binding to IL-13Rα2 than IL-13Rα1 relative to the wild-type IL-13 sequence (e.g., SEQ ID NO: 200).

[0302] 2. IL-13 mutant protein with extended half-life

[0303] In some embodiments, the IL-13 mutant protein of the subject IL-2 fusion protein 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 the Fc region of serum albumin (e.g., human serum albumin), PEG, a PEG derivative, or an IgG subclass lacking the variable region of the IgG heavy chain.

[0304] In some embodiments, the half-life-enhancing peptide is an Fc region. The Fc region may include the entire Fc region or a smaller portion thereof, which retains the ability to extend the circulating half-life of the IL-2 fusion protein to which it resides. Furthermore, the full-length or fragmented Fc region may be a variant of the wild-type molecule. In some embodiments, the IL-13 mutant protein is linked to an IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region is a human IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the Fc region includes a monomer comprising a CH2-CH3 domain of a human IgG1, IgG2, IgG3, or IgG4 region. In some embodiments, the Fc region is dimerized and comprises two monomers, each of which comprises a CH2-CH3 domain of a human IgG1, IgG2, IgG3, or IgG4 region. In some embodiments, the Fc region comprises two monomers, each comprising an IL-13 mutant protein linked to an Fc region monomer comprising a CH2-CH3 domain. In such embodiments, the Fc region monomers dimerize to form a dimerized Fc region linked to two IL-13 mutant proteins. In embodiments including the dimer Fc region, each of the two monomers may include an amino acid substitution that favors the formation of the heterodimer (e.g., a "mortar and pestle" or "mortar and pestle" amino acid substitution, see, for example, U.S. Patent No. 8,216,805, which is incorporated herein by reference for its disclosure regarding "mortar and pestle" amino acid substitution). In some embodiments, the Fc region contains a substituted N297A.

[0305] In some embodiments, the half-life-enhancing polypeptide is serum albumin. In an exemplary embodiment, the serum albumin is human serum albumin or a variant thereof capable of extending the serum half-life of the linked IL-13 mutant protein. In some embodiments, human serum albumin has the following amino acid sequence:

[0306] (SEQ ID NO:400).

[0307] In some embodiments, the IL-13 mutant protein is directly or indirectly linked to the serum half-life enhancing peptide. In some embodiments, the half-life enhancing peptide is linked to the N-terminus of the IL-13 mutant protein. In some embodiments, the half-life enhancing peptide is linked to the C-terminus of the IL-13 mutant protein. In some embodiments, the IL-13 mutant protein is directly linked to the half-life enhancing peptide. In some embodiments, the IL-13 mutant protein is linked to the half-life enhancing peptide via a linker peptide. Any suitable linker may be used to link the IL-13 mutant protein to the half-life enhancing peptide, including the linkers described herein (e.g., the gly-ser linker described herein).

[0308] Table 8 shows exemplary IL-13 mutant proteins comprising a half-life-enhancing peptide for the subject matter IL-2 fusion peptide described herein. In some embodiments, the IL-13 mutant protein is linked to the half-life-enhancing peptide and has the amino acids of one of SEQ ID NO: 242-281. In some embodiments, the IL-13 mutant protein is linked to the half-life-enhancing peptide and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with one of the sequences in Table 8 (SEQ ID NO: 242-281). In some embodiments, the IL-13 mutant protein is linked to the half-life-enhancing peptide and has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to any of the sequences in Table 8 (SEQ ID NO: 242-281). In the examples, the IL-13 mutant protein was linked to a half-life-enhancing peptide and exhibited greater selectivity for binding to IL-13Rα2 than IL-13Rα1 relative to the wild-type IL-13 sequence (e.g., SEQ ID NO: 200).

[0309] Table 8: IL-13 Extended Half-Life Fusion Compounds

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] 3. IL-13 mutant protein / antibody fusion

[0322] In other embodiments, the IL-13 mutant protein of the IL-2 fusion protein provided herein includes an antibody or its antigen-binding portion.

[0323] In some embodiments, the IL-13 mutant protein / antibody fusion comprises three polypeptides. The first polypeptide comprises an IL-13 mutant protein linked to a first Fc region. The second polypeptide comprises a heavy chain variable region (VH) and a second Fc region. The third polypeptide comprises a light chain variable region (VL) and a light chain constant region. In this embodiment, the two Fc regions of the first and second polypeptides dimerize, and the VH and VL form an antigen-binding domain.

[0324] In some embodiments, the IL-13 mutant protein / antibody fusion comprises four polypeptides. A first polypeptide comprises a first antibody heavy chain linked to the IL-13 mutant protein, wherein the first antibody heavy chain includes a first heavy chain variable region (VH1) and a first Fc region. A second polypeptide comprises a second antibody heavy chain, wherein the second antibody heavy chain includes a second heavy chain variable region (VH2) and a second Fc region. A third polypeptide comprises a first variable light chain variable region (VL1) and a light chain constant region. A 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 VH1 and VL1, as well as VH2 and VL2, each form an antigen-binding domain.

[0325] An antibody or antigen-binding component is linked to the IL-13 mutant protein using any suitable technique. In some embodiments, the antibody or antigen-binding component is directly linked to the IL-13 mutant protein. In some embodiments, a connector is used to link the antibody or antigen-binding component to the IL-13 mutant protein. Any suitable connector, including the connector described herein, can be used to link the IL-13 mutant protein to the antibody or antigen-binding component. In embodiments, the connector is of formula (GGGGS). n The linker peptide, of which n It is an integer between 1 and 10. In some embodiments, the connector is GGGGSGGGGS (SEQ ID NO: 402). In some embodiments, the connector is GGGGSGGGGSGGGGS (SEQ ID NO: 403). In some embodiments, the connector is GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 404). In some embodiments, the connector is GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 405). In some embodiments, the IL-13 mutant protein is linked to the C-terminus of the Fc domain of the antibody or antigen-binding component. In some embodiments, the IL-13 mutant protein is linked to the N-terminus of the antibody or antigen-binding component.

[0326] In some embodiments, the IL-13 mutant protein comprises a fusion with an antibody or its antigen-binding portion, the fusion disrupting the interaction between the PD-1 receptor and its ligand PD-L1, and / or an antibody targeting a component of the PD-1 / PD-L1 signaling pathway. Exemplary IL-13 mutant protein / anti-PD1 fusions are depicted in Table 9 below.

[0327] Table 9: IL-13 anti-PD1 fusion complex

[0328]

[0329]

[0330]

[0331]

[0332]

[0333] 4. IL-13Ra2 antibody

[0334] In some embodiments, the subject IL-2 masking portion comprises an IL-13Ra2 antibody or an antigen-binding fragment thereof. Suitable IL-13Ra2 antibodies or antigen-binding fragments thereof include, but are not limited to, SAB1406031 (Sigma), HPA067363 (Sigma), AV53557 (Sigma), WH0003598M1 (Sigma), AP1145 (Sigma), and E7U7B (Cell Signaling Technologies). The documents disclosed in Signaling, 2H25L68 (Thermo Fisher Scientific), 018 (Thermo Fisher Scientific), PA5-96045v (Thermo Fisher Scientific), PA5-106798 (Thermo Fisher Scientific), PA5-46976 (Thermo Fisher Scientific), PA5-44066 (Thermo Fisher Scientific), PA5-47732 (Thermo Fisher Scientific), PA5-143269 (Thermo Fisher Scientific), PA5-86898 (Thermo Fisher Scientific), PA5-87205 (Thermo Fisher Scientific), BS-2461R (Thermo Fisher Scientific), 2E10 (Thermo Fisher Scientific), and WO2008146911 and WO2014072888, are incorporated herein by reference in their entirety. As used herein, “its antigen-binding fragment” refers to an antigen-binding fragment of an antibody, that is, an antibody fragment that retains the ability to specifically bind to the antigen to which the full-length antibody is bound, such as a fragment retaining one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; biantibodies; single-chain antibody molecules, such as sc-Fv; nanobodies; and multispecific antibodies formed from antibody fragments.

[0335] 5. Other components

[0336] In some embodiments, the topic IL-2 masking portion further includes an extracellular domain of CD122, CD132, or CD25.

[0337] Suitable extracellular domains of CD122, CD132, or CD25 include those that can reduce the activity of the IL-2 moiety when linked to an IL-2 fusion protein via PSL.

[0338] In some embodiments, the extracellular domain of CD122 has the following amino acid sequence:

[0339] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAP ISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT (SEQ IDNO: 350).

[0340] In some embodiments, the extracellular domain of CD132 has the following amino acid sequence:

[0341] LPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWA PENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKEN (SEQ ID NO: 351).

[0342] In some embodiments, the extracellular domain of CD25 has the following amino acid sequence:

[0343] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 352).

[0344] C. Protease-sensitive linker (PSL)

[0345] The IL-2 fusion protein described herein includes a protease-sensitive linker (PSL) that links an IL-2 moiety to an IL-13 mutant protein. In some embodiments, the PSL is capable of being cleaved in the tumor microenvironment, thereby demasking the IL-2 moiety. Demasking the IL-2 moiety then promotes antitumor activity in the tumor microenvironment. Exemplary PSLs that may be included in the subject IL-2 fusion protein 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).

[0346] PSLs may contain one or more protease cleavage sites or be sensitive to cleavage by oxidation and / or reduction. In one example, a peptide linker may be used that is readily cleaved by enzymes of the complement system, urokinase, tissue plasminogen activator, trypsin, plasmin, caspase, kallikrein, cathepsin, podocyte oleanolic acid, MMP, thrombin, urokinase-type plasminogen activator (uPA), mastrotaase, PSA, or another proteolytically active enzyme. According to another example, the linker may contain a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). According to another example, PSL can contain protease-cleavable Val-Cit (VC) linkers, Phe-Arg linkers, Val-Lys linkers, Val-Ala linkers, Val-Arg linkers, Val-Leu-Lys linkers, Gly-Phe-Leu-Gly linkers, Ala-Phe-Lys linkers, pol-L-lysine linkers, β-Ala-Leu-Ala-Leu linkers, Arg-Arg-Ala-Leu-Ala-Leu linkers, peptide mimic linkers, podin-cleavable Ala–Ala–Asn tripeptide linkers, peptide linkers cleaved by cathepsin B and other lysosomal proteases (such as Gly–Phe–Leu–Gly and Ala–Leu–Ala–Leu, caspase 3 DEVD sequences), or self-canceling linkers. This disclosure contemplates, for example, the adapter disclosed in Poreba, M, *Journal of the Federation of European Biochemical Societies* 287(10):1936-1969 (2020), which is incorporated herein by reference. Many tumors naturally release high levels of glutathione (a reducing agent), which can reduce disulfide bonds and subsequently release cargo portions at the delivery site. In some embodiments, PSL is a protease-cleavable adapter, a adapter that can be cleaved by matrix metalloproteinases (MMPs). MMPs are overexpressed in situ at tumor sites, and this disclosure contemplates adapters cleavable in such cases. For example, Hsu, EJ et al., *Nature* communicationThe connectors disclosed in 12(2768):1-13 (2021) are incorporated herein by reference. In some embodiments, the MMP connector 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 NO: 410). In some embodiments, the MMP connector sequence is SGARYRWLTA (SEQ ID NO: 411). In some embodiments, the MMP connector sequence is SGRSYAILTA (SEQ ID NO: 412). In some embodiments, the MMP connector sequence is SRSGRSPAIFTATG (SEQ ID NO: 413). In some embodiments, the MMP connector sequence is GSSGRSPAIFTAGS (SEQ ID NO: 414). In some embodiments, the MMP connector sequence is SGFIANPVTA (SEQ ID NO: 415). In some embodiments, the MMP connector sequence is PLGLVVAPLGLVVAPLGLVVA (SEQ ID NO: 416). In some embodiments, the MMP connector sequence is PLGLWAPLGLWAPLGLWA (SEQ ID NO: 417). In some embodiments, the MMP connector sequence is GGSGGTPLGLWAGGSGGT (SEQ ID NO: 418). In some embodiments, the MMP connector sequence is GGSGGTPAGLIGGGSGGT (SEQ ID NO: 419). In some embodiments, the MMP connector sequence is GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NO: 420). In some embodiments, the PSA connector sequence is GGSGGTHSSKLQGGSGGT (SEQ ID NO: 421).

[0347] D. Exemplary IL-2 fusion protein

[0348] In some embodiments, the IL-2 fusion protein comprises the IL-2 portion of the IL-2 mutant protein according to SEQ ID NO: 5 (MDNA109) or SEQ ID NO: 9 (MDNA109FEAA). In some embodiments, the IL-2 mutant protein is a variant of MDNA109 (SEQ ID NO: 5) or MDNA109FEAA (SEQ ID NO: 9) further comprising T3A and / or C125S substitutions.

[0349] In some embodiments, the IL-2 portion comprises a human albumin polypeptide, optionally wherein the human albumin is recombinant human albumin. In exemplary embodiments, the IL-2 portion comprises an anti-PD-1 antibody or its antigen-binding portion. In some IL-2 portions, a half-life-enhancing polypeptide such as human serum albumin polypeptide or an Fc region is linked.

[0350] In some embodiments, the IL-2 fusion protein comprises IL-13 having the amino acid sequence of MDNA132 (SEQ ID NO: 216) or MDNA132.15 (SEQ ID NO: 228). In an exemplary embodiment, 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).

[0351] Exemplary IL-2 fusion proteins are shown in Tables 10a and 10b.

[0352] Table 10a: Exemplary topic: IL-2 fusion protein

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377] Table 10b: Description and basic principles of the construct designed for testing masking efficiency

[0378]

[0379]

[0380]

[0381]

[0382] E. Nucleic acid molecules encoding IL-2 fusion protein

[0383] In some embodiments, the subject IL-2 fusion protein (as described above) can be obtained by expressing one or more nucleic acid molecules. For example, in an embodiment where the IL-2 fusion protein is a single polypeptide, the IL-2 fusion protein is encoded by a single nucleic acid molecule. In other embodiments, the IL-2 fusion protein consists of two or more polypeptides, each of which is encoded by a nucleic acid molecule.

[0384] The provided nucleic acid molecules may contain naturally occurring sequences or sequences different from those naturally occurring sequences but encoding the same polypeptide due to the degeneracy of the genetic code. These nucleic acid molecules may consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as DNA produced through phosphoramide-based synthesis) or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, nucleic acid molecules may be double-stranded or single-stranded (i.e., sense strands or antisense strands).

[0385] Nucleic acid molecules are not limited to sequences encoding polypeptides; they can also include some or all of the non-coding sequences located upstream or downstream of the coding sequence (e.g., the coding sequences for IL-2 or IL-13). Those generally skilled in molecular biology are familiar with routine procedures for isolating nucleic acid molecules. For example, they can be generated by treating genomic DNA with restriction endonucleases or by performing polymerase chain reaction (PCR). In the case where the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be generated, for example, through in vitro transcription.

[0386] The exemplary isolated nucleic acid molecules of this disclosure may include fragments not found in their natural state. Therefore, this disclosure covers recombinant molecules, such as those in which a nucleic acid sequence (e.g., a sequence encoding a mutant IL-2 or IL-13) is incorporated into a vector (e.g., a plasmid or viral vector) or the genome of a heterologous cell (or the genome of a homologous cell, at a location other than its natural chromosomal location).

[0387] Subject nucleic acid molecules may contain sequences encoding "markers" or "reporter genes." Examples of markers or reporter genes include β-lactamases, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), and aminoglycoside phosphotransferase (Neo-β-lactamase). r G418 r The enzymes used include dihydrofolate reductase (DHFR), hygromycin-β-phosphotransferase (HPH), thymidine kinase (TK), lacz (encoding β-galactosidase), and xanthine-guanine phosphoribosyltransferase (XGPRT). Those skilled in the art will recognize other useful agents, such as additional sequences that can function as markers or reporter genes.

[0388] The subject nucleic acid (and its encoded polypeptide) can be a nucleic acid from a mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog, or cat. In one embodiment, the nucleic acid molecule will be a human nucleic acid molecule.

[0389] F. Expression of mutant IL-2, IL-4, or IL-13 gene products

[0390] The nucleic acid molecules described above can be contained in a vector that can direct the expression of these nucleic acid molecules in cells, for example, transduced with the vector. Therefore, in addition to the subject IL-2 fusion protein, expression vectors containing one or more nucleic acid molecules encoding the subject IL-2 fusion protein and cells transfected with these expression vectors are also in preferred embodiments.

[0391] Of course, it should be understood that not all vectors and expression control sequences function equally well to express the DNA sequences described herein. Not all hosts will function equally well in the same expression system. However, those skilled in the art can select from these vectors, expression control sequences, and hosts without excessive experimentation. For example, when selecting a vector, the host must be considered, as the vector must replicate within it. The copy number of the vector, the ability to control the 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 amplify at copy number. Such amplifiable vectors are well known in the art. These include, for example, amplification via DHFR (see, for example, Kaufman, U.S. Patent Nos. 4,470,461, Kaufman and Sharp, “Construction of a Modular Dihydrafolate Reductase cDNA: Analysis of Signals Utilized for Efficient Expression”, Molecular Cell Biology, 2, pp. 1304-19 (1982)) or amplification via glutamine synthase (“GS”). See For example, U.S. Patent No. 5,122,464 and European Publication No. 338,841) are used to amplify vectors.

[0392] In some embodiments, the subject IL-2 fusion protein is expressed from a vector, preferably an expression vector. The vector can be used for autonomous replication in the host cell, or it can be integrated into the host cell's genome after introduction into the host cell and thereby replicate along with the host genome (e.g., a non-free mammalian vector). The expression vector directs the expression of the coding sequence operatively linked to it. Generally, expression vectors used in recombinant DNA technologies are typically in plasmid (vector) form. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0393] Exemplary recombinant expression vectors may include one or more regulatory sequences selected based on the host cell to be used for expression, which are operatively linked to the nucleic acid sequence to be expressed.

[0394] Expression constructs or vectors can be designed to express IL-2 fusion proteins in prokaryotic or eukaryotic host cells.

[0395] Vector DNA can be introduced into prokaryotic or eukaryotic cells using conventional transformation or transfection techniques. Appropriate methods for transforming or transfecting host cells can be found in Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory Press, Plainview, NY), and other standard molecular biology laboratory manuals.

[0396] Protein expression in prokaryotes is typically performed in *E. coli* using vectors containing constitutive or inducible promoters. Strategies for maximizing recombinant protein expression in *E. coli* can be found, for example, in Gottesman (1990), *Gene Expression Techniques: Enzymatic Approaches*, 185 (Academic Press, San Diego, CA), pp. 119-128, and in Wada et al. (1992), *Nucleic Acid Research*, 20: 2111-2118. Procedures for culturing, harvesting, disrupting, or extracting IL-2 mutant proteins or variants thereof from cells are described in detail in, for example, U.S. Patent 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, all of which are incorporated herein by reference in their entirety.

[0397] In some embodiments, the recombinant IL-2 fusion protein can also be prepared in eukaryotes such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors that can be used to express proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Molecular Cell Biology 3:2156-2165 and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors used for expression in Saccharomyces cerevisiae include pYepSec1 (Baldari et al. (1987) Journal of the European Society for Molecular Biology 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), and pJRY88 (Schultz et al. (1987) Gene). 54:113-123), pYES2 (Invitrogen Corporation, San Diego, California) and pPicZ (Invitrogen Corporation, San Diego, California)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) Journal of the European Society for Molecular Biology 6:187:195)). Suitable mammalian cells include Chinese hamster ovary cells (CHO) or COS cells. In mammalian cells, the control function of the expression vector is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyomavirus, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for prokaryotic and eukaryotic cells, see Chapters 16 and 17 (1989) of Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory, Plainview, NY). See Goeddel (1990) Gene Expression Technology: Enzymatic Approaches, 185 (San Diego Academic Press, California).

[0398] The sequence encoding the disclosed IL-2 fusion protein can be optimized for expression in host cells of interest. The GC content of the sequence can be adjusted to the average level of a given host cell, such as by calculating it using 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 host cells such that approximately 1%, approximately 5%, approximately 10%, approximately 25%, approximately 50%, approximately 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a specific host cell.

[0399] Suitable vectors include T7-based vectors for bacteria ( See For example, Rosenberg et al., Gene 56:125, 1987, pMSXND expression vector for mammalian cells (Lee and Nathans, Journal of Biochemistry 263:3521, 1988) and baculovirus-derived vectors for insect cells (e.g., pBacPAK9 expression vector from Clontech, Palo Alto, California).

[0400] In some embodiments, the nucleic acid insert encoding the subject IL-2 fusion protein in such vectors can be operatively linked to a promoter selected based, for example, the cell type for which expression is sought.

[0401] Several factors should be considered when selecting expression control sequences. These factors 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 potential secondary structures. Host selection should also consider host compatibility with the chosen vector, the toxicity of the products encoded by the DNA sequences of this invention, their secretion characteristics, their ability to correctly fold polypeptides, their fermentation or culture requirements, and the ease of purification of the products encoded by the DNA sequences.

[0402] Within these parameter ranges, those skilled in the art can select various vector / expression control sequence / host combinations to express the desired DNA sequence in fermentation or large-scale animal culture, such as using CHO cells or COS 7 cells.

[0403] In some embodiments, the choice of expression control sequences and expression vectors will depend on the choice of host. Multiple expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors having expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from *E. coli*, including colE1, pCRI, pER32z, pMB9, and their derivatives; plasmids with broader host ranges, such as RP4; phage DNA, such as many derivatives of phage λ, such as NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells include 2 Plasmids and their derivatives. 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).

[0404] Furthermore, any of a variety of expression control sequences can be used in these vectors. Such useful expression control sequences include those associated with the structural genes of the aforementioned expression vectors. Examples of useful expression control sequences include, for example, early and late promoters of SV40 or adenoviruses, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of bacteriophage λ such as PL, the control region of the fd capsid protein, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatases such as PhoA, promoters of yeast α-mating systems, polyhedral promoters of baculoviruses, and other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0405] The T7 promoter can be used in bacteria, the polyhedrosis protein promoter in insect cells, and the cytomegalovirus or metallothionein promoter in mammalian cells. Furthermore, in higher eukaryotes, tissue-specific and cell-type-specific promoters are widely available. These promoters are so named because they direct the expression of nucleic acid molecules in a given tissue or cell type in vivo. Skilled technicians are well aware of the many promoters and other regulatory elements that can be used to direct nucleic acid expression.

[0406] In addition to sequences that promote transcription of the inserted nucleic acid molecules, vectors can contain other genes encoding origin of replication and selectivity markers. For example, neomycin resistance (neomycin resistance...) r The gene confers G418 resistance to cells that express it, and thus allows for phenotypic selection of transfected cells. Those skilled in the art can readily determine whether a given regulatory element or selectivity marker is suitable for a particular experimental setting.

[0407] Viral vectors that can be used in this invention include, for example, retroviruses, adenoviruses and adeno-associated vectors, herpesviruses, simian virus 40 (SV40) and bovine papillomavirus vectors (…). See For example, Gluzman (ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY.

[0408] Prokaryotic or eukaryotic cells containing and expressing nucleic acid molecules encoding the IL-2 mutant protein disclosed herein are also characteristic of this invention. The cells of this invention are transfected cells, i.e., cells into which nucleic acid molecules, such as those encoding IL-2, IL-4, or IL-13 mutant proteins or bifunctional molecules, have been introduced via recombinant DNA technology. Progeny of such cells are also considered to be within the scope of this invention.

[0409] The exact components of the expression system are not important. For example, the IL-2 fusion protein can be produced in a prokaryotic host such as the bacterium *Escherichia coli*, or in a eukaryotic host such as insect cells (e.g., Sf21 cells) 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, Virginia). When choosing an expression system, only the compatibility between the components is important. This decision can be made by a technician or a person with general technical skills. In addition, if guidance is needed when selecting an expression system, technicians can refer to Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Supplement 1987).

[0410] As described in this article, expressed peptides can be purified from the expression system using conventional biochemical procedures and can be used as, for example, therapeutic agents.

[0411] In some embodiments, depending on the host organism used to produce the mutant protein, the resulting IL-2 fusion protein will be glycosylated or non-glycosylated. If bacteria are chosen as the host, the resulting IL-2 fusion protein will be non-glycosylated. On the other hand, eukaryotic cells will glycosylate both IL-2 and IL-13 in the fusion protein, but may do so differently from the glycosylation of native IL-2 or IL-13. IL-2 fusion proteins produced by the transformed host can be purified according to any suitable method. Various methods are known for purifying IL-2 fusion proteins. See, for example, the Latest Laboratory Guide to Protein Science (…). Current Protocols in Protein Science ), Volume 2. Edited by John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, John W. & Son Publishing). IL-2 fusion proteins can be isolated from inclusion bodies produced in *E. coli* or from conditioned media of mammalian or yeast cultures producing a given mutant protein using cation exchange, gel filtration, and / or reversed-phase liquid chromatography.

[0412] Another exemplary method for constructing the DNA sequence encoding the subject IL-2 fusion protein is through chemical synthesis. This involves the direct chemical synthesis of peptides by means of a protein sequence encoding an IL-2 mutant protein exhibiting the aforementioned properties. This method can incorporate both natural and non-natural amino acids at the sites where the IL-2 or IL-13 mutant protein interacts with its corresponding receptor, affecting the IL-2 fusion protein. Alternatively, the gene for the IL-2 fusion protein can be chemically synthesized using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the desired amino acid sequence of the IL-2 fusion protein and preferably selected from those codons that are advantageous in the host cell from which the recombinant mutant protein will be produced. In this respect, it is well known that the genetic code is degenerate—an amino acid may be encoded by more than one codon. For example, Phe (F) is encoded by two codons, TIC or TTT, Tyr (Y) by TAC or TAT, and his (H) by CAC or CAT. Trp (W) is encoded by a single codon, TGG. Therefore, it should be understood that for a given DNA sequence encoding a particular IL-2 fusion protein, there will be many degenerate DNA sequences encoding said IL-2 fusion protein. For example, it should be understood that, in addition to the preferred DNA sequence of the mutant protein H9, there are many degenerate DNA sequences encoding the IL-2 fusion protein shown. These degenerate DNA sequences are considered to be within the scope of this disclosure. Therefore, in the context of this invention, "degenerate variants thereof" means all DNA sequences encoding a particular mutant protein and thus enabling its expression.

[0413] The biological activity of the IL-2 fusion protein can be determined by any suitable method known in the art. Such assays include PHA-embryonic cell proliferation and NK cell proliferation.

[0414] G. Treatment methods

[0415] In some embodiments, the subject IL-2 fusion protein and / or the nucleic acid expressing it may be administered to a subject to treat a condition associated with abnormal apoptosis or differentiation processes (e.g., cell proliferative disorders or cell differentiation disorders such as cancer, by, for example, generating active or passive immunity). In the treatment of such diseases, the disclosed IL-2 fusion protein may have advantageous properties, such as reducing vascular leakage syndrome.

[0416] Examples of proliferative and / or differentiated diseases include cancers (e.g., carcinomas, sarcomas, metastatic diseases, or hematopoietic neoplasia such as leukemia). Metastatic tumors may be caused by a variety of primary tumor types, including but not limited to those listed below: sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoma, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumors, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, stomach cancer, brain cancer, and CNS tumors. In some embodiments, the cancer being treated is a solid tumor. In some embodiments, the cancers treated include, but are not limited to, cancers that can generally include solid tumors as well as sarcomas, carcinomas, head and neck cancers, glioblastomas, bladder cancers, oral cancers, mesotheliomas, pancreatic cancers, liver cancers, colorectal cancers, lung cancers, skin cancers, lymphomas, gastrointestinal cancers, prostate cancers, ovarian cancers, breast cancers, basaloid breast tumors, endometrial cancers, multiple myelomas, melanomas, lymphomas, lung cancers (including small cell lung cancers), kidney cancers, stomach cancers, brain cancers, and CNS tumors. CNS tumors include gliomas, glioblastomas, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, meningiomas, neuroblastomas, retinoblastomas, medulloblastomas, adult pituitary adenomas, O6-methylguanine-methyltransferase (MGMT) positive or negative CNS tumors, and furin-positive CNS tumors.

[0417] The IL-2 fusion protein can be used to treat patients who have, are suspected of having, or may have a high risk of developing any type of cancer, including renal cell 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 cancers include those forming in the cervix, lungs, prostate, breast, head and neck, colon, and ovaries. The term also includes carcinosarcoma, a malignant tumor composed of cancerous and sarcomatous tissue.

[0418] Other examples of proliferative conditions include hematopoietic systemic neoplasia.

[0419] Alternatively, or in addition to methods of direct administration to a patient, in some embodiments, the subject IL-2 fusion protein can be used in an in vitro method. For example, cells (e.g., peripheral blood lymphocytes isolated from a patient or a purified population of lymphocytes placed or maintained in a culture) can be cultured in vitro in a culture medium, and the contact step can be influenced by adding the subject IL-2 fusion protein to the culture medium. The culture step may include additional steps in which the cells are stimulated or treated with other agents, for example, to stimulate proliferation or to expand a population of cells that are responsive to an antigen of interest (e.g., a cancer antigen or a viral antigen). These cells are then administered to a patient after treatment.

[0420] Anti-PD-1 antibodies used in combination with the subject IL-2 fusion protein disclosed herein for the treatment of cancer and / or proliferative disorders include, but are not limited to, nivolumab (OPDIVO). ® BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA) ® ), cimiprimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872).

[0421] In some embodiments, the subject IL-2 fusion protein is used in combination with nivolumab for cancer treatment. In some embodiments, the subject IL-2 fusion protein is used in combination with pembrolizumab for cancer treatment. In some embodiments, the subject IL-2 fusion protein is used in combination with BMS-936558 for cancer treatment. In some embodiments, the subject IL-2 fusion protein is used in combination with MDX-1106 for cancer treatment. In some embodiments, the subject IL-2 fusion protein is used in combination with ONO-4538 for cancer treatment. In some embodiments, the subject IL-2 fusion protein is used in combination with AMP224 for cancer treatment. In some embodiments, the subject IL-2 fusion protein is used in combination with CT-011 for cancer treatment. In some embodiments, the subject IL-2 fusion protein is used in combination with MK-3475 for cancer treatment.

[0422] In some embodiments, the subject IL-2 fusion protein is used in combination with antibodies and / or immunotherapies for the treatment of cancer. These antibodies and / or immunotherapies include, but are not limited to, anti-CTLA4 mAbs, such as ipilimumab and trimelimumab; anti-PD-L1 antagonist antibodies, such as BMS-936559 / MDX-1105, MEDI4736, and RG-7446 / MPDL3280A; anti-LAG-3 antibodies, such as IMP-321; and agonist antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs, such as CP-870, 893, lucaizumab, and dacetuzumab; and anti-CD137 mAbs. mAbs (anti-4-1-BB antibodies), such as BMS-663513 urelumab (anti-4-1BB antibody; see, for example, U.S. Patent Nos. 7,288,638 and 8,962,804, which are incorporated herein by reference in their entirety); lirilumab (anti-KIR mAb; IPH2102 / BMS-986015; blocking NK cell inhibitory receptors) and PF-05082566 (utomilumab; see, for example, U.S. Patent Nos. 8,821,867; 8,337,850; and 9,468,678; and International Patent Application Publication No. WO 2012 / 032433, which are incorporated herein by reference in their entirety); anti-OX40 mAb (see, for example, WO 2006 / 029879 or WO 2010 / 096418, which is incorporated herein by reference in its entirety; anti-GITR mAbs, such as TRX518 (see, for example, U.S. Patent No. 7,812,135, which is incorporated herein by reference in its entirety); anti-CD27 mAbs, such as varlilumab CDX-1127 (see, for example, WO 2016 / 145085 and U.S. Patent Publications Nos. US 2011 / 0274685 and US 2012 / 0213771, which are incorporated herein by reference in their entirety), anti-ICOS mAbs (e.g., MEDI-570, JTX-2011), and anti-TIM-3 antibodies (see, for example, WO 2013 / 006490 or U.S. Patent Publication No. US 2016 / 0257758, which are incorporated herein by reference in their entirety).

[0423] In some embodiments, the subject IL-2 fusion protein is used in combination with another antibody (which may include a monoclonal antibody) for the treatment of solid tumors. In some embodiments, the monoclonal antibody is used for the treatment of sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoma, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumors, 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 gliomas, glioblastomas, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, meningiomas, neuroblastomas, retinoblastomas, medulloblastomas, adult pituitary adenomas, O6-methylguanine-methyltransferase (MGMT) positive or negative CNS tumors, and furin-positive CNS tumors.

[0424] In some embodiments, the subject IL-2 fusion protein is used in combination with an antibody against antibody-dependent cell-mediated cytotoxicity (ADCC) for the treatment of cancer.

[0425] In some embodiments, the subject IL-2 fusion protein described herein is used in combination with an antibody, said antibody including dupilumab and nivolumab. ® BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA) ®), cimiprimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872). In some embodiments, mutant IL-2, IL-4, or IL-13 proteins and / or bifunctional molecules as described herein are used in combination with antibodies, said antibodies including anti-CTLA4 mAbs, such as ipilimumab and trimemumab; anti-PD-L1 antagonistic antibodies, such as BMS-936559 / MDX-1105, MEDI4736, and RG-7446 / MPDL3280A; anti-LAG-3 antibodies, such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs, such as CP-870,893, rucalizumab, and dasizumab; anti-CD137 mAbs (anti-4-1-BB antibodies), such as BMS-663513 urerutumab (anti-4-1BB antibody; see, for example, U.S. Patent Nos. 7,288,638 and 8,962,804, which are incorporated herein by reference in their entirety); and lirelurumab (anti-KIR). mAb; IPH2102 / BMS-986015; blocking NK cell inhibitory receptors) and PF-05082566 (utomilumab; see, for example, U.S. Patent Nos. 8,821,867; 8,337,850; and 9,468,678; and International Patent Application Publication No. WO 2012 / 032433, which are incorporated herein by reference in their entirety); anti-OX40 mAb (see, for example, WO 2006 / 029879 or WO2010 / 096418, which are incorporated herein by reference in their entirety); anti-GITR mAb, such as TRX518 ( SeeFor example, U.S. Patent No. 7,812,135, which is incorporated herein by reference in its entirety; anti-CD27 mAbs, such as lincomycin CDX-1127 (see, for example, WO 2016 / 145085 and U.S. Patent Publications Nos. US 2011 / 0274685 and US 2012 / 0213771, which are incorporated herein by reference in their entirety), anti-ICOS mAbs (e.g., MEDI-570, JTX-2011) and anti-TIM-3 antibodies (see, for example, WO 2013 / 006490 or U.S. Patent Publication No. US 2016 / 0257758, which are incorporated herein by reference in their entirety), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-sirs-α and / or anti-CD112R.

[0426] In some embodiments, the subject IL-2 fusion protein described herein is fused with an antibody selected from: dupilumab, nivolumab (OPDIVO2 max), etc. ® BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA) ®), cimiprimab (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 described herein is fused with an antibody selected from: anti-CTLA4 mAb, such as ipilimumab, trimemumab; 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 mAb, such as CP-870,893, rucalizumab, dasizumab; anti-CD137 mAb (anti-4-1-BB antibody), such as BMS-663513 urerutumab (anti-4-1BB antibody; see, for example, U.S. Patent Nos. 7,288,638 and 8,962,804, which are incorporated herein by reference in their entirety); lireruzumab (anti-KIR). mAb; IPH2102 / BMS-986015; blocking NK cell inhibitory receptors) and PF-05082566 (utomilumab; see, for example, U.S. Patent Nos. 8,821,867; 8,337,850; and 9,468,678; and International Patent Application Publication No. WO 2012 / 032433, which are incorporated herein by reference in their entirety); anti-OX40 mAb (see, for example, WO2006 / 029879 or WO 2010 / 096418, which are incorporated herein by reference in their entirety); anti-GITR mAb, such as TRX518 ( See For example, U.S. Patent No. 7,812,135, which is incorporated herein by reference in its entirety; anti-CD27 mAbs, such as lincomycin CDX-1127 (see, for example, WO 2016 / 145085 and U.S. Patent Publications Nos. US 2011 / 0274685 and US 2012 / 0213771, which are incorporated herein by reference in their entirety), anti-ICOS mAbs (e.g., MEDI-570, JTX-2011) and anti-TIM-3 antibodies (see, for example, WO 2013 / 006490 or U.S. Patent Publication No. US 2016 / 0257758, which are incorporated herein by reference in their entirety), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-sirs-α and / or anti-CD112R.

[0427] H. Pharmaceutical Composition and Administration

[0428] In some embodiments, the subject IL-2 fusion protein may be incorporated into the composition, including a pharmaceutical composition. Such compositions typically comprise a peptide or nucleic acid molecule and a pharmaceutically acceptable carrier. Such compositions may also contain an anti-PD-1 antibody. In some embodiments, the composition comprises an IL-2 mutant protein, said IL-2 mutant protein being a fusion protein and / or associated with a CAR-T construct and / or expressed by or associated with an oncolytic virus.

[0429] The anti-PD-1 antibody and the subject IL-2 fusion protein can be administered as a co-composition, as two separate compositions simultaneously, and / or as two separate compositions sequentially. In some embodiments, the anti-PD-1 antibody or inhibitor and the subject IL-2 fusion protein are administered together as a single co-composition (i.e., co-formulated). In some embodiments, the anti-PD-1 antibody or inhibitor and the subject IL-2 fusion protein are administered simultaneously as two separate compositions (i.e., separate formulations). In some embodiments, the anti-PD-1 antibody or inhibitor and the subject IL-2 fusion protein are administered sequentially as separate compositions (i.e., separate formulations). In some embodiments, when the anti-PD-1 antibody or inhibitor and the 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 the 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, anti-PD-1 antibodies include, but are not limited to, nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475.

[0430] Other immunotherapeutic agents and the IL-2 fusion protein, as described above, can be administered as a co-composition, as two separate compositions simultaneously, and / or as two separate compositions sequentially. In some embodiments, the other immunotherapeutic agents and the IL-2 fusion protein are administered together as a single co-composition (i.e., co-formulated). In some embodiments, the other immunotherapeutic agents and the IL-2 fusion protein are administered simultaneously as two separate compositions (i.e., separate formulations). In some embodiments, the other immunotherapeutic agents and the IL-2 fusion protein are administered sequentially as separate compositions (i.e., separate formulations). In some embodiments, when the other immunotherapeutic agents and the IL-2 fusion protein are administered sequentially as separate compositions, an anti-PD-1 antibody or inhibitor is administered before the IL-2 fusion protein. In some embodiments, when the other immunotherapeutic agents and the IL-2 fusion protein are administered sequentially as separate compositions, the IL-2 fusion protein is administered before the other immunotherapeutic agents.

[0431] The pharmaceutical composition is formulated to be compatible with its intended route of administration. Anti-PD-1 antibodies and / or IL-2 fusion proteins may be administered orally, but are more likely to be administered via 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 intended for parenteral administration may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or dextran. The pH may be adjusted with an acid or base (such as monosodium phosphate and / or disodium phosphate, hydrochloric acid, or sodium hydroxide) to (e.g., to a pH of about 7.2-7.8, such as 7.5). Parenteral preparations can be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0432] Suitable pharmaceutical compositions for injectable applications include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition should be sterile and should have a flowability sufficient for easy injection. It should be stable under the conditions of manufacture and storage and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants (e.g., sodium dodecyl sulfate). Microbial activity can be prevented by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyols like mannitol, sorbitol, and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0433] Sterile injectable solutions can be prepared by incorporating an active compound in the desired amount with one or a combination of the ingredients listed above in a suitable solvent, followed by filtration sterilization if necessary. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other desired ingredients from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which produce powders of the active ingredient and any other desired ingredients from their previous sterile filtered solutions.

[0434] Oral compositions (if used) typically include an inert diluent or an edible carrier. For oral therapeutic administration, the active compound may be incorporated into excipients and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions may also be prepared using liquid carriers used as mouthwashes. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel™, or corn starch; lubricants, such as magnesium stearate or Sterates™; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavorings.

[0435] In the case 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 a pressurized container or dispenser containing a suitable propellant, such as a gas (e.g., carbon dioxide) or from a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0436] Systemic administration of anti-PD-1 antibodies and / or IL-2 fusion proteins or nucleic acids can also be performed transmucosally or transdermally. For transmucosally or transdermally administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art and, for example, for transmucosally administration, include detergents, bile salts, and clostridial acid derivatives. Transmucosally administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated as an ointment, cream, gel, or lotion as commonly known in the art.

[0437] In some embodiments, the compounds (anti-PD-1 antibodies and / or IL-2 fusion proteins or nucleic acids) may also be formulated as suppositories (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0438] In some embodiments, the compound (the subject IL-2 fusion protein or nucleic acid) may also be administered via transfection or infection using methods known in the art, including but not limited to those described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnology 20:1006-1010, 2002), or Putnam (Am.J. Health Syst. Pharm. 53:151-160, 1996, Errata 53:325, 1996).

[0439] In one embodiment, an anti-PD-1 antibody and / or IL-2 fusion protein or nucleic acid is prepared together with a carrier that protects the anti-PD-1 antibody and / or IL-2 fusion protein from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811.

[0440] The dosage, toxicity, and therapeutic efficacy of such anti-PD-1 antibodies, IL-2 fusion proteins, or nucleic acid compounds can be determined through standard pharmaceutical procedures in cell cultures or laboratory animals, such as by determining the LD50. 50 (The dose that is lethal to 50% of the population) and ED 50 The dose effective for 50% of the population is determined by [the specific dose]. The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50. 50 / ED 50 Compounds exhibiting a high therapeutic index are preferred. Although compounds exhibiting toxic side effects can be used, care should be taken to design delivery systems that target such compounds to the site of the affected tissue, thereby minimizing potential damage to uninfected cells and thus reducing side effects.

[0441] A range of dosages for human use can be formulated using data obtained from cell culture assays and animal studies. The dosages of such compounds are preferably within the range of ED. 50The dosage is within a range of circulating concentrations with very low or no toxicity. The dosage can vary within this range depending on the dosage form and route of administration used. For any compound used in the methods of this invention, the therapeutically effective dose can be initially estimated based on cell culture assays. In animal models, the dose can be formulated to achieve IC50 values ​​as determined in cell culture. 50 The range of circulating plasma concentrations, including the concentration at which the test compound achieves half-maximal inhibition of symptoms. This information can be used to more accurately determine the effective dose in the human body. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0442] As defined herein, the therapeutically effective amount (i.e., effective dose) of the subject IL-2 fusion protein and / or anti-PD-1 antibody or inhibitor depends on the selected peptide or antibody. In some embodiments, a single dose of the IL-2 fusion protein may be administered in the range of about 0.001 mg / kg to 0.1 mg / kg of patient body weight. In some embodiments, a single dose of the anti-PD-1 antibody or inhibitor may be administered in the range of about 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. In some embodiments, doses of the anti-PD-1 antibody or inhibitor and / or IL-2 fusion protein may be administered at doses 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, or 10.0 mg / kg. In some embodiments, 600,000 IU / kg is administered (IU can be determined by lymphocyte proliferation bioassay and is expressed in International Units (IU) established by the World Health Organization International Standard 1 for Interleukin-2 (Human)). The dosage may be similar to, but is expected to be less than, that of PROLEUKIN®. The composition may be administered once or more daily to once or more weekly; including every other day. Those skilled in the art will understand that certain factors may influence the dosage and timing required for effective treatment of a subject, including but not limited to the severity of the disease or condition, prior treatment, the subject’s overall health and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the subject IL-2 fusion protein may include a single treatment or may include a series of treatments. In one embodiment, the composition is administered for five consecutive days every 8 hours, followed by a rest period of 2 to 14 days (e.g., 9 days), followed by another five consecutive days every 8 hours. In some embodiments, administration is performed as three doses every four days.

[0443] The pharmaceutical composition may be included in a container, package, or dispenser along with the instructions for use.

[0444] The following examples are provided to describe certain embodiments of the invention provided herein, and these examples should not be construed as limiting.

[0445] Other embodiments

[0446] In this embodiment, an IL-2 cytokine fusion protein is provided, comprising: (a) an IL-2 moiety comprising IL-2 or an IL-2 mutant protein, optionally an IL-2 mutant protein fusion; (b) a protease-sensitive linker (PSL); and (c) an IL-2 masking moiety, wherein the PSL links the IL-2 masking moiety to the IL-2 moiety.

[0447] In this embodiment, an IL-2 cytokine fusion protein is provided, comprising: (a) an IL-2 moiety comprising IL-2 or an IL-2 mutant protein, optionally an IL-2 mutant protein fusion; (b) at least one protease-sensitive linker (PSL); and (c) at least one IL-2 masking moiety; wherein the at least one PSL links the IL-2 masking moiety to the at least one IL-2 moiety.

[0448] In an embodiment, the masking portion includes IL-13, an IL-13 mutant protein, an IL-13Ra2 binding mutant protein, an IL-13Ra2 antibody or an antigen-binding fragment thereof, wherein the IL-2 masking portion is capable of binding to IL-13Ra2 but not to IL-13Ra1.

[0449] In an embodiment, the masking portion comprises an IL-13 mutant protein having an amino acid sequence of any of SEQ ID NO: 200-241.

[0450] In an embodiment, the masking portion comprises an IL-13 mutant protein with the following amino acid substitutions compared to wild-type human IL-13 (SEQ ID NO: 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.

[0451] In an embodiment, the masking portion comprises an IL-13 mutant protein having the amino acid sequence of SEQ ID NO: 216 or SEQ ID NO: 228.

[0452] In an embodiment, the masking portion further includes an extracellular domain of CD122, an extracellular domain of CD132, or an extracellular domain of CD25.

[0453] In an embodiment, the IL-2 portion comprises an IL-2 mutant protein having an amino acid sequence having any one of SEQ ID NO: 5-24 and 105.

[0454] In the embodiments, the IL-2 mutant protein has the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

[0455] In the embodiments, the IL-2 mutant protein further comprises T3A and C125S amino acid substitutions.

[0456] In an embodiment, the IL-2 portion further comprises albumin, an Fc domain, or an antibody linked to the IL-2 or an IL-2 mutant protein.

[0457] In an embodiment, the albumin is human albumin, optionally wherein the human albumin is recombinant human albumin.

[0458] In an embodiment, the IL-2 portion comprises an IL-2 x anti-PD1 fusion protein, the IL-2 x anti-PD1 fusion protein comprising: (a) a first polypeptide comprising IL-2 or an IL-2 mutant protein linked to a first Fc domain; (b) a second polypeptide comprising a 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 the VL form a PD-1 binding domain.

[0459] In this embodiment, the second polypeptide is an antibody heavy chain.

[0460] In an embodiment, the IL-2 portion comprises an IL-2 x anti-PD1 fusion protein, the IL-2 x anti-PD1 fusion protein comprising: (a) a first polypeptide comprising a first antibody heavy chain linked to IL-2 or an IL-2 mutant protein, 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.

[0461] In the embodiments, the first PD-1 binding domain and the second PD-1 binding domain have the same amino acid sequence.

[0462] In the embodiments, the first PD-1 binding domain and the second PD-1 binding domain have different amino acid sequences.

[0463] In an embodiment, the antibody was engineered to have a mutated "mortar and pestle" (KiH) in constant region 3 (CH3) of its heavy chain.

[0464] In an embodiment, the IL-2 portion comprises an IL-2 x antibody (KiH) fusion protein, the IL-2 x antibody (KiH) fusion protein comprising: (a) a first polypeptide comprising an IL-2 or IL-2 mutant protein, optionally an IL-2 fusion, linked to a second constant region (CH2 and CH3) of the "palm" heavy chain of the antibody (KiH); (b) a second polypeptide comprising the "mortar" heavy chain of the antibody (KiH); and (c) a third polypeptide comprising the light chain of the antibody (KiH).

[0465] In an embodiment, the IL-2 portion comprises an IL-2 x antibody (KiH) fusion protein, the IL-2 x antibody (KiH) fusion protein comprising: (a) a first polypeptide comprising a "pestle" heavy chain of the antibody (KiH) linked to IL-2 or an IL-2 mutant protein, optionally an IL-2 fusion body; (b) a second polypeptide comprising a "mortar" heavy chain of the antibody (KiH); and (c) a third polypeptide comprising a light chain of the antibody (KiH).

[0466] In an embodiment, the IL-2 portion comprises an IL-2 x antibody (KiH) fusion protein, the IL-2 x antibody (KiH) fusion protein comprising: (a) a first polypeptide comprising a "pestle" heavy chain of the antibody (KiH); (b) a second polypeptide comprising a "mortar" heavy chain of the antibody (KiH) linked to IL-2 or an IL-2 mutant protein or an IL-2 mutant protein fusion; and (c) a third polypeptide comprising a light chain of the antibody (KiH).

[0467] In an embodiment, the IL-2 masking portion is linked to the IL-2 or the IL-2 mutant protein.

[0468] In an embodiment, the IL-2 masking portion is linked to the antibody heavy chain that is not connected to the IL-2 or the IL-2 mutant protein.

[0469] In this embodiment, the antibody (KiH) is anti-PD1 (KiH).

[0470] In the embodiments, the IL-2 mutant protein has the amino acid sequence of any one of SEQ ID NO: 5-24 and 105.

[0471] In the embodiments, the IL-2 mutant protein further comprises T3A and C125S amino acid substitutions.

[0472] In one embodiment, the PSL can be cleaved within the tumor microenvironment.

[0473] In the embodiments, the PSL has the amino acid sequence PLGLVVAPLGLVVAPLGLVVA, PLGLWAPLGLWAPLGLWA, GGSGGTPLGLWAGGSGGT, GGSGGTPAGLIGGGSGGT, GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT, or GGSGGTHSSKL QGGSGGT.

[0474] In this embodiment, an IL-2 cytokine fusion protein comprising sequences selected from the group consisting of: (a) SEQ ID NO: 302, 303, and 304; (b) SEQ ID NO: 299, 300, and 301; (c) SEQ ID NO: 305, 306, and 307; (d) SEQ ID NO: 308, 309, and 310; (e) SEQ ID NO: 311, 312, and 313; (f) SEQ ID NO: 314, 315, and 316; (g) SEQ ID NO: 317, 318, and 319; (h) SEQ ID NO: 320, 321, and 322; (i) SEQ ID NO: 323; (j) SEQ ID NO: 329, 330, and 331; (k) SEQ ID NO: 332, 333, and 334; (l) SEQ ID NO: 335, 336 and 337; (m) SEQ ID NO: 338, 339 and 340; (n) SEQ ID NO: 353, 354 and 355; (o) SEQ ID NO: 359, 360 and 361; (p) SEQ ID NO: 362, 363 and 364; (q) SEQ ID NO: 365, 366 and 367; (r) SEQ ID NO: 325, 326 and 327; (s) SEQ ID NO: 353, 354 and 355; and (t) SEQ ID NO: 324.

[0475] In the embodiments, this document provides a pharmaceutical composition comprising the IL-2 cytokine fusion protein described herein and a pharmaceutically acceptable carrier.

[0476] In the embodiments, this document provides a nucleic acid composition encoding the IL-2 cytokine fusion protein described herein.

[0477] In the embodiments, this document provides an expression vector composition comprising the nucleic acid composition described herein.

[0478] In the embodiments, this document provides a method for preparing the IL-2 cytokine fusion protein described herein, the method comprising culturing the nucleic acid composition or the expression vector composition described herein under conditions in which the IL-2 cytokine fusion protein is expressed, and recovering the IL-2 cytokine fusion protein.

[0479] In this embodiment, a method for treating a subject with cancer expressing IL-13Ra2 is provided, the method comprising administering to the subject the IL-2 cytokine fusion protein described herein or the pharmaceutical composition described herein.

[0480] In the embodiments, the IL-2 cytokine fusion protein comprises one or more of SEQ ID NO: 1-367.

[0481] In this embodiment, a method of treating cancer in a subject of need is provided, the method comprising administering to the subject an IL-2 cytokine fusion protein comprising: (a) an IL-2 mutant protein; and (b) an albumin or Fc domain or antibody; wherein the IL-2 mutant protein has an amino acid sequence of any one of SEQ ID NO: 5-24 and 105 and optionally further comprises T3A and / or C125S amino acid substitutions.

[0482] In the embodiments, the IL-2 cytokine fusion protein comprises one or more of SEQ ID NO: 1-367.

[0483] In the embodiments, the IL-2 cytokine fusion protein has the amino acid sequence of SEQ ID NO: 53.

[0484] In one embodiment, the IL-2 cytokine fusion protein was administered as a novel adjuvant prior to surgical procedures for tumor removal.

[0485] In one embodiment, 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 another embodiment, 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.

[0486] In one embodiment, the IL-2 cytokine fusion protein was administered as an adjuvant after surgery to remove a tumor.

[0487] In some embodiments, the IL-2 cytokine fusion protein is administered at most one week after the surgery, optionally at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 1 day, or less than 1 day after the surgery. In some embodiments, administration of the IL-2 cytokine fusion protein is initiated at least two weeks after the surgery.

[0488] In one embodiment, the IL-2 cytokine fusion protein was administered as a novel adjuvant prior to and after surgery for tumor removal.

[0489] In one embodiment, 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 prior to the surgery. In another embodiment, 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 two weeks after the surgery.

[0490] In the embodiments, the cancer is sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoma, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, small cell lung cancer, kidney cancer, stomach cancer, brain cancer, or CNS tumor.

[0491] Example

[0492] Example 1: Evaluation of the masking effect of IL-13 superfactor on IL-2 superagonist activity in in vitro IL-2 reporter gene assay

[0493] A. Introduction

[0494] MDNA132 is an IL-13 superfactor that selectively binds to the IL-13 decoy receptor (IL-13Ra2) but not to the IL-13 functional receptor (IL-13Ra1).

[0495] The mDNA132.15-PSL-MDNA109-Alb (SEQ ID NO: 298; also referred to herein as mDNA213-PSL-MDNA109-alb) was generated. T3 / C125The basic principle is to use a protease-sensitive linker (PSL) to mask the IL-2 "β-only" superagonist MDNA109-Alb with MDNA132.15 (also referred to herein as "MDNA213"). The assumption is that once MDNA132.15 docks MDNA109-Alb with an IL-13 decoy receptor-overexpressing tumor, the PSL will be cleaved. Masking facilitates peripheral T cell activation, thereby increasing tolerance. MDNA109-Alb was used as a comparative on-plate for this construct. MDNA132-PSL-MDNA11 T3 / C125 (SEQ ID NO: 294) was also expressed and purified in CHO, and IL-2 activity was tested in the Jurkat IL-2Rβγ bioassay. An additional construct, mDNA213-PSL-mDNA109-alb, was designed and generated. T3 / C125 (SEQ ID NO: 298) and MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin (SEQ ID NO: 299-301) to further validate the proof of concept. In mDNA223 A3 / S125 -fPSL2f-MDNA213 Lin In, mDNA213-PSL-mDNA109-alb T3 / C125 It has three copies of PSL, none of which are side-attached to 'GS' residues, while a single copy of PSL is side-attached to a 'GS' residue.

[0496] B. Method

[0497] The JURKAT IL2Rβ Bioassay – from Promega, the Jurkat IL2Rβγ bioassay is designed for use with novel IL-2 and IL-15 molecules engineered to reduce CD25 binding, as Jurkat cells lack CD25 expression. This is a simple, homogeneous, sensitive, and reproducible assay that can be completed within one day. Due to the lack of the CD25 receptor in the cells, the engineered IL-2 molecule binds to the CD122 receptor expressed on the cells and triggers signal transduction via pSTAT5, which is detected by luminescence in the Jurkat reporter gene cells.

[0498] Following the manufacturer's recommendations, seed 50 µL of Jurkat IL-2Rβγ cells into 96-well plates. Dilute the construct to a 3-fold solution and then add 25 µL to each well (Table 11). Treat the cells for 6 hours, then add the luciferase substrate and incubate for 10 minutes. Measure the luminescence using an iD5 plate reader. Run two plates for assays, with each plate containing a series of dilutions of the test construct in four copies of the wells.

[0499] Table 11: Constructs used in the Jurkat IL-2Rβγ reporter gene assay

[0500]

[0501] C. Result

[0502] Both mDNA109-Alb and mDNA132.15-PSL-mDNA109-Alb produced significant dose responses. Figure 1A EC50 of these dose-response curves 50 The difference was 18.8-fold, with mDNA109-Alb exhibiting higher potency (lower EC50). 50 ). Using MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin The masked version (which showed reduced potency in the Jurkat IL2Rβγ reporter gene assay compared to the unmasked version) achieved similar results. MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin The EC50 of MDNA223 (1624 pM) was 12.3 times lower than that of MDNA223 (131.9 pM). This is consistent with the intentional masking of the MDNA109FEAA portion of MDNA223 by MDNA213.

[0503] Table 12 summarizes EC 50 .

[0504] Table 12: EC50 and EC50 of mDNA132.15-PSL-mDNA109-Alb in Jurkat IL-2Rβγ assay 50 Summary of change factors

[0505]

[0506] mDNA11 (SEQ ID NO: 53) and mDNA132-PSL-mDNA11 T3 / C125 (SEQ ID NO: 294) Both produced a significant dose response ( Figure 1B EC50 of the two dose-response curves 50 The difference was approximately 5.6 times, with mDNA11 exhibiting higher potency (lower EC50). 50 (509 pM), and mDNA132-PSL-mDNA11 T3 / C125 EC showed 2835 pM 50 .

[0507] D. Conclusion

[0508] MDNA132.15-PSL-MDNA109-Alb exhibited 18.8-fold lower potency, indicating that MDNA132.15 can mask the activity of MDNA109-Alb.

[0509] Example 2: Evaluation of the masking effect of IL-13 superfactor on the binding of IL-2 superagonist to CD122

[0510] A. Introduction

[0511] The binding affinity experiment presented in this example confirms Example 1 to verify the masking effect of the mDNA132.15 IL-13 superfactor. Observations in the previous experiments showed that mDNA132.15 masks mDNA109-albumin by reducing the potency of IL-2-induced pSTAT5.

[0512] B. Method

[0513] The Ni-NTA biosensor (Sartorius, catalog number 18-5101) was hydrated in a 96-well plate (Greiner, part number 655209) as required by Sartorius, the manufacturer of the Octet RH16 Biolayer Interferometer (BLI) instrument. The ligand (200 nM His-CD122) was immobilized onto the Ni-NTA biosensor and then immersed in titration solutions of either MDNA109-Alb (construction number 7741-044, 82.8 kDa) or MDNA132.15-PSL-MDNA109-Alb (construction number 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 kinetic reagent (Sartorius, catalog 18-1105). Assays were performed in 384-well tilt-well plates (Sartorius, catalog 18-5080) at 25°C with a volume of 50 μL per well. Protein-binding biosensors immersed in buffer were used instead of the corresponding analytes to subtract baseline drift. Negative control experiments were included, where the ligands were not loaded onto the biosensors, and intermediate concentrations of the analytes were tested to determine the binding profile of the “naked” biosensors. No significant nonspecific binding was observed in any of the experiments. A blocking step using Superblock (Thermo Fisher Scientific, catalog 37515) was added.

[0514] The general BLI procedure conditions used are as follows:

[0515] Biosensor test in buffer solution (30 seconds)

[0516] His-CD122 (ligand) fixation (12 minutes)

[0517] Block using Superblock (2 minutes)

[0518] Determine the baseline in the buffer solution (3 minutes).

[0519] Analyte titration association (5 minutes)

[0520] Dissociate back into baseline buffer (15 minutes)

[0521] C. Result

[0522] Using MDNA109-albumin as an experimental control, the binding affinity of the construct MDNA132.15-PSL-MDNA109-albumin to human CD122 was tested. Figure 2 The data was presented as a sensor image.

[0523] As observed, both mDNA109-albumin and mDNA132.15-PSL-mDNA109-albumin exhibit affinity for human CD122, but their unique... k on The value resulted in a 6.9-fold increase in the binding affinity constant K. D The differences were observed, with mDNA109-albumin and mDNA132.15-PSL-mDNA109-albumin showing K+ values ​​of 4.6 nM and 31.6 nM, respectively. D The change in the binding affinity constant indicates a masking effect of MDNA132.15, which is fused together via PSL (protease-sensitive linker), on the binding of MDNA109-albumin.

[0524] D. Conclusion

[0525] The masking effect was clearly confirmed by the affinity data, observed through a 6.9-fold difference in the KD constant between the construct mDNA109-albumin and mDNA132.15-PSL-mDNA109-albumin. The data will be further validated by cleaving the construct with MMP9 and testing the recovery of mDNA109-albumin binding.

[0526] Example 3: Characterization of tumor-targeting and activatable T-MASK platforms to enhance tumor accumulation and tolerance to potent immunomodulators.

[0527] The novel T-MASK (superfactor targeting metalloproteinase activation) platform involves fusing a dual-targeting / masking tumor domain with an effective immunomodulator via a metalloproteinase (MMP)-sensitive linker (PSL) to achieve the following objectives: (1) to reduce / fine-tune the efficacy of the immunomodulator through steric hindrance to increase systemic tolerability, and (2) to promote retention in the tumor microenvironment (TME) to maximize MMP cleavage and restore full efficacy at the intended target site. As a proof of concept, an IL-13 superfactor (MDNA213, also referred to herein as “MDNA132.15”) with high selectivity and affinity for the IL-13 decoy receptor IL-13Rα2 (a tumor-associated antigen expressed in many aggressive solid tumors) was selected as the targeting / masking tumor domain.

[0528] Example 3a: Evaluation of the masking effect of IL-2 superagonists on the binding of IL-13 superfactor to IL-13Ra2

[0529] A. Introduction

[0530] MDNA132 is an IL-13 superfactor that selectively binds to the IL-13 decoy receptor (IL-13Ra2) but not to the IL-13 functional receptor (IL-13Ra1).

[0531] The basic principle of generating MDNA132.15-PSL-MDNA109-Alb is to use a protease-sensitive linker (PSL) to mask MDNA109-Alb with MDNA132.15. It is assumed that once MDNA132.15 docks MDNA109-Alb with an IL-13 decoy receptor overexpressing tumor, the PSL will be cleaved.

[0532] B. Method

[0533] The Ni-NTA biosensor (Sartorius, catalog 18-5101) was hydrated in a 96-well plate (Greiner part number 655209) as required by the manufacturer of the Octet RH16 Biolayer Interferometer (BLI) instrument (Sartorius). The ligand (200 nM His-CD122) was immobilized onto the Ni-NTA biosensor and then immersed in titration of either mDNA109-Alb (construction 7741-044, 82.8 kDa) or mDNA132.15-PSL-MDNA109-Alb (construction 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 kinetic reagent (Sartorius, catalog 18-1105). Assays were performed in 384-well tilt-well plates (Sartorius, catalog 18-5080) at 25°C with a volume of 50 μL per well. Protein-binding biosensors immersed in buffer were used instead of the corresponding analytes to subtract baseline drift. Negative control experiments were included, where ligands were not loaded onto the biosensors, and moderate concentrations of analytes were tested to determine the binding profile of the “naked” biosensors. No significant nonspecific binding was observed in any of the experiments. A blocking step using Superblock (Thermo Fisher Scientific, catalog 37515) was added.

[0534] The general BLI procedure conditions used are as follows:

[0535] Biosensor test in buffer solution (30 seconds)

[0536] His-CD122 (ligand) fixation (12 minutes)

[0537] Block using Superblock (2 minutes)

[0538] Determine the baseline in the buffer solution (3 minutes).

[0539] Analyte titration association (5 minutes)

[0540] Dissociate back into baseline buffer (15 minutes)

[0541] C. Result

[0542] Using Fc-MDNA132.15 (KIH) as an experimental control, the binding affinity of the construct MDNA132.15-PSL-MDNA109-albumin to human IL-13Ra2 was tested. Figure 3 The data was presented as a sensor image.

[0543] As observed, both Fc-MDNA132.15 (KIH) and MDNA132.15-PSL-MDNA109-albumin showed affinity for human IL-13Ra2, but their unique... k on and k off The value resulted in an 11.5-fold increase in the binding affinity constant K. D The differences were observed, with Fc-MDNA132.15 (KIH) and MDNA132.15-PSL-MDNA109-albumin showing K+ values ​​of 0.32 nM and 3.7 nM, respectively. D The change in the binding affinity constant indicates a masking effect of the binding of MDNA109-albumin, which is fused together via PSL (protease-sensitive linker), to MDNA132.15.

[0544] D. Conclusion

[0545] The masking effect was clearly confirmed by the affinity data, which was observed through an 11.5-fold difference in the KD constant between the construct Fc-MDNA132.15 and MDNA132.15-PSL-MDNA109-albumin.

[0546] Example 3b: MMP9 proteolytic activation restored the IL-2 activity of mDNA132.15-PSL-mDNA109-albumin.

[0547] The ability of proteolytic cleavage of mDNA132.15-PSL-MDNA109-albumin (also known as “MDNA213-PSL-MDNA109-Alb”) to restore IL-2 activity of mDNA109 was evaluated.

[0548] Preactivated MMP9 was purchased from Sigma Millipore (SAE0078). The construct was incubated for 1 hour at 37°C in a reaction solution containing 9.5 µg of protein (including or excluding 2 µg / mL MMP9) in 160 µL of digestion buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (w / v), pH 7.5). After digestion, the reaction solution was placed on ice and diluted in culture medium for testing in the Jurkat IL2Rβγ reporter gene assay. Aliquots were also removed, inactivated by adding SDS-PAGE loading buffer, and stored at -80°C until analysis. Samples were analyzed by SDS-PAGE under reducing conditions using a 4–12% Bis-Tris Bolt polyacrylamide gel (Ingenieur). Molecular weight protein markers (Novex™ Sharp pre-stained protein standard LC5800) were also run on the gel. After electrophoresis, the gel was stained with Simply Blue Safe Stain (Ingenieur).

[0549] mDNA213-PSL-MDNA109-alb T3 / C125 The MMP9 cutting data is shown in Figure 4 Left side. MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-alb) excluding MMP9. T3 / C125 (Lane 2) resolved as a single band at 109 kDa. The presence of MMP9 (lane 3) resulted in cleavage producing an additional band at 83 kDa corresponding to MDNA109-alb (unmasked version, lane 1). However, the presence of MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-alb) was also observed. T3 / C125 The evidence is incomplete, with nearly 50% still uncut.

[0550] Subsequently, the reaction mixture after MMP9 cleavage was tested in a Jurkat IL-2 assay. Figure 4 (Right side). MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-albT3 / C125 The active portion of the MMP9 cleavage product was restored to the level of mDNA109-albumin. The potency of the MMP9 cleavage product (EC50 = 1144 pM) was higher than that of the simulated cleavage reaction (EC50 = 5108 pM), but still 3-fold lower than that of mDNA109-albumin (EC50 = 337 pM). This is consistent with the reaction of MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-alb). T3 / C125 The incomplete cut is consistent.

[0551] Example 3c: Functional characterization of mDNA223-PSL-mDNA132.15 in IL-2 and PD-1 reporter gene assays

[0552] A. Introduction

[0553] mDNA132 is an IL-13 superfactor that selectively binds to the IL-13 decoy receptor (IL-13Rα2) but not to functional IL-13Rα1. IL-13Rα2 is overexpressed in a variety of tumors, including glioblastoma, pancreatic, bladder, and colon cancer, but not in normal tissues except the testes, suggesting it is a tumor-associated antigen (TAA). Therefore, it is possible to utilize mDNA132 to selectively deliver therapeutic payloads to IL-13Rα2-overexpressing tumors without evoking undesirable responses in normal tissues (i.e., reduced toxicity risk).

[0554] MDNA223 (also known as anti-mPD1-MDNA109FEAA KIH) is a bifunctional superfactor immunotherapy (BiSKIT) involving the fusion of anti-PD1 and MDNA109FEAA. The latter is an IL-2 superfactor with enhanced affinity for IL-2Rβ but does not bind to IL-2Rα, leading to selective activation of immune effector cells (CD8+ T cells and NK cells) and a reduced ability to stimulate immunosuppressive Tregs. The lack of binding to IL-2Rα also reduces the risk of toxicity. MDNA223 is engineered to cis-bind to PD-1 and IL-2R expressed on CD8+ T cells to induce immune checkpoint blockade (via binding to PD-1) and functional stimulation (via binding to IL-2R) on the same cells. In vivo studies in a mouse syngeneic tumor model have shown that MDNA223 exhibits superior anticancer activity compared to the combination of anti-PD1 + MDNA19 (i.e., MDNA109FEAA-Fc), thus identifying this BiSKIT as a potential candidate for further development.

[0555] To target mDNA223 to the tumor microenvironment (TME) with the potential to enhance efficacy and reduce the risk of systemic toxicity (i.e., expand the therapeutic index), mouse (m) mDNA223 was generated based on the following fundamental principles. -PSL-MDNA132.15 construct:

[0556] T3A and C125S mutations were added to the FEAA portion of mDNA109 (i.e., the IL-2 portion of mDNA11). The T3A mutation is used in other IL-2 drugs, including Proleukin, to eliminate glycosylation, which can reduce the homogeneity of the drug product. The C125S mutation is used to eliminate the risk of disulfide bridge rearrangement at this residue, thereby reducing the risk of spreadability issues. These mutations are indicated in the name of this construct as ' '.

[0557] The fusion of mDNA132.15 and mDNA223 can target tumors expressing IL-13Rα2. Direct fusion of mDNA132.15 to the mDNA109FEAA moiety of BiSKIT via a protease-sensitive linker (PSL) may potentially reduce the activity of mDNA109FEAA due to steric hindrance. The fusion is not expected to affect PD-1 blockade. MDNA223 is released from mDNA132.15 by cleaving the PSL within the TME via the protease. This removes steric hindrance and restores mDNA223. This allows mDNA223 to target the TME while simultaneously reducing its activity in circulation to mitigate the risk of peripheral toxicity.

[0558] B. Method

[0559] Jurkat IL2Rβγ assay

[0560] Following the manufacturer's recommendations, seed 50 µL of cells into 96-well plates. Serially dilute the test sample and add (25 µL) to each well to achieve the final test concentration listed in Table 13. Incubate the cells for 6 hours, then add the luciferase substrate and incubate for 10 minutes. Measure the luminescence on an iD5 plate reader.

[0561] Table 13: Constructs tested in the Jurkat IL2Rβγ reporter gene assay.

[0562]

[0563] PD-1 reporter gene assay

[0564] In the PD-1 / PD-L1 blocking bioassay, PD-L1 aAPC / CHO-K1 cells are used to conjugate PD-1 effector cells via the T-cell receptor (TCR) or PD-1 receptor. When PD-L1 conjugates with PD-1, TCR signaling and downstream luciferase reporter gene activation driven by NFAT response elements are inhibited. PD-1 blocking antibodies prevent the interaction between PD-1 and PD-L1, allowing TCR signaling and subsequent NFAT reporter gene luminescence.

[0565] Following the manufacturer's recommendations, 100 µL of mouse PD-L1 aAPC / CHO-K1 T&U cells were seeded into 96-well plates 16 hours prior to the assay setup. The next day, the test sample was serially diluted to 2x. The culture medium was removed from the pre-coated reporter cells and 40 µL of the 2x test sample, along with 40 µL of mouse PD-1 T&U effector cells, and added to each well to achieve the concentrations listed in Table 14. The cells were incubated for 6 hours, and then the luciferase substrate was added and incubated for 15 minutes. Luminescence was measured on an iD5 reader.

[0566] Table 14: Constructs tested in PD-1 reporter gene assays.

[0567]

[0568] C. Result

[0569] Jurkat IL2Rβγ reporter gene assay measures the efficacy of IL-2R signaling.

[0570] like Figure 5 As shown, mMDNA223-PSL-MDNA132.15 (SEQ ID NO: 299-301, masked version) exhibited reduced potency in the Jurkat IL2Rβγ reporter gene assay compared to mDNA223 (unmasked version). EC50 of mMDNA223-PSL-MDNA132.15 50 (1624 pM) was 12.3 times lower than mMDNA223 (131.9 pM). The reduced efficacy of mMDNA223-PSL-MDNA132.15 in inducing IL-2R signaling was consistent with the masking effect of mDNA132.15 fused to the MDNA109FEAA portion of the construct.

[0571] PD-1 / PD-L1 reporter gene assays assess the efficacy of immune checkpoint blockade:

[0572] In PD-1 reporter gene assays ( Figure 5Both mMDNA223 (EC50 = 3.7 nM) and mMDNA223-PSL-MDNA132.15 (EC50 = 3.9 nM) exhibited similar potency for PD-1 / PD-L1 immune checkpoint blockade. The potency of both constructs was highly comparable to that of the parental anti-mPD1 antibody (EC50 = 3.4 nM). These data suggest that the fusion of mDNA132.15 with the mDNA109FEAA moiety of BiSKIT does not affect the function of the anti-PD1 moiety.

[0573] D. Conclusion

[0574] Compared to mMDNA223 (EC50 = 131.9 pM), mMDNA223 -PSL- mDNA132.15 (EC50 = 1624pM) showed reduced potency in IL-2R signaling. mMDNA223 Both -PSL-mDNA132.15 (EC50 = 3.9 nM) and mMDNA223 (EC50 = 3.7 nM) maintain similar potency for PD-1 / PD-L1 immune checkpoint blockade. Therefore, the fusion of mDNA132.15 with mMDNA223 attenuates (i.e., masks) the activity of mDNA109FEAA (IL-2R signaling) but has no effect on PD1 (immune checkpoint blockade), which is consistent with the intent of the construct design.

[0575] Example 3d: MMP9 proteolytic activation restored IL-2 activity in mDNA223-PSL-mDNA132.15.

[0576] The evaluation of “mDNA223-fPSLf-mDNA213” or “mDNA223” was conducted. A3 / S125 - fPSL2f-MDNA213 Lin The ability of protein hydrolysis of (SEQ ID NO: 299-301) to restore IL-2 activity of MDNA109.

[0577] mDNA223 A3 / S125 -fPSL2f-MDNA213 Lin The results of MMP9 cutting are presented in Figure 6Left side. MDNA223 (anti-mPD1-MDNA109FEAA KIH, lane 1) resolved to three bands under reducing conditions, corresponding to heavy chain 1 (HC1; fused with MDNA109FEAA; approximately 65 kDa), heavy chain 2 (HC2, approximately 52 kDa), and light chain (LC, approximately 25 kDa). Lane 2 shows a simulated cleavage of MDNA223-fPSLf-MDNA213 KIH, where the size of HC1 (approximately 90 kDa) was increased by the addition of fPSLf-MDNA213. When MMP9 was used at a concentration of 2 µg / mL, incubation at 37°C for 1 hour (lane 3) resulted in MDNA223... A3 / S125 -fPSL2f-MDNA213 Lin Nearly complete cleavage. These results indicate that mDNA223 A3 / S125 -fPSL2f-MDNA213 Lin It can be efficiently cleaved by MMP9 to release the mDNA213 masking domain without affecting the integrity of the remaining construct (e.g., based on size).

[0578] Cut mDNA223 A3 / S125 -fPSL2f-MDNA213 Lin The construct further exhibited IL-2 activity ( Figure 6 (Right side).

[0579] In MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin Following MMP9 cleavage to remove the mDNA213 domain, IL-2 activity was restored to a similar potency to that of mDNA223, which was obtained from mDNA223 (607 pM) compared to the cleaved mDNA223. A3 / S125 -fPSL2f-MDNA213 Lin (419 pM) similar EC 50 It is clearly visible. Therefore, MMP9 cleavage removes the masking effect of MDNA213 in MDNA223-fPSLf-MDNA213.

[0580] Example 3e: Further functional characterization of mDNA223-PSL-mDNA132.15 in IL-2 and PD-1 reporter gene assays

[0581] As shown in Table 15 below, the functional properties of additional mDNA223-PSL-MDNA132.15 (also referred to as "MDNA223-PSL-MDNA213") constructs were evaluated using different PSL adapters. For each of these constructs, IL-2-mediated signaling and PD-1 / PD-L1 immune checkpoint blockade were evaluated.

[0582] Table 15: Exemplary PSL Connectors

[0583]

[0584] like Figure 7-10 As shown, each of these MDNA223-PSL-MDNA132.15 constructs exhibited reduced IL-2-mediated signaling efficacy compared to MDNA223, and there was no change in PD-1 / PD-L1 blockade.

[0585] Example 3f: mDNA223 in IL-2 and PD-1 reporter gene assays T3AC125S Anti-mPD1(H)-fPSL2f-MDNA213 and anti-mPD1(K)-fPSL2f-MDNA213:MDNA223 T3AC125S Functional representation

[0586] MDNA223 was evaluated T3AC125S Anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NO: 314-316) and anti-mPD1(K)-fPSL2f-MDNA213:MDNA223 T3AC125S Functional characteristics of (SEQ ID NO: 317-319). In mDNA223 T3AC125S In the anti-mPD1(H)-fPSL2f-MDNA213 construct, MDNA213 is linked to the C-terminus of the homodimer of the mortise Fc domain via PSL. In the anti-mPD1(K)-fPSL2f-MDNA213:MDNA223 T3AC125S In the constructs, the C-terminus of the mDNA213 "pike" homodimer was linked. For each of these constructs, IL-2-mediated signaling and PD-1 / PD-L1 immune checkpoint blockade were evaluated.

[0587] like Figure 11 and 12 As shown, compared to mDNA223, mDNA223 T3AC125S Anti-mPD1(H)-fPSL2f-MDNA213 and anti-mPD1(K)-fPSL2f-MDNA213:MDNA223 T3AC125SThe IL-2-mediated signal transduction ability of the constructs was reduced by 3-fold and 2-fold, respectively. Furthermore, no changes in PD-1 / PD-L1 blockade were observed for either construct.

[0588] Example 3g: mDNA223 in IL-2 and PD-1 reporter gene assays T3AC125S - fPSL2f-MDNA213: Functional characterization of anti-mPD1(H)-fPSL2f-MDNA213

[0589] MDNA223 was evaluated T3AC125S -fPSL2f-MDNA213: Functional characteristics of anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NO:320-322). (The text abruptly ends here, likely due to an incomplete sentence or missing information.) T3AC125S -fPSL2f-MDNA213: In the anti-mPD1(H)-fPSL2f-MDNA213 construct, the first MDNA213 is linked to the C-terminus of the homodimer of the "mortar" Fc domain via PSL, and the second MDNA213 is linked to MDNA109FEAA. 223T3AC125S Connections. IL-2-mediated signal transduction and PD-1 / PD-L1 immune checkpoint blockade were evaluated.

[0590] like Figure 13 As shown, compared to mDNA223, mDNA223 T3AC125S -fPSL2f-MDNA213: The IL-2-mediated signal transduction efficacy of the anti-mPD1(H)-fPSL2f-MDNA213 construct was reduced by 39-fold, while PD-1 / PDL-1 blockade remained unchanged. Furthermore, as... Figure 14 As shown, mDNA223 T3AC125S -fPSL2f-MDNA213: The anti-mPD1(H)-fPSL2f-MDNA213 construct is sensitive to MMP9 cleavage in vitro and shows activity upon MMP9 cleavage in IL-2R reporter gene assays. Figure 15 ).

[0591] Example 3h: MDNA213-fPSL2f-MDNA11 T3AC125S Functional characterization of IL-2 and PD-1 reporter gene assays

[0592] The mDNA213-fPSL2f-mDNA11 was evaluated. T3AC125S Functional characteristics of (SEQ ID NO: 323). In mDNA213-fPSL2f-mDNA11 T3AC125S In the construct, mDNA213 interacts with mDNA111 via CSL.23T3AC125S The construct's MDNA109FEAA 223T3AC125S Connections. IL-2-mediated signal transduction and PD-1 / PD-L1 immune checkpoint blockade were evaluated.

[0593] like Figure 16 As shown, compared with mDNA223, mDNA213-fPSL2f-mDNA11 T3AC125S The IL-2-mediated signal transduction efficiency of the construct was reduced by 21-fold. Furthermore, as... Figure 17 As shown, mDNA213-fPSL2f-mDNA11 T3AC125S The construct was sensitive to MMP9 cleavage in vitro and showed activity upon MMP9 cleavage in IL-2R reporter gene assays. Figure 18 ).

[0594] Example 4: Characterization of tumor-targeting and activatable T-MASK platforms to enhance tumor accumulation and tolerance to potent immunomodulators.

[0595] A. Background

[0596] A novel T-MASK (superfactor targeting metalloproteinase activation) platform involves fusing a dual-targeting / masking tumor domain with a potent immunomodulator via a metalloproteinase (MMP)-sensitive linker (PSL) to achieve the following objectives: (1) to reduce / fine-tune the efficacy of the immunomodulator through steric hindrance to increase systemic tolerability, and (2) to promote retention in the tumor microenvironment (TME) to maximize MMP cleavage and restore full efficacy at the intended target site. As a proof of concept, an IL-13 superfactor (MDNA213) with high selectivity and affinity for the IL-13 decoy receptor IL-13Rα2 (a tumor-associated antigen expressed in many aggressive solid tumors) was selected as the targeting / masking tumor domain. MDNA213 was fused via PSL with MDNA11 and MDNA223, both of which contain non-α,β-enhanced IL-2 fused with albumin or an anti-PD1 antibody, respectively. Preliminary results of characterization of the two T-MASK constructs are presented, demonstrating conditional fine-tuning of IL-2R agonism.

[0597] B. Method

[0598] T-MASK optimization included evaluating the PSL linker and targeting / masking of tumor domains. In vitro IL-2 and PD-1 / PDL-1 reporter gene assays were performed to evaluate IL-2R stimulation and anti-PD1 blockade, respectively. In vitro MMP assays were used to validate the cleavability and full potency recovery of the T-MASK construct.

[0599] C. Result

[0600] In IL-2R-induced p-STAT5 reporter gene assays, the T-MASK construct showed a potency reduction of approximately 10-40 fold compared to the corresponding unmasked version. The degree of fine-tuning can be adjusted via the length and composition of the PSL and the orientation of the domains, providing the T-MASK platform with versatility and the potential to bind a complete library of peripheral circulating immune cells. In mDNA223 (anti-PD1-IL-2...) 超级因子 In the case of [specific details missing], fusion of mDNA213 to generate the mDNA223-CSL-MDNA213T-MASK construct resulted in reduced IL-2R agonism, but had no effect on the expected efficacy of PD1 / PDL-1 blockade. Demasking of the mDNA223-PSL-MDNA213 construct (also known as the "MDNA113" construct), for example by MMP-mediated cleavage, completely restored its IL-2R signaling activity to the same level as the unmasked mDNA223 construct. Similar data were obtained using the masked version of mDNA11, demonstrating the robustness of the T-MASK platform. In vivo studies are underway to evaluate the effects of the T-MASK construct on peripheral immune cell expansion (systemic response), tumor preservation (maximized activation), and tumor growth inhibition (targeted response).

[0601] Example 5: Recovery of IL-2R signaling activity after in vitro protein hydrolysis and cleavage

[0602] A. Method

[0603] MMP9 cleavage: The construct was incubated in a reaction solution containing 9.5 µg of the construct (with or without MMP9) in 160 µL of cleavage buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (w / v), pH 7.5). Four different conditions were tested to observe differences in cleavage efficiency, if any. After digestion, the reaction solution was placed on ice and diluted in culture medium for testing in Jurkat IL2Rβγ reporter cell assays. Aliquots were also rapidly frozen in liquid nitrogen before storage at -80°C for subsequent SDS-PAGE analysis.

[0604] Jurkat IL2Rβγ reporter assay: Plate 50 µL of cells into 96-well plates according to the manufacturer's instructions. Serially dilute the test sample and add (25 µL) to each well. Incubate the cells for 6 hours and then add the luciferase substrate and incubate for an additional 10 minutes. Measure the luminescence on an iD5 reader.

[0605] B. Result

[0606] The results are shown in Figure 19 and 20 Except for MASK A4, all BiSKITs were digested to varying degrees (60-70%) by MMP9 under the conditions described in the methods. MASK A4 was designed to have no cleavage site to demonstrate the selectivity of MMP protein hydrolysis.

[0607] MDNA223 (also known as anti-mPD1-MDNA109FEAA KIH) resolves as three bands under reducing conditions, corresponding to heavy chain 1 (HC1; fused with MDNA109FEAA; approximately 65 kDa), heavy chain 2 (HC2, approximately 52 kDa), and light chain (LC, approximately 25 kDa).

[0608] Simulated cleavage resolution of constructs MASK A1 to MASK A4 yielded three bands, with the size of HC1 (approximately 90 kDa) increased by the addition of fPSLf-MDNA213. As expected, incubation with MMP9 resulted in cleavage of the constructs, with the band corresponding to MDNA223, except for MASK A4, which lacked the PSL, i.e., the MMP9 cleavage site.

[0609] Simulation and MMP9 cleavage resolution of MASK A5 and MASK A6 yielded three bands, with the size of HC1 (approximately 70 kDa) increased by the addition of fPSLf-MDNA213. As expected, incubation with MMP9 resulted in cleavage of the construct, with the band corresponding to MDNA223.

[0610] Simulated cleavage of MASK A7 resolved into three bands, with the sizes of HC1 (approximately 90 kDa) and HC2 (approximately 70 kDa) increased by the addition of fPSLf-MDNA213. As expected, incubation with MMP9 resulted in cleavage of the construct, with the band corresponding to MDNA223. Simulated cleavage of MASK B1 resolved into a single band of approximately 110 kDa, while digestion with MMP9 resolved into two bands of approximately 84 kDa and approximately 12 kDa, corresponding to MDNA11 and MDNA213, respectively.

[0611] C. IL-2 activity restoration

[0612] We reported that the MASK-IT constructs A7 and B1 exhibited approximately 40-fold and 20-fold reductions in IL-2 activity and sensitivity to MMP9 digestion, respectively, in Jurkat IL2Rβγ reporter gene assays, as described above. Further testing was conducted to assess the recovery of IL-2 activity in MASK A7 and MASK B1 after MMP9 cleavage.

[0613] In the Jurkat IL-IL2Rβγ cell reporter gene assay ( Figure 21A and 21B In the absence of MMP9 (mimetic digestion), both MASK A7 and MASK B1 exhibited reduced IL-2 activity. However, under all test conditions, in the presence of MMP9, the activity recovered to a level similar to that of mDNA223 or mDNA11. Therefore, MMP9 cleavage removed the masking effect on mDNA213 by both constructs, consistent with the design of this MASK-IT construct. The EC50 for mDNA223 and mDNA11 was reported to be 0.5 nM. MASK A7 and MASK B1 exhibited reduced potency, with EC50s of 2.9 nM for both. For different cleavage conditions, MASK A7 and MASK B1 recovered their potency after MMP9 cleavage, with EC50s ranging from 0.5 to 0.6 nM.

[0614] D. In mouse CTLL-2 and human PBMC proliferation assays, the IL-2 function of mDNA113 was masked.

[0615] CTLL2 cells were seeded at a density of 30,000 cells per well in 96-well plates containing T-STIM proliferation supplement. After seeding, cells were treated with increased concentrations of test or control samples for 48 hours. Following treatment, Cell Titer Blue viability reagent (Promega G8080) was added to each well, and the plate was scanned at 560Ex / 590Em after fluorescence activity was observed.

[0616] like Figure 22 As shown, compared to mDNA223, mDNA223 A3 / S125 -fPSL2f- MDNA213 Lin / ParH The rightward shift of the curves for (MASK A7; SEQ ID NO: 320, 321, and 322) indicates lower potency. EC50 of mDNA113 (4.3 nM) 50 It is 14 times lower than mDNA223 (0.3 nM). Similarly, mDNA213-fPSL2f-MDNA11 A3 / S125 (SEQ ID NO: 323) (at 14 nM) showed that its activity was 1 / 113 of that of MDNA11 (0.12 nM).

[0617] Figure 23 Data on human PBMC proliferation assays are presented. Three human PBMC donors were stimulated with masked and unmasked versions of mDNA223 for 48 hours, and BrDU was analyzed and incorporated as proliferation readings. (mDNA223 (unmasked) and mDNA223...) A3 / S125-fPSL2f- MDNA213 Lin / ParH (Masking) Both induce PBMC proliferation, with the latter showing reduced potency. Although EC 50 The values ​​were derived based on logistic curve fitting, but the failure to reach a clear upper limit within the tested dose range makes these values ​​unreliable and unsuitable for comparison. (mDNA223 and mDNA223) A3 / S125 -fPSL2f-MDNA213 Lin / ParH The comparison of the stimulation index clearly shows the attenuation of the latter at all tested concentrations. Similar observations were made with masked and unmasked MDNA11.

[0618] E.mDNA113 attenuates Th1 responses through IL-2-mediated induction of human pSTAT5 in human PBMCs.

[0619] Using MDNA223 (unmasked) and MDNA223 A3 / S125 -fPSL2f- MDNA213 Lin / ParH Human PBMCs were (masked) and treated for 15 minutes, then harvested for flow cytometry analysis of immune subsets. Results are shown in Figure 24-28 middle.

[0620] mDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH Reduce the original CD8 in human PBMC + IL-2-mediated pSTAT5 induction in both T cells and Tregs. 50 It was revealed that in the original CD8 + In T cells, pSTAT5 induction decreased by approximately 7-fold, including mDNA223 and mDNA223. A3 / S125 -fPSL2f-MDNA213 Lin / ParH EC values ​​of 776.9 pM and 5402 pM, respectively. 50 (Average from 3 independent donors). In Treg, pSTAT5 induction was observed to be reduced by approximately 10-fold, with mDNA223 showing a decrease compared to 829.4 pM. A3 / S125 -fPSL2f-MDNA213 Lin / ParH average EC 50 It is 8504 pM.

[0621] Original CD8 + ECG of T cells and Tregs 50 Ratios are considered a useful metric for evaluating the therapeutic potential of constructs because they reflect the balance between antitumor and pretumor immune responses. The comparable ratio between unmasked and masked mDNA223 demonstrates the effectiveness of mDNA223...A3 / S125 -fPSL2f-MDNA213 Lin / ParH The therapeutic potential was not impaired, but IL-2 activity was masked.

[0622] Similar observations were made with masked and unmasked mDNA11. EC 50 It was revealed that in the original CD8 + In T cells, pSTAT5 induction decreased by approximately 7-fold, particularly in MDNA11 and MDNA213-fPSL2f-MDNA11. A3 / S125 EC values ​​of 512.8 pM and 3674.3 pM, respectively. 50 (Average from 3 independent donors). In Treg, pSTAT5 induction was observed to be reduced by approximately 5-fold, with MDNA213-fPSL2f-MDNA11 showing a decrease in activity compared to 611.6 pM for MDNA11. A3 / S125 average EC 50 It is 2825 pM.

[0623] F. Receptor binding affinity

[0624] EMT6 (WT) and EMT6 / IL-13Ra2 cells were thawed and cultured in T75 flasks with Waymouths MB752 / 1 (w / L-Glu) containing 15% FBS. Cells were passaged at 80% confluence using 0.05% trypsin EDTA. Cells were counted on a hemocytometer and seeded at a 1:10 passage ratio into 6-well TC-treated plates. Cells were removed at 80% confluence by incubation with a cell stripping agent (non-enzymatic dissociation solution) for 20 minutes. Cells were transferred to 96°C U-bottom plates and treated with the construct for 30 minutes (5-fold 6-point curve). Fc-MDNA213 was used as a control. After incubation, cells were stained with anti-Fc antibody to detect cell receptor binding. Cell receptor binding of the construct and anti-Fc was determined by flow cytometry. Affinity (EC50) was measured using a GraphPad Prism.

[0625] Figure 29 The data presented are shown. At two test concentrations, Fc-MDNA213 (positive control) binds to the cell receptor. MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH It bound to EMT6 / IL-13Ra2 cells in vitro, but with lower affinity compared to Fc-MDNA213. Binding was observed to be dose-dependent. The reduced binding compared to Fc-MDNA213 could be explained by steric hindrance from the use of a protease-sensitive linker in the MDNA223 fusion.

[0626] Pharmacodynamic evaluation of G.MDNA113

[0627] The study investigated mDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH The pharmacodynamic response of the drug, compared with unmasked mDNA223, reduced the expansion of peripheral lymphocytes in mice upon intraperitoneal (IP) administration.

[0628] Figure 30 and 31 The use of masked and unmasked mDNA223 was demonstrated. A3 / S125 and MDNA11 A3 / S125 Peripheral lymphocyte expansion was measured by CBC analysis 72 hours after treatment. Equivalent doses of unmasked and masked mDNA223 were compared. A3 / S125 or mDNA11 A3 / S125 Peripheral lymphocyte expansion demonstrated a significant reduction in the corresponding masked version. These data indicate that mDNA223 A3 / S125 and MDNA11 A3 / S125 The circulating masking domain mDNA213 was efficiently shielded, successfully reducing peripheral lymphocyte proliferation. However, increasing mDNA223... A3 / S125 -fPSL2f-MDNA213 Lin / ParH and mDNA213-fPSL2f-mDNA11 A3 / S125 The dosage (increased from 2 mg / kg to 5 mg / kg, 7.5 mg / kg and 10 mg / kg) resulted in peripheral lymphocyte expansion, similar to that in animals treated with the cadaver.

[0629] Other blood cells, including neutrophils (PMNs), monocytes, basophils, and eosinophils, as expected, were detected with masked or unmasked mDNA223. A3 / S125 and MDNA11 A3 / S125 It will not be affected after processing.

[0630] In vivo tumor growth inhibition in the H.MC-38 colon cancer model

[0631] like Figure 32 As shown, the masking of mDNA223 (mDNA223) A3 / S125 -fPSL2f- MDNA213 Lin / ParH The unmasked version showed considerable tumor growth inhibition, providing strong evidence that mDNA113 is cleaved by proteases in the TME, leading to mDNA223. A3 / S125 -fPSL2f-MDNA213 Lin / ParHReleased from the masking domain (mDNA213), thereby restoring its full potency and therapeutic potential. MDNA223 A3 / S125 -fNCLf- MDNA213 Lin / ParH It exhibited moderate tumor growth inhibition, which supports the shielding of MDNA223 in vivo when it is masked by MDNA213 through an uncleavable linker.

[0632] In vivo tumor growth inhibition in an I.EMT6 / IL13Rα2 breast tumor model

[0633] like Figure 33 As shown, the data indicates that, compared with mDNA223 A3 / S125 -fPSL2f- MDNA213 Lin / ParH Compared to mDNA223, using an equimolar dose of mDNA23 A3 / S125 -fNCLf-MDNA213 Lin / ParH The treatment showed significantly weaker inhibition of tumor growth. (mDNA223) A3 / S125 -fPSL2f-MDNA213 Lin / ParH Its therapeutic efficacy is comparable to that of mDNA223. Furthermore, mDNA223... A3 / S125 -fPSL2f-MDNA213 Lin / ParH The efficacy of MDNA223 is significantly superior to that of MDNA11 alone (an IL-2 super agonist) or its co-administration with anti-mPD1.

[0634] In vivo tumor growth inhibition in the J.MC-38 colon cancer model

[0635] like Figure 34 As shown, in the MC-38 model, compared with MDNA223 (in which animals showed a significant reduction in body weight after the first dose), MDNA223... A3 / S125 -fPSL2f- MDNA213 Lin / ParH Systemic administration of the drug demonstrated tumor growth inhibition without weight loss. The mean tumor size was 40 mm. 3 Dosing should begin at [time]. MDNA223 and MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH It was administered at equimolar doses of 7.5 mg / kg and 8.8 mg / kg, respectively.

[0636] in conclusion:

[0637] MDNA223-PSL-MDNA213 is a novel T-MASK construct designed to enhance tolerability while leveraging the synergistic effect between PD1 / PDL-1 blockade and IL-2R agonism for immunotherapy. Ongoing research is exploring alternative targeting / masking of tumor domains and immunomodulators, including other cytokines and potent therapeutic agents, to potentially broaden the utility of the T-MASK platform.

[0638] Example 6: MTD Study Comparing MDNA113 and Unmasked MDNA223

[0639] A. Naming

[0640] The example references the following builder:

[0641] mDNA113 A3 / S125 or mDNA113A A3 / S125 = MDNA223 A3 / S125 - fPSL2f-MDNA213 Lin / ParH

[0642] mDNA113B = mDNA223 A3 / S125 -fPSL2f-ecCD122: MDNA213

[0643] mDNA223 A3 / S125 = MDNA223

[0644] mDNA113 NC = MDNA223-2x(GS-MDNA213) [SEQ ID NO: 325 -327]

[0645] mDNA113B NC =anti-mPD1(K)-MDNA109FEAA-T3AC125S-fPSL2f-ecCD122:anti-mPD1(H)-GS-MDNA213 [SEQ ID NO: 353 – 355]

[0646] B. Objective

[0647] The goal of this study is to evaluate mDNA113 A3 / S125 The maximum tolerated dose (MTD) in a commonly used mouse strain C57Bl / 6 was used to observe mDNA113. A3 / S125 Is it better than MDNA223? A3 / S125 Better tolerance.

[0648] C. Method

[0649] This study used female C57Bl / 6 mice that were 8 weeks old at the start of drug administration. The study design is outlined in Table 16 below.

[0650] Table 16: mDNA113 in MTD studies A3 / S125 and MDNA223 A3 / S125 Dosage of each group

[0651]

[0652] D. Result

[0653] For once-weekly and twice-weekly dosing regimens Figure 35 and Figure 36 The weights of all the animals are shown in the image.

[0654] Group 1 animals (MDNA223) A3 / S125 20 mg / kg, once weekly) showed transient weight loss after administration of all three doses, while group 5 animals (MDNA113) A3 / S125 (23 mg / kg, once a week) showed mild and transient weight loss after the first two doses.

[0655] Group 2 (MDNA223) A3 / S125 10 mg / kg, once a week) and group 6 (mDNA113) A3 / S125 (12 mg / kg, once a week) Animals in both groups showed transient weight loss after the first and third doses, which was recovered as the study progressed.

[0656] Group 3 (MDNA223) A3 / S125 7 mg / kg, once a week) and group 7 (mDNA113) A3 / S125 Animals in both groups (8 mg / kg, once weekly) showed mild, transient weight loss after the first and third doses, which recovered in subsequent study days.

[0657] Group 4 (MDNA223) A3 / S125 All animals administered 7 mg / kg twice weekly died after the second dose. Although group 8 (MDNA113) was found to have... A3 / S125 Of the three animals in the study (8 mg / kg, twice a week), two died after the second dose, but one animal survived after all four doses and gained weight for the remainder of the study period.

[0658] Group 9 (MDNA223) A3 / S125 All animals in group 10 (4 mg / kg, twice weekly) died after the second dose, while group 10 (MDNA223) died after the second dose. A3 / S125 All animals that received 4 mg / kg twice weekly survived to the end of the study. Animals in group 10 showed slight, transient weight loss after the second dose, which recovered and remained consistent throughout the study period.

[0659] Figure 37 The survival curve is shown in the figure.

[0660] Groups 1 to 3 (MDNA223) A3 / S125 Weekly group) and groups 5 through 7 (MDNA113) A3 / S125 All animals in the weekly group survived until the end of the study.

[0661] Group 4 (MDNA223) was discovered. A3 / S125 All animals and group 8 (MDNA113) were treated with 7 mg / kg twice weekly. A3 / S125 Two-thirds of the animals that received 8 mg / kg twice weekly died after the second dose. The remaining animals in group 8 survived to the end of the study. Therefore, a survival rate of 33% was observed in group 8.

[0662] Group 9 (MDNA223) A3 / S125 All animals in group 10 (4 mg / kg, twice weekly) died after the second dose, while group 10 (MDNA113) died after the second dose. A3 / S125 All animals survived to the end of the study (100% survival) at 4.7 mg / kg twice a week.

[0663] E. Conclusion

[0664] When administered via IP at a once-weekly dosing regimen, MDNA113 A3 / S125 Unmasked version MDNA223 A3 / S125 Both were well tolerated at all doses. Therefore, mDNA223 was not achieved. A3 / S125 or mDNA113 A3 / S125 MTD.

[0665] When administered via IP at either the test dose of 7 mg / kg or 4 mg / kg twice weekly, the unmasked version of MDNA223... A3 / S125Poor tolerability. All animals died within one week of the second dose. On the other hand, MDNA113... A3 / S125 Better tolerability was observed with twice-weekly dosing; one in three animals survived at a molar equivalent of 8 mg / kg, and all animals survived at a molar equivalent of 4.7 mg / kg. Therefore, MDNA113... A3 / S125 The MTD was determined to be 4.7 mg / kg, administered twice weekly. Data indicated that, with the twice-weekly dosing regimen, MDNA223... A3 / S125 The MTD is less than 4 mg / kg.

[0666] Example 7: In vitro evaluation of MASK-IT to determine reduced IL-2 activity and retained PD-1 blockade

[0667] A. Objective

[0668] To enhance the masking effect against mDNA223 or other peripheral IL-2 BiSKIT, several novel constructs with additional masking domains (i.e., extracellular domains of human CD122 or CD25) were designed. The reduced IL-2 activity and retention of PD-1 blockade of the constructs compared to unmasked mDNA223 were evaluated.

[0669] B. Method

[0670] These constructs were tested in the Jurkat IL2Rβγ reporter assay (cells lacking the CD25 receptor) and the HEK Blue IL-2 assay (cells expressing the trimeric receptors CD25 / CD122 / CD132).

[0671] These constructs were tested in a PD-1 / PDL-1 blocking reporter gene assay to validate their anti-PD1 efficacy.

[0672] We tested the recovery of IL-2R agonism in these constructs after MMP9 cleavage.

[0673] C. Result

[0674] Unmasked MDNA223 A3 / S125 and MDNA113 A3 / S125 Used as a baseline and experimental control, respectively. Due to anti-mPD1-mDNA109 A3 / S125 -fPSL2f-ecCD25:mDNA213 and anti-mPD1-H9T A3 / S125 -fPSL2f-ecCD25:mDNA213 could not be purified to the oligomer-forming monomer fraction during the purification process; therefore, the P1 and P2 fractions of the construct were tested in the assay. Although anti-mPD1-mDNA109 A3 / S125The P1 fraction of -fPSL2f-ecCD25:mDNA213 showed complete loss of IL-2 activity, but compared with unmasked mDNA223... A3 / S125 In comparison, P2-level fractionation showed a reduction of approximately 41-fold in IL-2 signaling. It also showed a reduction compared to mDNA113. A3 / S125 It has approximately 5 times the masking efficiency, where the masking domain contains only mDNA213, instead of anti-mPD1-mDNA109. A3 / S125 -fPSL2f- ecCD25: The extracellular domains of MDNA213 and CD25 in MDNA213 ( Figure 38A ).

[0675] With unmasked mDNA223 A3 / S125 In comparison, mDNA223 A3 / S125 -fPSL2f- ecCD122:mDNA213 showed a 453-fold reduction in IL-2 signaling and better masking than mDNA113. A3 / S125 Increased by approximately 47 times ( Figure 38B ).

[0676] Table 17 summarizes EC 50 .

[0677] Table 17: EC5 values ​​of individual test samples in the Jurkat IL2Rβγ bioassay 50 Measured values ​​(in pM).

[0678]

[0679] The HEK Blue IL-2 reporter assay tests the presence of trimeric receptor complexes (CD25 / CD122 / CD132) in constructs (including constructs containing IL-2 mutant proteins that retain CD25 binding).

[0680] Fc-MDNA109 T3 / C125 and anti-hPD1-mDNA109 A3 / S125 Used as an experimental control, it demonstrated similar potency. The potency of Rh IL-2 used as a baseline control was approximately 3.5 times lower, consistent with the fact that mDNA109 is a β-enhanced IL-2 mutant protein. MASK-IT anti-mPD1MDNA109 A3 / S125 The two fractions of -fPSL2f-ecCD25:mDNA213, P1 and P2, showed reduced potency, decreasing by approximately 11-fold and 8-fold, respectively. Figure 39A ).

[0681] To compare the efficacy of mask-IT, anti-mPD1-H9T was used. A3 / S125- fPSL2f-ecCD25:mDNA213,H9T T3 / C125 -Albumin and anti-hPD1-H9T A3 / S125 As an anti-mPD1-H9T A3 / S125 Unavailable experimental control. H9T compared to rhIL-2 used as a baseline. T3 / C125 -Albumin and anti-hPD1-H9T A3 / S125 It showed lower efficacy. However, the anti-mPD1-H9T... A3 / S125 The IL-2 signaling efficacy of the P1 and P2 fractions of -fPSL2f-ecCD25:mDNA213 was masked by approximately 124-fold and 43-fold, respectively. Figure 39B ).

[0682] Unmasked mDNA223 A3 / S125 and anti-hPD1-H9TFEAA A3 / S125 Used as an experimental control to compare mDNA223 A3 / S125 -fPSL2f-ecCD122:mDNA213 and anti-hPD1-H9TFEAA A3 / S125 A corresponding decrease in the IL-2 potency of -fPSL2f-ecCD122:mDNA213 was observed. Attenuations of approximately 65-fold and 688-fold were observed, respectively. Notably, in the assay, anti-hPD1-H9TFEAA... A3 / S125 The efficacy exhibited is MDNA223 A3 / S125 Approximately 1 / 7, indicating that H9TFEAA is a weaker IL-2 agonist than mDNA109FEAA. Figure 39C ).

[0683] Table 18 summarizes all EC50 values.

[0684] Table 18: EC values ​​of individual test samples in HEK Blue IL-2 assay 50 Measured values ​​(in pM).

[0685]

[0686] Testing MASK-IT for PD-1 / PDL-1 blockade demonstrated its effectiveness against unmasked mDNA223. A3 / S125 Similar potency to the baseline anti-mPD1 antibody ( Figure 40 ).

[0687] MASK-IT was digested with MMP9 and analyzed by SDS-PAGE. MDNA223 (also known as anti-mPD1-MDNA109FEAAKIH) resolved into three bands under reducing conditions, corresponding to heavy chain 1 (HC1; fused with MDNA109FEAA; approximately 65 kDa), heavy chain 2 (HC2, approximately 52 kDa), and light chain (LC, approximately 25 kDa).

[0688] All tested constructs (i.e., MASK-IT of mDNA223, anti-mPD1-H9TFEAA, and anti-mPD1-MDNA109 fractions 1 and 2) showed near-complete cleavage in the MMP9 reaction. In the absence of rMMP9, the constructs resolved to three bands, with the sizes of HC1 (approximately 90 kDa) and HC2 (approximately 70 kDa) increasing due to the fusion fPSL2f-ecCD122:MDNA213. MMP9 cleavage releases the fPSL2f-ecCD122:MDNA213 masking domain, as indicated by the presence of a protein band of similar size to that of mDNA223. Figure 41 ).

[0689] Recovery of IL-2 activity after MMP9 cleavage

[0690] In Jurkat In the assay of IL2Rβγ or HEK Blue IL-2 reporter genes, Tested MASK-IT after MMP9 cutting IL-2 activity. These studies show that after being cleaved by MMP9, IL-2 activity recovers to a similar potency to its corresponding unmasked version. Figure 42A and 42B Table 19), (except for mPD1-H9T) A3 / S125 -fPSL2f-ecCD25:mDNA213 (excluding P2 fraction).

[0691] Table 19: EC5 values ​​of each construct tested in IL-2 reporter gene assays with or without MMP9. 50 Measured values ​​(in pM)

[0692]

[0693]

[0694] D. Conclusion

[0695] Compared with unmasked control and mDNA113 A3 / S125Compared to using only mDNA213 and IL-13 superfactors as masking domains, the designed constructs exhibited reduced IL-2 activity when tested in the Jurkat IL2Rβγ reporter assay or the HEK Blue IL-2 assay. The data are summarized below, with the masking domains of each construct underlined for inference:

[0696] In Jurkat IL2Rβγ assay, compared with unmasked mDNA223 A3 / S125 In comparison, mDNA223 A3 / S125 -fPSL2f- ecCD122:mDNA213 It showed a 453-fold reduction in IL-2 activity and better masking properties than mDNA113. A3 / S125 (MDNA223) A3 / S125 -fPSL2f- MDNA213 Lin / ParH It increased by about 47 times.

[0697] With unmasked mDNA223 A3 / S125 In comparison, anti-mPD1-mDNA109 A3 / S125 - fPSL2f- ecCD25:mDNA213 The P1 level showed elimination, while the P2 level showed a reduction of approximately 41-fold in IL-2 signaling. In the Jurkat IL2Rβγ assay, the masking effect was better than that of mDNA113. A3 / S125 (MDNA223) A3 / S125 -fPSL2f- MDNA213 Lin / ParH It increased by about 5 times.

[0698] In HEK Blue IL-2 assays, compared with unmasked anti-hPD1-H9T... A3 / S125 Compared to anti-mPD1-H9T A3 / S125 -fPSL2f- ecCD25:mDNA213 The IL-2 signal transduction efficiency was reduced by approximately 124 times and approximately 43 times in the P1 and P2 fractions, respectively.

[0699] In HEK Blue IL-2 assays, a correlation was observed with anti-hPD1-H9TFEAA. A3 / S125 Compared to anti-hPD1-H9TFEAA A3 / S125 -fPSL2f- ecCD122:mDNA213 The activity of IL-2 decreased by 688 times.

[0700] It was observed that MASK-IT was sensitive to protein hydrolysis in vitro and that the weakened IL-2 function after cleavage by MMP9 was restored.

[0701] Example 8: In vivo imaging of labeled mDNA113 and unmasked mDNA223 to verify accumulation in tumors expressing IL-13Rα2.

[0702] A. Objective

[0703] In vivo imaging systems (IVIS) are used to visualize labeled proteins or other analytes in mice to understand their distribution and accumulation. Multiple images are acquired over time to provide a longitudinal view of the biodistribution kinetics of drugs in living animals. This study investigates the potential for persistent and selective tumor accumulation of mDNA113 in mice carrying tumors expressing IL-13Rα2 to generate a superior antitumor response. This study will validate the IL-13Rα2 targeting of mDNA213 within mDNA113.

[0704] B. Method

[0705] Markers for masked and unmasked mDNA223: MDNA113 and MDNA223 were labeled using VivoTag800 IN VIVO NIR fluorescent dye (Perkin Elmer, catalog number NEV11107) according to the manufacturer’s protocol.

[0706] Combined analysis using biolayer interferometry: To ensure that the labeling does not alter receptor binding properties, the binding of MDNA113 and MDNA223 to mouse CD122, one of their homologous receptors, was tested using biological layer interferometry (BLI) / Octet.

[0707] The conditions for the BLI steps used are as follows: Biosensor detection in PBS kinetic buffer (0.5 min), ligand protein fixation (10 min), blocking with Superblock (2 min), baseline in PBS kinetic buffer (2 min), analyte titration association (5 min), and analyte dissociation back to baseline buffer (15 min).

[0708] In vivo imaging: Several human cell lines are known to express IL-13Rα2, including melanoma cell lines. The A375 (human melanoma) xenograft model, expressing IL-13Rα2, was used to evaluate the targeting and accumulation of mDNA113 and mDNA223 in athymic nude mice. A549, a human lung tumor with low / no IL-13Rα2 expression, was used as a control in the contralateral flank of mice.

[0709] Animal information (supplier, species, strain, sex, age or weight, and quantity):

[0710] Supplier: Charles River Laboratories

[0711] Species: Mouse

[0712] Strain: Athymic nude mice (strain code 490)

[0713] Sex: Female

[0714] Age / weight: 5-6 weeks

[0715] Quantity: 12 animals (9 + 3 extra) for purposeful breeding, free of specific pathogens, and experimentally immature at the start of the study. Age / weight refers to age / weight at the time of supplier delivery.

[0716] The study design is shown in Table 20.

[0717] Table 20: Study Design for In Vivo Imaging Using Labeled MDNA113 and MDNA223

[0718]

[0719] Supine mice were imaged using epifluorescence IVIS with excitation at 785 nm and emission at 810 nm before administration (baseline images) and at 4, 24, 72, 120, and 168 hours after injection of the labeled construct. Mice were euthanized and tumors were collected for ex vivo imaging at the last imaging time point (168 hours after injection).

[0720] C. Result

[0721] VivoTag800 tag: Each construct in a total of 1.5–1.8 mg was subjected to a labeling reaction. The labeling results are summarized in Table 21 below. These calculations do not account for the increased molecular weight due to the VivoTag800 fluorescent dye. A protein:vivotag800 ratio greater than 1 indicates successful labeling.

[0722] Table 21: Protein labeling and absorbance data of mDNA223 and mDNA113

[0723]

[0724] Prior to in vivo imaging studies, the binding of the labeled construct to the mouse CD122 receptor was tested using Octet.

[0725] Receptor binding analysis via BLI / Octet: Binding affinity analysis was performed using BLI / Octet to confirm that the receptor binding profiles of mDNA113 and mDNA223 were unaffected by vivot800 labeling. The unlabeled and labeled versions of both constructs showed similar binding properties to mouse CD122, indicating that binding affinity remained unchanged after vivot800 labeling.

[0726] For both unlabeled and labeled mDNA223, the binding affinity constant (Kbinding) calculated based on steady-state kinetics is... D The K+ values ​​were 320 nM and 240 nM, respectively. Similarly, the unlabeled and labeled versions of mDNA113 showed K+ values ​​of 1.8 uM and 4.0 uM, respectively. D ( Figure 43 ).

[0727] It is worth noting that the K of mDNA113 D It is about 10 times higher than MDNA223, which is consistent with the reduced binding affinity of MDNA113 to mouse CD122, due to steric hindrance caused by the fusion of the masking / targeting domain MDNA213.

[0728] In vivo imaging: A375 (IL-13Rα2 positive; right flank) and A549 (IL-13Rα2 negative; left flank) cells were transplanted into the flanks of athymic nude mice and allowed to grow into established tumors. Mice carrying established tumors were injected with labeled mDNA223 or mDNA113 IV, and IVIS images were acquired before administration, at 4, 24, 72, 120, and 168 hours after treatment, as well as in vitro imaging after euthanasia. Compiled images are shown in... Figure 44 middle.

[0729] In some mice, accumulation of labeled mDNA113 was observed in A375 tumors (right flank) compared to A549 tumors (left flank). At earlier time points (i.e., up to 24 hours), mDNA113 was distributed throughout the mice. As the systemic distribution of mDNA113 was cleared at later time points (72 to 168 hours), accumulation in IL-13Rα2-expressing tumors (A375) was clearly visible in at least one of three mice. The localization of mDNA113 to A375 tumors is consistent with the hypothesis that mDNA213 mediates mDNA113 targeting of IL-13Rα2-expressing tumors. Following in vivo imaging at the 168-hour time point, mice were euthanized, and their intact tumors were excised for ex vivo imaging. Ex vivo images confirmed the selective accumulation of mDNA113 in IL-13Rα2-expressing tumors (A375).

[0730] As clearly seen in the 72-hour data images, transient accumulation of labeled mDNA223 (experimental control) was observed in vivo. Furthermore, post-termination ex vivo images showed low accumulation in A549 (IL-13Rα2 negative) and A375 (IL-13Rα2 positive) tumors, indicating a lack of specific targeting in the absence of the mDNA213 domain.

[0731] D. Conclusion

[0732] The BLI / Octet study showed that labeling of mDNA223 and mDNA113 did not change their binding profile.

[0733] In vivo and ex vivo imaging showed persistent and targeted accumulation of mDNA113 in A375 tumors expressing IL-13Rα2 in mice for approximately 10 days. Accumulation of mDNA223 in vivo was observed to be transient; however, ex vivo images showed non-targeted accumulation in both A549 (IL-13Rα2 negative) and A375 (IL-13Rα2 positive) tumors.

[0734] Example 9: The MC-38 tumor growth inhibition (TGI) study to evaluate the efficacy of mDNA113 and mDNA223-fPSL2f-ecCD122:mDNA213 (mDNA113B).

[0735] A. Objective

[0736] The goal of this study was to investigate the therapeutic efficacy of MDNA113 and MDNA223-fPSL2f-ecCD122:MDNA213 (MDNA113B).

[0737] B. Method

[0738] The study design is shown in Table 22.

[0739] Table 22: Study Design of MC38 TGI Study

[0740]

[0741]

[0742] C. Result

[0743] When treated weekly, mDNA113 exhibited superior tumor growth inhibition compared to unmasked mDNA223. This is likely due to conditional activation of mDNA113 at the tumor site / TME following proteolytic cleavage by the protease. When treated with mDNA113, four out of seven mice showed complete tumor regression, compared to one out of seven when treated with mDNA223. Figure 45 ).

[0744] However, the potency of mDNA223-fPSL2f-ecCD122:MDNA213 was lower than that of unmasked mDNA223, which may be attributed to incomplete proteolytic activation at the tumor site. When treated with mDNA223-fPSL2f-ecCD122:MDNA213, two out of seven mice showed complete tumor regression.

[0745] Based on a twice-weekly dosing schedule, mDNA113 showed significant tumor growth inhibition compared to the non-cleavable version of mDNA113. When treated with mDNA113, two out of seven mice showed complete tumor regression. Figure 46 ).

[0746] D. Conclusion

[0747] Both MDNA113 and MDNA223-fPSL2f-ecCD122:MDNA213 showed significant tumor growth inhibition comparable to unmasked MDNA223.

[0748] Example 10: PD (pharmacodynamic) study to evaluate peripheral lymphocyte expansion in mice

[0749] A. Objective

[0750] The goal of this study was to investigate peripheral lymphocyte expansion in response to unmasked MDNA223 and masked MDNA223 (i.e., MDNA113A and MDNA113B).

[0751] B. Method

[0752] Balb / c was used in this study. Animals were administered the drugs according to Table 23.

[0753] Table 23: Study Design of Mouse PD Study

[0754]

[0755] Animals were allowed to acclimatize to the environment for 3 days before the study began.

[0756] After the animals have adapted to the environment, administer the medication to them according to the table above.

[0757] Collect samples and send them for CBC analysis.

[0758] C. Result

[0759] Compared to unmasked mDNA223, MASK-IT mDNA113A and mDNA113B showed attenuated peripheral lymphocyte expansion. However, increasing the dose of the constructs rescued the masked response. MDNA113A showed a similar response at 5-fold higher doses (i.e., 11.7 mg / kg), while MDNA113B showed a similar response to unmasked mDNA223 at 10-fold higher doses (i.e., 24 mg / kg). However, even at 24 mg / kg, lymphocyte expansion was significantly (p = 0.02) lower than that of unmasked mDNA223. Figure 47 ).

[0760] D. Conclusion

[0761] Compared to unmasked mDNA223, MASK-IT showed reduced peripheral lymphocyte expansion, providing strong evidence for reduced systemic toxicity. The data are consistent with the intent of the construct design.

[0762] Example 11: MTD study to measure the maximum tolerated dose of mDNA113A and mDNA113B

[0763] A. Objective

[0764] The purpose of this study was to investigate the tolerance of MDNA113B compared to MDNA113A and the unmasked version MDNA223.

[0765] B. Method

[0766] This study used C57Bl / 6 mice. The study design is shown in Table 24.

[0767] Table 24: MTD Study Design for mDNA113A and mDNA113B

[0768]

[0769] C. Result

[0770] Mice tolerated all doses well. Figure 48 The results of body weight measurements are presented. Sharp weight loss was observed in the MDNA223 (30 mg / kg, once weekly) and MDNA113A (8 mg / kg, twice weekly) groups. However, the mice regained their body weight in subsequent study days. No weight loss was observed in any of the MDNA113B groups.

[0771] D. Conclusion

[0772] All tested doses were well tolerated, and no deaths were observed in any group. Therefore, the mean time to death (MTD) was not achieved.

[0773] Example 12: IL-2 activity before and after MMP-9 digestion (non-cleavable mask-it)

[0774] A. Objective

[0775] The goal of this study is to investigate the restorative effect of proteolytic activation (if present) of cleavable and non-cleavable versions of MASK-IT on IL-2 activity.

[0776] B. Method

[0777] The constructs were subjected to simulated (without MMP9) or MMP9 digestion at 37°C for 1 hour at 5 ug / mL, and the constructs were then processed to assess IL-2 activity in the Jurkat IL-2by reporter assay.

[0778] C. Result

[0779] Figure 49 The data presented show that the cleavable version of MASK-IT, MDNA113, regained its IL-2 activity upon proteolytic activation in the presence of MMP9. As expected, neither the non-cleavable versions, MDNA113A nor MDNA113B, showed a recovery of IL-2 activity. The non-cleavable versions lack the PSL site for cleavage and therefore demask MDNA223.

[0780] Table 25 summarizes EC 50 ,and Figure 50 The document shows evidence of whether or not a cut exists.

[0781] Table 25: EC50 of MASK-IT with or without MMP9

[0782]

[0783] D. Conclusion

[0784] The non-cleavable version was not activated after being hydrolyzed by the MMP9 protein.

[0785] Example 13: Cell receptor binding assay to verify the affinity of mDNA113 for cells expressing IL-13Rα2 in vitro.

[0786] A. Objective

[0787] This study used A375 human melanoma cells (positive for IL-13Rα2) and A549 human lung epithelial cells (negative for IL-13Rα2) to evaluate the binding of MDNA113 to cells expressing IL-13Rα2 in order to determine the presence of MDNA213 (IL-13 superfactor).

[0788] B. Method

[0789] Builder:

[0790] 1. Control (tested only on A549 cells): Fc-MDNA213

[0791] Only at 1800 nM and 600 nM

[0792] 2. Test sample (tested on A375 cells):

[0793] (i)MDNA223

[0794] (ii)Fc-MDNA213

[0795] (iii) MDNA113A

[0796] (iv) MDNA113B

[0797] All titrations were performed using a 3x 8-point curve, starting from 1800 nM.

[0798] Wash the cells with 2 mL of DPBS at 70-80% confluence.

[0799] 1 mL of Corning cell stripping agent will be added.

[0800] Cells were incubated at 37°C for 20 minutes to separate them.

[0801] Gently reverse the flow of the cells and wash them thoroughly with 2 mL of culture medium.

[0802] Transfer the cells to a 50 mL test tube and centrifuge at 400 G for 10 minutes.

[0803] The cells were resuspended in 4 mL of culture medium, and equal aliquots were counted on a hemocytometer.

[0804] Cells were seeded in 96-well U-shaped plates at a dose of 100K cells (100 µL).

[0805] Centrifuge the cells at 400 G for 3 minutes and aspirate the supernatant.

[0806] The cells were resuspended in 100 µL of the construct (in the culture medium).

[0807] Incubate the cells at 4°C for 30 minutes.

[0808] Coloring

[0809] Centrifuge the cells at 400 G for 3 minutes and aspirate the supernatant.

[0810] The cells were resuspended in 200 µL of PBS.

[0811] Centrifuge the cells at 400 G for 3 minutes and aspirate the supernatant.

[0812] Wash the cells with 200 µL of PBS at least twice.

[0813] The cells were resuspended in 100 µL of anti-Fc PE (Abcam, catalog number AB98596) staining solution.

[0814] Incubate the cells at 4°C for 30 minutes.

[0815] Add 100 µL of MACS buffer to each well.

[0816] Centrifuge the plate at approximately 400 xg for 3 minutes.

[0817] Wash the cells at least twice with 200 µL MACS buffer.

[0818] The cells were resuspended in MACS / PBS buffer containing 200 µL of 0.5% PFA.

[0819] Unstained cells will be used in the negative population.

[0820] Cells will be collected using a flow cytometer.

[0821] Flow cytometry

[0822] Construct anti-Fc PE histograms and observe the mean fluorescence intensity (MFI) for each sample.

[0823] The relative average fluorescence intensity of each sample was calculated relative to the untreated (construction-free) control.

[0824] C. Result

[0825] Figure 51 The study data are presented in the table. Fc-MDNA213 binds strongly to IL-13Rα2 expressed on A375 cells, but no binding was observed on A549 cells. MASK-IT, MDNA113A, and MDNA113B tested on A375 cells showed similar binding patterns to MDNA213, but with slight reductions. MDNA223 showed nonspecific binding at the highest tested dose.

[0826] D. Conclusion

[0827] MDNA113A and MDNA113B showed dose-dependent binding to IL-13Rα2 expressed on A375 cells, with a moderate reduction in binding compared to Fc-MDNA213.

[0828] Example 14: MC-38 tumor growth inhibition study to determine the efficacy of mDNA113A and mDNA113B.

[0829] A. Objective

[0830] The goal of this study was to determine the in vivo efficacy of mDNA113A and mDNA113B.

[0831] B. Method

[0832] This study used eighty-eight C57Bl / 6 mice. The study design is shown in Table 26.

[0833] Table 26: Study Design (IP Implementation) of the MC38 TGI Study

[0834]

[0835] C. Result

[0836] Figure 52 The data showed that mDNA223 treatment was superior to monotherapy with mDNA11 or anti-mPD1, as well as their combination. The data are consistent with previous observations in the CT26 colon model and the B16F10 melanoma model.

[0837] Figure 53Data from groups treated with cleavable and non-cleavable mDNA113A are presented. At a dose of 18 mg / kg, the results showed [data missing - likely related to mDNA113A treatment]. NC In comparison, mDNA113A showed superior efficacy.

[0838] Figure 54 Data from groups treated with cleavable and non-cleavable mDNA113B are presented. At a dose of 37 mg / kg, compared with mDNA113A... NC In comparison, mDNA113B demonstrated superior efficacy. This provides strong evidence that mDNA113B is activated at the tumor site, thereby releasing mDNA223, which is not masked by ecCD122 and mDNA213.

[0839] D. Conclusion

[0840] Both MDNA113A and MDNA113B are activated by proteolytic activity at the tumor site and exhibit similar efficacy to unmasked MDNA223.

[0841] Example 15: Adjuvant effect of mDNA223 and novel adjuvant effect of mDNA113 and unmasked mDNA223 in a 4T1.2 orthotopic breast tumor model.

[0842] A. Objective

[0843] The goal of this study was to investigate novel adjuvant or adjuvant effects of MDNA223 and MDNA113 in a 4T1.2 breast orthotopic tumor model.

[0844] B. Method

[0845] This study used Balb / c mice. The study design is shown in Table 27.

[0846] Table 27: Study design of adjuvant / novel adjuvant effects of mDNA113 in a 4T1.2 orthotopic breast tumor model.

[0847]

[0848] C. Result

[0849] like Figure 55 As shown, both MDNA223 and MDNA113 provided significant survival benefits when used as novel adjuvants. The deaths observed in the untreated control and the anti-mPD1 novel adjuvant treatment were caused by distal migration not present in the MDNA113 or MDNA223 novel adjuvant treatments.

[0850] However, for mDNA223, the survival benefit of adjuvant treatment was not significant, but it was superior to anti-mPD1 treatment alone. Figure 56 ).

[0851] MDNA113 cannot be used as an adjuvant because it requires proteolytic activation at the tumor site.

[0852] D. Conclusion

[0853] In the 4T1.2 orthotopic breast tumor model, MDNA223 exhibited a significant novel adjuvant effect when used as an adjuvant, and provided a significant survival benefit when used as a novel adjuvant.

[0854] In the 4T1.2 orthotopic breast tumor model, the use of mDNA113 as a novel adjuvant also prolonged survival, and distant metastasis was observed in untreated controls and mice treated with anti-mPD1.

[0855] Example 16: Assessment of the binding affinity of mDNA113 to CD122 (IL-2Rβ)

[0856] A. Naming

[0857] The example references the following builder:

[0858] mDNA113 A3 / S125 or mDNA113A A3 / S125 = MDNA223 A3 / S125 -fPSL2f- MDNA213 Lin / ParH [SEQ ID NO: 320 -322]

[0859] mDNA113B = mDNA223 A3 / S125 -fPSL2f-ecCD122:MDNA213 [SEQ ID NO: 338 -340]

[0860] mDNA223 A3 / S125 = MDNA223 [SEQ ID NO: 107 -109]

[0861] B. Objective

[0862] The goal of this study was to evaluate the binding affinity of masked (MDNA113) to CD122 (IL-2Rβ) compared to unmasked MDNA223.

[0863] C. Method

[0864] Binding affinity was assessed on the BLI Octet. The general BLI procedure conditions used are as follows:

[0865] Determine the biosensor in the buffer solution (30 seconds).

[0866] His-CD25 / His-CD122 (ligand) fixation (12 minutes).

[0867] Block using Superblock (2 minutes).

[0868] Determine the baseline in the buffer solution (3 minutes).

[0869] Test the association of the construct variant (analyte) by titration (5 minutes).

[0870] Dissociate back into baseline buffer (15 minutes)

[0871] D. Result

[0872] Compared to unmasked mDNA223, MD113A and mDNA113B showed reduced binding to CD122, but with different affinities. Compared to unmasked mDNA223, mDNA113A showed approximately 1.6-fold reduced affinity, while mDNA113B showed 163-fold reduced affinity. Figure 57 ).

[0873] E. Conclusion

[0874] Compared to unmasked mDNA223, mDNA113B exhibited significantly reduced binding affinity.

[0875] Example 17: In vitro evaluation of mask-IT (including mDNA223T3 / C125) to determine reduced IL-2 activity

[0876] A. Naming

[0877] The example references the following builder:

[0878] mDNA223 T3 / C125 [SEQ ID NO: 74-76]

[0879] mDNA113B T3 / C125 [SEQ ID: 365-367]

[0880] B. Objective

[0881] The goal of this study was to verify the reduced IL-2 activity of mDNA113BT3 / C125 compared to unmasked mDNA223T3 / C125, and that this reduced activity was reversible via proteolytic cleavage.

[0882] C. Method

[0883] The construct was tested in the Jurkat IL2Rβγ reporter gene assay (cells lacking the CD25 receptor) with and without MMP9.

[0884] D. Result

[0885] Compared to unmasked mDNA223T3 / C125, mDNA113BT3 / C125 exhibited approximately 630-fold attenuation of IL-2 activity. IL-2 activity recovered upon in vitro MMP9 cleavage. Figure 58 ).

[0886] E. Conclusion

[0887] MDNA113BT3 / C125 is activated during proteolytic cleavage.

[0888] Example 18: Administration of novel adjuvants for mDNA11

[0889] A. Background

[0890] MDNA11 is an albumin-fused "β-enhancing non-α" IL-2 superfactor that preferentially amplifies and activates immune effector cells rather than immunosuppressive Tregs. MDNA11 effectively inhibits subcutaneously implanted syngeneic tumors, resulting in complete tumor regression and a potent memory response. Mice treated with MDNA11 exhibited increased tumor infiltration of CD8+ T and NK cells, as well as elevated circulating memory and antigen-specific CD8+ T cell counts. Based on these results, this study investigated whether pretreatment of immune cells with MDNA11 against cancer cells prior to tumor resection could prevent metastasis and prolong survival. This study compared the efficacy of MDNA11 after tumor removal in a novel adjuvant and adjuvant-adjuvant settings in an aggressive orthotopic breast cancer model prone to distant metastasis.

[0891] B. Objective

[0892] In a homogeneous 4T1.2 orthotopic breast cancer model, the effects of a novel adjuvant using MDNA11 and adjuvant treatment were compared. The model underwent complete surgical resection of the tumor, and the effects of the novel adjuvant and adjuvant MDNA11 treatment on providing long-term protection against subsequent tumor attack were evaluated.

[0893] C. Method

[0894] 4T1.2 tumor cells were implanted into the mammary fat pads of female Balb / c mice, and the tumors were allowed to grow to a palpable size. Mice were either untreated (control), treated with surgery alone to remove the tumors, or administered a single dose of mDNA11 intraperitoneally as a novel adjuvant 4 days before surgery or as an adjuvant 2 days after surgery. Surviving mice from all treatment groups were re-excited with 4T1.2 cells in the right and left hind legs at days 65 and 98 of the study. Throughout the re-excitation phase of the study, survival, tumor volume, and signs of metastasis at necropsy were measured.

[0895] The collected data included survival rates, endpoint autopsies to record signs of metastasis, and multiplex immunofluorescence (MIF) analysis of immune cell infiltration in the resected tumor.

[0896] D. Result

[0897] As shown in Table 28 and Figures 59-62 As shown, all control mice that underwent tumor resection but received no treatment died on day 54 of the study, with severe metastases in multiple organs. In the adjuvant group, which received a single dose of mDNA11 after tumor resection, 3 out of 8 mice (37.5%) survived to the end of the study (day 134). Of the 5 mice in the adjuvant group that died during the study, 4 developed distant metastases. In contrast, of the 8 mice that received a single dose of mDNA11 before tumor resection, 7 (87.5%) survived to day 134 of the study without observable clinical symptoms. The mouse that was found to have died did not develop metastases. On day 65, the remaining mice in the novel adjuvant group (n = 7) and the adjuvant group (n = 3) were re-excited by subcutaneous implantation of 4T1.2 cells into the flanks. A second re-excitation was performed on day 98 of the study. The mice did not receive any additional treatment and did not exhibit tumor growth or metastasis in either of the two re-provocations, while the untreated control mice, which were also implanted with 4T1.2 cancer cells, showed robust tumor growth at the implantation site.

[0898] Table 28: Summary of Autopsy Results

[0899]

[0900]

[0901] E. Conclusion

[0902] In the 4T1.2 orthotopic breast cancer model, a single dose of mDNA11 in a novel adjuvant setting provided immediate and long-term protection against metastatic disease.

[0903]

[0904] The examples provided above are intended to provide a complete disclosure and description of embodiments of how to manufacture and use the compositions, systems, and methods of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention. It will be apparent to those skilled in the art that modifications to the above-described mode of carrying out the invention are intended to fall within the scope of the appended claims. All patents and publications referenced in this specification indicate the level of skill of those skilled in the art to which this invention pertains. All references cited in this disclosure are incorporated by reference as if each reference had been individually incorporated in its entirety by reference.

[0905] All headings and section titles are for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will recognize the appropriate combination of various aspects from different headings and sections in accordance with the spirit and scope of the invention as set forth herein.

[0906] All references cited in this article are incorporated herein by reference in their entirety and for all purposes are as if each individual publication or patent or patent application were specifically or individually indicated to be incorporated herein by reference in their entirety for all purposes.

[0907] Many modifications and variations can be made to this application without departing from the spirit and scope thereof, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are provided by way of example only, and this application is limited only to the terms of the appended claims and the full scope of the equivalents entitled thereto.

Claims

1. An IL-2 cytokine fusion protein, comprising: a) The IL-2 portion, wherein the IL-2 portion comprises IL-2 or an IL-2 mutant protein, optionally an IL-2 mutant protein fusion; b) Protease-sensitive linker (PSL); and c) IL-2 shielding section The PSL connects the IL-2 masking portion to the IL-2 portion.

2. The IL-2 cytokine fusion protein according to claim 1, wherein the masking portion comprises IL-13, an IL-13 mutant protein, an IL-13Ra2 binding mutant protein, an IL-13Ra2 antibody or an antigen-binding fragment thereof, wherein the IL-2 masking portion is capable of binding to IL-13Ra2 but not to IL-13Ra1.

3. The IL-2 cytokine fusion protein according to claim 1 or 2, wherein the masking portion comprises an IL-13 mutant protein having the amino acid sequence of any one of SEQ ID NO: 200-241.

4. The IL-2 cytokine fusion protein according to any one of claims 1 to 3, wherein the masking portion comprises an IL-13 mutant protein comprising the following amino acid substitutions compared to wild-type human IL-13 (SEQ ID NO: 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 according to any one of claims 1 to 4, wherein the masking portion comprises an IL-13 mutant protein having the amino acid sequence of SEQ ID NO: 216 or SEQ ID NO:

228.

6. The IL-2 cytokine fusion protein according to any one of claims 1 to 5, wherein the masking portion 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 according to any one of claims 1 to 6, wherein the IL-2 portion comprises an IL-2 mutant protein having an amino acid sequence having any one of SEQ ID NO: 5-24 and 105.

8. The IL-2 cytokine fusion protein according to claim 7, wherein the IL-2 mutant protein has the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:

9.

9. The IL-2 cytokine fusion protein according to claim 8, wherein the IL-2 mutant protein further comprises T3A and C125S amino acid substitutions.

10. The IL-2 cytokine fusion protein according to any one of claims 1 to 9, wherein the IL-2 portion further comprises albumin, an Fc domain, or an antibody linked to the IL-2 or an IL-2 mutant protein.

11. The IL-2 cytokine fusion protein of claim 10, wherein the albumin is 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 to have a mutated "knob-in-hole" (KiH) in constant region 3 (CH3) of its heavy chain.

13. The IL-2 cytokine fusion protein of claim 12, wherein the IL-2 portion comprises an IL-2 x antibody (KiH) fusion protein, the IL-2 x antibody (KiH) fusion protein comprising: a) A first polypeptide comprising IL-2 or an IL-2 mutant protein, optionally an IL-2 fusion protein, linked to a second constant region and a third constant region (CH2 and CH3) of the "clump" heavy chain of the antibody (KiH); b) A second polypeptide comprising the mortar 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 portion comprises an IL-2 x antibody (KiH) fusion protein, the IL-2 x antibody (KiH) fusion protein comprising: a) A first polypeptide comprising a "pestle" heavy chain of the antibody (KiH) linked to IL-2 or an IL-2 mutant protein, optionally an IL-2 fusion complex; b) A second polypeptide comprising the mortar 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 portion comprises an IL-2 x antibody (KiH) fusion protein, the IL-2 x antibody (KiH) fusion protein comprising: a) A first polypeptide comprising the "pepper" heavy chain of the antibody (KiH); b) A second polypeptide comprising a mortar heavy chain of the antibody (KiH) linked to IL-2 or an IL-2 mutant protein or an IL-2 mutant protein fusion; c) A third polypeptide comprising the light chain of the antibody (KiH).

16. The IL-2 cytokine fusion protein according to any one of claims 12 to 15, wherein the IL-2 masking portion is linked to the IL-2 or the IL-2 mutant protein.

17. The IL-2 cytokine fusion protein according to any one of claims 12 to 16, wherein the IL-2 masking portion is linked to the antibody heavy chain not connected to the IL-2 or the IL-2 mutant protein.

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

19. The IL-2 cytokine fusion protein according to any one of claims 12 to 18, wherein the IL-2 mutant protein has the amino acid sequence of any one of SEQ ID NO: 5-24 and 105.

20. The IL-2 cytokine fusion protein of claim 19, wherein the IL-2 mutant protein further comprises T3A and C125S amino acid substitutions.

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

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

324.

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

25. A nucleic acid composition encoding an IL-2 cytokine fusion protein according to any one of claims 1 to 23.

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

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

28. A method of treating a subject with cancer expressing IL-13Ra2, the method comprising administering to the subject an IL-2 cytokine fusion protein according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24.

29. The method of claim 27, wherein the IL-2 cytokine fusion protein comprises one or more of SEQ ID NO: 1-367.

30. A method of treating cancer in a subject of need, the method comprising administering an IL-2 cytokine fusion protein to the subject, the IL-2 cytokine fusion protein comprising: a) IL-2 mutant protein; and b) Albumin or Fc domain or antibody; The IL-2 mutant protein described herein has the amino acid sequence of any one of SEQ ID NO: 5-24 and 105 and optionally further comprises T3A and / or C125S amino acid substitutions.

31. The method of claim 29, wherein the IL-2 cytokine fusion protein comprises one or more of SEQ ID NO: 1-367.

32. The method according to claim 29 or 30, wherein the IL-2 cytokine fusion protein has the amino acid sequence of SEQ ID NO:

53.

33. The method according to any one of claims 27 to 31, wherein the IL-2 cytokine fusion protein is administered as a novel adjuvant prior to surgical removal of a tumor.

34. The method of claim 32, wherein the IL-2 cytokine fusion protein is administered at most one week prior to the surgery, optionally at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 1 day, or less than 1 day prior to the surgery, or wherein the IL-2 cytokine fusion protein is administered at most 9 weeks prior to the surgery, optionally at most 8 weeks, at most 7 weeks, at most 6 weeks, at most 5 weeks, at most 4 weeks, at most 3 weeks, at most 2 weeks, at most 1 week, or less than 1 week prior to the surgery.

35. The method according to any one of claims 27 to 31, wherein the IL-2 cytokine fusion protein is administered as an adjuvant after surgical removal of a tumor.

36. The method of claim 34, wherein the IL-2 cytokine fusion protein is administered at most one week after the surgery, optionally at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 1 day or less after the surgery, or wherein the IL-2 cytokine fusion protein is administered at least two weeks after the surgery.

37. The method according to any one of claims 32 to 35, wherein the IL-2 cytokine fusion protein is administered as a novel adjuvant prior to and after surgical removal of the tumor.

38. The method of claim 36, wherein the IL-2 cytokine fusion protein is administered at most one week prior to the surgery, optionally at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 1 day, or less than 1 day prior to the surgery, and wherein the IL-2 cytokine fusion protein is administered at most one week after the surgery, optionally at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 1 day, or less than 1 day after the surgery, or wherein the IL-2 cytokine fusion protein is administered at most 9 weeks prior to the surgery, optionally at most 8 weeks, at most 7 weeks, at most 6 weeks, at most 5 weeks, at most 4 weeks, at most 3 weeks, at most 2 weeks, at most 1 week, or less than 1 week prior to the surgery, and wherein the IL-2 cytokine fusion protein is administered starting at least two weeks after the surgery.

39. The method according to any one of claims 27 to 37, wherein the cancer is sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoma, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basaloid breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, small cell lung cancer, kidney cancer, gastric cancer, brain cancer, or CNS tumor.

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