IL-2 fusion protein

Abstract

This specification provides novel IL-2 fusion proteins comprising an IL-2 masking moiety containing IL-13 mutein, IL-13Ra2-binding mutein, an IL-13Ra2 antibody or its antigen-binding fragment, the extracellular domain of CD122, the extracellular domain of CD132, or the extracellular domain of CD25, and an IL-2 moiety conjugated by a protease-sensitive linker (PSL). In some embodiments, the IL-2 fusion protein comprises an IL-13 mutein or an IL-13Ra2 antibody or its fragment that can bind to IL-13Rα2 but not to IL-13Rα1, and the protease-sensitive linker is cleavable by a protease in the tumor microenvironment. Such IL-2 fusion proteins are useful, for example, in the treatment of IL-13Rα2-expressing cancers.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under Section 119 of the United States Patent Act to U.S. Patent Applications No. 63 / 510,580 filed on 27 June 2023, No. 63 / 594,694 filed on 31 October 2023, No. 63 / 595,044 filed on 1 November 2023, No. 63 / 595,271 filed on 1 November 2023, and No. 63 / 575,960 filed on 8 April 2024 (all of which are expressly incorporated herein by reference in their entirety). [Background technology]

[0002] Interleukin-2 (IL-2) is a pluripotent cytokine primarily produced by activated CD4+ T cells and plays a crucial role in the production of a normal immune response. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances B cell growth, and activates monocytes and natural killer cells. Thanks to these activities, IL-2 has been tested and approved for use as a 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).

[0003] Interleukin-2 (IL-2) is one of four α-helix bundle type I cytokines that were first identified as T cell growth factors (Morgan et al., Science 193:1007 (1976)), but it has since been shown to have a wide range of effects. IL-2 promotes CD4+ T helper differentiation (Zhu et al., Annual review of immunology 28:445 (2010), Liao et al., Nat Immunol 9:1288 (2008), and Liao et al., Nat Immunol 12:551 (2011)) and regulatory T (Treg) cell development (Cheng et al., Immunol Rev 241:63 (2011)), induces natural killer cells and cytotoxic CD8+ T cells (Liao et al., Immunity 38:13 (2013)), and mediates activation-induced cell death (AICD) (Lenardo et al., Nature 353:858 (1991)).

[0004] IL-2 acts by interacting with three different receptors: interleukin-2 receptor alpha (IL-2Rα; CD25), interleukin-2 receptor beta (IL-2Rβ; CD122), and interleukin-2 receptor gamma (IL-2Rγ; CD132; the common gamma chain). The first receptor identified was IL-2Rα, a 55kD polypeptide (p55) that appears upon T cell activation and was originally called the Tac (T-activating) antigen. IL-2Rα is approximately 10 -8 M's K dIt binds to IL-2 and is also known as the "high affinity" IL-2 receptor. IL-2 binding to cells expressing only IL-2Rα does not elicit any detectable biological response. In most environments, IL-2 acts via three different receptors: IL-2Rα, IL-2Rβ, and IL-2Rγ. Most cells, such as resting T cells, do not respond 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. IL-2 binding to IL-2Rα triggers sequential engagement of IL-2Rβ and IL-2Rγ, leading to T cell activation. An IL-2 "superkine" with enhanced binding affinity to IL-2Rβ has been previously developed (Levin et al., Nature 484:529 (2012)).

[0005] Despite the great potential of IL-2 for use in cancer therapy, its clinical application remains relatively limited, partly due to the severe toxicity associated with high-dose IL-2 administration. Because IL-2 has a short serum half-life of several minutes, high doses of IL-2 are typically required to achieve optimal immunomodulatory effects. However, such high doses inevitably result in severe toxicity, including vascular leak syndrome (VLS), pulmonary edema, hypotension, and cardiotoxicity. Therefore, novel and effective IL-2 cancer therapies that minimize IL-2-related toxicity are still needed. [Overview of the initiative]

[0006] This specification provides a novel IL-2 fusion protein comprising an IL-2 moiety bound to at least one IL-2 masking moiety containing IL-13 mutein, IL-13Ra2-binding mutein, or an IL-13Ra2 antibody or its antigen-binding fragment, via a protease-sensitive linker (PSL). In embodiments, the IL-2 fusion protein comprises at least one IL-13 mutein, IL-13Ra2-binding mutein, or IL-13Ra2 antibody or its antigen-binding fragment that can bind to IL-13Rα2 but not to IL-13Rα1, and the protease-sensitive linker is cleavable by a protease in the tumor microenvironment. When IL-13 mutein or IL-13Ra2 antibody or its antigen-binding fragment is bound to IL-2 by PSL, the IL-13 mutein 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. In addition, IL-13 mutain or IL-13Ra2 antibody or its antigen-binding fragment enables the target IL-2 fusion protein to bind to IL-13Rα2-expressing tumors. Upon localization to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing IL-13 mutain or IL-13Ra2 antibody or its antigen-binding fragment, and "demasking" the IL-2 portion. The demasked IL-2 portion can then provide antitumor activity at the localized tumor site. Therefore, in embodiments, the target IL-2 fusion protein provided herein advantageously exhibits reduced IL-2-related toxicity while enabling targeted activity in IL-13Rα2-expressing tumors. Such IL-2 fusion proteins are particularly useful for the treatment of IL-13Rα2-expressing cancers.

[0007] In one embodiment, the Specified Information provides an IL-2 cytokine fusion protein comprising: a) an IL-2 moiety comprising IL-2 or IL-2 mutein, optionally an IL-2 mutein fusion; b) at least one protease-sensitive linker (PSL); and c) at least one IL-2 masking moiety comprising IL-13 mutein, IL-13Ra2-binding mutein, or an IL-13Ra2 antibody or its antigen-binding fragment, wherein the PSL binds the IL-2 masking moiety to the IL-2 moiety, and optionally the IL-2 masking moiety can bind to IL-13Ra2 but not to IL-13Ra1.

[0008] In some embodiments, the masking moiety comprises an IL-13 mutain having one amino acid sequence from SEQ ID NOs. 200 to 241. In some embodiments, the masking moiety comprises at least one IL-13 mutain 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 moiety comprises at least one IL-13 mutain having the amino acid sequence of SEQ ID NO: 216 or SEQ ID NO: 228.

[0009] In some embodiments, novel IL-2 fusion proteins are provided herein, comprising an IL-2 moiety bound to at least one IL-2 masking moiety, including IL-13 mutein, IL-13Ra2-binding mutein, or IL-13Ra2 antibody or its antigen-binding fragment, by a protease-sensitive linker (PSL). In embodiments, the IL-2 fusion protein comprises at least one IL-13 mutein, IL-13Ra2-binding mutein, or IL-13Ra2 antibody or its antigen-binding fragment, which can bind to IL-13Rα2 but not to IL-13Rα1, and the protease-sensitive linker is cleavable by a protease in the tumor microenvironment. When IL-13 mutein or IL-13Ra2 antibody or its antigen-binding fragment is bound to IL-2 by PSL, the IL-13 mutein 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. In addition, IL-13 mutain or IL-13Ra2 antibody or its antigen-binding fragment enables the target IL-2 fusion protein to bind to IL-13Rα2-expressing tumors. Upon localization to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing IL-13 mutain or IL-13Ra2 antibody or its antigen-binding fragment, and "demasking" the IL-2 portion. The demasked IL-2 portion can then provide antitumor activity at the localized tumor site. Therefore, in embodiments, the target IL-2 fusion protein provided herein advantageously exhibits reduced IL-2-related toxicity while enabling targeted activity in IL-13Rα2-expressing tumors. Such IL-2 fusion proteins are particularly useful for the treatment of IL-13Rα2-expressing cancers.

[0010] In one embodiment, the Specified Information provides an IL-2 cytokine fusion protein comprising: a) an IL-2 moiety comprising IL-2 or IL-2 mutein, optionally an IL-2 mutein fusion; b) at least one protease-sensitive linker (PSL); and c) at least one IL-2 masking moiety comprising IL-13 mutein, IL-13Ra2-binding mutein, or an IL-13Ra2 antibody or its antigen-binding fragment, wherein the PSL binds the IL-2 masking moiety to the IL-2 moiety, and optionally the IL-2 masking moiety can bind to IL-13Ra2 but not to IL-13Ra1.

[0011] In some embodiments, the masking moiety comprises an IL-13 mutain having one amino acid sequence from SEQ ID NOs. 200 to 241. In some embodiments, the masking moiety comprises at least one IL-13 mutain 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 moiety comprises at least one IL-13 mutain having the amino acid sequence of SEQ ID NO: 216 or SEQ ID NO: 228.

[0012] In some embodiments, the masking portion further includes the extracellular domain of CD122, the extracellular domain of CD132, or the extracellular domain of CD25.

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

[0014] In some embodiments, the IL-2 moiety further comprises albumin or an Fc domain or antibody conjugated to IL-2 or IL-2 mutein. In certain embodiments, the albumin is human albumin, and optionally, the human albumin is recombinant human albumin. In certain embodiments, the antibody is manipulated as a “nob-in-hole” (KiH) having a mutation in the constant region 3 (CH3) of the antibody heavy chain.

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

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

[0017] In some embodiments, the IL-2 moiety is an IL-2x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising IL-2 or IL-2 mutein or IL-12 fusion product bound to the second and third constant regions (CH2 and CH3) of the "knob" heavy chain of the antibody (KiH); b) a second polypeptide comprising the "hole" heavy chain of the antibody (KiH); and c) a third polypeptide comprising the light chain of the antibody (KiH).

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

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

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

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

[0022] In some embodiments, IL-2 mutein has one of the amino acid sequences from SEQ ID NOs. 5-24 and 105. In some embodiments, IL-2 mutein further comprises T3A and C125S amino acid substitutions.

[0023] In some embodiments, PSL can be cleaved in the tumor microenvironment. In certain embodiments, PSL has the amino acid sequence PLGLVVAPLGLVVAPLGLVVA, PLGLWAPLGLWAPLGLWA, GGSGGTPLGLWAGGSGGT, GGSGGTPAGLIGGGSGGT, GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT, or GGSGGTHSSKLQGGSGGT.

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

[0025] In another embodiment, the Specified herein provides a pharmaceutical composition comprising one of the IL-2 cytokine fusions disclosed herein and a pharmaceutically acceptable carrier.

[0026] Furthermore, this specification provides a nucleic acid composition and an expression vector composition containing a nucleic acid encoding a target IL-2 cytokine fusion protein, a host cell containing the nucleic acid composition or the expression vector composition, and a method for producing an IL-2 cytokine fusion protein using the host cell.

[0027] In another embodiment, the Specified provides a method for treating a subject requiring treatment for IL-13Ra2-expressing cancer, comprising administering an IL-2 cytokine fusion protein to the subject.

[0028] In another aspect, a method for treating cancer in a subject requiring treatment, wherein the subject is given an IL-2 cytokine fusion protein, a) IL-2 mutain and, b) comprising albumin or Fc domain or antibody, A method is provided comprising administering an IL-2 cytokine fusion protein having one of the amino acid sequences of SEQ ID NOs. 5-24 and 105, and optionally further comprising a T3A and / or C125S amino acid substitution.

[0029] In another embodiment, the IL-2 cytokine fusion protein comprises one or more of sequence numbers 1 to 367.

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

[0031] In another embodiment, IL-2 cytokine fusion proteins are administered as neoadjuvants prior to surgery to remove tumors.

[0032] In another embodiment, the IL-2 cytokine fusion protein may be administered up to one week prior to surgery, optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day prior to surgery, or up to nine weeks prior to surgery, optionally up to eight weeks, seven weeks, six weeks, five weeks, four weeks, three weeks, two weeks, one week, or one week prior to surgery.

[0033] In another embodiment, IL-2 cytokine fusion proteins are administered as adjuvants after surgery to remove tumors.

[0034] In another embodiment, the IL-2 cytokine fusion protein may be administered up to one week after surgery, optionally up to seven days, six days, five days, four days, three days, two days, one day, or within one day after surgery, or the IL-2 cytokine fusion protein may be administered starting at least two weeks after surgery.

[0035] In another embodiment, the IL-2 cytokine fusion protein is administered both as a neoadjuvant before surgery to remove the tumor and as an adjuvant after surgery to remove the tumor.

[0036] In another embodiment, the IL-2 cytokine fusion protein may be administered up to one week prior to surgery, optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day prior to surgery; or it may be administered up to one week after surgery, optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day prior to surgery; or it may be administered up to nine weeks prior to surgery, optionally up to eight weeks, seven weeks, six weeks, five weeks, four weeks, three weeks, two weeks, one week, or one week prior to surgery; and administration of the IL-2 cytokine fusion protein may be initiated at least two weeks after surgery.

[0037] In another embodiment, cancer is sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoid cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, small cell lung cancer, kidney cancer, stomach cancer, brain cancer, or CNS tumors. [Brief explanation of the drawing]

[0038] [Figure 1A] This document provides graphs summarizing the Jarcut IL2Rβγ reporter analysis of constructs MDNA109-Alb (SEQ ID NO: 56) and MDNA132.15-PSL-MDNA109-Alb (SEQ ID NO: 298) (also known as "MDNA213-PSL-MDNA109-Albumin"). The assays were performed independently on two plates. Plots and curve fittings are presented for each construct on each plate. [Figure 1B] This paper provides graphs of Jarcut IL2Rβγ reporter analysis of constructs MDNA11 (SEQ ID NO: 53) and MDNA132-PSL-MDNA11T3 / C125 (SEQ ID NO: 294). The assays were performed independently on two plates. Plots and curve fittings are presented for each construct on each plate. [Figure 2] For the constructs shown, sensorograms from BLI are provided, using 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. [Figure 3] We provide sensorograms for BLI performed using constructs shown with receptors, specifically human IL13Ra2 as the ligand and the constructs (Fc-MDNA132.15 (SEQ ID NO: 270) and MDNA132.15-PSL-MDNA109-albumin (SEQ ID NO: 298)) as the analytes. [Figure 4]This provides a summary of a study demonstrating that MMP9-mediated proteolytic activation restores IL-2 activity of MDNA213-PSL-MDNA109-albumin (SEQ ID NO: 298). [Figure 5] This provides a summary of studies showing that MDNA223T3AC125S-fPSL2f-MDNA213 (SEQ ID NOs. 299-301) remains unchanged in reduced IL-2 activity and PD-1 / PDL-1 blockade. [Figure 6] This provides a summary of studies demonstrating that MMP9-mediated proteolytic activation restores IL-2 activity in MDNA223T3AC125S-fPSL2f-MDNA213 (SEQ ID NOs. 299-301). [Figure 7] This provides a summary of studies showing that MDNA223T3AC125S-fPSAf-MDNA213:anti-mPD1(H) (SEQ ID NOs. 302-304) exhibits a 12-fold reduction in potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade. [Figure 8] This provides a summary of studies showing that MDNA223T3AC125S-fPSL3f-MDNA213:anti-mPD1(H) (SEQ ID NOs. 305-307) exhibits an 11-fold reduction in potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade. [Figure 9] This provides a summary of studies showing that MDNA223T3AC125S-fPSL2fPSL3f-MDNA213:Anti-mPD1(H) (SEQ ID NOs. 308-310) exhibits a 10-fold reduction in potency (i.e., masked activity) in IL-2-mediated signaling and no change in PD-1 / PDL-1 blockade. [Figure 10] This provides a summary of studies showing that MDNA223T3AC125S-GS-MDNA213:anti-mPD1(H) (SEQ ID NOs. 311-313) exhibits an 11-fold reduction in potency (i.e., masked activity) in IL-2-mediated signaling and no change in PD-1 / PDL-1 blockade. [Figure 11]This provides a summary of studies showing that MDNA223T3AC125S: anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NOs. 314-316) exhibits a 3-fold reduction in potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade. [Figure 12] This provides a summary of studies showing that anti-mPD1(K)-fPSL2f-MDNA213:MDNA223T3AC125S (SEQ ID NOs. 317-319) exhibits a 2-fold reduction in potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade. [Figure 13] This provides a summary of studies showing that MDNA223T3AC125S-fPSL2f-MDNA213: anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NOs. 320-322) exhibits a 39-fold reduction in potency (i.e., masked activity) in IL-2 mediated signaling and no change in PD-1 / PDL-1 blockade. [Figure 14] This provides a summary of studies demonstrating that MDNA223T3AC125S:anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NOs. 320-322) is susceptible to MMP9 cleavage in vitro. [Figure 15] This provides a summary of studies showing that MDNA223T3AC125S: anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NOs. 320-322) is activated by MMP9 cleavage when observed in an IL-2R reporter assay. [Figure 16] This provides a summary of studies showing that MDNA213-fPSL2f-MDNA11T3AC125S (SEQ ID NO: 323) exhibits approximately 10-fold reduced potency (i.e., masked activity) in IL-2 mediated signaling. [Figure 17] This provides a summary of tests demonstrating that MDNA213-fPSL2f-MDNA11T3AC125S (SEQ ID NO: 323) is susceptible to MMP9 cleavage in vitro. [Figure 18] This provides a summary of studies showing that MDNA213-fPSL2f-MDNA11T3AC125S (SEQ ID NO: 323) is activated by MMP9 cleavage when observed in an IL-2 reporter assay. [Figure 19] The image shows SDS-PAGE analysis. Images of reduced SDS-PAGE gels from various BiSKIT samples are shown. The location of the molecular weight marker is indicated on the left. [Figure 20] The image shows SDS-PAGE analysis. Images of reduced SDS-PAGE gels from various BiSKIT samples are shown. The location of the molecular weight marker is indicated on the left. [Figure 21A] The graphs of the Jarcut IL2Rβγ receptor analysis are shown. Plots and curve fittings are presented for each construct on each plate. Data are presented as mean ± SEM. [Figure 21B] The graphs of the Jarcut IL2Rβγ receptor analysis are shown. Plots and curve fittings are presented for each construct on each plate. Data are presented as mean ± SEM. [Figure 22] The graphs of CTLL-2 proliferation analysis for the indicated constructs are shown. Plots and curve fittings are presented for each construct on each plate. Data are presented as mean + SEM. [Figure 23] The results of a human PBMC proliferation assay are shown. Three PBMC donors were stimulated for 48 hours with constructs of different concentrations. Proliferation was analyzed by BrDU uptake. Data are presented as mean + SEM. [Figure 24] The results of a human pSTAT5 assay are shown. Three PBMC donors were stimulated for 15 minutes with constructs at different concentrations. pSTAT5 induction is shown in naive CD8+ T cells, NK cells, and Treg cells. [Figure 25] This document presents a compilation of EC50 data for masked and unmasked MDNA223 tested across PBMC samples from three human donors for naive CD8+ T cells and Treg cells. [Figure 26]For various constructs, the frequencies of pSTAT5-expressing cells—CD8+ T cells, NK cells, and Tregs—in three donors are shown. Human PBMCs were stimulated with indicated masking and unmasking MDNA11 for 15 minutes and analyzed for pSTAT5 by flow cytometry. Data are presented as single measures. [Figure 27] This document presents edited EC50 data for masked and unmasked MDNA11 tested across PBMC samples from three human donors for naive CD8+ T cells and Treg cells. [Figure 28] This shows the EC50 ratio of naive CD8+ T cells to Tregs. The EC50 ratio of naive CD8 T cells to Tregs is the average of the individual ratios from different PMBC samples used. [Figure 29] This study shows the binding kinetics of the cell receptor (IL13Ra2) to Fc-MDNA213 (SEQ ID NOs. 270-271) and MDNA223A3 / S125-fPSL2f-MDNA213Lin / ParH (SEQ ID NOs. 320-322). IL-13Ra2-expressing EMT6 / IL13Ra2 cells were subjected to increasing concentrations of the constructs shown, and binding was detected by anti-Fc antibodies. [Figure 30] CBC analysis: Complete blood counts are shown 3 days after treatment with MDNA223A3 / S125 masking and non-masking methods. Mice were treated with the indicated constructs (IP). Blood was collected for CBC analysis 72 hours after treatment. Data are presented as mean ± SEM. [Figure 31] CBC analysis: Complete blood counts are shown 3 days after treatment with MDNA11A3 / S125 masking and non-masking methods. Mice were treated with the indicated constructs (IP). Blood was collected for CBC analysis 72 hours after treatment. Data are presented as mean ± SEM. [Figure 32] The study using the MC-38 colon cancer model is shown: C57Bl / 6 mice were treated with the indicated construct at 15 μg / tumor in a twice-weekly regimen, for a total of four doses. Data are presented as mean tumor measurements and mean ± SEM at different days. [Figure 33] The study using the EMT6 / IL13Rα2 mammary tumor model is shown: Balb / c mice were treated with IP therapy using equimolar doses of the construct shown, administered once weekly for a total of two doses, 4 days after cell transplantation. Data are presented as mean tumor measurements and mean ± SEM at different days. [Figure 34] The study using the MC-38 colon cancer model is shown: C57Bl / 6 mice were treated with IP using equimolar doses of the constructs shown, administered once weekly in a total of two doses. Data are presented as mean ± SEM, with mean body weight (left) and tumor measurements (right) at different days. [Figure 35] This shows the body weight of animals in a once-weekly administration regimen. The mean body weight for each group is shown. Data are presented as mean ± SD. [Figure 36] This shows the body weight of animals in a twice-weekly administration regimen. Mean body weight measurements for each group are shown. Data are presented as mean ± SD. [Figure 37] This shows the survival curves of animals in an MTD (Module-to-Dose) study using MDNA113AA3 / S125. Mice were treated with the indicated doses and administration schedules. The Kaplan-Meier plots show overall survival for each group. Mice that survived until the end of the study were censored on day 18. N=3 mice per group. [Figure 38] The graphs of the Jarcut IL2Rβγ receptor analysis are shown. Plots and curve fittings are presented for each construct on each plate. Data are presented as mean ± SEM. [Figure 39] The graph shows the results of the HEK Blue IL-2 reporter analysis. OD650nm data were plotted as a function of cytokine concentration (nM) on a semi-log graph. The 4-parameter logistic curve fitting is presented as a solid line. Error bars represent the standard error of the mean of the repeated wells. [Figure 40] The graphs of the mouse PD-1 / PD-L1 blockade assay are shown. Plots and curve fittings are presented for each construct in each plate. [Figure 41]The image shows SDS-PAGE analysis. Images of reduced SDS-PAGE gels using various MASK-ITs are shown. The location of the molecular weight marker is indicated on the left. [Figure 42A] Graphs of Jarcut IL2Rβγ or HEK Blue IL-2 reporter analysis are shown. Plots and curve fittings are presented for each construct on each plate. Data are presented as mean ± SEM. [Figure 42B] Graphs of Jarcut IL2Rβγ or HEK Blue IL-2 reporter analysis are shown. Plots and curve fittings are presented for each construct on each plate. Data are presented as mean ± SEM. [Figure 42C] Graphs of Jarcut IL2Rβγ or HEK Blue IL-2 reporter analysis are shown. Plots and curve fittings are presented for each construct on each plate. Data are presented as mean ± SEM. [Figure 43] Representative sensorgrams of unlabeled and labeled MDNA223 and MDNA113, showing their binding to mouse CD122, are shown. [Figure 44] The in vivo imaging data at the indicated time is shown. Tumors A549 and A375 are located in the left and right flanks, respectively, as can be seen. [Figure 45] The results of the MC-38 TGI study are shown: Mice were treated as indicated. Data are expressed as mean ± SEM. [Figure 46] The results of the MC-38 TGI study are shown: Mice were treated as indicated. Data are expressed as mean ± SEM. [Figure 47] The mice exhibited a PD response. Mice were treated with the indicated construct. Samples were collected on day 3 for CBC analysis. Data are expressed as mean ± SEM. [Figure 48] Body weight is indicated. Animals in different groups were treated with either a once-weekly or twice-weekly dosing regimen, as indicated. Body weight was measured twice a week during the study. [Figure 49]The Jarcut IL-2 reporter assay is shown. Data are presented as mean ± SEM. [Figure 50] The SDS-PAGE for detecting disconnection using MMP9 is shown. [Figure 51] Flow cytometry demonstrates IL-13Ra2 binding. The shown construct was titrated in A375 cells. Fc-MDNA213 was titrated in both A375 and A549 cells. [Figure 52] The results of the MC38 tumor growth inhibition study are shown: C57Bl / 6 mice were treated with IP as shown. Data are presented as mean ± SEM. [Figure 53] The results of the MC38 tumor growth inhibition study are shown. C57Bl / 6 mice were treated with IP as shown. Data are presented as mean ± SEM. [Figure 54] The results of the MC38 tumor growth inhibition study are shown. C57Bl / 6 mice were treated with IP as shown. Data are presented as mean ± SEM. [Figure 55] Survival curves for the neoadjuvant effects of MDNA113, MDNA223, or anti-mPD1 in a 4T1.2 breast tumor model are shown. [Figure 56] Survival curves for the adjuvant effects of MDNA223 or anti-mPD1 in a 4T1.2 breast tumor model are shown. [Figure 57] This document provides sensorograms of BLI performed using constructs that use human CD122 (IL-2Rβ) as the ligand and MDNA223A3 / S125 (SEQ ID NOs. 107-109), MDNA113AA3 / S125 (SEQ ID NOs. 320-322), and MDNA113BA3 / S125 (SEQ ID NOs. 335-337) as the analytes. [Figure 58] This provides an overview of a study demonstrating that MDNA113BT3 / C125 is activated by MMP9 cleavage when observed in an IL-2R reporter assay. [Figure 59]The Kaplan-Meier survival plot shows the total number of surviving mice (n / 8) over 65 study days. Mice were orthotopically transplanted with 4T1.2 tumor cells and treated with neoadjuvant and adjuvant MDNA11 (5 mg / kg) with surgical tumor resection (day 16). *Comparison with control (surgery, no treatment). Mantel-Cox test. N=8 per group. [Figure 60] Individual tumor volumes in mice after initial tumor transplantation with 4T1.2 tumors and subsequent reloading are shown. N=5 in the control group. N=7 and 3 in the neoadjuvant and adjuvant groups, respectively. [Figure 61] Multiplex immunofluorescence images of 4T1.2 tumors excised from control mice and mice treated with MDNA11 (5 mg / kg) showing immune cell infiltration are shown. N=4 per group. [Figure 62] This shows individual quantification of immune cell infiltration across the total tumor area using multiplex immunofluorescence imaging of 4T1.2 tumors excised from control mice and mice treated with MDNA11 (5 mg / kg). N=4 per group. [Modes for carrying out the invention]

[0039] To facilitate understanding of this disclosure, certain terms and phrases are defined below and throughout this specification.

[0040] definition All references cited herein are incorporated by reference in their entirety as if they were fully described. Unless otherwise defined, technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, NY 2001), and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide a general guide to many of the terms used in this disclosure. Procedures involving the use of commercially available kits and reagents, where necessary, are generally carried out according to the manufacturer's defined protocols and / or parameters unless otherwise specified.

[0041] As used herein, "IL-2" refers to wild-type IL-2, whether natural or recombinant. Mature human IL-2 arises as a 133-amino acid sequence (less than the signal peptide consisting of an additional 20 N-terminal amino acids), as described in Fujita, et.al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of human IL-2 (SEQ ID NO: 1, full length) is found in Genbank accession locator NP_000577.2. The amino acid sequence of mature human IL-2 is shown in SEQ ID NO: 2 (human wild-type mature; substitution position numbering is based on this sequence). The amino acid sequence of mouse (Mus musculus) IL-2 is found in Genbank accession locator (SEQ ID NO: 3). The amino acid sequence of mature mouse IL-2 is shown in SEQ ID NO: 4. Sequence ID 1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT Sequence ID 2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT Sequence ID 3 MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ Sequence ID 4 APTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0042] As used herein, “IL-2 mutein” means an IL-2 polypeptide in which specific substitutions have been made to the interleukin-2 protein. IL-2 muteins are characterized by the insertion, deletion, substitution, and modification of amino acids at one or more sites or other residues of the native IL-2 polypeptide chain. In some embodiments, the insertion, deletion, substitution, and / or modification result in an IL-2 mutein that retains IL-2Rβ binding activity. Exemplary muteins may include the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. The phrase “includes an” can also be used interchangeably with “includes at least one” when referring, for example, to the IL-2 muteins of this disclosure.

[0043] Mutein also includes conserved modifications and substitutions at other positions in IL-2 (i.e., those that have minimal impact on the secondary or tertiary structure of mutein). Such conserved substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and by Argos in EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conserved modifications: 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.

[0044] "Numbered according to IL-2" means identifying amino acids selected by referring to the positions in which those amino acids normally exist in the mature sequence of wild-type IL-2, for example, wild-type IL-2 having the sequence of SEQ ID NO: 2. For example, in the embodiment, R81 refers to arginine, which is the 81st amino acid in SEQ ID NO: 2; L80 refers to leucine, which is the 80th amino acid in SEQ ID NO: 2; L85 refers to leucine, which is the 85th amino acid in SEQ ID NO: 2; I86 refers to isoleucine, which is the 86th amino acid in SEQ ID NO: 2; I92 refers to isoleucine, which is the 92nd amino acid in SEQ ID NO: 2; F42 refers to phenylalanine, which is the 42nd amino acid in SEQ ID NO: 2; and K43 refers to lysine, which is the 43rd amino acid in SEQ ID NO: 2.

[0045] As used herein, the abbreviations for genetically encoded L-enantiomers used in the methods disclosed herein are conventional and are as follows in Table 1. [Table 1]

[0046] "Hydrophilic amino acids" refer to amino acids that exhibit hydrophobicity less than zero, according to the standardized consensus hydrophobicity scale of Eisenberg et al., 1984, J.Mol.Biol.179:125-142. Genetically encoded hydrophilic amino acids include Thr(T), Ser(S), His(H), Glu(E), Asn(N), Gln(Q), Asp(D), Lys(K), and Arg(R).

[0047] The term "cell type possessing the IL-2Rαβγ receptor" refers to cells known to possess this receptor type, namely T cells, activated T cells, B cells, activated monocytes, and activated NK cells.

[0048] As used herein with respect to polypeptide or DNA sequences, the term “identity” refers to subunit sequence identity between two molecules. Molecules are identical at a subunit position when that position is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide). The similarity between two amino acid or nucleotide sequences is a direct function of the number of identical positions. Generally, sequences are aligned to obtain the highest-order match. Identity can be calculated, if necessary, using published methods and widely available computer programs such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package from the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with its default parameters.

[0049] 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).

[0050] If the variant IL-2 polypeptides of this disclosure are "substantially pure," they may be at least about 60% by weight (dry weight) of the polypeptide of interest, e.g., the polypeptide containing the variant IL-2 amino acid sequence. For example, the polypeptide may 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 polypeptide of interest. Purity can be measured by any suitable standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0051] An "agonist" is a compound that interacts with a target to cause or promote an increase in the activation of that target.

[0052] A "partial agonist" is a compound that interacts with the same target as an agonist, but even when the dose of the partial agonist is increased, it does not produce the same large biochemical and / or physiological effects as the agonist.

[0053] A "superagonist" (also called a "superkine") is a type of agonist that can produce a larger, more powerful response than the endogenous agonist of the target receptor, and therefore has a potency exceeding 100%.

[0054] "Operatively linked" means that the target nucleotide sequence (i.e., the sequence encoding IL-2 mutein) is linked to a regulatory sequence in a manner that enables the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system, or in the host cell if the vector is introduced into the host cell). "Regulatory sequences" include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequences include those that direct the constitutive expression of the nucleotide sequence in many types of host cells, and those that direct the expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will understand that the design of an expression vector may depend on factors such as the selection of the host cell to be transformed and the desired level of protein expression. The expression construct of the present invention can be introduced into host cells to produce human IL-2 mutein as disclosed herein, or a biologically active variant thereof.

[0055] As used herein, the term “IL-2 masking moiety” refers to a polypeptide component that reduces the activity of the IL-2 moiety. In some embodiments, the IL-2 masking moiety includes the IL-13 protein, its variants, or fragments. In some embodiments, the IL-2 masking moiety further includes the extracellular domain of CD122, CD132, or CD25. The phrase “includes an” can also be used interchangeably with “includes at least one” when referring, for example, to the IL-2 mutain, IL-2 masking moiety, IL-13 mutain, or PSL of this disclosure.

[0056] The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to a specific target cell but also to its offspring or potential offspring. Such offspring may not be identical to the parent cell in practice, because certain modifications may occur in subsequent generations due to mutation or environmental influences, but they are still included within the scope of the terms used herein.

[0057] As used herein, the terms “transformation” and “transfection” refer to a variety of industry-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, particle gun, or electroporation.

[0058] As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, physiological salines, solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders that are compatible with pharmaceutically acceptable administration. Auxiliary active compounds (e.g., antibiotics) may also be incorporated into the composition.

[0059] 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, among others.

[0060] As used herein, the terms “cancer” (or “cancerous”), “hyperproliferative,” “tumor,” and / or “neoplastic” refer to cells capable of autonomous growth (i.e., an abnormal appearance or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic disease conditions may be classified as pathological (i.e., characterizing or constituting a disease condition), or they may be classified as nonpathological (i.e., deviations from normal but not related to a disease condition). This term means that it includes all types of cancerous growth or carcinogenic processes, metastatic tissue, or malignant transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. “Pathological hyperproliferative” cells occur in disease conditions characterized by the growth of malignant tumors. Examples of nonpathological hyperproliferative cells include the proliferation of cells associated with wound repair. The terms “cancer” or “neoplasm” are used to refer to malignant tumors of various organ systems, including those affecting the lungs, breasts, thyroid gland, lymph nodes and lymphoid tissues, the reproductive system, the gastrointestinal tract, and the urogenital tract, as well as adenocarcinomas, which are generally considered to include most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung cancers, small intestine cancers, and esophageal cancers. Cancer 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, lymphoid cancers, gastrointestinal cancers, prostate cancers, ovarian cancers, breast cancers, basal 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 glioblastoma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendrocyte, menangioma, meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, O6-methylguanine-methyltransferase (MGMT) positive or negative CNS tumors, and furin-positive CNS tumors.

[0061] The term "cancer" is recognized in the industry as referring to malignant tumors of epithelial or endocrine tissue, including respiratory carcinomas, gastrointestinal carcinomas, genitourinary carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine carcinomas, and melanomas. "Adenocarcinoma" refers to carcinomas that originate from glandular tissue or in which tumor cells form recognizable glandular structures.

[0062] As used herein, the term “hematopoietic neoplasm” refers to a disease involving hematopoietic hyperplasia / neoplasmic cells arising from, for example, the bone marrow, lymphoid system, or erythroid system, or their progenitor cells. Preferably, the disease arises from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Additional exemplary bone marrow disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11:267-97); and lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), including lineage B ALL and lineage T ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), pilocytic cell leukemia (HLL), and Waldenstrom macroglobulinemia (WM). Additional 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 lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Stemberg disease.

[0063] As used herein, terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures a disease and / or side effects resulting from the disease. “Treatment,” as used herein, encompasses all treatments of diseases in mammals, particularly humans, and includes (a) preventing the development of a disease in subjects who have been prediagnosed as having a disease or being at risk of acquiring a disease, but who have not yet been diagnosed as having a disease; (b) suppressing a disease, i.e., inhibiting its development; and (c) alleviating a disease, i.e., causing its regression. A therapeutically effective dose may be a dose that reduces the number of tumors, tumor size, and / or increases survival.

[0064] The terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to mammals including, but not limited to, humans and non-human primates, including monkeys and humans; sports mammals (e.g., horses); domestic mammals (e.g., sheep, goats, etc.); pet mammals (e.g., dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0065] The terms “pharmaceutically acceptable” and “physiologically acceptable” mean a bioacceptable 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 not a material that is biologically or otherwise undesirable; for example, the material can be administered to a subject without causing substantially undesirable biological effects. Such a pharmaceutically acceptable composition can therefore be used, for example, when administering IL-2 mutein to a subject. In particular, IL-2 mutein containing substitutions L80F, R81D, L85V, I86V, and I92F is administered in combination with anti-PD-1 to subjects with cancer. In some embodiments, the administered IL-2 mutein further includes a substitution at position F42A. In some embodiments, the administered IL-2 mutein further includes a substitution at position K43N.

[0066] As used herein, the term “unit dosage form” refers to a physically distinct unit suitable as a unit dose for the subject to be treated, each unit comprising a predetermined amount of a pharmaceutical carrier (excipient, diluent, vehicle, or filler) and optionally associated with producing a desired effect (e.g., prophylactic or therapeutic effect) when administered in one or more doses. In some embodiments, the therapeutic effect is to reduce the number of tumors. In some embodiments, the therapeutic effect is to reduce the size of tumors. In some embodiments, the therapeutic effect is to increase survival rates.

[0067] In some embodiments, the unit dosage form may be in ampoules and vials containing, for example, a liquid composition or a composition in a freeze-dried or lyophilized state, and a sterile liquid carrier may be added before in vivo administration or delivery. Individual unit dosage forms may be included in multi-dose kits or containers. IL-2 mutein combined with an anti-PD-1 antibody, and its pharmaceutical composition, may be packaged in one or more unit dosage forms for ease of administration and uniformity of dosage.

[0068] The "therapeutic effective dose" falls within a relatively broad range that can be determined through experiments and / or clinical trials. For example, in the case of in vivo injection, this would be, for instance, direct injection into the target tissue or vascular structure (e.g., liver tissue or veins). Other effective doses can be readily established by those skilled in the art through routine trials to establish dose-response curves.

[0069] "Effective dose" or "sufficient dose" means an amount, administered in a single dose or multiple doses, alone or in combination with one or more other compositions (therapeutic drugs, etc.), treatments, protocols, or therapeutic regimens (e.g., including vaccine regimens) that provides a detectable response, a measurable or detectable degree, or an expected or desirable outcome or benefit to a subject over any period of time (e.g., minutes, hours, days, months, years, or until cure).

[0070] An “effective dose” or “sufficient dose” for treatment (e.g., to provide improvement or therapeutic benefit or enhancement) is also a satisfactory outcome if it reduces, mitigates, inhibits, suppresses, limits, or controls the progression or worsening of the disease, but typically it is effective in providing a measurable response to one, more, or all adverse symptoms, consequences, or complications of the disease, e.g., one or more adverse symptoms, disorders, illnesses, pathologies, or complications caused by or associated with the disease. In some embodiments, an effective dose is sufficient to reduce the number of tumors. In some embodiments, an effective dose is sufficient to reduce the size of tumors. In some embodiments, an effective dose is sufficient to increase survival rates.

[0071] "Prevention" and its grammatical variations mean methods in which contact, administration, or in vivo delivery to a subject precedes the onset of the disease. Administration or in vivo delivery to a subject may be carried out before the onset of any adverse symptoms, conditions, or complications caused by or associated with the disease. For example, screening (e.g., genetic) may be used to identify such subjects as candidates for the described methods and uses, even if the subjects do not develop the disease. Thus, even if such subjects do not develop the symptoms of the disease, such subjects include those screened positive for a deficiency or absence of a functional gene product (protein) leading to the disease, or for the production of abnormal partially functional or non-functional gene products (proteins), and subjects screened positive for abnormal or defective (mutant) gene products (proteins) leading to the disease.

[0072] I. Detailed explanation This specification provides novel IL-2 fusion protein compositions comprising an IL-2 moiety bound to IL-13 mutein, IL-13Ra2-binding mutein, or an IL-13Ra2 antibody or its antigen-binding fragment, via a protease-sensitive linker (PSL). In embodiments, the IL-2 fusion protein composition comprises IL-13 mutein, IL-13Ra2-binding mutein, or an IL-13Ra2 antibody or its antigen-binding fragment, which can bind to IL-13Rα2 but not to IL-13Rα1, and the protease-sensitive linker is cleavable by a protease in the tumor microenvironment. Although not based on any specific operating theory, it is believed that the IL-13 mutein or IL-13Ra2 antibody or its antigen-binding fragment in the IL-2 fusion protein compositions described herein binds to and masks the activity of the IL-2 moiety. In addition, IL-13 mutain or IL-13Ra2 antibody or its antigen-binding fragment enables the target IL-2 fusion protein composition to bind to IL-13Rα2-expressing tumors. Upon localization to the tumor microenvironment, the PSL of the IL-2 fusion protein undergoes proteolytic cleavage, thereby releasing IL-13 mutain or IL-13Ra2 antibody or its antigen-binding fragment, and "demasking" the IL-2 portion. The demasked IL-2 portion can then act at the tumor site. Therefore, in embodiments, the target IL-2 fusion protein provided herein advantageously exhibits reduced IL-2-related toxicity while enabling targeted activity in IL-13Rα2-expressing tumors. Such IL-2 fusion protein compositions are useful for the treatment of IL-13Rα2-expressing cancers. Embodiments of the target IL-2 fusion protein are described in further detail below.

[0073] A. IL-2 section In embodiments, the IL-2 fusion protein provided herein comprises an IL-2 moiety containing IL-2 or IL-2 mutein. 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 mutein 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, conservative substitutions, including substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. In some embodiments, these mutations are located at amino acid residues in contact with IL-2Rβ and / or IL-2Rγ.

[0074] More specifically, mutations (conservative or non-conservative, by addition or deletion) can occur at one or more positions. In some embodiments, the mutations in IL-2 mutain, 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 mutains that can be included in the target IL-2 fusion protein are shown in Table 2 below. [Table 2-1] [Table 2-2]

[0075] In some embodiments, substitutions in IL-2 mutein include L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, IL-2 mutein further includes the F42A substitution, numbered according to wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, IL-2 mutein further includes the Y45A substitution, numbered according to wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, IL-2 mutein further includes the E62A substitution, numbered according to wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, substitutions in IL-2 mutein include F42A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, substitutions in IL-2 mutain include F42A, Y45A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of Sequence ID No. 2. In some embodiments, substitutions in IL-2 mutain include F42A, E62A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of Sequence ID No. 2. In some embodiments, substitutions in IL-2 mutain include F42A, Y45A, E62A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of Sequence ID No. 2. In some embodiments, substitutions in IL-2 mutain include E62A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, substitutions in IL-2 mutain include Y45A, E62A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, substitutions in IL-2 mutain include Y45A and E62A, numbered according to wild-type human IL-2 of SEQ ID NO: 2.

[0076] In some embodiments, IL-2 mutein 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 IL-2 mutein resulting in increased and / or enhanced IL-2Rβ binding include L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, IL-2 mutein for use in the IL-2 fusion protein described in the present invention comprises L80F, R81D, L85V, I86V, and I92F and exhibits increased IL-2Rβ binding. In some embodiments, IL-2 mutein for use in the present invention further includes a substitution at position F42A. In some embodiments, IL-2 mutein for use in the present invention further includes a substitution at position K43N. In some embodiments, mutain includes one or more substitutions selected from the group consisting of substitutions L80F, R81D, L85V, I86V, and I92F, as well as F42A, Y45A, and E62A, all of which are compared to wild-type human IL-2 (SEQ ID NO: 2).

[0077] In some embodiments, amino acid substitutions that increase IL-2 Rβ binding affinity include L80F, R81D, L85V, I86V, and I92F.

[0078] In some embodiments, target IL-2 muteins having a higher binding affinity to IL-2Rβ compared to wild-type human IL-2 include amino acid substitutions L80F, R81D, L85V, I86V, and I92F. In some embodiments, IL-2 muteins have the following amino acid sequences: [ka] (Sequence ID 5).

[0079] In some embodiments, IL-2 mutein is IL-2Rβ / IL-2Rγ c The ability to stimulate one or more signaling pathways dependent on heterodimerization was increased. In some embodiments, the subject IL-2 mutein had an enhanced ability to stimulate STAT5 phosphorylation in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, IL-2 mutein stimulated STAT5 phosphorylation in IL-2Rβ+ cells at levels of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher than the levels at which wild-type IL-2 stimulates STAT5 phosphorylation in the same cells. In some embodiments, IL-2 mutein stimulates STAT5 phosphorylation in IL-2Rβ+ cells at levels of 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or higher, compared to the level at which wild-type IL-2 stimulates STAT5 phosphorylation in the same cells. In some embodiments, IL-2Rβ+ cells are T cells. In certain embodiments, T cells are CD8+ T cells. In some embodiments, CD8+ T cells are newly isolated CD8+ T cells. In other embodiments, CD8+ T cells are activated CD8+ T cells. In other embodiments, IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, IL-2 mutein includes substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0080] In some embodiments, mutein has an enhanced ability to stimulate ERK1 / ERK2 signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, IL-2 mutein stimulates pERK1 / ERK2 signaling in IL-2Rβ+ cells at levels of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher than the levels at which wild-type IL-2 stimulates pERK1 / ERK2 signaling in the same cells. In some embodiments, IL-2 mutein stimulates pERK1 / ERK2 phosphorylation in IL-2Rβ+ cells at levels of 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or higher, compared to the level at which wild-type IL-2 stimulates pERK1 / ERK2 phosphorylation in the same cells. In some embodiments, IL-2Rβ+ cells are T cells. In certain embodiments, T cells are CD8+ T cells. In some embodiments, CD8+ T cells are newly isolated CD8+ T cells. In other embodiments, CD8+ T cells are activated CD8+ T cells. In other embodiments, IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, IL-2 muteins include substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0081] STAT5 and ERK1 / 2 signaling can be measured, for example, by phosphorylation of STAT5 and ERK1 / 2 using any suitable method known in the art. For example, STAT5 and ERK1 / 2 phosphorylation can be measured using antibodies specific to the phosphorylated versions of these molecules in combination with flow cytometry analysis as described herein. In some embodiments, mutain has an enhanced ability to stimulate PI 3-kinase signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, IL-2 mutain stimulates PI 3-kinase signaling in IL-2Rβ+ cells at levels of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or higher than the levels at which wild-type IL-2 stimulates PI 3-kinase signaling in the same cells. In some embodiments, IL-2 mutein stimulates PI 3-kinase signaling in IL-2Rβ+ cells at levels of 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or higher, compared to the level at which wild-type IL-2 stimulates PI 3-kinase signaling in the same cells. In some embodiments, IL-2Rβ+ cells are T cells. In certain embodiments, T cells are CD8+ T cells. In some aspects, CD8+ T cells are activated CD8+ T cells. In other embodiments, IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, IL-2 mutein includes substitutions 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 an antibody specific to phospho-S6 ribosomal protein in conjunction with flow cytometry analysis as described herein.

[0082] In some embodiments, IL-2 mutein is a stimulator of IL-2 and / or IL-15 STAT5 phosphorylation in CD8+ T cells. In some embodiments, mutein is a promoter of IL-2 and / or IL-15-induced CD8+ T cell proliferation. In some embodiments, mutein is a stimulator of IL-2-dependent TCR-induced cell proliferation. In some aspects, IL-2 mutein includes substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0083] IL-2 promotes the differentiation of Th1, Th9, and Treg T cells and inhibits the differentiation of Th17 cells. Therefore, although not bound by a specific operating theory, IL-2 mutein, acting as an IL-2 superagonist, is thought to be able to promote the differentiation of Th1, Th9, and / or Treg cells or inhibit the differentiation of Th17 cells. In some embodiments, IL-2 mutein is a promoter of IL-2-dependent Th1, Th9, and / or Treg differentiation. In some embodiments, mutein is an inhibitor of Th17 differentiation. In some embodiments, IL-2 mutein includes substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0084] In some embodiments, IL-2 mutein signals less and / or independently of CD25 compared to wild-type human IL-2 (e.g., reduced or removed CD25 binding). In some embodiments, reduced and / or independent signaling with respect to CD25 allows for preferential activation of effector T cells while limiting Treg stimulation. In some embodiments, reduced and / or independent signaling with respect to CD25 allows for reduced toxicity. In some embodiments, mutein comprises one or more substitutions selected from the group consisting of substitutions L80F, R81D, L85V, I86V, and I92F, and F42A, Y45A, and E62A, all of which are compared to wild-type human IL-2 (SEQ ID NO: 2). In some embodiments, IL-2 mutein comprises SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0085] In some embodiments, IL-2 mutein can increase and / or restore responsiveness to anerogenic NK cells. In some embodiments, IL-2 mutein can increase and / or restore responsiveness to anerogenic NK cells within the tumor microenvironment. In some embodiments, IL-2 mutein includes substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0086] In some embodiments, mutaine is an inhibitor of IL-2-dependent activation of natural killer (NK) cells. IL-2 activation of NK cells can be measured by any preferred method known in the art, for example, by measuring IL-2-induced CD69 expression and / or cytotoxicity as described herein.

[0087] In some embodiments, the increase in IL-2Rβ binding affinity is any binding affinity to IL-2Rβ that is greater than the wild-type human IL-2 binding affinity to IL-2Rβ. In some embodiments, the binding affinity is a 2x, 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 120x, 150x, 170x, 190x, 200x, 220x, 240x, or greater increase in binding affinity to IL-2Rβ compared to the wild-type human IL-2 binding affinity to IL-2Rβ.

[0088] In some embodiments, the increase in binding ability to IL-2Rβ is any binding ability to IL-2Rβ that is greater than the wild-type human IL-2 binding ability to IL-2Rβ. In some embodiments, the binding ability is a 2x, 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 120x, 150x, 170x, 190x, 200x, 220x, 240x, or greater increase in binding ability to IL-2Rβ compared to the wild-type human IL-2 binding ability to IL-2Rβ.

[0089] In some embodiments, target IL-2 mutains exhibiting higher binding affinity to IL-2Rβ compared to wild-type human IL-2 also show reduced binding to CD25 and include amino acid substitutions F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the reduction in binding affinity is approximately 220-fold, i.e., from approximately 6.6 nM Kd for wild-type human IL-2 to approximately 1.4 μM for mutains containing F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutain has the following amino acid sequence: [ka] (Also known as sequence number 6; H9-F42A).

[0090] In some embodiments, target IL-2 mutains having a higher binding affinity to IL-2Rβ compared to wild-type human IL-2 also exhibit reduced binding to CD25 and include amino acid substitutions K43N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the reduced binding affinity is due to the possibility of glycosylation with the K43N substitution at position 43. By replacing asparagine with lysine (K43N), CD25 binding in IL-2 mutains including the amino acid substitutions K43N, L80F, R81D, L85V, I86V, and I92F is reduced and / or eliminated. In some embodiments, IL-2 mutains have the following amino acid sequence: [ka] (Also known as sequence number 7; H9-K43N).

[0091] In some embodiments, the decrease in binding affinity to CD25 is any binding affinity to CD25 that is lower than the wild-type human IL-2 binding affinity. In some embodiments, the binding affinity is a decrease of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170, 190, 200, 220, 240, or more in terms of binding affinity to CD25 compared to the wild-type human IL-2 binding affinity to CD25.

[0092] In some embodiments, target IL-2 muteins having higher binding affinity to IL-2Rβ and lower binding affinity to CD25 compared to wild-type human IL-2 include amino acid substitutions F42A, Y45A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, IL-2 muteins have the following amino acid sequences: [ka] (Sequence ID 8; H9-F42A / Y45A; H9-FYAA).

[0093] In some embodiments, target IL-2 muteins having higher binding affinity to IL-2Rβ and lower binding affinity to CD25 compared to wild-type human IL-2 include amino acid substitutions F42A, E62A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, IL-2 muteins have the following amino acid sequences: [ka] (Sequence ID 9; H9-F42A / E62A; H9-FEAA).

[0094] In some embodiments, target IL-2 muteins having higher binding affinity to IL-2Rβ and lower binding affinity to CD25 compared to wild-type human IL-2 include amino acid substitutions F42A, Y45A, E62A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, IL-2 muteins have the following amino acid sequences: [ka] (Sequence ID 10; H9-F42A / Y45A / E62A; H9-FYEAAA).

[0095] In some embodiments, IL-2 mutein includes a T3A amino acid substitution to eliminate glycosylation of IL-2 mutein, thereby reducing the homogeneity between IL-2 mutein and IL-2 fusion protein products. In some embodiments, IL-2 mutein includes a C125S amino acid substitution to eliminate the risk of disulfide bond shuffling at this residue, thereby reducing developmental risk. In some embodiments, IL-2 mutein is a variant of MDNA109 (SEQ ID NO: 5) or MDN109FEAA (SEQ ID NO: 9), further comprising a T3A and / or C125S substitution.

[0096] In some embodiments, the IL-2 mutein of the IL-2 fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with one of the IL-2 muteins in Table 2. In some embodiments, the IL-2 mutein of the IL-2 fusion protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to any one of the IL-2 muteins (SEQ ID NOs. 5-24) in Table 2. In some embodiments, the variant exhibits a higher binding affinity to IL-2Rβ compared to wild-type human IL-2. In embodiments, the variant also exhibits reduced binding to CD25.

[0097] 1. Extended half-life IL-2 portion In some embodiments, the IL-2 moiety comprises a polypeptide that increases the in vivo serum half-life of IL-2 contained in the IL-2 fusion protein (i.e., a “serum half-life enhancing” or “half-life enhancing” polypeptide). In various embodiments, the half-life enhancing polypeptide is serum albumin (e.g., human serum albumin), PEG, a PEG derivative, or the Fc region of an IgG subclass of an antibody lacking an IgG heavy chain variable region. Exemplary Fc regions may contain mutations that inhibit complement fixation and Fc receptor binding, or they may be soluble, i.e., capable of binding to complement or lysing cells via other mechanisms such as antibody-dependent complement lysis (ADCC; USSN 08 / 355,502, filed December 12, 1994).

[0098] In some embodiments, the half-life-enhancing polypeptide is the Fc region. The “Fc region” may be a naturally occurring or synthetic polypeptide homologous to the C-terminal domain of IgG produced by digesting IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. The IL-2 portion of the IL-2 fusion protein in question may include the entire Fc region, or a smaller portion that retains the ability to extend the cyclic half-life of the IL-2 portion. In addition, the full-length or fragmented Fc region may be a variant of the wild-type molecule. In some embodiments, the IL-2 portion of the IL-2 fusion protein in question includes an IgG1, IgG2, IgG3, or IgG4Fc region. In some embodiments, the Fc region is a human IgG1, IgG2, IgG3, or IgG4Fc region. In some embodiments, the Fc region of the IL-2 portion includes a monomer containing the CH2-CH3 domain of a human IgG1, IgG2, IgG3, or IgG4 region. In some embodiments, the Fc region of the IL-2 moiety is dimerized, each containing two monomers, each containing a CH2-CH3 domain in the human IgG1, IgG2, IgG3, or IgG4 region. In some embodiments, the IL-2 moiety contains two monomers, each monomer containing IL-2 or IL-2 mutein linked to an Fc region monomer containing a CH2-CH3 domain. In such embodiments, the Fc region monomers dimerize to form a dimerized IL-2 moiety. In some embodiments, the IL-2 moiety contains two monomers, one monomer containing IL-2 or IL-2 mutein linked to a first Fc region monomer containing a CH2-CH3 domain, and the second monomer containing a second Fc region monomer but not IL-2 or IL-2 mutein. The first and second Fc regions dimerize to form a dimerized Fc region. In embodiments including a dimeric Fc region, each of the two monomers may include amino acid substitutions that are convenient for heterodimer formation (e.g., "knob-in-hole" or "knob and hole" amino acid substitutions; see, for example, U.S. Patent No. 8,216,805, incorporated herein by reference for disclosures relating to "knob and hole" amino acid substitutions). In some embodiments, the Fc region includes substitution N297A.

[0099] The Fc region can be "soluble" or "insoluble," but is typically insoluble. Insoluble Fc regions usually lack a high-affinity Fc receptor binding site and a C'1q binding site. The high-affinity Fc receptor binding site of mouse IgG Fc contains a Leu residue at position 235 of IgG Fc. Therefore, the Fc receptor binding site can be disrupted by mutation or deletion of Leu 235. For example, substituting 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 the Glu 318, Lys 320, and Lys 322 residues of IgG. For example, substituting Glu 318, Lys 320, and Lys 322 with Ala residues prevents IgG1 Fc from directing antibody-dependent complement lysis. In contrast, the soluble IgG Fc region possesses a high-affinity Fc receptor binding site and a C'1q binding site. The high-affinity Fc receptor binding site contains the Leu residue at position 235 of IgG Fc, and the C'1q binding site contains the Glu 318, Lys 320, and Lys 322 residues of IgG1. Soluble IgG Fc has wild-type residues or conserved amino acid substitutions at these sites. Soluble IgG Fc can target cells for antibody-dependent cytotoxicity or complement-targeted cytolysis (CDC). Appropriate mutations for human IgG are also known (see, e.g., Morrison et al., The Immunologist 2:119-124, 1994, and Brekke et al., The Immunologist 2:125, 1994).

[0100] In some embodiments, the half-life-enhancing polypeptide is serum albumin. In exemplary embodiments, serum albumin is human serum albumin or a variant thereof that can extend the serum half-life of bound IL-2 or IL-2 mutein. In some embodiments, human serum albumin has the following amino acid sequence: (Sequence ID 400).

[0101] In some embodiments, the IL-2 moiety (e.g., wild-type IL-2 or IL-2 mutein provided herein) is directly or indirectly linked to a serum half-life enhancing polypeptide. In some embodiments, the half-life enhancing polypeptide is linked to the N-terminus of IL-2 (e.g., wild-type IL-2 or IL-2 mutein provided herein). In some embodiments, the half-life enhancing polypeptide is linked to the C-terminus of IL-2 (e.g., wild-type IL-2 or IL-2 mutein provided herein). In some embodiments, IL-2 and / or IL-2 mutein is directly linked to the half-life enhancing polypeptide. In some embodiments, IL-2 or IL-2 mutein is linked to the half-life enhancing polypeptide via a linker peptide such as GGGGS. In some embodiments, the linker is (GGGGS)n, where n is an integer from 1 to 10. In some embodiments, the linker is GGGGS (SEQ ID NO: 401). In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO: 402). In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 403). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 404). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 405).

[0102] In some embodiments, the linker comprises one or more protease cleavage sites (e.g., a protease-cleavable linker). The linker may additionally comprise one or more protease cleavage sites or be sensitive to cleavage by oxidation and / or reduction. Examples of peptide linkers that are susceptible to cleavage by complement system enzymes, urokinases, tissue plasminogen activators, trypsins, plasmins, caspases, kallikrein, cathepsins, regmine, MMPs, thrombin, urokinase-type plasminogen activators (uPAs), matryptases, prostate-specific antigens (PSAs), or other enzymes with proteolytic activity may be used. In another example, the linker may comprise a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Another example suggests that linkers may include 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, beta-Ala-Leu-Ala-Leu linkers, Arg-Arg-Ala-Leu-Ala-Leu linkers, peptide-mimetic linkers, regmine-cleavable Ala-Ala-Asn tripeptide linkers, peptide linkers such as Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu that are cleaved by cathepsin B and other lysosomal proteases, caspase 3 DEVD sequences, or autodegradable linkers. For example, a linker disclosed in Poreba, M, FEBS J.287(10):1936-1969(2020), which is incorporated herein by reference, is intended by this disclosure. Many tumors naturally release high levels of glutathione (a reducing agent), which can reduce disulfide bonds, and the cargo portion is subsequently released at the delivery site. In some embodiments, the linker is a protease-cleavable linker, which is a linker that can be cleaved by a matrix metalloproteinase (MMP). MMPs are overexpressed in tumors, and a cleavable linker in such a context is intended by this disclosure.For example, the linker disclosed in Hsu, EJ, et al., Nat.Commun. 12(2768):1-13(2021), which is incorporated herein by reference, is intended by this disclosure. In some embodiments, the MMP linker sequence is selected from the group consisting of SGARYRWLTA (SEQ ID NO: 406), SGRSYAILTA (SEQ ID NO: 407), SRSGRSPAIFTATG (SEQ ID NO: 408), GSSGRSPAIFTAGS (SEQ ID NO: 409), and SGFIANPVTA (SEQ ID NO: 410). In some embodiments, the MMP linker sequence is SGARYRWLTA (SEQ ID NO: 411). In some embodiments, the MMP linker sequence is SGRSYAILTA (SEQ ID NO: 412). In some embodiments, the MMP linker sequence is SRSGRSPAIFTATG (SEQ ID NO: 413). In some embodiments, the MMP linker sequence is GSSGRSPAIFTAGS (SEQ ID NO: 414). In some embodiments, the MMP linker sequence is SGFIANPVTA (SEQ ID NO: 415). In some embodiments, the MMP linker sequence is PLGLVVAPLGLVVAPLGLVVA (SEQ ID NO: 416). In some embodiments, the MMP linker sequence is PLGLWAPLGLWAPLGLWA (SEQ ID NO: 417). In some embodiments, the MMP linker sequence is GGSGGTPLGLWAGGSGGT (SEQ ID NO: 418). In some embodiments, the MMP linker sequence is GGSGGTPAGLIGGGSGGT (SEQ ID NO: 419). In some embodiments, the MMP linker sequence is GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NO: 420). In some embodiments, the PSA linker sequence is GGSGGTHSSKLQGGSGGT (SEQ ID NO: 421).

[0103] In some embodiments, the IL-2 moiety includes a polypeptide that functions as an antigen tag, such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated, highly specific anti-FLAG antibody, as described herein (see also Blanar et al., Science 256:1014, 1992 and LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In some embodiments, the IL-2 moiety further includes a C-terminal c-myc epitope tag.

[0104] In other embodiments, the IL-2 moiety includes a heterologous polypeptide, such as an Aga2p aglutinin subunit, which functions to enhance the expression of the IL-2 moiety or to direct its cellular localization (see, for example, Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0105] Table 3 shows exemplary IL-2 moieties, including half-life-enhancing polypeptides for use in the target IL-2 fusion polypeptides described herein. In some embodiments, the IL-2 moiety of the IL-2 fusion protein has one amino acid from SEQ ID NOs. 25-73. In some embodiments, the IL-2 moiety of the IL-2 fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with one of the IL-2 moieties (SEQ ID NOs. 25-67) in Table 3. 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 one of the IL-2 moieties (SEQ ID NOs. 25-67) in Table 3. In embodiments, the IL-2 exhibits a higher binding affinity to IL-2Rβ compared to wild-type human IL-2. In embodiments, the variant also exhibits reduced binding to CD25. [Table 3-1] [Table 3-2] Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15

[0106] 2.IL-2 / antibody fusion part 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").

[0107] In some embodiments, the IL-2 / antibody fusion comprises three polypeptides. The first polypeptide comprises IL-2 or IL-2 mutein bound 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 are dimerized, and VH and VL form an antigen-binding domain.

[0108] In some embodiments of the IL-2 / antibody fusion moiety, the IL-2 / antibody fusion moiety comprises four polypeptides. The first polypeptide comprises a first antibody heavy chain conjugated to IL-2 or IL-2 mutaine, the first antibody heavy chain comprising a first heavy chain variable region (VH1) and a first Fc region. The second polypeptide comprises a second antibody heavy chain, the second antibody heavy chain comprising a second heavy chain variable region (VH2) and a second Fc region. The third polypeptide comprises a first variable light chain variable region (VL1) and a light chain constant region. The fourth polypeptide comprises a second variable light chain variable region (VL2) and a light chain constant region. In this embodiment, the two Fc regions of the first and second polypeptides dimerize, and VH1 and VL1, and VH2 and VL2, respectively, form antigen-binding domains (i.e., a first antigen-binding domain and a second antigen-binding domain). In some embodiments, the first and second antigen-binding domains are the same. In some embodiments, the first and second antigen-binding domains are different.

[0109] The antibody or antigen-binding component is conjugated to IL-2 or IL-2 mutaine using any preferred technique. In some embodiments, the antibody or antigen-binding component is directly linked to IL-2 or IL-2 mutaine. In some embodiments, the antibody or antigen-binding component is linked to IL-2 or IL-2 mutaine using a linker. IL-2 or IL-2 mutaine can be conjugated to the antibody or antigen-binding component using any preferred linker, including the linkers described herein. In some embodiments, the linker is a linker peptide having the formula (GGGGS)n, where n is an integer from 1 to 10. In some embodiments, the linker is GGGGS (SEQ ID NO: 401). In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO: 402). In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 403). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 404). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 405). In some embodiments, IL-2 or IL-2 mutein is bound to the C-terminus of the Fc domain of the antibody or antigen-binding component. In some embodiments, IL-2 or IL-2 mutein is bound to the N-terminus of the antibody or antigen-binding component.

[0110] The antibody or antigen-binding component of the IL-2 moiety can function as a targeting moiety. For example, it can be used to localize a chimeric protein to a specific cell subset or target molecule. A method for producing cytokine antibody chimeric polypeptides is described, for example, in U.S. Patent No. 6,617,135.

[0111] In some embodiments, the IL-2 moiety includes fusion to an antibody or its antigen-binding moiety, which is an antibody against a component of the PD-1 / PD-L1 signaling pathway, disrupting the interaction between the PD-1 receptor and its ligand, PD-L1. Antibodies known in the art that bind to PD-1, disrupt the interaction between PD-1 and its ligand, PD-L1, and stimulate an antitumor immune response are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the antibody or its antigen-binding moiety specifically binds to PD-1. For example, antibodies that target PD-1 and can be used in this invention include, for example, 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.), pizilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (semiprimab, Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers This includes, but is not limited to, Squibb, and / or the humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is 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, inhibiting its interaction with PD-1 and thereby increasing immune activity. Any antibody known in the art that binds to PD-L1, disrupts the interaction between PD-1 and PD-L1, and stimulates an antitumor immune response is suitable for use in the chimeric polypeptides 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 antitumor immune response is suitable for use in the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-PD-1 antibody. In some embodiments, the IL-2 moiety includes fusion to an anti-PD-L1 antibody.

[0112] Table 4 shows exemplary IL-2 / anti-PD-1 antibody fusion regions that can be included in the target IL-2 fusion protein. In some embodiments, the IL-2 region of the IL-2 fusion protein has one amino acid from Table 4. In some embodiments, the IL-2 region of the IL-2 fusion protein is a variant of one of the IL-2 regions in Table 4. In some embodiments, the IL-2 region 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 regions 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 one of the IL-2 regions in Table 4. In some embodiments, the variant exhibits a higher binding affinity to IL-2Rβ compared to wild-type human IL-2. In some embodiments, the variant also exhibits reduced binding to CD25. In some embodiments, the variant can bind to PD-1 (human PD-1). [Table 4-1] [Table 4-2] Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 4-11 Table 4-12

[0113] In some embodiments, the IL-2 moiety includes an antibody or its antigen-binding moiety that targets CTLA-4 and disrupts its interactions with CD80 and CD86. Exemplary antibodies targeting CTLA-4 include FDA-approved ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb) and tremelimumab (tisilimubab, CP-675, 206, Pfizer) currently in human trials. Other suitable antibodies targeting CTLA-4 are disclosed in WO2012 / 120125, U.S. Patent No. 6,984720, U.S. Patent No. 6,682,7368, and U.S. Patent Applications 2002 / 0039581, 2002 / 0086014, and 2005 / 0201994, 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 antitumor immune response is suitable for use in the chimeric polypeptide disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-CTLA-4 antibody.

[0114] In some embodiments, the IL-2 moiety includes a fusion to an antibody or its antigen-binding moiety that targets LAG-3 and disrupts its interaction with MHC class II molecules. An exemplary antibody targeting LAG-3 is IMP321 (Immutep), currently in human trials. Other suitable antibodies targeting LAG-3 are disclosed in U.S. Patent Application No. 2011 / 0150892, 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 use in the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes a fusion to an anti-LAG-3 antibody.

[0115] In some embodiments, the IL-2 moiety includes fusion to an antibody or its antigen-binding moiety that targets TIGIT and disrupts its interaction with CD155 (PVR) and / or CD112 (PVRL2, Nectin-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, Nectin-2), and stimulates an antitumor immune response or immunostimulatory response resulting in overall antitumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-TIGIT antibody.

[0116] In some embodiments, the IL-2 moiety includes a fusion to an antibody or its antigen-binding moiety that targets CD112R (also known as PVRIG) and disrupts its interaction with CD112 and / or PVRL2 / nectin-2. Those skilled in the art will understand that any antibody that binds to CD112R, disrupts its interaction with CD112 and / or PVRL2 / nectin-2, and stimulates an antitumor immune response or immunostimulatory response resulting in overall antitumor activity is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the IL-2 moiety includes a fusion to an anti-CD112R antibody.

[0117] In some embodiments, the IL-2 moiety includes fusion to an antibody targeting B7-H3 or B7-H4, or to its antigen-binding moiety. While the B7 family has no arbitrarily defined receptor, these ligands are upregulated in tumor cells or tumor-infiltrating cells. An exemplary antibody targeting B7-H3 is MGA271 (Macrogenics), currently in human trials. Other suitable antibodies targeting B7 family members are disclosed in U.S. Patent Application No. 2013 / 0149236, 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 use in the IL-2 moieties disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-B7-H3 or B7-H4 antibody.

[0118] In some embodiments, the IL-2 moiety includes fusion to an antibody or its antigen-binding moiety that targets TIM-3 and disrupts its interaction with galectin-9. Suitable antibodies targeting TIM-3 are disclosed in U.S. Patent Application No. 2013 / 0022623, 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 use in the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-TIM-3 antibody.

[0119] In some embodiments, the IL-2 moiety includes fusion to 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 resulting in overall antitumor activity is suitable for use in the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-4-1BB / CD137 antibody.

[0120] In some embodiments, the IL-2 moiety includes fusion to 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 resulting in overall antitumor activity is suitable for use with the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-GITR antibody.

[0121] In some embodiments, the IL-2 moiety includes fusion to an antibody or its antigen-binding moiety 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 resulting in overall antitumor activity is suitable for use in the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-OX40 antibody.

[0122] In some embodiments, the IL-2 moiety includes fusion to an antibody or its antigen-binding moiety 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 resulting in overall antitumor activity is suitable for use in the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-CD40 antibody.

[0123] In some embodiments, the IL-2 moiety includes fusion to an antibody or its antigen-binding moiety that targets ICOS and disrupts its interaction with its ligand. Those skilled in the art will understand that any antibody that binds to ICOS, disrupts its interaction with its ligand, and stimulates an antitumor immune response or immunostimulatory response resulting in overall antitumor activity is suitable for use with the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-ICOS antibody.

[0124] In some embodiments, the IL-2 moiety includes fusion to an antibody or its antigen-binding moiety 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 resulting in overall antitumor activity is suitable for use in the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-CD28 antibody. In some embodiments, the IL-2 moiety includes fusion to an anti-CD3 antibody, including a T-cell engager anti-CD3 antibody.

[0125] In some embodiments, the IL-2 moiety includes fusion to an antibody or its antigen-binding moiety that targets IFNα and disrupts its interaction with its ligand. Those skilled in the art will understand that any antibody that binds to IFNα, disrupts its interaction with its ligand, and stimulates an antitumor immune response or immunostimulatory response resulting in overall antitumor activity is suitable for use with the IL-2 moiety disclosed herein. In some embodiments, the IL-2 moiety includes fusion to an anti-IFNα antibody.

[0126] 3. Additional IL-2 fusion portion In some embodiments, the IL-2 moiety comprises a fusion to a tumor antigen or a polypeptide that targets a tumor antigen. Generally, tumor antigens enable the distinction of tumor cells from their normal cell counterparts and can include, for example, tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). In some embodiments, tumor antigens are oncogenes and / or tumor suppressors, as well as overexpressed or abnormally expressed cellular proteins, tumor antigens produced by oncogenic viruses, carcinoembryonic antigens, changes in cell surface glycolipids and glycoproteins, and / or cell type-specific differentiation antigens. Such tumor antigens can include melanoma antigens, cancer testis antigens, epithelial tumor antigens, cell cycle regulatory proteins, prostate-specific antigens (including, for example, prostate cancer tumor antigens as disclosed in U.S. Patent No. 5,538,866), lymphomas (U.S. Patents Nos. 4,816,249, 5,068,177, and 5,227,159). Tumor antigens can include, for example, HMW mucin bound to 2G3 and 369F10, c-erbB-2 related tumor antigen (a glycoprotein of approximately 42 kD or about 55 kD), antigens of about 40, 60, 100, and 200 kD bound to approximately 113F1, 9-O-acetyl GD3, p97, alpha-fetoprotein (AFP) (for example, for germ cell tumors and / or hepatocellular carcinoma), carcinoembryonic antigen (CEA) (for example, for intestinal cancer, sometimes for lung cancer or breast cancer), CA-125 (for example, for ovarian cancer), MUC-1 (for example, for breast cancer), epithelial tumor antigen (ETA) (for example, for breast cancer), tyrosinase (for example, for malignant melanoma), melanoma-associated antigen (MAGE) (for example, for malignant melanoma), cancer / testis antigen 1 (CTAG1B), melanoma-associated antigen 1 (MAGEA1), abnormal Ras products, abnormal p53 products, overexpression of cyclins (including, for example, cyclin B1), mutations in fibronectin, post-translational changes in MUC1 glycoprotein, secreted tumor antigens (including, for example, gangliosides), but are not limited thereto.

[0127] Other fusions may include fusion with a pro-apoptotic payload. Exemplary sequences are provided in the following table. In some embodiments, the IL-2 moiety described herein is fused to a pro-apoptotic payload, such as BAD, BAX, BAK, BIK, and / or BID sequences. In some embodiments, the pro-apoptotic payload is a Bcl-2 domain-containing peptide and / or a sequence of BAD, BAX, BAK, BIK, and / or BID sequences. Exemplary pro-apoptotic fusions are provided in Table 5 below. [Table 5]

[0128] Other fusions may include fusion with anti-apoptotic payloads for use in extending the activation of CD8 cells, NK cells, and anergistic NK cells, and exemplary such sequences are shown in the table below. Such extension of T cell activation can demonstrate its benefit in cancer therapy methods. [Table 6-1] [Table 6-2]

[0129] B.IL-2 masking area The IL-2 fusion proteins provided herein include IL-13 mutain, IL-13Ra2-binding mutain, or IL-13Ra2 antibody or its antigen-binding fragment, which are linked to the IL-2 moiety by a protease-sensitive linker (PSL). When bound to IL-13 mutain or IL-13Ra2 antibody or its antigen-binding fragment, the IL-2 moiety has reduced IL-2 activity; that is, the IL-2 moiety is "masked" by IL-13 mutain, IL-13Ra2-binding mutain, IL-13Ra2-binding mutain, or IL-13Ra2 antibody or its antigen-binding fragment. In some embodiments, IL-13 mutain, IL-13Ra2-binding mutain, IL-13Ra2-binding mutain, or IL-13Ra2 antibody or its antigen-binding fragment can bind to IL-13Rα2-expressing tumors, thereby localizing the IL-2 fusion protein to the tumor microenvironment. Upon localization to the tumor microenvironment, the protease-sensitive linker is cleaved by the protease, thereby releasing IL-13 mutain, IL-13 Ra2-binding mutain, or IL-13 Ra2 antibody or its antigen-binding fragment from the IL-2 fusion protein. In some embodiments, the "unmasked" IL-2 portion is then active in the tumor microenvironment, thereby promoting IL-2 antitumor activity.

[0130] In some embodiments, IL-13 mutein or IL-13Ra2 antibody or its antigen-binding fragment is bound to the IL-2 mutein of the IL-2 moiety by a PSL linker. In some embodiments, IL-13 mutein or IL-13Ra2 antibody or its antigen-binding fragment is bound to a region of the IL-2 moiety that is not IL-2 mutein. For example, in some embodiments where the IL-2 moiety further comprises a half-life-enhancing polypeptide (e.g., serum albumin or an Fc region), IL-13 mutein or IL-13Ra2 antibody or its antigen-binding fragment is bound to the half-life-enhancing polypeptide of the IL-2 moiety by PSL. In some embodiments where the IL-2 moiety comprises a dimeric Fc region, IL-2 mutein is bound to one of the Fc region monomers, and IL-13 mutein or IL-13Ra2 antibody or its antigen-binding fragment is bound to the other Fc region monomer by PSL.

[0131] In some embodiments, the IL-2 fusion protein comprises two or more IL-13 mutains or IL-13Ra2 antibodies or their antigen-binding fragments. In some embodiments, at least one of the IL-13 mutains or IL-13Ra2 antibodies or their antigen-binding fragments is bound to the IL-2 mutain of the IL-2 moiety by PSL. In some embodiments, at least one of the IL-13 mutains or IL-13Ra2 antibodies or their antigen-binding fragments is bound to a region of the IL-2 moiety that is not IL-2 mutain (e.g., serum albumin or Fc region).

[0132] 1. IL-13 Mutein In this embodiment, IL-13 mutein contains one or more amino acid substitutions compared to wild-type human IL-13:

[0133] PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN(Sequence ID 200)

[0134] Preferred IL-13 muteins include those that can reduce the IL-2 activity of the IL-2 moiety when bound to an IL-2 fusion protein via PSL. In some embodiments, IL-13 muteins can bind to the IL-13Rα2 receptor. In some embodiments, IL-13 muteins exhibit enhanced binding to the IL-13Rα2 receptor and reduced binding to the IL-13Rα1 receptor compared to wild-type IL-13 (e.g., SEQ ID NO: 200). In exemplary embodiments, IL-13 muteins can bind to the IL-13Rα2 receptor but cannot bind to the IL-13Rα1 receptor.

[0135] In some embodiments, the IL-13 mutain of the target IL-2 fusion protein is substituted with amino acids: (1) L10F, L10I, L10V, L10A, L10D, L10T, L10H; (2) R11S, R11N, R11H, R11L, R11I; (3) I14L, I14F, 114V, I14M; (4) V18L, V18F, V18I; (5) E12A, (6) R65D, (7) R86K, R86T, R86M; (8) D87E, D87K, D87R, D87G, D87S; (9) T88I, T88K, T8 8R; (10)K89R, K89T, K89M; (11)L101F, 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, one or more of which these substitutions result in altered affinity for one or both of IL-13Rα1 and IL-13Rα2. In other embodiments, the modified residues are two or more, three or more, four or more, or five or more of the contact residue combination sets defined above, but with no more than 14 amino acids. This is described in International Patent Publication WO2013 / 112871, the disclosure of which is incorporated herein by reference in its entirety.

[0136] The set of modifications may include the following specific changes compared to wild-type human IL-13 (SEQ ID NO: 200): (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 changes. 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.

[0137] In some embodiments, the IL-13 mutein of the IL-2 fusion protein exhibits higher selectivity for binding to IL-13Rα2 compared to IL-13Rα1 compared to the wild-type IL-13 sequence (SEQ ID NO: 200). A specific set of modifications that provide higher selectivity for binding to IL-13Rα2 compared to IL-13Rα1 compared to the wild-type IL-13 sequence is, but is not limited to, • [L10H, E15R, R86T, D87G, T88R, R108K] (MDNA132+E15R or MDNA132.15, e.g., Sequence ID 228) • [L10H, R86T, D87G, T88R, R108K] (C11, e.g., sequence number 229) • [L10D, R11I, V18I, R86K, D87K, K89R, R108K] (e.g., C2, e.g., sequence number 224) • [L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, R108K] (e.g., C3, e.g., sequence number 225) • [L10V, K89R, L101N, K105E, R108T] (e.g., C4, e.g., Sequence ID 211) • [R11S, I14M, T88S, L101N, K105A, R108K] (e.g., C7, e.g., sequence number 212) • [L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, R108K] (C9, for example, sequence number 226) • [L10A, V18F, R86K, D87K, K89R, L101I, K104R, R108K] (D7, for example, sequence number 231) ·[L10T / D, R11I, V18I, R86K, D87K / G, T88S, K89R, L101Y, K104R, K105T, R108K] [L10A / V, R86T, D87G, T88K, K89R, L101N, K104R, K105A / E, R108K / T] may be included.

[0138] In some embodiments, the set of modifications includes L10H, R86T, D87G, T88R, and R108K (C11, e.g., sequence number 229). In some embodiments, the set of modifications includes L10V, K89R, L101N, K105E, and R108T (C4, e.g., sequence number 211). In some embodiments, the set of modifications includes R11S, I14M, T88S, L101N, K105A, and R108K (C7, e.g., sequence number 212). In some embodiments, the set of modifications includes L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, and R108K (C9, e.g., sequence number 226). In some embodiments, the set of modifications includes L10H, E15R, R86T, D87G, T88R, and R108K (MDNA132+E15R, e.g., SEQ ID NO: 228). In some embodiments, the set of modifications includes L10A, V18F, R86K, D87K, K89R, L101I, K104R, and R108K (D7, e.g., SEQ ID NO: 231). In some embodiments, the set of modifications includes L10T / D, R11I, V18I, R86K, D87K / G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications includes L10T, R11I, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications includes L10T, R11I, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications includes L10D, R11I, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications includes L10D, R11I, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications includes L10A / V, R86T, D87G, T88K, K89R, L101N, K104R, K105A / E, and R108K / T.In some embodiments, the set of modifications includes L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108K. In some embodiments, the set of modifications includes L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108K. In some embodiments, the set of modifications includes L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108T. In some embodiments, the set of modifications includes L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108T. In some embodiments, the set of modifications includes L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108K. In some embodiments, the set of modifications includes L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108K. In some embodiments, the set of modifications includes L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and dR108T. In some embodiments, the set of modifications includes L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108T.

[0139] In some embodiments, the IL-13 mutein of the IL-2 fusion protein exhibits higher selectivity for binding to IL-13Rα1 compared to IL-13Rα2 compared to the wild-type IL-13 sequence (e.g., SEQ ID NO: 200). A specific set of modifications that provide higher selectivity for binding to IL-13Rα1 compared to IL-13Rα2 compared to the wild-type IL-13 sequence is, but is not limited to, ·[L10V, V18I, D87S, D88S, L101F, K104R, K105T] ·[R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T] ·[L10V, V18I, D87S, T88S, L101F, K104R, K105T] ·[L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T] ·[L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A] ·[L10V, V18I, D87S, T88S, L101F, K104R, K105T] ·[V18I, R86T, D87G, T88S, L101Y, K104R, K105A] • [R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M] may be included. These substitutions may be combined with substitutions [E12A / G / S, R65D / E] in some cases.

[0140] In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, and K105T. In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, and K105T. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, and K105T. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, and K105A. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, and K105A. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, and F107M. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A / G / S, and R65D / E.In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A / G / S, and R65D / E. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A / G / S, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D / E. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65D / E.In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D / E. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D / E.In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65D / E. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D / E. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65D. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65D.In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D.In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65D. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications is R11S, V18I, R86K, D87G, T88S, K89M, L. This includes 101Y, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65E. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65E. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65E. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65E.In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65E. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65E. In some embodiments, the set of modifications includes L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications includes R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications includes L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications includes L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65E. In some embodiments, the set of modifications includes L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications includes V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65E. In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65E. In some embodiments, the set of modifications includes L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T (see, for example, IL-13dn; Sequence ID 232).

[0141] Table 7 shows exemplary IL-13 mutaines suitable for use in the target IL-2 fusion proteins described herein. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6]

[0142] In some embodiments, the IL-13 mutain 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 respect to one of the IL-13 mutains in Table 7. In some embodiments, the IL-13 mutain of the IL-2 fusion protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to any one of the IL-2 mutains in Table 7 (SEQ ID NOs. 200-241). In embodiments, the IL-13 mutain exhibits higher selectivity for binding to IL-13Rα2 compared to IL-13Rα1 compared to wild-type IL-13 sequences (e.g., SEQ ID NO. 200).

[0143] 2. Extended half-life IL-13 mutaine In some embodiments, the IL-13 mutein of the target IL-2 fusion protein comprises 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 may be any of the half-life enhancing polypeptides described herein. In various embodiments, the half-life enhancing polypeptide is serum albumin (e.g., human serum albumin), PEG, a PEG derivative, or the Fc region of an IgG subclass of an antibody lacking an IgG heavy chain variable region.

[0144] In some embodiments, the half-life-enhancing polypeptide is an Fc region. The Fc region may include the entire Fc region or a smaller portion of it that retains the ability to extend the cyclic half-life of the IL-2 fusion protein. In addition, the full-length or fragmented Fc region may be a variant of the wild-type molecule. In some embodiments, the IL-13 mutein is bound to an IgG1, IgG2, IgG3, or IgG4Fc region. In some embodiments, the Fc region is a human IgG1, IgG2, IgG3, or IgG4Fc region. In some embodiments, the Fc region contains a monomer containing the CH2-CH3 domain of a human IgG1, IgG2, IgG3, or IgG4 region. In some embodiments, the Fc region is a dimer, each containing two monomers, each containing the CH2-CH3 domain of a human IgG1, IgG2, IgG3, or IgG4 region. In some embodiments, the Fc region contains two monomers, each monomer containing IL-13 mutein linked to an Fc region monomer containing the CH2-CH3 domain. In such embodiments, the Fc region monomer dimerizes to form a dimeric Fc region that binds to two IL-13 muteins. In embodiments including a dimeric Fc region, each of the two monomers may include amino acid substitutions that are convenient for heterodimer formation (e.g., "knob-in-to-hole" or "knob and hole" amino acid substitutions; see, for example, U.S. Patent No. 8,216,805, incorporated herein by reference for disclosures relating to "knob and hole" amino acid substitutions). In some embodiments, the Fc region includes substitution N297A.

[0145] In some embodiments, the half-life-enhancing polypeptide is serum albumin. In exemplary embodiments, serum albumin is human serum albumin or a variant thereof that can extend the serum half-life of bound IL-13 mutein. In some embodiments, human serum albumin has the following amino acid sequence: (Sequence ID 400).

[0146] In some embodiments, IL-13 mutein is directly or indirectly linked to a serum half-life enhancing polypeptide. In some embodiments, the half-life enhancing polypeptide is linked to the N-terminus of IL-13 mutein. In some embodiments, the half-life enhancing polypeptide is linked to the C-terminus of IL-13 mutein. In some embodiments, IL-13 mutein is directly linked to the half-life enhancing polypeptide. In some embodiments, IL-13 mutein is linked to the half-life enhancing polypeptide via a linker peptide. Using any suitable linker, IL-13 mutein can be linked to a half-life enhancing polypeptide containing a linker described herein (e.g., the gly-ser linker described herein).

[0147] Table 8 shows exemplary IL-13 muteins, including half-life-enhancing polypeptides for use in the target IL-2 fusion polypeptides described herein. In some embodiments, the IL-13 mutein is bound to the half-life-enhancing polypeptide and has one amino acid from SEQ ID NOs. 242-281. In some embodiments, the IL-13 mutein is bound to the half-life-enhancing polypeptide and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to one of the sequences (SEQ ID NOs. 242-281) in Table 8. In some embodiments, the IL-13 mutein is bound to the half-life-enhancing polypeptide and has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions compared to any one of the sequences (SEQ ID NOs. 242-281) in Table 8. In this embodiment, IL-13 mutein is bound to a half-life-enhancing polypeptide and exhibits higher selectivity for binding to IL-13Rα2 compared to IL-13Rα1 compared to wild-type IL-13 sequences (e.g., SEQ ID NO: 200). [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9]

[0148] 3. IL-13 mutain / antibody fusion In other embodiments, the IL-13 mutaine of the IL-2 fusion protein provided herein comprises an antibody or its antigen-binding moiety.

[0149] In some embodiments, the IL-13 mutain / antibody fusion comprises three polypeptides. The first polypeptide comprises IL-13 mutain bound 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 are dimerized, and VH and VL form an antigen-binding domain.

[0150] In some embodiments, the IL-13 mutain / antibody fusion comprises four polypeptides. The first polypeptide comprises a first antibody heavy chain conjugated to IL-13 mutain, the first antibody heavy chain comprising a first heavy chain variable region (VH1) and a first Fc region. The second polypeptide comprises a second antibody heavy chain, the second antibody heavy chain comprising a second heavy chain variable region (VH2) and a second Fc region. The third polypeptide comprises a first variable light chain variable region (VL1) and a light chain constant region. The fourth polypeptide comprises a second variable light chain variable region (VL2) and a light chain constant region. In this embodiment, the two Fc regions of the first and second polypeptides are dimerized, and VH1 and VL1, and VH2 and VL2, each form an antigen-binding domain.

[0151] The antibody or antigen-binding component is conjugated to IL-13 mutaine using any preferred technique. In some embodiments, the antibody or antigen-binding component is directly linked to IL-13 mutaine. In some embodiments, the antibody or antigen-binding component is linked to IL-13 mutaine using a linker. IL-13 mutaine can be conjugated to the antibody or antigen-binding component using any preferred linker, including the linkers described herein. In some embodiments, the linker is a linker peptide having the formula (GGGGS)n, where n is an integer from 1 to 10. In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO: 402). In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 403). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 404). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 405). In some embodiments, IL-13 mutaine is conjugated to the C-terminus of the Fc domain of the antibody or antigen-binding component. In some embodiments, IL-13 mutein is bound to the N-terminus of an antibody or antigen-binding component.

[0152] In some embodiments, IL-13 mutein involves fusion to an antibody or its antigen-binding moiety, which disrupts the interaction between the PD-1 receptor and its ligand, PD-L1, and / or is an antibody against a component of the PD-1 / PD-L1 signaling pathway. Exemplary IL-13 mutein / anti-PD1 fusions are shown in Table 9 below. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4]

[0153] 4.IL-13Ra2 antibody In some embodiments, the target IL-2 masking moiety includes an IL-13Ra2 antibody or its antigen-binding fragment. Suitable IL-13Ra2 antibodies or their antigen-binding fragments include SAB1406031 (Sigma), HPA067363 (Sigma), HPA067363 (Sigma), AV53557 (Sigma), WH0003598M1 (Sigma), AP1145 (Sigma), E7U7B (CellSignaling), 2H25L68 (ThermoFisher), 018 (ThermoFisher), PA5-96045v (ThermoFisher), PA5-106798 (ThermoFisher), PA5-46976 (Therm Examples include, but are not limited to, those disclosed in WO2008 / 146911 and WO2014 / 072888 (all incorporated herein by reference), PA5-44066(ThermoFisher), PA5-47732(ThermoFisher), PA5-143269(ThermoFisher), PA5-86898(ThermoFisher), PA5-87205(ThermoFisher), BS-2461R(ThermoFisher), 2E10(ThermoFisher), and WO2008 / 146911 and WO2014 / 072888 (all incorporated herein by reference). As used herein, “its antigen-binding fragment” refers to an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen to which it is bound by a full-length antibody, for example, a fragment that retains one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies; and multispecific antibodies formed from antibody fragments.

[0154] 5. Additional ingredients In some embodiments, the target IL-2 masking region further includes the extracellular domain of CD122, CD132, or CD25.

[0155] Preferred extracellular domains of CD122, CD132, or CD25 include those capable of reducing the IL-2 activity of the IL-2 moiety when bound to an IL-2 fusion protein via PSL.

[0156] In some embodiments, the extracellular domain of CD122 has the following amino acid sequence:

[0157] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT (Sequence ID 350).

[0158] In some embodiments, the extracellular domain of CD132 has the following amino acid sequence:

[0159] LPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKEN (Sequence ID 351).

[0160] In some embodiments, the extracellular domain of CD25 has the following amino acid sequence:

[0161] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (Sequence ID 352).

[0162] C. protease-sensitive linker (PSL) The IL-2 fusion proteins provided herein comprise an IL-2 moiety and a protease-sensitive linker (PSL) that binds to IL-13 mutein. In some embodiments, the PSL can be cleaved in the tumor microenvironment, thereby "demasking" the IL-2 moiety. The demasked IL-2 moiety then promotes antitumor activity in the tumor microenvironment. Exemplary PSLs that may be included in the target IL-2 fusion proteins include PLGLVVAPLGLVVAPLGLVVA (SEQ ID NO: 416), PLGLWAPLGLWAPLGLWA (SEQ ID NO: 417), GGSGGTPLGLWAGGSGGT (SEQ ID NO: 418), GGSGGTPAGLIGGGSGGT (SEQ ID NO: 419), GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NO: 420), or GGSGGTHSSKLQGGSGGT (SEQ ID NO: 421).

[0163] PSLs may additionally contain one or more protease cleavage sites or be sensitive to cleavage by oxidation and / or reduction. Examples of peptide linkers that are susceptible to cleavage by complement system enzymes, urokinases, tissue plasminogen activators, trypsins, plasmins, caspases, kallikrein, cathepsins, regmine, MMPs, thrombin, urokinase-type plasminogen activators (uPAs), matryptases, PSAs, or other enzymes with proteolytic activity may be used. In another example, the linker may contain disulfide bonds (e.g., disulfide bonds on a cysteine ​​molecule). Another example suggests that PSL may include 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, beta-Ala-Leu-Ala-Leu linkers, Arg-Arg-Ala-Leu-Ala-Leu linkers, peptide-mimicking linkers, regmine-cleavable Ala-Ala-Asn tripeptide linkers, peptide linkers such as Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu that are cleaved by cathepsin B and other lysosomal proteases, caspase 3 DEVD sequences, or autodegradable linkers. For example, a linker disclosed in Poreba, M, FEBS J.287(10):1936-1969(2020), incorporated herein by reference, is intended by this disclosure. Many tumors naturally release high levels of glutathione (a reducing agent), which can reduce disulfide bonds, and subsequently release the cargo portion at the delivery site. In some embodiments, PSL is a protease-cleavable linker, which is a linker that can be cleaved by a matrix metalloproteinase (MMP). MMPs are overexpressed in tumors, and a cleavable linker in such a context is intended by this disclosure.For example, the linker disclosed in Hsu, EJ, et al., Nat.Commun. 12(2768):1-13(2021), which is incorporated herein by reference, is intended by this disclosure. In some embodiments, the MMP linker sequence is selected from the group consisting of SGARYRWLTA (SEQ ID NO: 406), SGRSYAILTA (SEQ ID NO: 407), SRSGRSPAIFTATG (SEQ ID NO: 408), GSSGRSPAIFTAGS (SEQ ID NO: 409), and SGFIANPVTA (SEQ ID NO: 410). In some embodiments, the MMP linker sequence is SGARYRWLTA (SEQ ID NO: 411). In some embodiments, the MMP linker sequence is SGRSYAILTA (SEQ ID NO: 412). In some embodiments, the MMP linker sequence is SRSGRSPAIFTATG (SEQ ID NO: 413). In some embodiments, the MMP linker sequence is GSSGRSPAIFTAGS (SEQ ID NO: 414). In some embodiments, the MMP linker sequence is SGFIANPVTA (SEQ ID NO: 415). In some embodiments, the MMP linker sequence is PLGLVVAPLGLVVAPLGLVVA (SEQ ID NO: 416). In some embodiments, the MMP linker sequence is PLGLWAPLGLWAPLGLWA (SEQ ID NO: 417). In some embodiments, the MMP linker sequence is GGSGGTPLGLWAGGSGGT (SEQ ID NO: 418). In some embodiments, the MMP linker sequence is GGSGGTPAGLIGGGSGGT (SEQ ID NO: 419). In some embodiments, the MMP linker sequence is GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NO: 420). In some embodiments, the PSA linker sequence is GGSGGTHSSKLQGGSGGT (SEQ ID NO: 421).

[0164] D. Exemplary IL-2 fusion protein In some embodiments, the IL-2 fusion protein comprises an IL-2 moiety containing IL-2 mutain according to SEQ ID NO: 5 (MDNA109) or SEQ ID NO: 9 (MDNA109FEAA). In some embodiments, the IL-2 mutain is a variant of MDNA109 (SEQ ID NO: 5) or MDNA109FEAA (SEQ ID NO: 9), further comprising a T3A and / or C125S substitution.

[0165] In some embodiments, the IL-2 moiety comprises a human albumin polypeptide, and optionally, the human albumin is recombinant human albumin. In exemplary embodiments, the IL-2 moiety comprises an anti-PD-1 antibody or its antigen-binding moiety. In some cases, the IL-2 moiety is conjugated to a human serum albumin polypeptide or a half-life-enhancing polypeptide such as an Fc region.

[0166] In some embodiments, the IL-2 fusion protein includes IL-13 having the amino acid sequence of MDNA132 (SEQ ID NO: 216) or MDNA132.15 (SEQ ID NO: 228). In exemplary embodiments, PSL has the amino acid sequence of PLGLWAPLGLWAPLGLWA (SEQ ID NO: 417), or GGSGGTPLGLWAGGSGGT (SEQ ID NO: ), or GGSGGTPAGLIGGGSGGT (SEQ ID NO: ), or GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT (SEQ ID NO: 420), or GGSGGTHSSKLQGGSGGT (SEQ ID NO: 421).

[0167] Examples of target IL-2 fusion proteins are shown in Tables 10a and 10b. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] Table 10-5 Table 10-6 Table 10-7 Table 10-8 Table 10-9 Table 10-10 Table 10-11 Table 10-12 Table 10-13 Table 10-14 Table 10-15 Table 10-16 Table 10-17 [Table 10-18] [Table 10-19] [Table 10-20] [Table 10-21] [Table 10-22] [Table 10-23] [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

[0168] Nucleic molecule encoding E.IL-2 fusion protein In some embodiments, the target IL-2 fusion protein, such as those described above, can be obtained by the expression of one or more nucleic acid molecules. For example, in embodiments where the IL-2 fusion is a single polypeptide, the IL-2 fusion protein is encoded by a single nucleic acid molecule. In other embodiments where the IL-2 fusion protein consists of two or more polypeptides, each of the two or more polypeptides is encoded by a nucleic acid molecule.

[0169] The nucleic acid molecules provided may contain sequences that occur naturally or sequences that differ from those that occur naturally but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules may consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA such as that produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. In addition, nucleic acid molecules may be double-stranded or single-stranded (i.e., either sense strand or antisense strand).

[0170] Nucleic acid molecules are not limited to sequences that encode polypeptides, but can also include some or all of non-coding sequences upstream or downstream of coding sequences (e.g., the coding sequences for IL-2 or IL-13). Those skilled in molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can be generated, for example, by processing genomic DNA with restriction endonucleases or by performing polymerase chain reactions (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be generated, for example, by in vitro transcription.

[0171] The exemplary isolated nucleic acid molecules of this disclosure may include fragments not found in their natural state. Accordingly, this disclosure includes recombinant molecules in which nucleic acid sequences (e.g., sequences encoding mutant IL-2 or IL-13) are incorporated into vectors (e.g., plasmids or viral vectors) or heterologous cell genomes (or homologous cell genomes at locations other than natural chromosomal locations).

[0172] The target nucleic acid molecule may contain a sequence encoding a "marker" or "reporter." Examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), and aminoglycoside phosphotransferase (NEO). r G418 rThis includes dihydrofolate reductase (DHFR), hygromycin B-phosphotransferase (HPH), thymidine kinase (TK), lacz (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art will recognize additional useful reagents, such as additional sequences that can function as markers or reporters.

[0173] The target nucleic acids (and the polypeptides they encode) may be from mice, rats, guinea pigs, cattle, sheep, horses, pigs, rabbits, monkeys, baboons, dogs, or cats. In one embodiment, the nucleic acid molecule is from a human.

[0174] Expression of mutant IL-2, IL-4, or IL-13 gene products. The nucleic acid molecules described above may be contained in a vector that can direct their expression in cells transduced by the vector, for example. Therefore, an expression vector containing one or more nucleic acid molecules encoding the target IL-2 fusion protein, in addition to the target IL-2 fusion protein, and cells transfected with these expression vectors, is one of the preferred embodiments.

[0175] Of course, it should be understood that not all vectors and regulatory sequences will function equally well to express the DNA sequences described herein. Furthermore, not all hosts will function equally well in the same expression system. However, those skilled in the art can select from these vectors, regulatory sequences, and hosts without excessive experimentation. For example, when selecting a vector, the host must be considered, as the vector needs to replicate within the host. The vector's copy number, its ability to control its copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. For example, usable vectors include those capable of amplifying the DNA encoding the IL-2 fusion protein by copy number. Such amplifiable vectors are known in the art. These include vectors that can be amplified by, for example, DHFR amplification (see, e.g., Kaufman, U.S. Patent No. 4,470,461; Kaufman and Sharp, “Construction of a Modular Dihydrafolate Reductase cDNA: Analysis of Signals Utilized for Efficient Expression”, Mol. Cell. Biol., 2, pp. 1304-19 (1982)) or glutamine synthetase ("GS") amplification (see, e.g., U.S. Patent No. 5,122,464 and European Publication No. 338,841).

[0176] In some embodiments, the target IL-2 fusion protein will be expressed from a vector, preferably an expression vector. The vector may be useful for autonomous replication in the host cell or, upon introduction into the host cell, be integrated into the host cell's genome and thereby replicated together with the host genome (e.g., a non-episomal mammalian vector). The expression vector can direct the expression of a coding sequence that is operably linked. Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids (vectors). However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0177] An exemplary recombinant expression vector may include one or more regulatory sequences that are selected based on the host cell used for expression and are operably ligated to the nucleic acid sequence being expressed.

[0178] Expression constructs or vectors can be designed for the expression of IL-2 fusion proteins in prokaryotic or eukaryotic host cells.

[0179] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0180] Protein expression in prokaryotes is most frequently performed in Escherichia 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) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.), pp. 119-128 and Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118. Processes for growing, harvesting, disrupting, or extracting IL-2 mutaine or its variants from cells are substantially described, for example, in U.S. Patents 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, which are incorporated herein by reference in their entirety.

[0181] In some embodiments, recombinant IL-2 fusion proteins can also be produced in eukaryotes such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors usable for protein expression in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in yeast S. cerenvisiae include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San This includes pPicZ (Invitrogen Corporation, San Diego, Calif.) and pPicZ (Invitrogen Corporation, San Diego, Calif.); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J.6:187:195)). Suitable mammalian cells include Chinese hamster ovary cells (CHO) or COS cells. In mammalian cells, the regulatory function of the expression vector is often provided by viral moduloviruses. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and Simianvirus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2 ndSee Chapters 16 and 17 of (ed., Cold Spring Harbor Laboratory Press, Plainview, NY). See Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0182] The sequences encoding the IL-2 fusion proteins of this disclosure can be optimized for expression in host cells of interest. The GC content of the sequences can be adjusted to the average level of a particular cell host, such that it is calculated by referencing known genes expressed in the host cells. Methods for codon optimization are known in the art. Codons within the IL-2 fusion protein can be optimized to enhance expression in host cells such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons in the coding sequence are optimized for expression in a particular host cell.

[0183] Suitable vectors for use include T7-based vectors for bacterial use (see, e.g., Rosenberg et al., Gene 56:125, 1987), pMSXND expression vectors for mammalian cell use (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors for insect cell use (e.g., the expression vector pBacPAK9 from Clontech, Palo Alto, Calif.).

[0184] In some embodiments, the nucleic acid insert encoding the target IL-2 fusion protein in such a vector can be operably linked to a promoter selected, for example, based on the cell type to which expression is desired.

[0185] When selecting an expression regulatory sequence, various factors must be considered. These include, for example, the relative strength of the sequence, its regulatory ability, and its compatibility with the actual DNA sequence encoding the target IL-2 fusion protein, particularly with respect to its potential secondary structure. The host should be selected considering its compatibility with the chosen vector, the toxicity of the product encoded by the DNA sequence of the present invention, its secretory properties, its ability to correctly fold the polypeptide, its fermentation or culture requirements, and the ease of purifying the product encoded by the DNA sequence.

[0186] Within these parameters, those skilled in the art can select various vector / expression regulatory sequence / host combinations to express a desired DNA sequence in fermentation or large-scale animal culture, for example, using CHO cells or COS7 cells.

[0187] In some embodiments, the selection of expression regulatory sequences and expression vectors may depend on the host selection. A wide variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include bacterial plasmids such as E. coli plasmids containing col El, pCRI, pER32z, pMB9, and their derivatives, a broader range of host plasmids, e.g., RP4, phage DNA, e.g., numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, e.g., M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells include 2μ plasmids and their derivatives. Vectors useful 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).

[0188] In addition, these vectors can utilize a variety of arbitrary expression regulatory sequences. Such useful expression regulatory sequences include those related to the structural genes of the aforementioned expression vectors. Examples of useful expression regulatory sequences include, for example, the early and late promoters of SV40 or adenoviruses, the lac system, trp system, TAC or TRC system, major operator and promoter regions of phage lambda, e.g., PL, regulatory regions of fd coat proteins, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatases, e.g., PhoA, promoter of the yeast α-mating system, polyhedral promoter of baculoviruses, and other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof.

[0189] The T7 promoter can be used in bacteria, the polyhedrin 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 for their ability to direct the expression of nucleic acid molecules in specific tissues or cell types within the body. Those skilled in the art will be familiar with the many promoters and other regulators that can be used to direct nucleic acid expression.

[0190] In addition to sequences that promote the transcription of inserted nucleic acid molecules, vectors can also contain other genes encoding origins of replication and selection markers. For example, neomycin resistance (neomycin resistance). r The gene confers G418 resistance to the cells in which it is expressed, enabling phenotypic selection of transfected cells. Those skilled in the art can easily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental situation.

[0191] Viral vectors that can be used in the present 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).

[0192] Prokaryotic or eukaryotic cells containing and expressing nucleic acid molecules encoding the target IL-2 mutein disclosed herein are also features of the present invention. The cells of the present invention are transfected cells, i.e., cells into which nucleic acid molecules encoding IL-2, IL-4, or IL-13 mutein, or bifunctional molecules, have been introduced by recombinant DNA techniques. Progeny of such cells are also considered to be within the scope of the present invention.

[0193] The exact components of the expression system are not important. For example, the target IL-2 fusion protein can be produced in prokaryotic hosts such as the bacterium E. coli, or in eukaryotic hosts 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, Va.). The only important factor in selecting an expression system is the compatibility of its components. Experts or those skilled in the art can make such a decision. Furthermore, if guidance is needed in selecting an expression system, those skilled in the art may 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 Suppl. 1987).

[0194] The expressed polypeptide can be purified from the expression system using standard biochemical procedures and can be used, for example, as a therapeutic agent as described herein.

[0195] In some embodiments, the resulting IL-2 fusion protein will be glycosylated or deglycosylated depending on the host organism used to produce mutaine. If bacteria are selected as the host, the produced IL-2 fusion protein will not be glycosylated. Eukaryotic cells, on the other hand, will glycosylate the IL-2 and IL-13 of the fusion protein, but probably not in the same way that native IL-2 or IL-13 are glycosylated. The IL-2 fusion protein produced by the transformed host can be purified according to any preferred method. Various methods are known for purifying IL-2 fusion proteins. For example, see Current Protocols in Protein Science, Vol 2, Eds: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, John Wiley and Sons, Inc.). IL-2 fusion proteins can be isolated from inclusion bodies produced in E. coli or from conditioned media from a given mutaine-producing mammalian or yeast culture using cation exchange, gel filtration, and / or reverse-phase liquid chromatography.

[0196] Another exemplary method for constructing the DNA sequence encoding the target IL-2 fusion protein is by chemosynthesis. This includes the direct synthesis of peptides by chemical means of protein sequences encoding IL-2 mutaines exhibiting the described properties. This method can incorporate both native and non-native amino acids at positions that influence the interaction between the IL-2 or IL-13 mutaines of the IL-2 fusion protein and their corresponding receptors. Alternatively, the gene for either IL-2 fusion protein can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired IL-2 fusion protein, preferably selecting codons that are favorable to the host cell in which the recombinant mutaine is produced. In this regard, it is well recognized that the genetic code is degenerate, meaning that an amino acid may be encoded by two or more codons. For example, Phe(F) may be encoded by the 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, for a given DNA sequence encoding a particular IL-2 fusion protein, it will be understood that there are many degenerate DNA sequences encoding that IL-2 fusion protein. For example, in addition to the preferred DNA sequence of mutain H9, it will be understood that there are many degenerate DNA sequences encoding the shown IL-2 fusion protein. These degenerate DNA sequences are considered to be within the scope of this disclosure. Therefore, in the context of this invention, “its degenerate variant” means all DNA sequences that encode a particular mutain and thereby enable its expression.

[0197] The biological activity of IL-2 fusion proteins can be assayed by any suitable method known in the art. Such assays include PHA blast proliferation and NK cell proliferation.

[0198] G. Treatment method In some embodiments, target IL-2 fusion proteins and / or nucleic acids expressing them can be administered to treat abnormal apoptosis or disorders associated with differentiation processes (e.g., cell proliferation disorders or cell differentiation disorders such as cancer, resulting in active or passive immunity). In the treatment of such diseases, the disclosed IL-2 fusion proteins may have advantageous properties such as reduced vasoleakage syndrome.

[0199] Examples of disorders of cell proliferation and / or differentiation include cancer (e.g., carcinoma, sarcoma, metastatic disorder, or hematopoietic neoplasm disorder, e.g., hematological disease). Metastatic tumors can arise from a number of primary tumor types, including but not limited to sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoid cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer (including small cell lung cancer), kidney cancer, gastric cancer, brain cancer, and CNS tumors. In some embodiments, the cancer for treatment is a solid tumor. In some embodiments, cancers for treatment include, but are not limited to, sarcomas, carcinomas, head and neck cancers, glioblastomas, bladder cancers, oral cancers, mesotheliomas, pancreatic cancers, liver cancers, colorectal cancers, lung cancers, skin cancers, lymphoid cancers, gastrointestinal cancers, prostate cancers, ovarian cancers, breast cancers, basal breast tumors, endometrial cancers, multiple myelomas, melanomas, lymphomas, lung cancers (including small cell lung cancers), kidney cancers, gastric cancers, brain cancers, and CNS tumors. CNS tumors include gliomas, glioblastomas, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastoma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendrocyte, menangioma, meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, O6-methylguanine-methyltransferase (MGMT) positive or negative CNS tumors, and furin-positive CNS tumors.

[0200] The target IL-2 fusion protein can be used to treat patients who have, are suspected of having, or are at 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, B, C, and / or D. Exemplary carcinomas include those formed from tissue of the cervix, lungs, prostate, breast, head and neck, colon, and ovaries. The term also includes carcinosarcoma, which includes malignant tumors composed of cancerous and sarcomatous tissue.

[0201] Additional examples of proliferative disorders include hematopoietic neoplasm disorders.

[0202] Alternatively, or in addition to direct administration to the patient, in some embodiments, the target IL-2 fusion protein can be used ex vivo. For example, cells (e.g., peripheral blood lymphocytes, or a purified population of lymphocytes isolated from a patient and placed in or maintained in culture) can be cultured in vitro in a culture medium, and the contact step can be achieved by adding the target IL-2 fusion protein to the culture medium. The culture step may include further steps of stimulating or treating cells with other agents to stimulate or expand the proliferation of a cell population that reacts to a target antigen (e.g., cancer antigen or viral antigen), for example. The cells are then administered to the patient after treatment.

[0203] Anti-PD-1 antibodies for use in combination with the subject IL-2 fusion proteins 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®), semiprimab (REGN2810), SHR-1210 (CTR20160175 and CTR2017090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), and BGB-A317 (CTR20160872).

[0204] In some embodiments, the target IL-2 fusion protein is used in combination with nivolumab for the treatment of cancer. In some embodiments, the IL-2 fusion protein is used in combination with pembrolizumab for the treatment of cancer. In some embodiments, the target IL-2 fusion protein is used in combination with BMS-936558 for the treatment of cancer. In some embodiments, the target IL-2 fusion protein is used in combination with MDX-1106 for the treatment of cancer. In some embodiments, the target IL-2 fusion protein is used in combination with ONO-4538 for the treatment of cancer. In some embodiments, the target IL-2 fusion protein is used in combination with AMP224 for the treatment of cancer. In some embodiments, the target IL-2 fusion protein is used in combination with CT-011 for the treatment of cancer. In some embodiments, the target IL-2 fusion protein is used in combination with MK-3475 for the treatment of cancer.

[0205] In some embodiments, the target IL-2 fusion protein is used in combination with antibodies and / or immunotherapies for the treatment of cancer, including anti-CTLA4 mAbs such as ipilimumab and tremelimumab; anti-PD-L1 antagonist antibodies such as BMS-936559 / MDX-1105, MEDI4736, and RG-7446 / MPDL3280A; anti-LAG-3 such as IMP-321; immunostimulatory protein-targeted agonist antibodies including anti-CD40 mAbs such as CP-870,893, lucatumumab, and dasetuzumab; anti-CD137 mAbs such as BMS-663513 urelumab (anti-4-1-BB antibody) (see, for example, U.S. Patent Nos. 7,288,638 and 8,962,804 (these are incorporated herein in their entirety by reference)); and lirirumab (anti-KIR). mAB; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors) and PF-05082566 (utomirumab; see, for example, U.S. Patent Nos. 8,821,867, 8,337,850, and 9,468,678, and International Patent Application Publication WO2012 / 032433 (these are incorporated herein by reference in their entirety)); anti-OX40 mAb (see, for example, WO2006 / 029879 or WO2010 / 096418 (these are incorporated herein by reference in their entirety)); anti-GITR mAb such as TRX518 (see, for example, U.S. Patent No. 7,812,135 (these are incorporated herein by reference in their entirety)); anti-CD27 Examples of mAbs include, but are not limited to, valirumab CDX-1127 (see, e.g., WO2016 / 145085 and U.S. Patent Publications US2011 / 0274685 and US2012 / 0213771, which are incorporated herein by reference in their entirety), anti-ICOS mAbs (see, e.g., MEDI-570, JTX-2011), and anti-TIM-3 antibodies (see, e.g., WO2013 / 006490 or U.S. Patent Publications US2016 / 0257758, which are incorporated herein by reference in their entirety).

[0206] In some embodiments, the target IL-2 fusion protein is used in combination with another antibody that may include a monoclonal antibody for the treatment of solid tumors. In some embodiments, the monoclonal antibody is for the treatment of sarcomas, carcinomas, head and neck cancers, glioblastomas, bladder cancers, oral cancers, mesotheliomas, pancreatic cancers, liver cancers, colorectal cancers, lung cancers, skin cancers, lymphoid cancers, gastrointestinal cancers, prostate cancers, ovarian cancers, breast cancers, basal breast tumors, endometrial cancers, multiple myelomas, melanomas, lymphomas, lung cancers (including small cell lung cancers), kidney cancers, gastric cancers, brain cancers, and CNS tumors. CNS tumors include gliomas, glioblastomas, glioblastoma multiforme (GBM), refractory glioblastoma multiforme (rGBM), recurrent glioblastoma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendrocyte, menangioma, meningioma, neuroblastoma, retinoblastoma, medulloblastoma, adult pituitary adenoma, O6-methylguanine-methyltransferase (MGMT) positive or negative CNS tumors, and furin-positive CNS tumors.

[0207] In some embodiments, the target IL-2 fusion protein is used in combination with an antibody for antibody-dependent cell-mediated cytotoxicity (ADCC) for the treatment of cancer.

[0208] In some embodiments, the target IL-2 fusion proteins described herein are used in combination with antibodies including dupilumab, nivolumab (OPDIVO®), BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA®), semiprimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), and BGB-A317 (CTR20160872).In some embodiments, the IL-2, IL-4, or IL-13 muteins and / or bifunctional molecules described herein include anti-CTLA4 mAbs such as ipilimumab and tremelimumab; anti-PD-L1 antagonist antibodies such as BMS-936559 / MDX-1105, MEDI4736, and RG-7446 / MPDL3280A; anti-LAG-3 such as IMP-321; immunostimulatory protein-targeted agonist antibodies including anti-CD40 mAbs such as CP-870, 893, lucatumumab, and dasetuzumab; and anti-CD137 such as BMS-663513 urelumab. mAb (anti-4-1BB antibody) (see, for example, U.S. Patent Nos. 7,288,638 and 8,962,804 (these are incorporated herein by reference in their entirety)); lirirumab (anti-KIR mAB; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors) and PF-05082566 (utomirumab; see, for example, U.S. Patent Nos. 8,821,867, 8,337,850 and 9,468,678, and International Patent Application Publication WO2012 / 032433 (these are incorporated herein by reference in their entirety)); anti-OX40 mAbs (see, e.g., WO2006 / 029879 or WO2010 / 096418 (these are incorporated herein by reference in their entirety)); anti-GITR mAbs such as TRX518 (see, e.g., U.S. Patent No. 7,812,135 (these are incorporated herein by reference in their entirety)); anti-CD27 mAbs such as valirumab CDX-1127 (see, e.g., WO2016 / 145085 and U.S. Patent Publications US2011 / 0274685 and US2012 / 0213771 (these are incorporated herein by reference in their entirety)); anti-ICOS It is used in combination with antibodies, including mAbs (e.g., MEDI-570, JTX-2011, and anti-TIM-3 antibodies (e.g., see WO2013 / 006490 or U.S. Patent Publication No. US2016 / 0257758 (these are incorporated herein by reference in their entirety)), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-sirs-alpha, and / or anti-CD112R).

[0209] In some embodiments, the target IL-2 fusion protein described herein is fused with an antibody selected from dupilumab, nivolumab (OPDIVO®), BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab or KEYTRUDA®), semiprimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), and BGB-A317 (CTR20160872).In some embodiments, the IL-2 fusion proteins described herein include anti-CTLA4 mAbs such as ipilimumab and tremelimumab; anti-PD-L1 antagonist antibodies such as BMS-936559 / MDX-1105, MEDI4736, and RG-7446 / MPDL3280A; anti-LAG-3 such as IMP-321; immunostimulatory protein-targeted agonist antibodies including anti-CD40 mAbs such as CP-870, 893, lucatumumab, and dasetuzumab; anti-CD137 mAbs such as BMS-663513 urelumab (anti-4-1-BB antibody) (see, for example, U.S. Patent Nos. 7,288,638 and 8,962,804 (these are incorporated herein by reference in their entirety)); and lirirumab (anti-KIR). mAB; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors) and PF-05082566 (utomirumab; see, for example, U.S. Patent Nos. 8,821,867, 8,337,850, and 9,468,678, and International Patent Application Publication WO2012 / 032433 (these are incorporated herein by reference in their entirety)); anti-OX40 mAb (see, for example, WO2006 / 029879 or WO2010 / 096418 (these are incorporated herein by reference in their entirety)); anti-GITR mAb such as TRX518 (see, for example, U.S. Patent No. 7,812,135 (these are incorporated herein by reference in their entirety)); anti-CD27 such as valirumab CDX-1127 mAbs (see, for example, WO2016 / 145085 and U.S. Patent Publications US2011 / 0274685 and US2012 / 0213771 (these are incorporated herein by reference in their entirety)) are fused with anti-ICOS mAbs (see, for example, MEDI-570, JTX-2011, and anti-TIM-3 antibodies (see, for example, WO2013 / 006490 or U.S. Patent Publications US2016 / 0257758 (these are incorporated herein by reference in their entirety)), Herceptin, anti-EGFR, anti-VEGF, anti-TIGIT, anti-LAG3, anti-CD8, anti-CD47, anti-sirs-alpha, and / or anti-CD112R).

[0210] H. Pharmaceutical composition and method of administration In some embodiments, the target IL-2 fusion protein can be incorporated into a composition comprising a pharmaceutical composition. Such a composition typically comprises a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. Such a composition may also include an anti-PD-1 antibody. In some embodiments, the composition comprises an IL-2 mutain that is a fusion protein and / or associated with a CAR-T construct and / or expressed by or associated with an oncolytic virus.

[0211] The anti-PD-1 antibody and the target IL-2 fusion protein can be administered as a co-composition, simultaneously as two separate compositions, and / or sequentially as two separate compositions. In some embodiments, the anti-PD-1 antibody or inhibitor and the target IL-2 fusion protein are administered together as a single co-composition (i.e., formulated together). In some embodiments, the anti-PD-1 antibody or inhibitor and the target 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 target 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.

[0212] The other immunotherapeutic agents and IL-2 fusion proteins described may be administered as a co-composition, simultaneously as two distinct compositions, and / or sequentially as two distinct compositions. In some embodiments, the other immunotherapeutic agents and IL-2 fusion proteins are administered together as a single co-composition (i.e., formulated together). In some embodiments, the other immunotherapeutic agents and IL-2 fusion proteins are administered simultaneously as two distinct compositions (i.e., distinct formulations). In some embodiments, the other immunotherapeutic agents and IL-2 fusion proteins are administered sequentially as distinct compositions (i.e., distinct formulations). In some embodiments, when the other immunotherapeutic agents and IL-2 fusion proteins are administered sequentially as distinct compositions, the anti-PD-1 antibody or inhibitor is administered before the IL-2 fusion protein. In some embodiments, when the other immunotherapeutic agents and IL-2 fusion proteins are administered sequentially as separate compositions, the IL-2 fusion protein is administered before the other immunotherapeutic agent.

[0213] Pharmaceutical compositions are formulated to suit their intended route of administration. The anti-PD-1 antibody and / or IL-2 fusion protein of the present invention may be administered orally, but is more likely to be administered via parenteral routes, including intravenous administration. Examples of parenteral administration routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral applications include the following components: sterile diluents such as water for injection, physiological saline, fixative oil, polyethylene glycol, glycerin, 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 acetates, citrates, or phosphates; and tonicity adjusters such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide (e.g., about 7.2 to 7.8, e.g., to pH 7.5). Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or vials for multiple doses.

[0214] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (if water-soluble), or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to facilitate injection. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants such as sodium dodecyl sulfate. Prevention of microbial action can be achieved 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, such as sugars and polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. Long-term absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0215] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, along with one or a combination of the components listed above as needed, into a suitable solvent, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield powders of the active ingredient and any additional desired components from the previously sterile filtered solution.

[0216] Oral compositions, when used, generally contain an inert diluent or food carrier. For the purpose of oral therapeutic administration, the active compound may be mixed with an excipient and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions may also be prepared using a liquid carrier for use as a mouthwash. 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 of similar properties: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel®, or corn starch; lubricants such as magnesium stearate or Sterotes®; flow enhancers such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; and flavorings such as peppermint, methyl salicylate, or orange flavor.

[0217] For administration by inhalation, the anti-PD-1 antibody and / or IL-2 fusion protein, or the nucleic acid encoding it, is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas like carbon dioxide, or from a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0218] Systemic administration of anti-PD-1 antibodies and / or IL-2 fusion proteins or nucleic acids may be by mucosal or transdermal means. In the case of mucosal or transdermal administration, a penetrating agent suitable for the barrier to penetration is used in the formulation. Such penetrating agents are generally known in the art and, for example, in the case of mucosal administration, include surfactants, bile salts, and fusidic acid derivatives. Mucosal administration can be achieved by the use of nasal sprays or suppositories. In the case of transdermal administration, the active compound is formulated into ointments, medicinal ointments, gels, or creams, as is generally known in the art.

[0219] In some embodiments, the compound (anti-PD-1 antibody and / or IL-2 fusion protein or nucleic acid) may also be prepared for rectal delivery in the form of a suppository (e.g., with a conventional suppository base such as cocoa butter and other glycerides) or a retained enema.

[0220] In some embodiments, the compound (target IL-2 fusion protein or nucleic acid) may also be administered by 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 Biotechnol. 20:1006-1010, 2002), or Putnam (Am.J. Health Syst. Pharm. 53:151-160, 1996, erratum at Am.J. Health Syst. Pharm. 53:325, 1996).

[0221] In one embodiment, an anti-PD-1 antibody and / or IL-2 fusion protein or nucleic acid is prepared 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 an implant and microencapsulation delivery system. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, 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 (containing liposomes targeted to 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 described in U.S. Patent No. 4,522,811.

[0222] The dosage, toxicity, and therapeutic effect of such anti-PD-1 antibodies, IL-2 fusion proteins, or nucleic acid compounds can be determined by standard pharmacological techniques in cell culture or experimental animals, e.g., to determine LD 50 (the 50% lethal dose of the population) or ED 50 (the dose that has a therapeutic effect on 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the ratio of LD 50 / ED 50 . Compounds showing a high therapeutic index are preferred. Compounds showing toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of the affected tissue in order to minimize potential damage to uninfected cells and thereby reduce side effects.

[0223] Data obtained from cell culture assays and animal studies can be used to formulate the dosage range for use in humans. The dosage of such compounds preferably lies within the range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the present invention, the therapeutically effective amount can first be estimated from cell culture assays. The dosage can be formulated in animal models to achieve the circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound that reaches the maximum half-inhibition of the symptoms) as determined in cell culture. Such information can be used to more accurately determine the dosage useful in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0224] As defined herein, the therapeutically effective dose (i.e., effective dose) of the target IL-2 fusion protein and / or anti-PD-1 antibody or inhibitor depends on the selected polypeptide or antibody. In some embodiments, a single dose of the IL-2 fusion protein may range from about 0.001 mg / kg to 0.1 mg / kg per kg of body weight of the patient to be administered. In some embodiments, a single dose of the anti-PD-1 antibody or inhibitor may range from about 1 mg / kg to 20 mg / kg, or about 5 mg / kg to about 15 mg / kg, or about 10 mg / kg per kg of body weight of the patient to be administered. In some embodiments, doses of anti-PD-1 antibody or inhibitor and / or IL-2 fusion protein of about 0.005 mg / kg, 0.01 mg / kg, 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 5.0 mg / kg, and 10.0 mg / kg can be administered. In some embodiments, 600,000 IU / kg is administered (IU can be determined by lymphocyte proliferation bioassay and is expressed in International Units (IU) as established by the World Health Organization's First International Standard for Interleukin-2 (Human)). The dosage may be equivalent to, but is expected to be less than, the amount prescribed for PROLEUKIN®. The composition may be administered once daily to once weekly, including once every other day. Those skilled in the art will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other pre-existing diseases, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective dose of the subject IL-2 fusion protein may consist of a single treatment or a series of treatments. In one embodiment, the composition is administered every 8 hours for 5 days, followed by a drug-free period of 2 to 14 days, for example, 9 days, followed by another 5 days of administration every 8 hours. In some embodiments, the administration is given three times every 4 days.

[0225] Pharmaceutical compositions may be included in a container, pack, or dispenser along with instructions for administration.

[0226] The following examples are provided to illustrate certain embodiments of the invention provided herein and should not be construed as limiting.

[0227] Additional Embodiments In embodiments, the following IL-2 cytokine fusion proteins are provided herein, comprising: (a) an IL-2 moiety comprising IL-2 or IL-2 mutein, or optionally an IL-2 mutein fusion; (b) a protease-sensitive linker (PSL); and (c) an IL-2 masking moiety, wherein the PSL binds the IL-2 masking moiety to the IL-2 moiety.

[0228] In embodiments, the IL-2 cytokine fusion protein is provided herein, comprising: (a) an IL-2 moiety comprising IL-2 or IL-2 mutein, optionally an IL-2 mutein fusion; (b) at least one protease-sensitive linker (PSL); and (c) at least one IL-2 masking moiety, wherein at least one PSL binds the IL-2 masking moiety to at least one IL-2 moiety.

[0229] In this embodiment, the masking portion includes IL-13, IL-13 mutain, IL-13Ra2-binding mutain, IL-13Ra2 antibody, or an antigen-binding fragment thereof, and the IL-2 masking portion can bind to IL-13Ra2 but does not bind to IL-13Ra1.

[0230] In this embodiment, the masking portion contains IL-13 mutain having one of the amino acid sequences from SEQ ID NOs. 200 to 241.

[0231] In the embodiment, the masking portion is IL-13 mutein, 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.

[0232] In this embodiment, the masking portion contains IL-13 mutain having the amino acid sequence of SEQ ID NO: 216 or SEQ ID NO: 228.

[0233] In this embodiment, the masking portion further includes the extracellular domain of CD122, the extracellular domain of CD132, or the extracellular domain of CD25.

[0234] In this embodiment, the IL-2 portion comprises IL-2 mutein having one of the amino acid sequences from SEQ ID NOs. 5-24 and 105.

[0235] In this embodiment, IL-2 mutein has the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 9.

[0236] In the embodiment, IL-2 mutein further comprises T3A and C125S amino acid substitutions.

[0237] In the embodiment, the IL-2 portion further comprises albumin, an Fc domain, or an antibody bound to IL-2 or IL-2 mutein.

[0238] In this embodiment, the albumin is human albumin, and optionally, the human albumin is recombinant human albumin.

[0239] In this embodiment, the IL-2 moiety is an IL-2x anti-PD1 fusion protein comprising: (a) a first polypeptide comprising IL-2 or IL-2 mutein bound 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 VL form a PD-1 binding domain.

[0240] In this embodiment, the second polypeptide is an antibody heavy chain.

[0241] In this embodiment, the IL-2 portion is an IL-2x anti-PD1 fusion protein comprising: (a) a first polypeptide comprising a first antibody heavy chain bound to IL-2 or IL-2 mutein, wherein the first antibody heavy chain comprises a first heavy chain variable region (VH1) and a first Fc domain; (b) a second polypeptide comprising a second antibody heavy chain, wherein the second antibody heavy chain comprises a second heavy chain variable region (VH2) and a second Fc domain; (c) a third polypeptide comprising a first variable light chain variable region (VL1) and a light chain constant region; and (d) a fourth polypeptide comprising a second variable light chain variable region (VL2) and a light chain constant region, wherein VH1 and VL1 form a first PD-1 binding domain, and VH2 and VL2 form a second PD-1 binding domain.

[0242] In this embodiment, the first PD-1 binding domain and the second PD-1 binding domain have the same amino acid sequence.

[0243] In this embodiment, the first PD-1 binding domain and the second PD-1 binding domain have different amino acid sequences.

[0244] In this embodiment, the antibody is manipulated as a "knob-in-hole" (KiH) having a mutation in the constant region 3 (CH3) of its heavy chain.

[0245] In this embodiment, the IL-2 portion is an IL-2x antibody (KiH) fusion protein comprising: a) a first polypeptide containing an IL-2 fusion product bound to IL-2 or IL-2 mutein, or optionally to the second and third constant regions (CH2 and CH3) of the "knob" heavy chain of the antibody (KiH); b) a second polypeptide containing the "whole" heavy chain of the antibody (KiH); and c) a third polypeptide containing the light chain of the antibody (KiH).

[0246] In the embodiment, the IL-2 portion comprises an IL-2x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising IL-2 or IL-2 mutein, or optionally a "knob" heavy chain of an antibody (KiH) bound to an IL-2 fusion; b) a second polypeptide comprising a "hole" heavy chain of an antibody (KiH); and c) a third polypeptide comprising a light chain of an antibody (KiH).

[0247] In the embodiment, the IL-2 portion comprises an IL-2x antibody (KiH) fusion protein comprising: a) a first polypeptide comprising a "knob" heavy chain of the antibody (KiH); b) a second polypeptide comprising a "hole" heavy chain of the antibody (KiH) bound to IL-2 or IL-2 mutein or an IL-2 mutein fusion; and c) a third polypeptide comprising a light chain of the antibody (KiH).

[0248] In this embodiment, the IL-2 masking portion is bonded to IL-2 or IL-2 mutain.

[0249] In this embodiment, the IL-2 masking portion is bound to an antibody heavy chain that is not bound to IL-2 or IL-2 mutain.

[0250] In this embodiment, the antibody (KiH) is anti-PD1 (KiH).

[0251] In this embodiment, IL-2 mutein has one of the amino acid sequences from SEQ ID NOs. 5-24 and 105.

[0252] In the embodiment, IL-2 mutein further comprises T3A and C125S amino acid substitutions.

[0253] In this embodiment, the PSL can be cleaved within the tumor microenvironment.

[0254] In the embodiment, PSL has the amino acid sequence PLGLVVAPLGLVVAPLGLVVA, PLGLWAPLGLWAPLGLWA, GGSGGTPLGLWAGGSGGT, GGSGGTPAGLIGGGSGGT, GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT, or GGSGGTHSSKLQGGSGGT.

[0255] In embodiments as described herein, an IL-2 cytokine fusion protein is (a) SEQ ID NOs: 302, 303, and 304, (b) SEQ ID NOs: 299, 300, and 301, (c) SEQ ID NOs: 305, 306, and 307, (d) SEQ ID NOs: 308, 309, and 310, (e) SEQ ID NOs: 311, 312, and 313, (f) SEQ ID NOs: 314, 315, and 316, (g) SEQ ID NOs: 317, 318, and 319, (h) SEQ ID NOs: 320, 321, and 322, (i) SEQ ID NOs: 323, (j) SEQ ID NOs: 329, 330, and 331, (k) An IL-2 cytokine fusion protein is provided, comprising a sequence selected from the group consisting of (l)SEQ ID NOs: 335, 336, and 337, (m)SEQ ID NOs: 338, 339, and 340, (n)SEQ ID NOs: 353, 354, and 355, (o)SEQ ID NOs: 359, 360, and 361, (p)SEQ ID NOs: 362, 363, and 364, (q)SEQ ID NOs: 365, 366, and 367, (r)SEQ ID NOs: 325, 326, and 327, (s)SEQ ID NOs: 353, 354, and 355, and (t)SEQ ID NO: 324.

[0256] In embodiments, a pharmaceutical composition is provided herein comprising an IL-cytokine fusion product described herein and a pharmaceutically acceptable carrier.

[0257] In embodiments thereof, nucleic acid compositions encoding the IL-2 cytokine fusion protein described herein are provided.

[0258] In some embodiments, an expression vector composition comprising the nucleic acid composition described herein is provided.

[0259] In embodiments, a method for producing an IL-2 cytokine fusion protein as described herein is provided, comprising culturing a nucleic acid composition or an expression vector composition as described herein under conditions in which an IL-2 cytokine fusion protein is expressed, and recovering the IL-2 cytokine fusion protein.

[0260] In embodiments, a method is provided herein for treating a subject requiring treatment for IL-13Ra2-expressing cancer, comprising administering to the subject an IL-2 cytokine fusion protein or a pharmaceutical composition described herein.

[0261] In this embodiment, the IL-2 cytokine fusion protein includes one or more of SEQ ID NOs: 1 to 367.

[0262] In embodiments, a method is provided herein for treating a subject in need of cancer treatment, comprising administering to the subject an IL-2 cytokine fusion protein comprising (a) IL-2 mutein and (b) albumin or an Fc domain or an antibody, wherein the IL-2 mutein has one of the amino acid sequences of SEQ ID NOs. 5-24 and 105, and optionally further comprising a T3A and / or C125S amino acid substitution.

[0263] In this embodiment, the IL-2 cytokine fusion protein includes one or more of SEQ ID NOs: 1 to 367.

[0264] In this embodiment, the IL-2 cytokine fusion protein has the amino acid sequence of SEQ ID NO: 53.

[0265] In this embodiment, the IL-2 cytokine fusion protein is administered as a neoadjuvant before surgery to remove the tumor.

[0266] In the embodiment, the IL-2 cytokine fusion protein is administered up to one week before surgery, or optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day before surgery. In the embodiment, the IL-2 cytokine fusion protein is administered up to nine weeks before surgery, or optionally up to eight weeks, seven weeks, six weeks, five weeks, four weeks, three weeks, two weeks, one week, or one week before surgery.

[0267] In this embodiment, the IL-2 cytokine fusion protein is administered as an adjuvant after surgery to remove the tumor.

[0268] In the embodiments, the IL-2 cytokine fusion protein is administered up to one week after surgery, optionally up to seven days, six days, five days, four days, three days, two days, one day, or within one day after surgery. In the embodiments, the IL-2 cytokine fusion protein is administered starting at least two weeks after surgery.

[0269] In this embodiment, the IL-2 cytokine fusion protein is administered both as a neoadjuvant before surgery to remove the tumor and as an adjuvant after surgery to remove the tumor.

[0270] In the embodiments, the IL-2 cytokine fusion protein is administered up to one week before surgery, or optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day before surgery; and the IL-2 cytokine fusion protein is administered up to one week after surgery, or optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day after surgery. In the embodiments, the IL-2 cytokine fusion protein is administered up to nine weeks before surgery, or optionally up to eight weeks, seven weeks, six weeks, five weeks, four weeks, three weeks, two weeks, one week, or one week before surgery; and the IL-2 cytokine fusion protein is administered starting at least two weeks after surgery.

[0271] In the embodiments, cancer is sarcoma, carcinoma, head and neck cancer, glioblastoma, bladder cancer, oral cancer, mesothelioma, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, skin cancer, lymphoid cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, basal breast tumor, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, small cell lung cancer, kidney cancer, stomach cancer, brain cancer, or CNS tumor. [Examples]

[0272] Example 1: Evaluate IL-13 superkine masking of IL-2 superagonist activity using an in vitro IL-2 reporter assay. A. Preface MDNA132 is an IL-13 superkine that selectively binds to the IL-13 decoy receptor (IL-13Ra2) but does not bind to the IL-13 functional receptor (IL-13Ra1).

[0273] MDNA132.15-PSL-MDNA109-Alb (SEQ ID NO: 298, referred to herein as MDNA213-PSL-MDNA109-alb) T3 / C125The construct (also known as MDNA132-PSL-MDNA11) was constructed using a protease-sensitive linker (PSL) based on the theoretical premise of masking the IL-2 "beta only" superagonist MDNA109-Alb with MDNA132.15 (also referred to herein as "MDNA213"). Once cleaved, MDNA132.15 is hypothesized to dock MDNA109-Alb to IL-13 decoy receptor overexpressing tumors. This masking facilitates peripheral T cell activation, thereby improving tolerability. MDNA109-Alb was used as a comparator to this construct in plates. T3 / C125 (SEQ ID NO: 294) was also expressed and purified in CHO, and its IL-2 activity was tested using the Jarcut IL-2Rβγ bioassay. The additional construct, MDNA213-PSL-MDNA109-alb T3 / C125 (Sequence ID 298) and MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin We designed and produced (SEQ ID NOs. 299-301) to further validate the proof of concept. MDNA213-PSL-MDNA109-alb T3 / C125 It has three copies of PSL that are not adjacent to the "GS" residue, while the single copy of PSL is MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin It was adjacent to the "GS" residue in [the specified location].

[0274] B. Method The Jarcut IL2Rβ-γ bioassay from Promega is designed for intended use in the IL2Rβγ bioassay, using novel IL-2 and IL-15 molecules engineered for reduced CD25 binding, as Jarcut cells lack CD25 expression. This is a simple homogeneous assay that is highly sensitive, reproducible, and can be completed in one day. Because the cells lack the CD25 receptor, the engineered IL-2 molecule binds to the CD122 receptor expressed on the cells and induces signaling via pSTAT5, which is detected via luminescence in Jarcut reporter cells.

[0275] Jarcut IL-2Rβγ cells were seeded in 50 μL volumes in 96-well plates according to the manufacturer's recommendations. The construct was diluted as a 3× solution and then 25 μL was added to each well (Table 11). Cells were treated for 6 hours, then luciferase substrate was added and incubated for 10 minutes. Luminescence was then measured using an iD5 plate reader. Two plates were run in the assay, with each plate containing serial dilutions of the test construct in 4-well sequences. [Table 12]

[0276] C. Results Both MDNA109-Alb and MDNA132.15-PSL-MDNA109-Alb elicited clear dose-responses (Figure 1A). EC in these dose-response curves 50 There was an 18.8-fold difference, with MDNA109-Alb showing higher potency (lower EC). 50 ) was found to have the same effect. Similar results were observed with MDNA223 (non-masking type), which exhibited reduced efficacy in the Jarcut IL2Rβγ reporter assay. A3 / S125 -fPSL2f-MDNA213 Lin (Obtained using masking method.) MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin The EC50 of (1624 pM) was 12.3 times lower than that of MDNA223 (131.9 pM). This is consistent with the intended masking of the MDNA109FEAA portion of MDNA223 by MDNA213.

[0277] EC 50 This is summarized in Table 12. [Table 13]

[0278] MDNA11 (SEQ ID NO: 53) and MDNA132-PSL-MDNA11 T3 / C125Both (SEQ ID NO: 294) showed a clear dose-response (Figure 1B). EC of the two dose-response curves. 50 There is a difference of approximately 5.6 times, with MDNA11 showing higher potency (lower EC at 509 pM). 50 ) exhibits MDNA132-PSL-MDNA11 T3 / C125 The EC is 2835 pM 50 This was shown.

[0279] D. Conclusion MDNA132.15-PSL-MDNA109-Alb exhibited 18.8 times lower potency, demonstrating that MDNA132.15 can mask the activity of MDNA109-Alb.

[0280] Example 2: Evaluating IL-13 superkine masking of IL-2 superagonist binding to CD122. A. Preface The binding affinity experiment shown in this example confirms Example 1 and verifies the masking effect of the pfMDNA132.15IL-13 superkine. Observations in the previous experiment showed that MDNA132.15 masks MDNA109-albumin through a decrease in the potency of IL-2-induced pSTAT5.

[0281] B. Method Ni-NTA biosensors (Sartorius catalog No. 18-5101) were hydrated in each experiment in 96-well plates (Greiner part No. 655209) as required by the Octet RH16 Biolayer Inferometer (BLI) instrument manufacturer (Sartorius). Ligand (200 nM His-CD122) was immobilized on the Ni-NTA biosensors, which were then immersed in titration of either MDNA109-Alb (construct No. 7741-044, 82.8 kDa) or MDNA132.15-PSL-MDNA109-Alb (construct No. 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 reaction rate reagent (Sartorius catalog No. 18-1105). The assay was performed on a 384-well tilt-well assay plate (Sartorius catalog No. 18-5080), with a volume of 50 μL per well at 25°C. Assay baseline drift was subtracted using protein-bound biosensors immersed in buffer instead of each analyte. Negative control experiments were included in which intermediate range concentrations of the analyte were tested to determine the "bare" biosensor binding profile without loading the ligand onto the biosensor. No significant nonspecific binding was observed in any experiment. The blocking step was added using Superblock (ThermoFisher catalog No. 37515).

[0282] The typical BLI process conditions used were as follows: • Biosensor check in assay buffer (30 seconds) • Immobilization with His-CD122 (ligand) (12 minutes) • Blocking using Superblock (2 minutes) • Baseline in assay buffer (3 minutes) ·Analyte titration meeting (5 minutes) • Dissociation back to baseline buffer (15 minutes)

[0283] C. Results The construct MDNA132.15-PSL-MDNA109-albumin was tested for its binding affinity to human CD122, with MDNA109-albumin used as the control. The data is presented as a sensorgram in Figure 2.

[0284] As observed, both MDNA109-albumin and MDNA132.15-PSL-MDNA109-albumin have a binding affinity constant K D A unique k that produces a 6.9 times difference on Despite the values, MDNA109-albumin and MDNA132.15-PSL-MDNA109-albumin showed affinity for human CD122, with K levels of 4.6 nM and 31.6 nM, respectively. D The results showed that the shift in the binding affinity constant indicates a masking effect on the binding of MDNA109-albumin by MDNA132.15, which is fused together via PSL (protease-sensitive linker).

[0285] D. Conclusion The binding affinity data clearly established a masking effect, observed by a 6.9-fold difference in the KD constant between the constructs MDNA109-albumin and MDNA132.15-PSL-MDNA109-albumin. The data will be further validated by cleaving the constructs using MMP9 and testing the recovery of MDNA109-albumin binding.

[0286] Example 3: Characterization of a tumor-targeting and activatable T-MASK platform that enhances tumor accumulation and tolerability of potent immunomodulators. The novel T-MASK (Targeted Metallo / Protease Activated Superkine) platform involves fusing a dual tumor targeting / masking domain to a potent immunomodulator via a metallo-protease (MMP)-sensitive linker (PSL) to achieve the following objectives: (1) to reduce / fine-tune the potency of the immunomodulator through steric hindrance to increase systemic tolerability, and (2) to promote retention in the tumor microenvironment (TME) to maximize MMP cleavage at the target site and restore the intended full potency. As proof of concept, the inventors selected an IL-13 superkine (MDNA213, also referred to herein as "MDNA132.15") which has high selectivity and affinity for the IL-13 decoy receptor IL-13Rα2, a tumor-associated antigen expressed in many invasive solid tumors, as the tumor targeting / masking domain.

[0287] Example 3a: Evaluate IL-2 superagonist masking of IL-13 superkine binding to IL-13Ra2. A. Preface MDNA132 is an IL-13 superkine that selectively binds to the IL-13 decoy receptor (IL-13Ra2) but does not bind to the IL-13 functional receptor (IL-13Ra1).

[0288] MDNA132.15-PSL-MDNA109-Alb is constructed using a protease-sensitive linker (PSL) based on the theoretical premise that MDNA109-Alb is masked by MDNA132.15. Once cleaved, it is hypothesized that MDNA132.15 docks MDNA109-Alb to IL-13 decoy receptor overexpressing tumors.

[0289] B. Method Ni-NTA biosensors (Sartorius catalog No. 18-5101) were hydrated in each experiment in 96-well plates (Greiner part No. 655209) as required by the Octet RH16 Biolayer Inferometer (BLI) instrument manufacturer (Sartorius). Ligand (200 nM His-CD122) was immobilized on the Ni-NTA biosensors, which were then immersed in titration of either MDNA109-Alb (construct No. 7741-044, 82.8 kDa) or MDNA132.15-PSL-MDNA109-Alb (construct No. 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 reaction rate reagent (Sartorius catalog No. 18-1105). The assay was performed on a 384-well tilt-well assay plate (Sartorius catalog No. 18-5080), with a volume of 50 μL per well at 25°C. Assay baseline drift was subtracted using protein-bound biosensors immersed in buffer instead of each analyte. Negative control experiments were included in which intermediate range concentrations of the analyte were tested to determine the "bare" biosensor binding profile without loading the ligand onto the biosensor. No significant nonspecific binding was observed in any experiment. The blocking step was added using Superblock (ThermoFisher catalog No. 37515).

[0290] The typical BLI process conditions used were as follows: • Biosensor check in assay buffer (30 seconds) • Immobilization with His-CD122 (ligand) (12 minutes) • Blocking using Superblock (2 minutes) • Baseline in assay buffer (3 minutes) ·Analyte titration meeting (5 minutes) • Dissociation back to baseline buffer (15 minutes)

[0291] C. Results The binding affinity of the construct MDNA132.15-PSL-MDNA109-albumin to human IL-13Ra2 was tested, with Fc-MDNA132.15(KIH) used as the experimental control. The data are presented as sensorgrams in Figure 3.

[0292] As observed, both Fc-MDNA132.15(KIH) and MDNA132.15-PSL-MDNA109-albumin showed affinity to human IL-13Ra2, but the binding affinity constant K D A unique k that produces an 11.5 times difference on and k off It contains Fc-MDNA132.15(KIH) and MDNA132.15-PSL-MDNA109-albumin, which have K levels of 0.32 nM and 3.7 nM, respectively. D The following was observed. The shift in the binding affinity constant indicates a masking effect of MDNA109-albumin on the binding of MDNA132.15 when fused together via PSL (protease-sensitive linker).

[0293] D. Conclusion The binding affinity data clearly established a masking effect, observed in the 11.5-fold difference in the KD constant between the construct Fc-MDNA132.15 and MDNA132.15-PSL-MDNA109-albumin.

[0294] Example 3b: MMP9-mediated proteolytic activation restores IL-2 activity of MDNA132.15-PSL-MDNA109-albumin. We evaluated the ability of MDNA132.15-PSL-MDNA109-albumin (also known as "MDNA213-PSL-MDNA109-Alb") to cleave and restore the IL-2 activity of MDNA109.

[0295] Pre-activated MMP9 was purchased from Sigma Millipore (SAE0078). The construct was incubated at 37°C for 1 hour in a reaction mixture containing 9.5 μg of protein with or without 2 μg / mL of MMP9 added in 160 μL total volume of cleavage buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (w / v), pH 7.5). After digestion, the reaction mixture was placed on ice and diluted in culture medium for testing with the Jarcut IL2Rβγ reporter cell assay. Portions were also collected, inactivated by adding SDS-PAGE loading buffer, and stored at -80°C until analysis. The samples were analyzed by SDS-PAGE using 4-12% Bis-Tris Bolt polyacrylamide gel (Invitrogen) under reducing conditions. A molecular weight protein marker (Novex® Sharp Pre-stained Protein Standard No. LC5800) was also run on the gel. After electrophoresis, the gel was stained with Simply Blue Safe Stain (Invitrogen).

[0296] MDNA213-PSL-MDNA109-alb T3 / C125 The MMP9 cleavage data for MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-albumin) is shown on the left of Figure 4. T3 / C125 ) was decomposed as a single band at 109 kDa. The presence of MMP9 (lane 3) resulted in a cleavage that produced an additional band at 83 kDa corresponding to MDNA109-alb (unmasked, lane 1). However, MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-alb T3 / C125 There was evidence of incomplete dissection, with nearly 50% remaining undissected.

[0297] Next, the reaction mixture after MMP9 cleavage was tested using the Jarcut IL-2 assay (Figure 4, right). MDNA132.15-PSL-MDNA109-albumin(MDNA213-PSL-MDNA109-alb T3 / C125 The activity of ) was partially restored to the level of MDNA109-albumin. The potency of the MMP9 cleavage product (EC50 = 1144 pM) was higher than that of the mock cleavage reaction (EC50 = 5108 pM), but remained approximately three times lower in potency compared to MDNA109-albumin (EC50 = 337 pM). This is because MDNA132.15-PSL-MDNA109-albumin (MDNA213-PSL-MDNA109-albumin) T3 / C125 This corresponds to an incomplete cut of ).

[0298] Example 3c: Functional characterization of MDNA223-PSL-MDNA132.15 in IL-2 and PD-1 reporter assays A. Preface MDNA132 is an IL-13 superkine that selectively binds to the IL-13 decoy receptor (IL-13Rα2) but not to functional IL-13Rα1. IL-13Rα2 is overexpressed in a wide range of tumors, including glioblastoma, pancreatic cancer, bladder cancer, and colon cancer, but not in normal tissues except the testes, suggesting it is a tumor-associated antigen (TAA). Therefore, MDNA132 may be used to selectively deliver therapeutic payloads to IL-13Rα2-overexpressing tumors without inducing undesirable responses in normal tissues (i.e., with reduced toxicity risk).

[0299] MDNA223 (also known as anti-mPD1-MDNA109FEAA KIH) is a bifunctional superkine immunotherapy (BiSKIT) involving the fusion of anti-PD1 and MDNA109FEAA. The latter is an IL-2 superkine that has enhanced affinity for IL-2Rβ, does not bind to IL-2Rα, and has a reduced ability to stimulate immunosuppressive Tregs, resulting in the selective activation of immune effector cells (CD8+ T and NK cells). The lack of binding to IL-2Rα can also reduce the risk of toxicity. MDNA 223 is designed to enable cis-binding with PD-1 and IL-2R expressed on CD8+ T cells, thereby inducing immune checkpoint blockade (via binding to PD-1) and functional stimulation (via binding to IL-2R) in the same cells. In vivo studies in a mouse syngeneic tumor model have shown that MDNA223 exhibits superior anticancer activity compared to the combination anti-PD1 + MDNA19 (i.e., MDNA109FEAA-Fc), thus identifying BiSKIT as a potential candidate for further development.

[0300] To potentially enhance efficacy and reduce the risk of systemic toxicity (i.e., broaden the therapeutic index) by targeting MDNA 223 to the tumor microenvironment (TME), we produced the mouse (m)MDNA 223*-PSL-MDNA132.15 construct based on the following rationale: The T3A and C125S mutations were added to the MDNA109FEAA portion (i.e., the IL-2 portion of MDNA11). The T3A mutation can be used in other IL-2 agents, including proleukin, to eliminate glycisolation and reduce the homogeneity of the drug product. The C125S mutation is used to eliminate the risk of disulfide bond shuffling at this residue and reduce developmental risk. These mutations are indicated by an asterisk (*) in the name of this construct. • Fusion of MDNA132.15 to MDNA223 to enable targeting of IL-13Rα2-expressing tumors. Direct fusion of MDNA132.15 and the MDNA109FEAA portion of BiSKIT by a protease-sensitive linker (PSL) can potentially reduce the activity of MDNA109FEAA as a result of steric hindrance. The fusion is not expected to affect PD-1 blockade. Cleavage of PSL by the protease within the TME releases MDNA223* from MDNA132.15, thereby mitigating steric hindrance and restoring full activity to MDNA223*. In effect, this allows MDNA223 to target the TME, reducing the risk of peripheral toxicity while attenuating its activity in circulation.

[0301] B. Method Jarcut IL2Rβγ assay Cells were seeded in 96-well plates at a volume of 50 μL, according to the manufacturer's recommendations. The test product was serially diluted and added to each well (25 μL) to achieve the final test concentrations listed in Table 13. Cells were incubated for 6 hours, followed by the addition of the luciferase substrate and incubation for 10 minutes. Luminescence was measured using an iD5 plate reader. [Table 14]

[0302] PD-1 reporter assay In PD-1 / PD-L1 blocking bioassays, PD-L1 aAPC / CHO-K1 cells are used to associate with PD-1 effector cells via the T cell receptor (TCR) or PD-1 receptor. When PD-L1 associates with PD-1, TCR signaling and downstream luciferase reporters 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 luminescence.

[0303] Mouse PD-L1 aAPC / CHO-K1 T&U cells were seeded in 100 μL volumes into 96-well plates 16 hours prior to assay setup, according to the manufacturer's recommendations. The following day, the test sample was serially diluted as 2× solution. The medium was removed from the pre-seeded reporter cells, and 40 μL of 2× test sample and 40 μL of mouse PD-1 T&U effector cells were added to each well to achieve the concentrations listed in Table 14. The cells were incubated for 6 hours, followed by the addition of luciferase substrate and incubation for 15 minutes. Luminescence was measured using an iD5 plate reader. [Table 15]

[0304] C. Results Efficacy in the Jarcut IL2Rβγ reporter assay for measuring IL-2R signaling. As shown in Figure 5, mMDNA223-PSL-MDNA132.15 (SEQ ID NOs. 299-301, masked type) showed reduced efficacy in the Jarcut IL2Rβγ reporter assay compared to MDNA223 (unmasked type). EC of mMDNA223-PSL-MDNA132.15 (1624 pM) 50 This was 12.3 times lower than mRNA223 (131.9 pM). The reduced efficacy of mRNA223-PSL-MDNA132.15 in inducing IL-2R signaling is consistent with the masking effect of MDNA132.15 fused to the MDNA109FEAA portion of the construct.

[0305] Efficacy of PD-1 / PD-L1 reporter assays to evaluate immune checkpoint blockade: In the PD-1 reporter assay (Figure 5), both mMDNA223 (EC50=3.7nM) and mMDNA223-PSL-MDNA132.15 (EC50=3.9nM) showed similar efficacy in blocking the PD-1 / PD-L1 immune checkpoint. The efficacy of both constructs was highly comparable to that of the parental anti-mPD1 antibody (EC50=3.4nM). These data indicate that the fusion of MDNA132.15 with the MDNA109FEAA portion of BiSKIT did not affect the functionality of the anti-PD1 portion.

[0306] D. Conclusion mMDNA223*-PSL-MDNA132.15 (EC50=1624pM) showed reduced efficacy in IL-2R signaling compared to mMDNA223 (EC50=131.9pM). Both mMDNA223*-PSL-MDNA132.15 (EC50=3.9nM) and mMDNA223 (EC50=3.7nM) maintained similar efficacy in PD-1 / PD-L1 immune checkpoint blockade. Therefore, the fusion of MDNA132.15 to mMDNA223 attenuated (i.e., masked) the activity of MDNA109FEAA (IL-2R signaling) but did not affect anti-PD1 (immune checkpoint blockade), which was consistent with the intent of the construct design.

[0307] Example 3d: MMP9-mediated proteolytic activation restores IL-2 activity of MDNA223-PSL-MDNA132.15. "MDNA223-fPSLf-MDNA213" or "MDNA223" restores the IL-2 activity of MDNA109. A3 / S125 -fPSL2f-MDNA213 Lin The proteolytic cleavage ability of (Sequence IDs 299-301) was evaluated.

[0308] MDNA223 A3 / S125 -fPSL2f-MDNA213 LinThe results from MMP9 cleavage are shown on the left of Figure 6. MDNA223 (anti-mPD1-MDNA109FEAAKIH, lane 1) was degraded under reducing conditions into three bands corresponding to heavy chain 1 (HC1, fused to MDNA109FEAA, approximately 65 kDa), heavy chain 2 (HC2, approximately 52 kDa), and light chain (LC, approximately 25 kDa). Lane 2 shows a mock cleavage of MDNA223-fPSLf-MDNA213 KIH, where the size of HC1 (approximately 90 kDa) is 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) yielded results for MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin This resulted in an almost complete cleavage of MDNA223. A3 / S125 -fPSL2f-MDNA213 Lin We demonstrated that MMP9 can efficiently cleave the MDNA213 masking domain, releasing it without affecting the integrity of the remaining construct as determined by size.

[0309] Severed MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin The construct exhibited further IL-2 activity (Figure 6, right).

[0310] MDNA223 removes the MDNA213 domain. A3 / S125 -fPSL2f-MDNA213 Lin After MMP9 cleavage, IL-2 activity is observed in MDNA223 (607 pM) and cleaved MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin (419 pM) Similar EC 50 As is evident, the potency was restored to a level similar to that of MDNA223. Therefore, the MMP9 cleavage released the masking effect of MDNA223-fPSLf-MDNA213.

[0311] Example 3e: Additional functional characterization of MDNA223-PSL-MDNA132.15 in IL-2 and PD-1 reporter assays The functional characterization of additional MDNA223-PSL-MDNA132.15 (also referred to as "MDNA223-PSL-MDNA213") constructs was evaluated using different PSL linkers, as shown in Table 15 below. IL-2-mediated signaling and PD-1 / PD-L1 immune checkpoint inhibition were evaluated for each of these constructs. [Table 16]

[0312] As shown in Figures 7-10, 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.

[0313] Example 3f: MDNA223 in IL-2 and PD-1 receptor assays T3AC125S :Anti-mPD1(H)-fPSL2f-MDNA213 and anti-mPD1(K)-fPSL2f-MDNA213:MDNA223 T3AC125S Functional characteristics evaluation MDNA223 T3AC125S :Anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NOs. 314-316) and anti-mPD1(K)-fPSL2f-MDNA213:MDNA223 T3AC125S The functional characteristics of (Sequence IDs 317-319) were evaluated. MDNA223 T3AC125S In the anti-mPD1(H)-fPSL2f-MDNA213 construct, MDNA213 is bound to the C-terminus of the "hole" Fc domain homodimer by PSL. (Anti-mPD1(K)-fPSL2f-MDNA213: MDNA223) T3AC125S In the constructs, MDNA213 is bound to the C-terminus of the "knob" homodimer. IL-2-mediated signaling and PD-1 / PD-L1 immune checkpoint inhibition were evaluated for each of these constructs.

[0314] As shown in Figures 11 and 12, MDNA223 T3AC125S :Anti-mPD1(H)-fPSL2f-MDNA213 and anti-mPD1(K)-fPSL2f-MDNA213:MDNA223 T3AC125S The IL-2-mediated signaling efficacy of the constructs was reduced 3-fold and 2-fold, respectively, compared to MDNA 223. Furthermore, no changes in PD-1 / PD-L1 blockade were observed for any of these constructs.

[0315] Example 3g: MDNA223 in IL-2 and PD-1 receptor assays T3AC125S -fPSL2f-MDNA213: Functional characterization of anti-mPD1(H)-fPSL2f-MDNA213 MDNA223 T3AC125S -fPSL2f-MDNA213: The functional properties of anti-mPD1(H)-fPSL2f-MDNA213 (SEQ ID NOs. 320-322) were evaluated. MDNA223 T3AC125S -fPSL2f-MDNA213: In the anti-mPD1(H)-fPSL2f-MDNA213 construct, the first MDNA213 is bound to the C-terminus of the "hole" Fc domain homodimer by PSL, and the second MDNA213 is bound to MDNA109FEAA 223T3AC125S It is bound to IL-2 signaling and PD-1 / PD-L1 immune checkpoint inhibition.

[0316] As shown in Figure 13, MDNA223 T3AC125S -fPSL2f-MDNA213: The IL-2 mediated signaling efficacy of the anti-mPD1(H)-fPSL2f-MDNA213 construct was 39-fold lower compared to MDNA223, and there was no change in PD-1 / PDL-1 blockade. Furthermore, as shown in Figure 14, MDNA223 T3AC125S -fPSL2f-MDNA213: The anti-mPD1(H)-fPSL2f-MDNA213 construct is sensitive to MMP9 cleavage in vitro and shows activity in MMP9 cleavage in an IL-2R reporter assay (Figure 15).

[0317] Example 3h: MDNA213-fPSL2f-MDNA11 in IL-2 and PD-1 reporter assays T3AC125S Functional characteristics evaluation MDNA213-fPSL2f-MDNA11 T3AC125S The functional characteristics of (Sequence ID 323) were evaluated. MDNA213-fPSL2f-MDNA11 T3AC125S In the construct, MDNA213 is MDNA111 by CSL. 23T3AC125S Construct MDNA109FEAA 223T3AC125S It is bound to IL-2 signaling and PD-1 / PD-L1 immune checkpoint inhibition.

[0318] As shown in Figure 16, MDNA213-fPSL2f-MDNA11 T3AC125S The IL-2 mediated signaling efficacy of the construct was reduced 21-fold compared to MDNA223. Furthermore, as shown in Figure 17, MDNA213-fPSL2f-MDNA11 T3AC125S The construct is sensitive to MMP9 cleavage in vitro and exhibits activity upon MMP9 cleavage in an IL-2R reporter assay (Figure 18).

[0319] Example 4: Characterization of a tumor-targeting and activatable T-MASK platform that enhances tumor accumulation and tolerability of potent immunomodulators. A. Background The novel T-MASK (Targeted Metallo / Protease Activated Superkine) platform involves fusing a dual tumor targeting / masking domain to a potent immunomodulator via a metallo-protease (MMP)-sensitive linker (PSL) to achieve the following: (1) to reduce / fine-tune the potency of the immunomodulator through steric hindrance to increase systemic tolerability, and (2) to promote retention in the tumor microenvironment (TME) to maximize MMP cleavage at the target site and restore the intended full potency. As proof of concept, the inventors selected the IL-13 superkine (MDNA213), which has high selectivity and affinity for the IL-13 decoy receptor IL-13Rα2, a tumor-associated antigen expressed in many invasive solid tumors, as the tumor targeting / masking domain. MDNA213 is fused to MDNA11 and MDNA223 via PSL, both containing non-alpha, beta-enhanced IL-2 fused to albumin or anti-PD1 antibody, respectively. We present preliminary results regarding the characterization of both T-MASK constructs and demonstrate a provisional fine-tuning of the IL-2R agonism.

[0320] B. Method T-MASK optimization included evaluation of the PSL linker and the direction of the tumor targeting / masking domain. In vitro IL-2 and PD-1 / PDL-1 reporter assays were performed to evaluate IL-2R stimulation and anti-PD1 blockade, respectively. In vitro MMP assays were used to verify the cleavage potential and full potency recovery of the T-MASK construct.

[0321] C. Results The T-MASK construct showed approximately 10-40-fold reduction in potency compared to its non-masking counterpart in IL-2R-induced p-STAT5 reporter cell assays. The degree of fine-tuning can be adjusted by the length and composition of PSL, as well as the orientation of the domain, providing versatility to the T-MASK platform and the potential to associate with the complete repertoire of peripheral circulating immune cells. MDNA223 (anti-PD1-IL-2 SuperkineIn the case of MDNA223-CSL-MDNA213, the fusion of MDNA213 to produce the MDNA223-CSL-MDNA213 T-MASK resulted in reduced IL-2R agonism, but, as expected, did not affect the efficacy of PD1 / PDL-1 blockade. For example, demasking of the MDNA223-PSL-MDNA213 construct (also referred to as the "MDNA113" construct), such as by MMP-mediated cleavage, fully restored its IL-2R signaling activity to the same level as the unmasked MDNA223 construct. Similar data were obtained with the masked form of MDNA11, demonstrating the robustness of the T-MASK platform. In vivo trials are underway to evaluate the effects of the T-MASK construct on peripheral immune cell expansion (systemic response), tumor retention (maximizing activation), and tumor growth inhibition (targeted response).

[0322] Example 5: Recovery of IL-2R signaling activity after proteolytic cleavage in vitro. A. Method MMP9 cleavage: Constructs were incubated in a reaction mixture containing 9.5 μg of construct with or without MMP9 in a total volume of 160 μL of cleavage buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (w / v), pH 7.5). Four discriminant conditions were tested, and any differences in cleavage efficiency were observed. After digestion, the reaction mixture was placed on ice and diluted in culture medium for testing with the Jarcut IL2Rβγ reporter cell assay. Portions were also collected and rapidly frozen in liquid nitrogen before storage at -80°C for subsequent SDS-PAGE analysis.

[0323] Jarcut IL2Rβγ Reporter Assay: Cells were seeded in 50 μL volumes in a 96-well plate according to the manufacturer's recommendations. The test sample was serially diluted and added (25 μL) to each well. Cells were incubated for 6 hours, then luciferase substrate was added and incubated for a further 10 minutes. Luminescence was measured using an iD5 plate reader.

[0324] B. Results The results are shown in Figures 19 and 20. All BiSKITs, except MASK A4, were digested by MMP9 to varying degrees (60-70%) under the conditions described in the methodology. MASK A4 was designed without cleavage sites to demonstrate the selectivity of MMP proteolysis.

[0325] MDNA223 (also known as anti-mPD1-MDNA109FEAAKIH) was degraded under reducing conditions into three bands corresponding to heavy chain 1 (HC1, fused to MDNA109FEAA, approximately 65 kDa), heavy chain 2 (HC2, approximately 52 kDa), and light chain (LC, approximately 25 kDa).

[0326] Mock cuts in constructs MASK A1–MASK A4 decomposed into three bands, and the size of HC1 (approximately 90 kDa) was increased by the addition of fPSLf-MDNA213. Incubation with MMP9 resulted in construct cuts corresponding to MDNA223 as expected, but MASK A4 was excluded because it did not have a PSL, i.e., an MMP9 cleavage site.

[0327] The mock and MMP9 cuts of MASK A5 and MASK A6 decomposed into three bands, with the size of HC1 (approximately 70 kDa) increased by the addition of fPSLf-MDNA213. Incubation with MMP9, as expected, resulted in a cut in the construct where the band corresponds to MDNA223.

[0328] The mock cut of MASK A7 decomposed into three bands, with the sizes of HC1 (approximately 90 kDa) and HC2 (approximately 70 kDa) increased by the addition of fPSLf-MDNA213. Incubation with MMP9 resulted in a construct cut where the band corresponds to MDNA223, as expected. The mock cut of MASK B1 decomposed into a single band of approximately 110 kDa, while MMP9 decomposed into two bands of approximately 84 kDa and 12 kDa, corresponding to MDNA11 and MDNA213, respectively.

[0329] C. IL-2 activity recovery As described above, MASK-IT constructs A7 and B1 were reported to exhibit approximately 40-fold and 20-fold decreases in IL-2 activity, respectively, and sensitivity to MMP9 digestion in the Jarcut IL2Rβγ reporter cell assay. MASK A7 and B1 were further tested for the recovery of IL-2 activity after MMP9 cleavage.

[0330] In the Jarcut IL-IL2Rβγ cell reporter assay (Figures 21A and 21B), both MASK A7 and MASK B1 exhibited reduced IL-2 activity in the absence of MMP9 (mock digestion). However, activity was restored to similar potency to MDNA223 or MDNA11 in the presence of MMP9 under all tested conditions. Therefore, MMP9 cleavage released both constructs from the masking effect of MDNA213, consistent with the design of this MASK-IT construct. EC50 for MDNA223 and MDNA11 was reported at 0.5 nM. MASK A7 and MASK B1 both exhibited reduced potency with an EC50 of 2.9 nM. Potency was restored by MMP9 cleavage of both MASK A7 and MASK B1, with EC50s of 0.5–0.6 nM under different cleavage conditions.

[0331] The IL-2 function of D.MDNA113 is masked in mouse CTLL-2 and human PBMC proliferation assays. CTLL2 cells were seeded in a medium containing T-STIM proliferation supplement in 96-well plates at a rate of 30,000 cells per well. After seeding, cells were treated for 48 hours with increasing concentrations of test or control samples. After treatment, Cell Titer Blue survival reagent (Promega G8080) was added to each well, and the plates were scanned at 560 Ex / 590 Em after the development of a fluorescent survival signal.

[0332] As shown in Figure 22, MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH(MASK A7, SEQ ID NOs: 320, 321, and 322) The rightward shift of the curve by MDNA223 showed its lower potency. The EC 50 of MDNA113 (4.3 nM) was 14-fold lower than that of MDNA223 (0.3 nM). Similarly, MDNA213-fPSL2f-MDNA11 A3 / S125 (SEQ ID NO: 323) showed an activity (14 nM) 113-fold lower than that of MDNA11 (0.12 nM).

[0333] Data from the human PBMC proliferation assay are presented in Figure 23. Three human PBMC donors were stimulated with masked and unmasked MDNA223 for 48 hours and subjected to analysis of BrDU incorporation as a readout for proliferation. Both MDNA223 (unmasked) and MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH (masked) induced PBMC proliferation, and the latter showed reduced potency. EC 50 values were correlated based on logistic curve fitting, but these values had low reliability for comparison because a clear upper threshold could not be achieved within the tested dose range. Comparison of the stimulation indices of MDNA223 and MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH clearly showed attenuation of the latter at all concentrations tested. Similar observations were made using masked and unmasked MDNA11.

[0334] E. Attenuation of Th1 response by MDNA113 in IL-2-mediated pSTAT5 induction in human PBMC Human PBMCs were treated with MDNA223 (unmasked) and MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH (masked) for 15 minutes and harvested for flow cytometry analysis of immune subsets. The results are shown in Figures 24 - 28.

[0335] MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParHNaive CD8 + attenuated IL-2-mediated pSTAT5 induction in human PBMCs in both naive CD8 + T cells and Tregs. EC 50 50 Naive CD8 + revealed an approximate 7-fold reduction in pSTAT5 induction in naive CD8 + T cells, and MDNA223 and MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH showed EC 50 values of 776.9 pM and 5402 pM, respectively (average from 3 independent donors). The decrease in pSTAT5 induction in Tregs was approximately 10-fold, as observed with an average EC Lin / ParH of 8504 pM for MDNA223-fPSL2f-MDNA213 compared to 829.4 pM for MDNA223. 50 The EC + ratio of naive CD8 + T cells to Tregs is considered a useful metric for evaluating the therapeutic potential of constructs, as it reflects the balance between anti-tumor and tumor-promoting immune responses. The equivalent ratio between unmasked and masked MDNA223 demonstrated that the therapeutic potential of MDNA223-fPSL2f-MDNA213 was not compromised, despite masking of IL-2 activity. A3 / S125 -fPSL2f-MDNA213 Lin / ParH values of 50

[0336] Naive CD8 + EC 50 50 A3 / S125 -fPSL2f-MDNA213 Lin / ParH

[0337] Similar observations were made using masked and unmasked MDNA11. EC 50 revealed an approximate 7-fold decrease in pSTAT5 induction in naive CD8 + T cells, and MDNA11 and MDNA213-fPSL2f-MDNA11 50 Naive CD8 + showed EC A3 / S125 values of 512.8 pM and 3674.3 pM, respectively (average from 3 independent donors). The decrease in pSTAT5 induction in Tregs was A3 / S125 50 compared to 611.6 pM for MDNA11, and A3 / S125 ​​​​Average EC at 2825 pM 50 It was observed that the ratio was approximately five times higher.

[0338] F. Receptor binding affinity EMT6 (WT) and EMT6 / IL-13Ra2 cells were thawed and cultured in T75 flasks with 15% FBS in Weymouth MB752 / 1 (with L-Glu supplementation). Cells were subcultured at 80% density using 0.05% trypsin EDTA. Cells were counted using a hemocytometer and seeded into 6-well TC treatment plates at a subculture ratio of 1:10. Cells were removed at 80% density by incubation for 20 minutes with the addition of cell stripper (non-enzymatic desorption solution). Cells were transferred to a 96U bottom and treated with a construct (5x 6-point curve) for 30 minutes. Fc-MDNA213 was used as a control. After incubation, cells were stained with an 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 by GraphPad Prism.

[0339] The data is shown in Figure 29. Fc-MDNA213 (positive control) binds to the cell receptor at the two concentrations tested. MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH In vitro, it binds to EMT6 / IL-13Ra2 cells with lower affinity compared to Fc-MDNA213. Binding was observed to be dose-dependent. The decrease in binding compared to Fc-MDNA213 may be explained by steric hindrance from MDNA223 fusion using a protease-sensitive linker.

[0340] Pharmacodynamic evaluation of G.MDNA113 MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH The pharmacodynamic response to was examined by comparing the reduction in in vivo peripheral lymphocyte expansion in mice administered intraperitoneally (IP) with that of non-masking MDNA223.

[0341] Masked and unmasked MDNA223 A3 / S125 and MDNA11 A3 / S125 Figures 30 and 31 show the peripheral expansion of lymphocytes measured by CBC analysis 72 hours after treatment. (Unmasked and masked MDNA223) A3 / S125 or MDNA11 A3 / S125 Peripheral lymphocyte expansion at equivalent doses showed a significant decrease in each masking type. These data were obtained from MDNA223 A3 / S125 and MDNA11 A3 / S125 However, by masking the circulating domain MDNA213, it was efficiently shielded and successfully reduced peripheral lymphocyte expansion. However, MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH and MDNA213-fPSL2f-MDNA11 A3 / S125 Increasing the dose (from 2 mg / kg to 5, 7.5, and 10 mg / kg) resulted in peripheral lymphocyte expansion similar to that observed in vehicle-treated animals.

[0342] Other blood cells, including neutrophils (PMNs), monocytes, basophils, and eosinophils, exhibited, as expected, masked or unmasked MDNA223. A3 / S125 and MDNA11 A3 / S125 The treatment by [company name] was unaffected.

[0343] In vivo tumor growth inhibition in the H.MC-38 colon cancer model As shown in Figure 32, the masking type of MDNA223 (MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH ) and non-masking types exhibit equivalent tumor growth inhibition, with MDNA113 being cleaved by proteases in the TME, and MDNA223 being cleaved from the masking domain (MDNA213). A3 / S125 -fPSL2f-MDNA213 Lin / ParH This provided strong evidence that it leads to the release of MDNA223, thereby restoring its full potency and therapeutic potential. A3 / S125 -fNCLf-MDNA213Lin / ParH When masked by MDNA213 via a non-cleavable linker, it exhibited moderate tumor growth inhibition, supporting in vivo shielding of MDNA223.

[0344] I. In vivo tumor growth inhibition in an EMT6 / IL13Rα2 breast tumor model. As shown in Figure 33, the data is obtained from equimolar doses of MDNA23 A3 / S125 -fNCLf-MDNA213 Lin / ParH Tumor growth inhibition with treatment using MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH It also showed that it was considerably weaker compared to MDNA223. A3 / S125 -fPSL2f-MDNA213 Lin / ParH The therapeutic efficacy of and MDNA223 was equivalent. In addition, MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH Furthermore, the efficacy of MDNA223 was significantly superior to that of MDNA11 (IL-2 superagonist) alone or its co-administration with anti-mPD1.

[0345] In vivo tumor growth inhibition in the J.MC-38 colon cancer model As shown in Figure 34, MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH Systemic administration of [the drug] exhibited tumor growth inhibition without weight loss in the MC-38 model, compared to MDNA223, in which animals showed a significant decrease in body weight after administration of the first dose. The administration was performed at 40 mm 3 The average tumor size was started with MDNA223 and MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH These were used in equimolar doses of 7.5 mg / kg and 8.8 mg / kg, respectively.

[0346] Conclusion: MDNA223-PSL-MDNA213 is a novel T-MASK construct designed to increase tolerability while leveraging the synergistic effect between PD1 / PDL-1 blockade and IL-2R agonism for immunotherapy. Ongoing trials will investigate alternative tumor targeting / masking domains and immunomodulators, including other cytokines and potent therapeutic agents, to potentially expand the utility of the T-MASK platform.

[0347] Example 6: MTD testing of MDNA113 compared to unmasked MDNA223 A. Terminology The following structures are mentioned in the examples. MDNA113 A3 / S125 or MDNA113A A3 / S125 =MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH MDNA113B=MDNA223 A3 / S125 -fPSL2f-ecCD122:MDNA213 MDNA223 A3 / S125 =MDNA223 MDNA113 NC =MDNA223-2x(GS-MDNA213)[Sequence IDs 325~327] MDNA113B NC =Anti-mPD1(K)-MDNA109FEAA-T3AC125S-fPSL2f-ecCD122:Anti-mPD1(H)-GS-MDNA213[Sequence IDs 353~355]

[0348] B. Purpose The purpose of the study was to identify MDNA113 in C57Bl / 6, one of the commonly used mouse strains. A3 / S125 Evaluate the maximum tolerated dose (MTD) of MDNA113 A3 / S125 MDNA223 A3 / S125 The goal was to investigate whether it would be better tolerated.

[0349] C. Method For this study, eight-week-old female C57Bl / 6 mice were used at the start of administration. The study design is outlined in Table 16 below. [Table 17]

[0350] D. Results The body weights of all animals are shown in Figures 35 and 36 for the once-weekly and twice-weekly administration regimens, respectively. • Animals in Group 1 (MDNA223) A3 / S125 The group (20 mg / kg, once weekly) showed transient weight loss after all three doses administered, and animals in group 5 (MDNA113) A3 / S125 The 23 mg / kg dose (once a week) resulted in mild, transient weight loss after the first two doses. ·Group 2 (MDNA223 A3 / S125 10 mg / kg, once a week) and group 6 (MDNA113 A3 / S125 Both animals (12 mg / kg, once weekly) showed transient weight loss after the first and third doses, which recovered as the study progressed. ·Group 3 (MDNA223 A3 / S125 7 mg / kg, once a week) and group 7 (MDNA113 A3 / S125 Both animals (8 mg / kg, once weekly) showed mild, transient weight loss after the first and third doses, which recovered on subsequent study days. ·Group 4 (MDNA223 A3 / S125 All animals in group 8 (7 mg / kg, twice a week) died after the second dose. On the other hand, group 8 (MDNA113) A3 / S125 Of the three animals treated with 8 mg / kg twice a week, two died after the second dose, but one animal survived all four doses and gained weight for the remainder of the study period. ·Group 9 (MDNA223 A3 / S125 All animals in group 10 (4 mg / kg, twice a week) died after the second dose, but group 10 (MDNA223) A3 / S125 All animals receiving 4 mg / kg twice a week survived until the end of the study. Animals in group 10 exhibited mild, transient weight loss after the second dose, which then recovered and remained consistent throughout the study period.

[0351] The survival curve is shown in Figure 37. ·Group 1~3 (MDNA223 A3 / S125 (Once a week group) and groups 5-7 ((MDNA113 A3 / S125 All animals in the group that received treatment once a week survived until the end of the experiment. ·Group 4 (MDNA223 A3 / S125 7 mg / kg, twice a week) All animals and group 8 (MDNA113 A3 / S125 Two-thirds of the animals receiving 8 mg / kg (twice a week) died after the second dose. The remaining animals in group 8 survived until the end of the study. Therefore, a survival rate of 33% was observed in group 8. ·Group 9 (MDNA223 A3 / S125 All animals in group 10 (4 mg / kg, twice weekly) died after the second dose, but group 10 (MDNA113) A3 / S125 All animals receiving 4.7 mg / kg twice a week survived until the end of the study (100% survival rate).

[0352] E. Conclusion MDNA113 A3 / S125 and non-masking type MDNA223 A3 / S125 Both were well tolerated at all doses when administered via IP on a once-weekly dosing schedule. Therefore, MDNA223 A3 / S125 or MDNA113 A3 / S125 None of these reached the MTD.

[0353] Non-masking type MDNA223 A3 / S125 When administered via IP at either the tested doses of 7 mg / kg or 4 mg / kg on a twice-weekly schedule, it was not well tolerated. All animals died within a week of the administration cycle after the second dose. Meanwhile, MDNA113 A3 / S125 It showed better tolerability, and with twice-weekly administration, one of three animals survived at a molar equivalent of 8 mg / kg, while all animals survived at a molar equivalent of 4.7 mg / kg. Therefore, MDNA113 A3 / S125 The MTD was established with a regimen of 4.7 mg / kg twice weekly. Data from MDNA223 A3 / S125 This suggests that the MTD (Mean Time Tolerance) is less than 4 mg / kg with a twice-weekly administration regimen.

[0354] Example 7: In vitro evaluation of MASK-IT to establish reduced IL-2 activity and retained PD-1 blockade. A.Purpose To enhance the masking effect against MDNA223 or other IL-2 BiSKITs in the periphery, several novel constructs were designed using additional masking domains, namely the extracellular domains of human CD122 or CD25. These constructs were evaluated compared to non-masking MDNA223 for their retention of reduced IL-2 activity and PD-1 blockade.

[0355] B. Method The constructs were tested using the Jarcat IL2Rβγ reporter assay (cells lacking the CD25 receptor) and the HEK Blue IL-2 assay (cells expressing the trimer receptors CD25 / CD122 / CD132). The constructs were tested using a PD-1 / PDL-1 blockade reporter assay to verify their anti-PD1 efficacy. The construct was tested for the recovery of IL-2R agonism after MMP9 cleavage.

[0356] C. Results Unmasked MDNA223 A3 / S125 and MDNA113 A3 / S125 These were used as benchmark control and experimental control, respectively. Anti-mPD1-MDNA109 A3 / S125 -fPSL2f-ecCD25:MDNA213 and anti-mPD1-H9T A3 / S125 -fPSL2f-ecCD25:MDNA213 could not be purified as a monomer fraction due to oligomer formation during the purification process, and both P1 and P2 fractions of the construct were tested in the assay. Anti-mPD1-MDNA109 A3 / S125 -fPSL2f-ecCD25: The P1 fraction of MDNA213 showed complete loss of IL-2 activity, while the P2 fraction showed unmasked MDNA223 A3 / S125 In comparison, it showed an approximately 41-fold decrease in IL-2 signaling. Furthermore, it was found to be related to anti-mPD1-MDNA109 A3 / S125-fPSL2f-ecCD25: In contrast to the extracellular domains of MDNA213 and CD25 in MDNA213, MDNA113 consists only of the masking domain MDNA213. A3 / S125 It showed approximately five times better masking efficiency than the previous method (Figure 38A).

[0357] MDNA223 A3 / S125 -fPSL2f-ecCD122:MDNA213 is unmasked MDNA223 A3 / S125 In comparison, there was a 453-fold attenuation in IL-2 signaling, and MDNA113 A3 / S125 This showed approximately 47 times better masking compared to the previous method (Figure 38B).

[0358] EC 50 This is summarized in Table 17. [Table 18]

[0359] Constructs containing IL-2 mutein with retained CD25 binding were tested for the presence of the trimer receptor complex (CD25 / CD122 / CD132) using the HEK Blue IL-2 reporter assay.

[0360] Fc-MDNA109 T3 / C125 and anti-hPD1-MDNA109 A3 / S125 Using [another substance] as an experimental control, it showed similar efficacy. Rh IL-2, used as a benchmark control, was approximately 3.5 times weaker in potency, consistent with the fact that MDNA109 is a beta-enhanced IL-2 mutain. MASK-IT anti-mPD1MDNA109 A3 / S125 The two fractions of -fPSL2f-ecCD25:MDNA213, P1 and P2, exhibited attenuated efficacy with approximately 11-fold and 8-fold decreases, respectively (Figure 39A).

[0361] To compare the efficacy of MASK-IT, anti-mPD1-H9T A3 / S125 -fPSL2f-ecCD25:MDNA213, H9T T3 / C125- Albumin, and anti-hPD1-H9T A3 / S125 to, anti-mPD1-H9T A3 / S125 It was used as an unavailable experimental control. Compared to rhIL-2, which is used as a benchmark, H9T T3 / C125 - Albumin and anti-hPD1-H9T A3 / S125 It showed lower efficacy. However, anti-mPD1-H9T A3 / S125 The IL-2 signaling efficacy of -fPSL2f-ecCD25:MDNA213 was masked approximately 124-fold and 43-fold for the P1 and P2 fractions, respectively (Figure 39B).

[0362] Unmasked MDNA223 A3 / S125 and anti-hPD1-H9TFEAA A3 / S125 Using MDNA223 as an experimental control, A3 / S125 -fPSL2f-ecCD122:MDNA213 and anti-hPD1-H9TFEAA A3 / S125 The reduction in IL-2 efficacy of -fPSL2f-ecCD122:MDNA213 was compared. Approximately 65-fold and 688-fold attenuations were observed, respectively. Notably, the anti-hPD1-H9TFEAA A3 / S125 In the assay, MDNA223 A3 / S125 It showed approximately seven times lower potency, suggesting that H9TFEAA is a weaker IL-2 agonist than MDNA109FEAA (Figure 39C).

[0363] Table 18 summarizes the EC50 values ​​for all items. [Table 19]

[0364] MASK-IT, which was tested for PD-1 / PDL-1 blockade, is non-masking MDNA223. A3 / S125 It also showed similar efficacy to the benchmark control anti-mPD1 antibody (Figure 40).

[0365] MASK-IT was digested using MMP9 and analyzed by SDS-PAGE. Under reducing conditions, MDNA223 (anti-mPD1-MDNA109FEAA KIH) was degraded into three bands corresponding to heavy chain 1 (HC1, fused to MDNA109FEAA, approximately 65 kDa), heavy chain 2 (HC2, approximately 52 kDa), and light chain (LC, approximately 25 kDa).

[0366] All constructs tested (i.e., MASK-IT of MDNA223, anti-mPD1-H9TFEAA, anti-mPD1-MDNA109 fractions 1 and 2) showed nearly complete cleavage in the MMP9 reaction. In the absence of rMMP9, the constructs were degraded as three bands whose size was increased by the fusion of HC1 (approximately 90 kDa) and HC2 (approximately 70 kDa) with fPSL2f-ecCD122:MDNA213. Cleavage by MMP9 released the fPSL2f-ecCD122:MDNA213 masking domain, as indicated by the presence of a protein band of similar size to MDNA223 (Figure 41).

[0367] Recovery of IL-2 activity after MMP9 cleavage MASK-IT was tested for IL-2 activity after MMP9 cleavage using either the Jarcut IL2Rβγ or HEK Blue IL-2 reporter assay. These tests were performed against anti-mPD1-H9T A3 / S125 Except for -fPSL2f-ecCD25:MDNA213 (P2 fraction), IL-2 activity was restored to the same potency as the unmasked forms after cleavage by MMP9 (Figures 42A and 42B, Table 19). [Table 20]

[0368] D. Conclusion The designed constructs, when tested with the Jarcut IL2Rβγ reporter assay or the HEK Blue IL-2 assay, showed no masking of the unmasked control and MDNA113 A3 / S125Compared to (using only MDNA213, the IL-13 superkine, as the masking domain), reduced IL-2 activity was observed. The data, along with the masking domains of each construct (underlined for inference), are summarized below. MDNA223 A3 / S125 -fPSL2f- ecCD122:MDNA213 In the Jarcut IL2Rβγ assay, non-masking MDNA223 A3 / S125 Compared to that, it showed a 453-fold decrease in IL-2 activity, and MDNA113 A3 / S125 (MDNA223 A3 / S125 -fPSL2f- MDNA213 Lin / ParH It showed approximately 47 times better masking than the previous method. ·Anti-mPD1-MDNA109 A3 / S125 -fPSL2f- ecCD25:MDNA213 The P1 fraction showed suppression, but the P2 fraction showed non-masking MDNA223 A3 / S125 In comparison, IL-2 signaling showed an approximately 41-fold decrease. Masking was observed in the Jarcut IL2Rβγ assay, specifically in MDNA113 A3 / S125 (MDNA223 A3 / S125 -fPSL2f- MDNA213 Lin / ParH This represents an improvement of approximately five times compared to the previous version. ·Anti-mPD1-H9T A3 / S125 -fPSL2f- ecCD25:MDNA213 The IL-2 signaling efficacy was observed in the HEK Blue IL-2 assay, which was unmasked against anti-hPD1-H9T A3 / S125 In comparison, the P1 and P2 fractions were attenuated by approximately 124 times and 43 times, respectively. • Anti-hPD1-H9TFEAA A3 / S125 -fPSL2f- ecCD122:MDNA213 A 688-fold decrease in IL-2 activity was observed in the HEK Blue IL-2 assay, indicating that anti-hPD1-H9TFEAA A3 / S125 This was observed in comparison to [another observation].

[0369] MASK-IT was observed to be sensitive to proteolytic cleavage in vitro and restored attenuated IL-2 function after cleavage by MMP9.

[0370] Example 8: In vivo imaging of labeled MDNA113 and unmasked MDNA223 to verify accumulation in IL-13Rα2-expressing tumors. A.Purpose Using an in vivo imaging system (IVIS), labeled proteins or other analytes were visualized in mice to understand their distribution and accumulation. Multiple images were acquired over time to provide a long-term view of the dynamics of drug biodistribution in living animals. This study investigated the potential for sustained and selective tumor accumulation of MDNA113 in mice carrying IL-13Rα2-expressing tumors that yield a superior antitumor response. The study also validates the role of MDNA213 in IL-13Rα2 targeting within MDNA113.

[0371] B. Method Labeling of masked and unmasked MDNA223: MDNA113 and MDNA223 were labeled using VivoTag800 IN VIVO NIR fluorescent dye (Perkin Elmer, catalog No. NEV11107) according to the manufacturer's protocol.

[0372] Biolayer Interference Binding Analysis: To confirm that the labeling does not alter receptor binding properties, MDNA113 and MDNA223 were tested for binding to mouse CD122, one of the congeneral receptors, using biolayer interference (BLI) / Octet.

[0373] The BLI process conditions used were as follows: biosensor check in PBS dynamic buffer (0.5 min), ligand protein immobilization (10 min), blocking with Superblock (2 min), baseline in PBS dynamic buffer (2 min), analyte titration association (5 min), and analyte dissociation back into baseline buffer (15 min).

[0374] In vivo imaging: Several human cell lines, including melanoma cell lines, are known to express IL-13Rα2. Using an A375 (human melanoma) xenograft model expressing IL-13Rα2, we evaluated the targeting and accumulation of MDNA113 and MDNA223 in athymic nude mice. A549, a human lung tumor with low / absent IL-13Rα2 expression, was used as a control in the contralateral flank of the mice.

[0375] Animal information (supplier, species, strain, sex, age, or weight, and number): • Supplier: Charles River Laboratories • Species: Mouse • Lineage: Athymoid nude (Lineage code 490) • Sex: Female • Age / Weight: 5-6 weeks • Number: 12 animals (9 + 3 additional) were purpose-bought, free of specific pathogens, and had no prior experimental experience at the start of the study. Age / weight refers to the age / weight at the time of delivery from the supplier.

[0376] The test design is shown in Table 20. [Table 21]

[0377] Epifluorescence IVIS imaging was performed using dorsal-positioned mice at pre-dose (baseline) and 4, 24, 72, 120, and 168 hours after injection of the labeled construct, with excitation at 785 nm and emission at 810 nm. Following the last imaging point (168 hours post-injection), the mice were euthanized and the tumors were collected for ex vivo imaging.

[0378] C. Results VivoTag 800 labeling: A total of 1.5–1.8 mg of each construct was subjected to the labeling reaction. Table 21 below summarizes the labeling results. Calculations do not consider the molecular weight with the VivoTag 800 fluorochrome added. Proteins greater than 1: VivoTag 800 ratio indicates successful labeling. [Table 22]

[0379] Prior to in vivo imaging studies, the labeled construct was tested for binding to the mouse CD122 receptor using Octet.

[0380] Receptor binding analysis using BLI / Octet: Binding affinity analysis using BLI / Octet confirmed that the receptor binding profiles of MDNA113 and MDNA223 were not affected by labeling with vivot800. Both unlabeled and labeled constructs showed similar binding characteristics to mouse CD122, indicating no change in binding affinity after labeling with vivotag800.

[0381] The binding affinity constant (K) calculated from the steady-state reaction rate. D The K2 levels for unlabeled and labeled MDNA223 were 320 nM and 240 nM, respectively. Similarly, for MDNA113, the K2 levels for unlabeled and labeled forms were 1.8 uM and 4.0 uM, respectively. D This was the result (Figure 43).

[0382] Notably, the K of MDNA113 D This was approximately 10 times higher than that of MDNA223, consistent with the decreased binding affinity of MDNA113 to mouse CD122 due to the fusion of the sterically hindrance-causing masking / targeting domain MDNA213.

[0383] In vivo imaging: A375 (IL-13Rα2 positive, right flank) and A549 (IL-13Rα2 negative, left flank) cells were engrafted in the flanks of athymic nude mice and allowed to grow into established tumors. Mice with established tumors were intravenously injected with labeled MDNA223 or MDNA113, and IVIS images were acquired pre-administration, 4, 24, 72, 120, and 168 hours post-treatment, and ex vivo after euthanasia. The edited images are shown in Figure 44.

[0384] Accumulation of labeled MDNA113 was observed in some mice in the A375 tumor (right flank) compared to the A549 tumor (left flank). At an earlier time point (i.e., up to 24 hours), MDNA113 was distributed throughout the body of the mice. Since MDNA113 disappeared from systemic distribution at a later time point (72–168 hours), accumulation in the IL-13Rα2-expressing tumor (A375) was evident in at least one of the three mice. The localization of MDNA113 to the A375 tumor is consistent with the hypothesis of MDNA213-mediated targeting of MDNA113 to IL-13Rα2-expressing tumors. Following in vivo imaging at 168 hours, the mice were euthanized and their intact tumors were excised for ex vivo imaging. Ex vivo imaging confirmed the selective accumulation of MDNA113 in IL-13Rα2-expressing tumors (A375).

[0385] Accumulation of labeled MDNA223 (experimental control) was observed to occur transiently in vivo, as is evident from the 72-hour data images. Additionally, post-treatment ex vivo images showed low accumulation in both A549 (IL-13Rα2 negative) and A375 cell (IL-13Rα2 positive) tumors, suggesting a lack of specific targeting in the absence of the MDNA213 domain.

[0386] D. Conclusion The BLI / Octet trial showed that labeling of MDNA223 and MDNA113 did not alter their binding profiles.

[0387] In vivo and ex vivo imaging demonstrated the durability and targeted accumulation of MDNA113 in IL-13Rα2-expressing A375 tumors in mice for approximately 10 days. While in vivo accumulation of MDNA223 was observed to be transient, ex vivo imaging showed untargeted accumulation in both A549 (IL-13Rα2-negative) and A375 cells (IL-13Rα2-positive).

[0388] Example 9: MC-38 tumor growth inhibition (TGI) study to evaluate the efficacy of MDNA113 and MDNA223-fPSL2f-ecCD122:MDNA213 (MDNA113B) A.Purpose The purpose of the study was to investigate the therapeutic efficacy of MDNA113 and MDNA223-fPSL2f-ecCD122:MDNA213 (MDNA113B).

[0389] B. Method The test design is shown in Table 22. [Table 23]

[0390] C. Results When treated once a week, MDNA113 exhibited superior tumor growth inhibition compared to non-masking MDNA223. This may be due to conditional activation of MDNA113 at tumor sites / TMEs during protease-mediated proteolytic cleavage. Compared to complete regression in 1 out of 7 mice treated with MDNA223, 4 out of 7 mice treated with MDNA113 showed complete tumor regression (Figure 45).

[0391] However, MDNA223-fPSL2f-ecCD122:MDNA213 was not as potent as non-masking MDNA223, which may be due to incomplete proteolytic activation at the tumor site. When treated with MDNA223-fPSL2f-ecCD122:MDNA213, 2 out of 7 mice showed complete tumor regression.

[0392] In a twice-weekly administration schedule, MDNA113 showed significant inhibition of tumor growth compared to the non-cleavable form of MDNA113. Two out of seven mice treated with MDNA113 showed complete tumor regression (Figure 46).

[0393] D. Conclusion Both MDNA113 and MDNA223-fPSL2f-ecCD122:MDNA213 showed significant tumor growth inhibition comparable to that of non-masking MDNA223.

[0394] Example 10: Pharmacodynamic (PD) study to evaluate peripheral lymphocyte expansion in mice A.Purpose The objective of the study was to investigate peripheral lymphocyte expansion in response to non-masking MDNA223 and masking MDNA223, namely MDNA113A and MDNA113B.

[0395] B. Method Balb / c was used for the study. Animals were administered according to Table 23. [Table 24]

[0396] C. Results MASK-IT constructs MDNA113A and MDNA113B showed attenuated peripheral lymphocyte expansion compared to non-masking MDNA223. However, the masking response could be rescued by increasing the construct dose. MDNA113A showed a similar response to non-masking MDNA223 at a 5-fold higher dose, i.e., 11.7 mg / kg, while MDNA113B showed a 10-fold higher dose, 24 mg / kg. However, even at 24 mg / kg, lymphocyte expansion was significantly lower than with non-masking MDNA223 (p=0.02) (Figure 47).

[0397] D. Conclusion MASK-IT exhibited attenuated lymphocyte expansion in the periphery compared to non-masking MDNA223, providing strong evidence for reduced systemic toxicity. The data were consistent with the intent of the construct design.

[0398] Example 11: MTD test to determine the maximum tolerated dose of MDNA113A and MDNA113B A.Purpose The trials are being conducted to investigate the tolerability of MDNA113B compared to MDNA113A and the non-masking MDNA223.

[0399] B. Method C57Bl / 6 mice were used for the test. The test design is shown in Table 24. [Table 25]

[0400] C. Results All doses were well tolerated by the mice. Body weight measurements are shown in Figure 48. Rapid weight loss was observed in the MDNA223 (30 mg / kg, once weekly) and MDNA113A (8 mg / kg, twice weekly) groups. However, the mice recovered the day after the test. No weight loss was observed in any of the MDNA113B groups.

[0401] D. Conclusion All doses tested were well tolerated, and no deaths were observed in any group. Therefore, the maximum dose (MTD) was not reached.

[0402] Example 12: IL-2 activity before and after MMP-9 digestion (non-cleavable MASK-IT) A.Purpose The purpose of the study was to investigate whether IL-2 activity was restored, if any, to the proteolytic activation of MASK-IT in both cleavable and non-cleavable forms.

[0403] B. Method The constructs were subjected to either a mock (absence of MMP9) or digestion with 5 μg / mL of MMP9 at 37°C for 1 hour, and then processed for evaluation of IL-2 activity in the Jarcut IL-2 reporter assay.

[0404] C. Results The data presented in Figure 49 show that MDNA113, a cleavable form of MASK-IT, recovers IL-2 activity after proteolytic activation in the presence of MMP9. Both the non-cleavable forms, MDNA113A and MDNA113B, do not show recovery of IL-2 activity, as expected. The non-cleavable forms lack PSL for cleavage and therefore lack demasking of MDNA223.

[0405] EC 50 This is summarized in Table 25, and evidence of the presence or absence of dissection is shown in Figure 50. [Table 26]

[0406] D. Conclusion The non-cleavable form was not activated after proteolytic cleavage by MMP9.

[0407] Example 13: Cell receptor binding assay to verify the affinity of MDNA113 to IL-13Rα2 expressing cells in vitro. A.Purpose The study was conducted using A375 human melanoma cells (positive for IL-13Rα2) and A549 human lung epithelial cells (negative for IL-13Rα2), and the presence of MDNA213 (IL-13 superkine) was evaluated by assessing the binding of MDNA113 to IL-13Rα2-expressing cells.

[0408] B. Method Structures: 1. Control (tested using only A549 cells): Fc-MDNA213 1800nM and 600nM only 2. Test sample (tested with A375 cells): (i) MDNA223 (ii) Fc-MDNA213 (iii) MDNA113A (iv) MDNA113B All titer measurements were performed using a 3x, 8-point curve starting from 1800 nM. Wash the cells, which are 70-80% densely packed, with 2 mL of DPBS. Add 1 mL of Corning Cell Stripper. • Incubate the cells at 37°C for 20 minutes to detach them. Gently reverse pipette the cells and wash them thoroughly with 2 mL of culture medium. Transfer the cells to a 50 mL tube and centrifuge at 400 G for 10 minutes. • Resuspend the cells in 4 mL of culture medium and take small portions for counting with a hemocytometer. Seed cells at a rate of 100K (per 100 μL) in a 96-well u-bottom plate. Centrifuge the cells at 400G for 3 minutes and aspirate the supernatant. • Resuspend the cells in 100 μL of the construct (culture medium). Incubate the cells at 4°C for 30 minutes.

[0409] staining Centrifuge the cells at 400G for 3 minutes and aspirate the supernatant. Resuspend the cells in 200 μL of PBS. Centrifuge the cells at 400G for 3 minutes and aspirate the supernatant. Wash the cells at least twice with 200 μL of PBS. • Resuspend the cells in 100 μL of anti-FcPE (Abcam No. AB98596) staining solution. Incubate the cells at 4°C for 30 minutes. Add 100 μL of MACS buffer to each well. • Centrifuge the plate at approximately 400 x g for 3 minutes. Wash the cells at least twice with 200 μL of MACS buffer. • Resuspend the cells in 0.5% PFA in 200 μL of MACS / PBS buffer. Unstained cells are used for the negative population. • Cells are obtained using a flow cytometer.

[0410] Flow analysis • For each sample, prepare an anti-FcPE histogram and observe the average fluorescence intensity (MFI).

[0411] For each sample, the relative average fluorescence intensity is calculated in comparison to the untreated (no construct) control.

[0412] C. Results The data from the tests are shown in Figure 51. Fc-MDNA213 strongly binds to IL-13Rα2 expressed in A375 cells, but no binding is observed in A549 cells. MASK-IT MDNA113A and MDNA113B, tested in A375 cells, showed a similar binding pattern to MDNA213, although slightly reduced. MDNA223 showed nonspecific binding at the highest test dose.

[0413] D. Conclusion MDNA113A and MDNA113B showed dose-dependent binding to IL-13Rα2 expressed in A375 cells, and their binding was moderately reduced compared to Fc-MDNA213.

[0414] Example 14: MC-38 tumor growth inhibition test to establish the efficacy of MDNA113A and MDNA113B A.Purpose The objective of the trial was to establish the in vivo efficacy of MDNA113A and MDNA113B.

[0415] B. Method 88 C57Bl / 6 mice were used for the experiment. The experiment design is shown in Table 26. [Table 27]

[0416] C. Results The data in Figure 52 demonstrate that MDNA223 treatment is superior to monotherapy with MDNA11 or anti-mPD1, and to concurrent administration of these. The data are consistent with our previous observations in the CT26 colon model and the B16F10 melanoma model.

[0417] Figure 53 shows data for the groups treated with cleavable and non-cleavable MDNA113A. At a dose of 18 mg / kg, MDNA113A NC It showed superior effectiveness compared to [another method].

[0418] Figure 54 shows data for the groups treated with cleavable and non-cleavable MDNA113B. At a dose of 37 mg / kg, MDNA113B was treated with MDNA113A NC It showed superior efficacy compared to [another method]. This provides strong evidence that MDNA113B is activated at tumor sites that release MDNA223 from masking by ecCD122 and MDNA213.

[0419] D. Conclusion Both MDNA113A and MDNA113B are activated by proteolysis at tumor sites and exhibit similar efficacy to non-masking MDNA223.

[0420] Example 15: Adjuvant effect of MDNA223 and neoadjuvant effect of MDNA113 and unmasked MDNA223 in a 4T1.2 orthotopic breast tumor model. A.Purpose The objective of the study was to investigate the neoadjuvant or adjuvant effects of MDNA223 and MDNA113 in a 4T1.2 orthotopic breast tumor model.

[0421] B. Method Balb / c mice were used for the experiment. The experiment design is shown in Table 27. [Table 28]

[0422] C. Results As shown in Figure 55, both MDNA223 and MDNA113 provided a significant survival benefit when used as neoadjuvants. Deaths observed in the untreated control and anti-mPD1 neoadjuvant-treated groups were due to distant metastases that were not present in MDNA113 or MDNA223 neoadjuvant-treated groups.

[0423] However, the survival benefit of adjuvant treatment was not as pronounced in MDNA223, even though it was superior to anti-mPD1 monotherapy (Figure 56).

[0424] MDNA113 cannot be used as an adjuvant because it requires proteolytic activation at the tumor site.

[0425] D. Conclusion MDNA223 exhibits a significant neoadjuvant effect compared to its use as an adjuvant treatment, and provides a significant survival benefit when used as a neoadjuvant in a 4T1.2 orthotopic breast tumor model.

[0426] MDNA113, when used as a neoadjuvant in the 4T1.2 orthotopic mammary tumor model, also extends survival and avoids distant metastasis observed in untreated controls and anti-mPD1 treated mice.

[0427] Example 16: Evaluation of binding affinity of MDNA113 to CD122(IL-2Rβ) A. Terminology The following structures are mentioned in the examples. MDNA113 A3 / S125 or MDNA113A A3 / S125 =MDNA223 A3 / S125 -fPSL2f-MDNA213 Lin / ParH [Sequence IDs 320-322] MDNA113B=MDNA223 A3 / S125 -fPSL2f-ecCD122:MDNA213[Sequence IDs 338~340] MDNA223 A3 / S125=MDNA223[Sequence IDs 107~109]

[0428] B. Purpose The purpose of the study was to evaluate the binding affinity of masked MDNA113 to CD122 (IL-2Rβ) compared to non-masked MDNA223.

[0429] C. Method Binding affinity was evaluated using BLI Octet. The typical BLI process conditions used were as follows: • Perform a biosensor check in assay buffer (30 seconds). • Immobilization with His-CD25 / His-CD122 (ligand) (12 minutes). Blocking with Superblock (2 minutes). • Baseline (3 minutes) in assay buffer. • Titration assembly of test construct variants (analytes) (5 minutes). • Dissociation back to baseline buffer (15 minutes)

[0430] D. Results Both MD113A and MDNA113B exhibited reduced binding to CD122 compared to unmasked MDNA223, despite varying affinity levels. MDNA113A showed approximately 1.6-fold reduced affinity, while MDNA113B showed 163-fold reduced affinity compared to unmasked MDNA223 (Figure 57).

[0431] E. Conclusion MDNA113B exhibits significantly reduced binding affinity compared to non-masking MDNA223.

[0432] Example 17: In vitro evaluation of MASK-IT (containing MDNA223T3 / C125) to establish reduced IL-2 activity A. Terminology The following structures are mentioned in the examples. MDNA223 T3 / C125 [Sequence IDs 74-76] MDNA113B T3 / C125 :[Sequence IDs 365-367]

[0433] B. Purpose The objective of the study was to verify the reduced IL-2 activity of MDNA113BT3 / C125, which is reversible by proteolytic cleavage, compared to that of unmasked MDNA223T3 / C125.

[0434] C. Method The constructs were tested using the Jarcat IL2Rβγ reporter assay (cells lacking the CD25 receptor) in the presence and absence of MMP9.

[0435] D. Results MDNA113BT3 / C125 showed approximately 630-fold attenuation of IL-2 activity compared to unmasked MDNA223T3 / C125. IL-2 activity was restored by in vitro MMP9 cleavage (Figure 58).

[0436] E. Conclusion MDNA113BT3 / C125 was activated by proteolytic cleavage.

[0437] Example 18: Neoadjuvant administration of MDNA11 A. Background MDNA11 is an albumin-fused "beta-enhanced, alpha-non-enhanced" IL-2 superkine that preferentially expands and activates immune effector cells over immunosuppressive Tregs. MDNA11 effectively inhibited subcutaneously transplanted syngeneic tumors with complete tumor regression and a robust memory response. Mice treated with MDNA11 showed increased tumor infiltration of CD8+ T and NK cells, as well as increased circulating memory and antigen-specific CD8+ T cells. Considering these results, we investigated whether preconditioning of immune cells against cancer cells with MDNA11 before tumor resection could prevent metastasis and extend survival. This study compared the efficacy of MDNA11 in neoadjuvant and adjuvant settings in an aggressive orthotopic breast cancer model prone to distant metastasis after tumor removal.

[0438] B. Purpose To compare the effects of neoadjuvant and adjuvant treatment with MDNA11 in a syngeneic 4T1.2 orthotopic breast cancer model involving surgery for complete tumor resection, and to evaluate the effects of neoadjuvant and adjuvant MDNA11 treatment in providing long-term protection from subsequent tumor burden.

[0439] C. Method Female Balb / c mice were transplanted with 4T1.2 tumor cells into the mammary fat tissue, and the tumors were allowed to grow to a palpable size. Mice were either untreated (control), treated only by surgery to remove the tumor, or given a single dose of MDNA11 intraperitoneally either as a neoadjuvant four days before surgery or as an adjuvant two days after surgery. Surviving mice from all treatment cohorts were reloaded with 4T1.2 cells in the right hind limb on day 65 of the study and in the left hind limb on day 98. Survival rates, tumor volume, and signs of metastasis at necropsy were measured throughout the reloading phase of the study.

[0440] The collected data included survival rates, autopsies at endpoints to record signs of metastasis, and multiplex immunofluorescence (MIF) analysis for immune cell infiltration in resected tumors.

[0441] D. Results As shown in Table 28 and Figures 59-62, all control mice that underwent tumor resection but received no treatment died by day 54 of the study and had macroscopic metastases in multiple organs. In the adjuvant group, where mice 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 had distant metastases. In contrast, of the 8 mice administered a single dose of MDNA11 before tumor resection, 7 (87.5%) survived to day 134 of the study without observable clinical symptoms. The one mouse that died did not have metastases. The remaining mice in the neoadjuvant (n=7) and adjuvant (n=3) groups were reloaded on day 65 by subcutaneous transplantation of 4T1.2 cells into their flanks. A second reload was performed on day 98 of the study. The mice, without any additional treatment, showed no tumor growth or metastasis upon reloading, while control naive mice similarly transplanted with 4T1.2 cancer cells exhibited robust tumor growth at the transplantation site. [Table 29]

[0442] E. Conclusion A single dose of MDNA11 in a neoadjuvant setting provided immediate and long-term protection from metastatic disease in a 4T1.2 orthotopic breast cancer model. ***

[0443] The above embodiments are provided to give those skilled in the art a complete disclosure and explanation of how embodiments of the compositions, systems, and methods of the present invention can be prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention. Modifications of the above forms for carrying out the invention that would be obvious to those skilled in the art are intended to fall within the scope of the following claims. All patents and publications referenced herein represent the state of the art to which the invention relates. All references cited herein are incorporated by reference to the same extent as each reference is incorporated by reference in whole.

[0444] All headings and section titles are used for clarity and reference purposes only and should not be considered limiting. For example, those skilled in the art will recognize the usefulness of combining various aspects from different headings and sections as needed, in accordance with the spirit and scope of the invention as described herein.

[0445] All references cited herein are incorporated herein by reference in their entirety for the same degree as each individual publication or patent or patent application is specifically and individually indicated by reference as if it were incorporated in its entirety for the sole purpose.

[0446] As will be apparent to those skilled in the art, many modifications and variations of this application may be made without departing from the spirit and scope of this application. The specific embodiments and examples described herein are provided only as examples, and this application should be limited only by the terminology of the appended claims, in addition to the entire scope of the equivalent for which the claims are granted.

Claims

1. It is an IL-2 cytokine fusion protein, a) IL-2 portion, which includes IL-2 or IL-2 mutaine, optionally an IL-2 mutaine fusion, b) Protease-sensitive linker (PSL), c) Including the IL-2 masking portion, The PSL is an IL-2 cytokine fusion protein in which the IL-2 masking portion is bound to the IL-2 portion.

2. The IL-2 cytokine fusion protein according to claim 1, wherein the masking portion comprises IL-13, IL-13 mutain, IL-13Ra2-binding mutain, IL-13Ra2 antibody, or an antigen-binding fragment thereof, and the IL-2 masking portion can bind to IL-13Ra2 but does not bind to IL-13Ra1.

3. The IL-2 cytokine fusion protein according to claim 1 or 2, wherein the masking portion comprises IL-13 mutain having one amino acid sequence from SEQ ID NOs. 200 to 241.

4. The masking portion, compared to wild-type human IL-13 (SEQ ID NO: 200), has the following amino acid substitutions: 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) An IL-2 cytokine fusion protein according to any one of claims 1 to 3, comprising IL-13 mutain, which includes 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 IL-13 mutain 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 the extracellular domain of CD122, the extracellular domain of CD132, or the 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 mutain having one amino acid sequence from SEQ ID NOs. 5 to 24 and 105.

8. The IL-2 cytokine fusion protein according to claim 7, wherein the IL-2 mutain 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 mutain 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 bound to IL-2 or IL-2 mutein.

11. The IL-2 cytokine fusion protein according to claim 10, wherein the albumin is human albumin, and optionally, the human albumin is recombinant human albumin.

12. The IL-2 cytokine fusion protein according to claim 10, wherein the antibody is manipulated as a "nob-in-hole" (KiH) having a mutation in the constant region 3 (CH3) of its heavy chain.

13. The IL-2 portion is an IL-2x antibody (KiH) fusion protein, a) A first polypeptide comprising IL-2 or IL-2 mutein, optionally an IL-2 fusion, bound to the second and third constant regions (CH2 and CH3) of the "knob" heavy chain of the antibody (KiH), b) A second polypeptide comprising the “whole” heavy chain of the antibody (KiH), c) The IL-2 cytokine fusion protein according to claim 12, comprising an IL-2x antibody (KiH) fusion protein comprising a third polypeptide comprising the light chain of the antibody (KiH).

14. The IL-2 portion is an IL-2x antibody (KiH) fusion protein, a) A first polypeptide comprising IL-2 or IL-2 mutein, and optionally a "knob" heavy chain of the antibody (KiH) conjugated to an IL-2 fusion, b) A second polypeptide comprising the "whole" heavy chain of the antibody (KiH), c) The IL-2 cytokine fusion protein according to claim 12, comprising an IL-2x antibody (KiH) fusion protein comprising a third polypeptide comprising the light chain of the antibody (KiH).

15. The IL-2 portion is an IL-2x antibody (KiH) fusion protein, a) A first polypeptide comprising the "knob" heavy chain of the antibody (KiH), b) A second polypeptide comprising the “whole” heavy chain of the antibody (KiH) conjugated to IL-2 or IL-2 mutein or IL-2 mutein fusion, c) The IL-2 cytokine fusion protein according to claim 12, comprising an IL-2x antibody (KiH) fusion protein comprising 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 bound to IL-2 or IL-2 mutain.

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

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 mutain has one amino acid sequence from among SEQ ID NOs. 5 to 24 and 105.

20. The IL-2 cytokine fusion protein according to claim 19, wherein the IL-2 mutain 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 can be 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 PLGLVVAAPGLGLVVAAPGLGLVVA, PLGLWAAPGLGLWAAPGLGLWA, GGSGGTPLGLWAGGSGGT, GGSGGTPAGLIGGGSGGT, GGSGGTPLGLWAGGSGGTPAGLIGGGSGGT, or GGGGTHSSKLQGGSGGT.

23. It is an IL-2 cytokine fusion protein, (a) Sequence IDs 302, 303, and 304, (b) Sequence IDs 299, 300, and 301, (c) Sequence IDs 305, 306, and 307, (d) Sequence IDs 308, 309, and 310, (e) Sequence IDs 311, 312, and 313, (f) Sequence IDs 314, 315, and 316, (g) Sequence IDs 317, 318, and 319, (h) Sequence IDs 320, 321, and 322, (i) Sequence ID 323, (j) Sequence IDs 329, 330, and 331, (k) Sequence IDs 332, 333, and 334, (l) Sequence IDs 335, 336, and 337, (m) Sequence IDs 338, 339, and 340, (n) Sequence IDs 353, 354, and 355, (o) Sequence IDs 359, 360, and 361, (p) Sequence IDs 362, 363, and 364, (q) Sequence IDs 365, 366, and 367, (r) Sequence IDs 325, 326, and 327, (s) Sequence IDs 353, 354, and 355, and (t) An IL-2 cytokine fusion protein comprising a sequence selected from the group consisting of sequence number 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 described in claim 25.

26. A method for producing an IL-2 cytokine fusion protein according to any one of claims 1 to 23, comprising: culturing a nucleic acid composition according to claim 25 or an 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.

27. A method for treating a subject requiring treatment for IL-13Ra2-expressing cancer, 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.

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

29. A method for performing cancer treatment on a subject requiring such treatment, wherein the subject is given an IL-2 cytokine fusion protein, a) IL-2 mutaine and, b) comprising albumin or Fc domain or antibody, A method comprising administering an IL-2 cytokine fusion protein in which the IL-2 mutain has one amino acid sequence from SEQ ID NOs. 5 to 24 and 105, and optionally further comprises a T3A and / or C125S amino acid substitution.

30. The method according to claim 29, wherein the IL-2 cytokine fusion protein comprises one or more of SEQ ID NOs: 1 to 367.

31. 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.

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

33. The method according to claim 32, wherein the IL-2 cytokine fusion protein is administered up to one week before the surgery, optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day before the surgery, or wherein the IL-2 cytokine fusion protein is administered up to nine weeks before the surgery, optionally up to eight weeks, seven weeks, six weeks, five weeks, four weeks, three weeks, two weeks, one week, or one week before the surgery.

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

35. The method according to claim 34, wherein the IL-2 cytokine fusion protein is administered up to one week after the surgery, optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day after the surgery, or the IL-2 cytokine fusion protein is administered starting at least two weeks after the surgery.

36. The method according to any one of claims 32 to 35, wherein the IL-2 cytokine fusion protein is administered both as a neoadjuvant before surgery to remove the tumor and as an adjuvant after surgery to remove the tumor.

37. The method according to claim 36, wherein the IL-2 cytokine fusion protein is administered up to one week before the surgery, optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day before the surgery, optionally up to one week after the surgery, or optionally up to seven days, six days, five days, four days, three days, two days, one day, or one day after the surgery, or optionally up to nine weeks before the surgery, optionally up to eight weeks, seven weeks, six weeks, five weeks, four weeks, three weeks, two weeks, one week, or one week before the surgery, and the administration of the IL-2 cytokine fusion protein is initiated at least two weeks after the surgery.

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

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