IL-2 fusion protein that preferentially binds to IL-2R alpha

Abstract

The present invention provides novel, isolated IL-2 fusion molecules that preferentially activate regulatory T cells (Tregs) in vitro and in vivo, as well as methods for making and using the novel fusion molecules to treat inflammatory and autoimmune diseases.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Application No. 62 / 885,471 filed on August 12, 2019, U.S. Provisional Application No. 63 / 015,644 filed on April 26, 2020, U.S. Provisional Application No. 63 / 019,319 filed on May 2, 2020, and U.S. Provisional Application No. 63 / 044,294 filed on June 25, 2020. The content of the priority - claiming applications is hereby incorporated by reference in its entirety into this specification.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format and is hereby incorporated by reference in its entirety into this specification. The ASCII copy created on August 12, 2020, has the name 025471_WO005_SL.txt and is 163,708 bytes in size.

Background Art

[0003] Background of the Invention Interleukin - 2 (IL - 2) plays a central role in lymphocyte production, survival, and homeostasis. It consists of 133 amino acids and is composed of four antiparallel, amphipathic α - helices that form an essential quaternary structure for its function (Smith, Science (1988) 240:1169 - 76; Bazan, Science (1992) 257:410 - 13). IL - 2 exerts its activity by binding to the interleukin - 2 receptor (IL - 2R), which consists of up to three independent subunits. The binding of the subunits α (CD25 or Tac antigen), β (CD122), and γ (γ c , common γ - chain or CD132) results in a trimeric, high - affinity receptor for IL - 2 (K D ~0.01 nM). A dimer IL - 2 receptor consisting of the β subunit and the γ subunit is called a medium - affinity IL - 2R (K D ~1 nM). The α subunit alone forms a monomeric low - affinity IL - 2 receptor (K D(about 10 nM). See, for example, Kim et al., Cytokine Growth Factor Rev. (2006) 17:349-66. The dimeric intermediate affinity IL-2 receptor binds IL-2 with an affinity approximately 100-fold lower than that of the trimeric high affinity receptor, but both the dimeric and trimeric IL-2 receptors can transmit signals upon binding of IL-2 (Minami et al., Annu Rev Immunol. (1993) 11:245-68). Thus, although the α subunit helps with high affinity binding of the receptor to IL-2, it is thought not to be essential for IL-2 signaling. However, the β and γ subunits are essential for IL-2 signaling (Krieg et al., Proc Natl Acad Sci. (2010) 107:11906-11). The trimeric IL-2 receptor is CD4 + FOXP3 + is expressed on regulatory T (Treg) cells. Treg cells constitutively express the highest levels of IL-2Rα in vivo (Fontenot et al., Nature Immunol. (2005) 6:1142-51). Also, in conventional activated T cells, the trimeric IL-2 receptor is transiently induced, but in the resting state these cells express only the dimeric IL-2 receptor.

[0004] Based on the published crystal structure of the IL-2 / IL-2R complex (Wang et al., Science (2005) 310:1159-63), researchers have mutated IL-2 to modulate its interaction with CD25, CD122, and / or CD132. As an example, mutations at D20, N88, or Q126 of human IL-2 have been reported to show a modification in the potency to activate T cells versus the potency to activate NK cells (U.S. Patent No. 6,955,807). In another example, IL-2 with mutations at positions 69 and 74 binds strongly to CD25, but mutations at positions 88 or 91 inhibit the interaction with CD122, and a mutation at position 126 inhibits the interaction with CD132 (PCT Publication WO2009 / 061853).

[0005] Treg cells are essential for suppressing autoimmunity and controlling inflammation. FOXP3 - CD25 + T effector cells (T eff ) can be CD4 + or CD8 + cells, either of which can cause inflammation, autoimmunity, organ transplant rejection, or graft-versus-host disease (GVHD). IL-2-stimulated STAT5 signaling is essential for normal Treg cell proliferation and survival and high FOXP3 expression. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] Despite the role of IL-2 in Treg activity, a safe and effective IL-2-based therapy for controlling Treg activity has not been clinically proven. Therefore, there is a need to develop an IL-2-based therapy that preferentially expands or stimulates Treg cells for the treatment of inflammatory and autoimmune diseases. MEANS FOR SOLVING THE PROBLEMS

[0007] SUMMARY OF THE INVENTION The present invention provides an isolated IL-2 fusion molecule comprising a carrier portion, a cytokine portion, and one or more masking portions, wherein the cytokine portion is fused to the carrier portion or the masking portion, and the one or more masking portions are fused to the carrier portion or the cytokine portion. The cytokine portion is an IL-2 polypeptide comprising an IL-2 amino acid sequence comprising (i) a C125A or C125S substitution or (ii) one or more substitutions selected from T3A, C125S, V69A, and Q74P (numbering according to SEQ ID NO: 1), and the one or more masking portions bind to the cytokine portion and inhibit binding of the cytokine portion to IL-2Rβ and / or IL-2Rγ but not to IL-2Rα on immune cells (e.g., T cells and NK cells). In certain embodiments, the IL-2 polypeptide binds to IL-2Rα with an affinity equal to or greater than that of wild-type IL-2.

[0008] The present invention also provides a method of treating an inflammatory condition or an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated IL-2 fusion molecule comprising a carrier moiety, a cytokine moiety, and one or more masking moieties, wherein the cytokine moiety is fused to the carrier moiety or to the masking moiety, one or more masking moieties are fused to the carrier moiety or the cytokine moiety, the cytokine moiety comprises an IL-2 polypeptide, and the one or more masking moieties bind to the cytokine moiety and inhibit binding of the cytokine moiety to IL-2Rβ and / or IL-2Rγ but not to IL-2Rα on immune cells (e.g., T cells and NK cells). In certain embodiments, the inflammatory condition or autoimmune disease is selected from the group consisting of asthma, type I diabetes, rheumatoid arthritis, allergy, systemic lupus erythematosus, multiple sclerosis, organ transplant rejection, and graft-versus-host disease.

[0009] In certain embodiments, the IL-2 polypeptide binds to IL-2Rα with an affinity equal to or greater than that of wild-type IL-2.

[0010] In certain embodiments, the IL-2Rβ ECD or a functional analog thereof has an amino acid sequence that is at least 95% (e.g., at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 3. In certain embodiments, the IL-2Rγ ECD or a functional analog thereof has an amino acid sequence that is at least 95% (e.g., at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 6. In certain embodiments, the IL-2 polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, and optionally, the amino acid sequence is SEQ ID NO: 2.

[0011] In certain embodiments, the IL-2 fusion molecule comprises a masking portion that includes the extracellular domain (ECD) of IL-2Rβ or IL-2Rγ or a functional analog thereof, and the masking portion is fused to the carrier portion with or without using a peptide linker. In other embodiments, the IL-2 fusion molecule comprises a first masking portion that includes the extracellular domain (ECD) of IL-2Rβ or IL-2Rγ or a functional analog thereof (wherein the first masking portion is fused to the carrier portion with or without using a peptide linker) and a second masking portion that includes the ECD of IL-2Rγ or IL-2Rβ or a functional analog thereof (wherein the second masking portion is fused to the cytokine portion or the first masking portion with or without using a peptide linker). In certain embodiments, the IL-2 fusion molecule of the present invention comprises at least two masking portions, one of which is the ECD of IL-2Rα or a functional analog thereof, and the ECD masking portion of IL-2Rα is fused to the cytokine portion, the carrier portion or another masking portion via a cleavable peptide linker. In a particular embodiment, the IL-2Rα ECD portion comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 7.

[0012] In certain embodiments, the cytokine portion is fused to the carrier portion or the masking portion via a non-cleavable peptide linker, and the masking portion is fused to the carrier portion or the cytokine portion via a non-cleavable peptide linker. In a particular embodiment, the masking portion is fused to the carrier portion or the cytokine portion via a peptide linker that comprises at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 22 amino acids, at least 25 amino acids, at least 30 amino acids or up to 44 amino acids.

[0013] In certain embodiments, the carrier moiety is selected from PEG molecules, albumin, albumin fragments, the Fc domain of an antibody, an antibody, or an antigen-binding fragment thereof. In certain embodiments, the carrier moiety is an antibody Fc domain, and the fusion molecule is a heterodimer comprising a first polypeptide chain that, in the N-terminal to C-terminal direction, comprises a molecular formula selected from F1-L1-E1, F1-L1-E1-L2-E2, and F1-L1-E2-L2-E1, and a second polypeptide chain that, in the N-terminal to C-terminal direction, comprises a molecular formula F2-L3-C, where F1 and F2 are subunits of the Fc domain, L1, L2, and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, E2 is an IL-2Rγ ECD or a functional analog thereof, and C is a cytokine moiety. In other embodiments, the carrier moiety is an antibody Fc domain, and the fusion molecule is a heterodimer comprising a first polypeptide chain that, in the N-terminal to C-terminal direction, comprises a molecular formula selected from E1-L1-F1, E1-L1-E2-L2-F1, and E2-L1-E1-L2-F1, and a second polypeptide chain that, in the N-terminal to C-terminal direction, comprises a molecular formula C-L3-F2, where F1 and F2 are subunits of the Fc domain, L1, L2, and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, E2 is an IL-2Rγ ECD or a functional analog thereof, and C is a cytokine moiety. In other embodiments, the carrier moiety is an antibody Fc domain, and the fusion molecule is, in the N-terminal to C-terminal direction, the following F1-L1-E1 and F2-L2-C-L3-E2, F1-L1-E1 and F2-L2-E2-L3-C, F1-L1-E2 and F2-L2-C-L3-E1, F1-L1-E2 and F2-L2-E1-L3-C, E1-L1-F1 and E2-L2-C-L3-F2, E1-L1-F1 and C-L2-E2-L3-F2, E2-L1-F1 and E2-L2-C-L3-F2 and E2-L1-F1 and C-L2-E1-L3-F2 A heterodimer comprising a first polypeptide chain and a second polypeptide chain composed of a molecular formula selected from the group consisting of, wherein F1 and F2 are subunits of the Fc domain, L1, L2 and L3 are peptide linkers, E1 is the IL-2Rβ ECD or a functional analog thereof, E2 is the IL-2Rγ ECD or a functional analog thereof, and C is a cytokine moiety. In certain embodiments, the peptide linkers L1, L2 and L3 are not cleavable. In certain embodiments, L1, L2 and L3 independently have an amino acid sequence selected from SEQ ID NOs: 40-46, 55-57 and 59. In other certain embodiments, at least one of L1, L2 and L3 has an amino acid sequence comprising 20-44 amino acids.

[0014] In certain embodiments, the IL-2 fusion molecule of the present invention comprises a first polypeptide chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 50, 51 or 52, and a second polypeptide chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 53 or 54. In certain embodiments, the IL-2 fusion molecule of the present invention comprises a first polypeptide chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 50, and a second polypeptide chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 53.

[0015] In certain embodiments, the IL-2 fusion molecule of the present invention has the following characteristics: (a) Binds to the high-affinity IL-2 receptor (IL-2Rαβγ) having α, β and γ subunits with an affinity more than 100 times that of the intermediate IL-2 receptor (IL-2Rβγ) having β and γ subunits. (b) Measured by surface plasmon resonance assay at 37°C, with a K of about 5 nM or more or 10 nM or more D for binding to IL-2Rβγ. (c) In a CTLL-2 cell proliferation assay, has an EC of less than about 1 nM and 0.01 nM, 0.25 nM or 0.05 nM or more 50 value. (d) In the NK92 cell proliferation assay, an EC value greater than about 0.05 nM, 0.1 nM, 0.25 nM or 0.5 nM 50 having, (e) In the NK92 cell proliferation assay in the presence of a neutralizing CD25 antibody, the Emax value is at least 5-fold or at least 10-fold lower compared to the absence of the neutralizing CD25 antibody, (f) preferentially stimulating FOXP3+ T regulatory cells over T effector cells and NK cells, (g) promoting the growth or survival of FOXP3+ regulatory T cells, and (h )F inducing phosphorylation of STAT5 in FOXP3+ T cells, but having a reduced ability to induce phosphorylation of STAT5 in FOXP3− T cells, having one or more of.

[0016] In another aspect, the present invention provides a pharmaceutical composition comprising an IL-2 fusion molecule of the present invention and a pharmaceutically acceptable excipient; one or more polynucleotides encoding the IL-2 fusion molecule, one or more expression vectors comprising the one or more polynucleotides; and a host cell comprising the vector, wherein the host cell can be a prokaryotic cell or a eukaryotic cell such as a mammalian cell. In certain embodiments, the mammalian host cell has a gene encoding uPA, MMP-2 and / or MMP-9 or the gene is knocked out (e.g., comprising one or more null mutations of these genes). Accordingly, the present invention also provides a method for producing an IL-2 fusion molecule, the method comprising culturing a host cell, which is a mammalian cell, under conditions that allow expression of the IL-2 fusion molecule and isolating the IL-2 fusion molecule.

[0017] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is not limiting, but is presented by way of illustration. Various changes and modifications within the scope of the present invention will be apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

Figure 1

[0019]

Figure 2

[0020]

Figure 3

[0021]

Figure 4

[0022]

Figure 5

[0023]

Figure 6

[0024]

Figure 7

[0025]

Figure 8

[0026]

Figure 9

[0027]

Figure 10

[0028]

Figure 11

[0029]

Figure 12

[0030]

Figure 13

[0031]

Figure 14

[0032]

Figure 15

[0033]

Figure 16

[0034]

Figure 17

[0035]

Figure 18

[0036]

Figure 19

[0037]

Figure 20

[0038]

Figure 21

Mode for Carrying Out the Invention

[0039] Detailed Description of the Invention The singular forms in this specification and the appended claims include plural forms unless the context clearly dictates otherwise. References to "about" a value or parameter in this specification include (and mean) deviations from that value or parameter itself. For example, the recitation "about X" includes the numerical value "X". Further, the use of "about" prior to a series of numbers includes "about" for each of the recited numbers in that series. For example, the recitation "about X, Y or Z" is intended to recite "about X, about Y or about Z".

[0040] The term "antigen-binding portion" refers to a polypeptide or set of interacting polypeptides that specifically binds to an antigen, an antibody (e.g., monoclonal antibody, polyclonal antibody, multispecific antibody, bispecific or bivalent antibody, anti-idiotype antibody or bifunctional hybrid antibody) or an antigen-binding fragment thereof (e.g., then, F ab, Fab’, F(ab’) 2, including but not limited to Fv, disulfide - linked Fv, scFv, single - domain antibodies (dAb) or diabodies, single - chain antibodies, and Fc - containing polypeptides such as immunoadhesins. In certain embodiments, the antibody can be of any heavy - chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD) or subtype (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ). In certain embodiments, the antibody can be of any light - chain isotype (e.g., kappa or lambda). The antibody can be human, non - human (e.g., derived from mouse, rat, rabbit, goat, or other non - human animals), chimeric (e.g., having non - human variable regions and human constant constant regions), or humanized (e.g., having non - human CDRs and human frameworks and constant constant regions). In certain embodiments, the antibody is a derivatized antibody.

[0041] The term "cytokine agonist polypeptide" refers to a wild - type cytokine or an analog thereof. An analog of a wild - type cytokine has the same biological specificity as the wild - type cytokine (e.g., binds to the same receptor and activates the same target cells), but the activity level of the analog can be different from that of the wild - type cytokine. The analog can be, for example, a mutein (i.e., a mutated polypeptide) of the wild - type cytokine and can contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 mutations relative to the wild - type cytokine.

[0042] The term "cytokine antagonist" or "cytokine mask" refers to a moiety (e.g., polypeptide) that binds to a cytokine and thereby inhibits the cytokine from binding to a receptor on the surface of a target cell and / or inhibits the cytokine from exerting its biological function while it is bound by the antagonist or mask. Examples of cytokine antagonists or masks include, but are not limited to, polypeptides derived from the extracellular domain of the natural receptor of the cytokine that contacts the cytokine.

[0043] The term "effective amount" or "therapeutically effective amount" refers to the amount of a compound or composition sufficient to treat a particular disorder, condition or disease, e.g., to ameliorate, alleviate, reduce and / or delay one or more of its symptoms.

[0044] The term "functional analog" refers to a molecule having the same biological specificity (e.g., binding to the same ligand) and / or activity (e.g., activating or inhibiting a target cell) as a reference molecule.

[0045] The terms "fused" or "fusion" in relation to two polypeptide sequences refer to the joining of the two polypeptide sequences via peptide bonds of the backbone. The two polypeptides can be fused directly or via a peptide linker having one or more amino acid lengths. The fusion polypeptide can be produced by recombinant techniques from a coding sequence containing the coding sequences of each of the two fusion partners, with or without the coding sequence of the intervening peptide linker. In certain embodiments, the fusion includes chemical conjugation.

[0046] The term "pharmaceutically acceptable excipient", when used to refer to a component included in a composition, means that the excipient is suitable for administration to a subject, including a human, without undue adverse side effects to the subject and without affecting the biological activity of the active pharmaceutical ingredient (API).

[0047] The term "subject" refers to a mammal and includes, but is not limited to, humans, pets (such as dogs, cats, etc.), farm animals (such as cows, horses, etc.), rodents, and primates.

[0048] As used herein, "treatment" or "treating" is an approach for obtaining a beneficial or desirable clinical outcome. Beneficial and desirable clinical outcomes include, but are not limited to, one or more of the following: alleviation of one or more symptoms attributable to a disease, reduction in the extent of a disease, improvement in the condition of a disease, stabilization of a disease (e.g., preventing or delaying worsening or progression of a disease), preventing or delaying spread of a disease (e.g., metastasis), preventing or delaying recurrence of a disease, partial or complete remission of a disease, stabilization of a disease (e.g., preventing or delaying worsening or progression of a disease), preventing or delaying expansion of a disease (e.g., metastasis), preventing or delaying recurrence of a disease, partial or complete remission of a disease, reduction in the dosage of one or more other agents required for treatment of the disease, improvement in the quality of life of a patient, and / or prolongation of the survival period. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0049] It is understood that one, several, or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the present invention. The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described thereunder.

[0050] isolated IL-2 fusion molecule The present invention provides an IL-2 fusion molecule useful for the treatment of inflammatory and autoimmune diseases. The inventors were surprised to find that the desired in vivo activity can be achieved without the need for cleavage or removal of the masking moiety. Masked IL-2 fusion molecules having a non-cleavable peptide linker have several important advantages compared to cleavably masked IL-2 fusion molecules. For example, cleavably masked IL-2 molecules require cleavage of the linker by a protease and removal of the masking moiety for activation. Since the distribution of proteases at the disease site is non-uniform, the activation level of the cytokine varies, which can further vary the therapeutic effect. Furthermore, non-specific activation may occur during circulation and / or manufacturing, adding concerns about safety and manufacturing complexity to cleavably masked molecules.

[0051] In certain embodiments, the IL-2 fusion molecule of the present invention has a reduced affinity for intermediate affinity IL-2Rβγ (e.g., greater than 1 nM, greater than 5 nM, greater than 10 nM, greater than 100 nM or greater than 1 μM K D ), while maintaining a wild-type affinity for IL-2Rα (CD25) (e.g., K D is about 10 nM), or having an affinity for IL-2Rα that is similar to (e.g., K D is about 1 - 20 nM) or higher (e.g., K D is less than 10 nM, less than 5 nM or less than 1 nM) wild-type affinity. The isolated IL-2 fusion molecule can comprise an IL-2 polypeptide (cytokine portion), a carrier (carrier portion) and an IL-2 antagonist (masking moiety or cytokine antagonist), wherein the IL-2 polypeptide is fused to the carrier directly or via a cleavable or non-cleavable peptide linker, and the IL-2 antagonist is linked to the IL-2 polypeptide or the carrier via a non-cleavable or cleavable peptide linker. In certain embodiments, the cytokine portion can be fused to the masking moiety, which can be fused to the carrier portion directly or via a cleavable or non-cleavable linker.

[0052] In a preferred embodiment, the IL-2 polypeptide is fused to the carrier via a non-cleavable peptide linker, and the IL-2 antagonist is bound to the carrier or the IL-2 polypeptide via a non-cleavable peptide linker. For example, the IL-2 antagonist may be fused to the carrier via the non-cleavable peptide linker of SEQ ID NO: 59. Also, the IL-2 polypeptide is a wild-type IL-2 polypeptide or does not contain mutations that reduce the binding affinity of the polypeptide for CD25.

[0053] The IL-2 fusion molecule of the present invention may comprise an IL-2 polypeptide (cytokine portion) bound to a carrier portion and masked (bound) by a cytokine antagonist (masking portion). The cytokine antagonist is selected from the extracellular domain (ECD) of IL-2Rβ (CD122), a functional analog of the IL-2Rβ ECD, the IL-2Rγ ECD (CD132), a functional analog of the IL-2Rγ ECD, and a combination of the IL-2Rβ ECD and the IL-2Rγ ECD. In certain embodiments, the cytokine antagonist inhibits the binding of the cytokine portion to IL-2Rγ and / or IL-2Rβ on T cells in a patient in need thereof, while the cytokine portion remains intact for binding to IL-2Rα (CD25). Since IL-2Rα (CD25) is preferentially expressed on Treg cells, the IL-2 fusion molecule of the present invention can preferentially stimulate the proliferation of Treg cells while minimizing the effect on non-Treg cells.

[0054] In certain embodiments, the carrier moiety is an Fc domain. In certain embodiments, the present IL-2 fusion molecule comprises, from the N-terminus to the C-terminus, a first polypeptide chain comprising a molecular formula selected from F1-L1-E1, F1-L1-E2-E2, and F1-L1-E2-L2-E1, and, from the N-terminus to the C-terminus, a second polypeptide chain comprising a molecular formula selected from F1-L1-E1, F1-L1-E2-E2, and F1-L1-E2-L2-E1, which is a heterodimer, wherein F1 and F2 are subunits of the heterodimeric Fc domain, L1, L2, and L3 are peptide linkers, E1 is the IL-2Rβ ECD or a functional analog thereof, E2 is the IL-2Rγ ECD or a functional analog thereof, and C is a cytokine moiety comprising an IL-2 polypeptide (e.g., wild-type human IL-2 or a mutein thereof).

[0055] In certain embodiments, the present IL-2 fusion molecule comprises, from the N-terminus to the C-terminus, a first polypeptide chain comprising a molecular formula selected from E1-L1-F1, E1-L1-E2-L2-F1, and E2-L1-E1-L2-F1, and, from the N-terminus to the C-terminus, a second polypeptide chain comprising a molecular formula selected from E1-L1-E2-F1 and E2-L1-E1-L2-F1, which is a heterodimer, wherein F1 and F2 are subunits of the heterodimeric Fc domain, L1, L2, and L3 are peptide linkers, E1 is the IL-2Rβ ECD or a functional analog thereof, E2 is the IL-2Rγ ECD or a functional analog thereof, and C is a cytokine moiety comprising an IL-2 polypeptide (e.g., wild-type human IL-2 or a mutein thereof).

[0056] In certain embodiments, the present IL-2 fusion molecule, from the N-terminus to the C-terminus, is as follows a. F1-L1-E1 and F2-L2-C-L3-E2, b. F1-L1-E1 and F2-L2-E2-L3-C, c. F1-L1-E2 and F2-L2-C-L3-E1, d. F1-L1-E2 and F2-L2-E1-L3-C, e. E1-L1-F1 and E2-L2-C-L3-F2, f. E1-L1-F1 and C-L2-E2-L3-F2, g. E2-L1-F1 and E2-L2-C-L3-F2, and h. E2-L1-F1 and C-L2-E1-L3-F2 a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the first and second polypeptide chains each comprising a molecular formula selected from the group consisting of: wherein F1 and F2 are subunits of the Fc domain of the heterodimer, L1, L2 and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, E2 is an IL-2Rγ ECD or a functional analog thereof, and C is a cytokine moiety.

[0057] In certain embodiments, peptide linkers L1, L2 and L3 independently have an amino acid sequence selected from SEQ ID NOs: 40-49 and 55-57.

[0058] In certain embodiments, at least one of peptide linkers L1, L2 and L3 has an amino acid sequence comprising at least 20-44 amino acids (e.g., at least 20, at least 25, at least 30, at least 35, at least 40, at least 45 or at least 55). In certain embodiments, at least one of the peptide linkers has at least 16, 18, 20, 22, 24, 26, 27, 28, 29, 31, 32, 33, 34, 36, 37, 38, 39, 41 or 42 amino acids.

[0059] In certain embodiments, the IL-2 fusion molecule has a structure exemplified in FIGS. 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 6A, 6B, 7A or 7B. In certain embodiments, the IL-2 fusion molecule has a structure shown in FIGS. 10A or 10B.

[0060] In certain embodiments, the isolated fusion molecule comprises a first polypeptide chain having an amino acid sequence that is at least 99% identical to one selected from SEQ ID NOs: 50, 51, and 52, and a second polypeptide chain having an amino acid sequence that is at least 99% identical to one selected from SEQ ID NOs: 53 and 54.

[0061] The isolated IL-2 fusion molecules 982 C1, C2, D1, D2, and 982 Ref comprise two polypeptide chains having the amino acid sequences shown in Table 1. Both molecules 982 C1 and 982 C2 contain two masking moieties that are the IL-2Rγ ECD and the IL-2Rβ ECD. Both 982 D1 and 982 D2 each contain one masking moiety that is the IL-2Rβ ECD. The IL-2 portions of both 982 C2 and 982 D2 contain the mutations T3A, V69A, P74Q, and C125S (numbering according to SEQ ID NO: 1).

[0062] [Table 1]

[0063] A. IL-2 polypeptide or mutein In the IL-2 fusion molecules of the present invention, the IL-2 polypeptide can be a wild-type IL-2 polypeptide such as the wild-type human IL-2 polypeptide (SEQ ID NO: 1) or an IL-2 mutein such as an IL-2 mutein derived from human IL-2. The IL-2 mutein is an IL-2 derivative that retains at least one aspect of the biological activity of IL-2. In certain embodiments, the IL-2 mutein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In certain specific embodiments, the IL-2 mutein has the same length as SEQ ID NO: 1 but has 7 or fewer (e.g., 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer) amino acid residues is differentIt may be that the IL-2 mutant has a reduced affinity for CD122 and / or CD132 and may contain one or more mutations selected from L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, d20g, d20t, d20w, m23r, r81a, r81g, r81s, r81t, d84a, d84e, d84g, d84i, d84m, d84q d84r, d84s, d84t, s87r, n88a, n88d, n88e, n88f, n88g, n88m, n88r, n88s, N88V, N88W, N90T, N90S, V91D, V91E, V91G, V91S, I92K, I92R, I92T, I92S, E95G, Q126E, Q126F, Q126G, Q126I, Q126L, Q126M, Q126N, Q126R, Q126V and Q126Y. Unless otherwise specified, all residue numbers of IL-2 follow the numbering of SEQ ID NO: 1. In certain embodiments, the mutant of IL-2 may have a mutation that results in an increased affinity for CD25. Such mutations can be selected from mutations at positions 69 and 74. In certain embodiments, the IL-2 mutant may contain one or more mutations selected from T3A, C125A, C125S and C125G. is a derivatized antibody.

[0064] B. Masking portion of the isolated IL-2 fusion molecule The cytokine antagonist, i.e., the masking portion, in the isolated IL-2 fusion molecule of the present invention is an extracellular domain of IL-2Rβ or IL-2Rγ or a functional analog thereof that is derived from human IL-2Rβ or IL-2Rγ (e.g., any of SEQ ID NOs: 3-6). In certain embodiments, the IL-2 fusion molecule comprises at least one masking portion. For example, the fusion molecule may comprise both the IL-2Rβ ECD and the IL-2Rγ ECD or only one of these ECDs. The ECD may be the entire extracellular domain of human IL-2Rβ or IL-2Rγ or only a portion thereof, as long as that portion is capable of maintaining the state where it can bind to the IL-2 portion or otherwise inhibits the binding of the IL-2 portion to IL-2Rβ or IL-2Rγ on T cells.

[0065] In certain embodiments, the IL-2 fusion molecule comprises an additional masking portion that is the ECD of IL-2Rα (e.g., SEQ ID NO: 7) or a functional analog thereof, wherein the ECD masking portion of IL-2Rα is fused to the cytokine portion, the carrier portion, or another masking portion within the fusion molecule via a cleavable peptide linker. The presence of the IL-2Rα masking portion attached to the fusion molecule via a cleavable linker allows the fusion molecule to home to the target site without binding to cells at non-target sites. Upon reaching the target site, the cleavable linker is cleaved by proteases that are present at high concentrations at the target site, and the activated fusion molecule binds to IL-2Rα on the cells at the target site (e.g., Treg cells) and can stimulate the bound cells.

[0066] A functional analog of the ECD of an IL-2R subunit (α, β, γ) refers to a polypeptide that has an affinity for IL-2 similar to that of the wild-type ECD. For example, the functional analog may comprise the core IL-2 binding region of the wild-type ECD and may have a sequence that is at least 95% (e.g., at least 96%, 97%, 98%, or 99%) identical to the wild-type ECD (e.g., SEQ ID NOs: 3-7, supra) over the entire length of the analog.

[0067] C. Carrier portion of the isolated IL-2 fusion molecule The carrier portion of the IL-2 fusion molecule of the present invention can be an antigen-binding portion or a site that is not an antigen-binding portion. The carrier portion improves the PK profile such as the serum half-life of the cytokine agonist polypeptide and can target the cytokine agonist polypeptide to a target site in the body such as a tumor site.

[0068] 1. Antigen-binding carrier portion The carrier portion can be an antibody or an antigen-binding fragment thereof, or an immunoadhesin. In certain embodiments, the antigen-binding portion is a full-length antibody having two heavy chains and two light chains, a Fab fragment, a Fab’ fragment, F(ab’) 2 fragment, an Fv fragment, a disulfide-bonded Fv fragment, a single-domain antibody, a nanobody, or a single-chain variable fragment (scFv). In certain embodiments, the antigen-binding portion is a bispecific antigen-binding portion and can bind to two different antigens or two different epitopes on the same antigen. The antigen-binding portion can provide an additional therapeutic effect or a synergistic therapeutic effect to the cytokine agonist polypeptide.

[0069] The IL-2 polypeptide and its mask can be fused to the N-terminus or C-terminus of the light chain and / or heavy chain of the antigen-binding portion. By way of example, the IL-2 polypeptide and its mask can be fused to an antibody heavy chain or an antigen-binding fragment thereof or an antibody light chain or an antigen-binding fragment thereof. In certain embodiments, the IL-2 polypeptide is fused to the C-terminus of one or both of the heavy chains of the antibody, and the mask of the cytokine is fused to the other end of the cytokine portion via a non-cleavable or cleavable peptide linker. In certain embodiments, the IL-2 polypeptide is fused to the C-terminus of one of the heavy chains of the antibody, and the cytokine'mask is fused to the C-terminus of the other heavy chain of the antibody via a non-cleavable or cleavable peptide linker, and the two heavy chains contain mutations that allow specific pairing of the two different heavy chains.

[0070] Strategies for forming heterodimers are well known (see, e.g., Spies et al., Mol Imm. (2015) 67(2)(A):95-106). For example, two heavy chain polypeptides of an isolated IL-2 fusion molecule can form a stable heterodimer by “knob-into-hole” mutations. “Knob-into-hole” mutations are made to promote the formation of heterodimers of antibody heavy chains and are commonly used to make bispecific antibodies (see, e.g., U.S. Patent No. 8,642,745). For example, the Fc domain of an antibody can consist of a T366W mutation in the CH3 domain of the “knob chain” and T366S, L368A, and / or Y407V mutations in the CH3 domain of the “hole chain”. Additional inter-chain disulfide bridges can also be used between the CH3 domains. For example, a Y349C mutation can be introduced into the CH3 domain of the “knob chain” and an E356C or S354C mutation can be introduced into the CH3 domain of the “hole chain” (see, e.g., Merchant et al., Nature Biotech (1998) 16:677-81). In other embodiments, the antibody portion may have a Y349C and / or T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A, and / or Y407V mutation in the other CH3 domain. In certain embodiments, the antibody portion has a Y349C and / or T366W mutation in one of the two CH3 domains and an S354C (or E356C), T366S, L368A, and / or Y407V mutation in the other CH3 domain, with an additional Y349C mutation in one CH3 domain and an additional E356C or S354C mutation in the other CH3 domain, capable of forming an inter-chain disulfide bridge (numbering always follows Kabat's EU index; Kabat; Kabat et al., “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).Other knob-into-hole technologies such as those described in EP1870459A1 can be used instead of or in addition to this. Thus, another example of a knob-into-hole mutation of the antibody site is to have an R409D / K370E mutation in the CH3 domain of the "knob chain" and a D399K / E357K mutation in the CH3 domain of the "hole chain" (EU numbering).

[0071] In certain embodiments, the antibody portion of the isolated IL-2 fusion molecule comprises the L234A and L235A ("LALA") mutations in its Fc domain. The LALA mutations eliminate complement binding and fixation as well as Fcγ-dependent ADCC (e.g., Hezareh et al. J. Virol. (2001) 75(24):12161-8). Further, the LALA mutations are present in the antibody portion in addition to the knob-into-hole mutations.

[0072] In certain embodiments, the antibody portion comprises the M252Y / S254T / T256E ("YTE") mutation of the Fc domain. The YTE mutation can simultaneously regulate the serum half-life, tissue distribution, and activity of IgG 1 (see Dall’Acqua et al., J Biol Chem. (2006) 281(33): 23514-24; and Robbie et al., Antimicrob Agents Chemother. (2013) 57(12):6147-53). In further embodiments, the YTE mutation is present in the antibody portion in addition to the knob-into-hole mutations. In certain embodiments, the antibody site has the YTE, LALA, knob-into-hole mutations or combinations thereof.

[0073] In certain embodiments, the antigen-binding portion binds to IL-1β, IL-1β receptor, IL-4, IL-4 receptor, IL-6, IL-6 receptor, IL-13, IL-13 receptor, IL-17, IL-17 receptor, IL-23, IL-23 receptor, TNFα or TNFα receptor.

[0074] 2. Other carrier portions Other non-antigen binding carrier moieties can be used in the isolated IL-2 fusion molecules of the present invention. For example, the Fc domain of an antibody (e.g., human IgG 1 , IgG 2 , IgG 3 , IgG 4 Fc), a polymer (e.g., PEG), albumin (e.g., human albumin) or a fragment or nanoparticle thereof can be used.

[0075] As an example, an IL-2 polypeptide and its antagonist can be fused to the Fc domain of an antibody to form an Fc fusion protein. In certain embodiments, the IL-2 polypeptide is fused (directly or via a peptide linker) to one of the C- or N-termini of the Fc domain polypeptide chain, and the cytokine mask is fused (via a non-cleavable or cleavable peptide linker) to the C- or N-terminus of the other Fc domain polypeptide chain, and the two Fc domain polypeptide chains contain mutations that allow for specific pairing of the two different Fc chains. In certain embodiments, the Fc domain contains the above-mentioned knob-into-hole mutations. In further embodiments, the Fc domain may also contain the above-mentioned YTE and / or LALA mutations. In certain embodiments, the Fc domain contains a mutation at N297 (EU numbering).

[0076] The carrier moiety of the isolated IL-2 fusion molecule can be composed of albumin (e.g., human serum albumin) or a fragment thereof. In certain embodiments, the albumin or albumin fragment is about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more identical to human serum albumin or a fragment thereof. About 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more or 99.8% or more identical.

[0077] In certain embodiments, the carrier portion is composed of albumin fragments (e.g., human serum albumin fragments) having an amino acid length of about 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 550 or more. In certain embodiments, the albumin fragment is between about 10 amino acids and about 584 amino acids in length (such as between about 10 and about 20, about 20 and about 40, about 40 and about 80, about 80 and about 160, about 160 and about 250, about 250 and about 350, about 350 and about 450, or about 450 and about 550 amino acids). In certain embodiments, the albumin fragment comprises a Sudlow I domain or a fragment thereof or a Sudlow II domain or a fragment thereof.

[0078] D. Linker Component of the Isolated Fusion Molecule The IL-2 polypeptide can be fused to a carrier moiety with or without using a peptide linker. The peptide linker can be cleavable or non-cleavable. In certain embodiments, the cytokine moiety is fused to the carrier via a peptide linker, and the peptide linker is selected from SEQ ID NOs: 40-46 and 55-57. In specific embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 42, 44, 45, 46, 55, 56 or 57. The masking moiety may be fused to the cytokine moiety or the carrier via a non-cleavable or cleavable linker or without a peptide linker. The cleavable linker may comprise one or more (e.g., two or three) cleavable sites (CM). Each CM can be a substrate for an enzyme or protease selected from legumain, plasmin, TMPRSS-3 / 4, MMP-2, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, β-secretase, uPA, PSA. In certain embodiments, the masking moiety is fused to the carrier via a peptide linker, and the peptide linker is selected from SEQ ID NOs: 40-46, 55, 56 and 57. In specific embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 42, 44, 45, 46, 55, 56 or 67. In certain embodiments, the peptide linker comprises at least 10 amino acids, 12 amino acids, 14 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 25 amino acids, 27 amino acids or 30 amino acids.

[0079] Specific non-limiting examples of the IL-2 polypeptide, cytokine mask, carrier, peptide linker and isolated IL-2 fusion molecule are shown in the sequences section below. Further, the isolated fusion molecules of the invention can be produced by well-known recombinant techniques. For example, one or more expression vectors containing the coding sequence of the polypeptide chain of the isolated fusion molecule can be transfected into mammalian host cells (e.g., CHO cells) and the cells can be cultured under conditions that allow for expression of the coding sequence and assembly of the expressed polypeptide into an isolated IL-2 fusion molecule complex.

[0080] Pharmaceutical composition A pharmaceutical composition containing the isolated IL-2 fusion molecule of the present invention (i.e., the active pharmaceutical ingredient (API)) prepares an API having a desired purity by mixing it with one or more arbitrary pharmaceutically acceptable excipients (see, for example, Remington’s Pharmaceutical Sciences, 16th Edition., Osol, A. Ed. (1980)) and is prepared in the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable excipients (or carriers) are generally harmless to the subject at the dosages and concentrations employed and include, for example, buffers containing inorganic or organic acids or their salts such as phosphates, citrates, succinates, histidines, acetates, etc., antioxidants such as ascorbic acid or methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, etc.), low molecular weight (about 10 residues or less) polypeptides, proteins such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, lysine, monosaccharides such as sucrose, glucose, mannose, dextrin, disaccharides and other carbohydrates, chelating agents such as EDTA, saccharides such as sucrose, mannitol, trehalose, sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes) and / or nonionic surfactants such as polyethylene glycol (PEG), but are not limited thereto.

[0081] Buffers are used to control the pH within a range that optimizes the therapeutic effect, especially when the stability is pH-dependent. The buffer is preferably present at a concentration in the range of about 50 mM to about 250 mM. Suitable buffers for use in the present invention include organic and inorganic acids and their salts such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate, etc. Further, the buffer can be composed of salts of histidine such as Tris and trimethylamine.

[0082] Preservatives are added to suppress the growth of microorganisms and are usually present in the range of 0.2% to 1.0% (w / v). Suitable preservatives for use in the present invention include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium halides (chloride, bromide, iodide, etc.), benzethonium chloride, thimerosal, phenol, butyl alcohol or benzyl alcohol, alkyl parabens such as methyl paraben or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

[0083] An isotonic agent, sometimes called a "stabilizer", is present to adjust or maintain the tonicity of the liquid in the composition. When used for large charged biomolecules such as proteins and antibodies, it can interact with the charged groups of the amino acid side chains to reduce the possibility of intermolecular and intramolecular interactions, and is often called a "stabilizer". The isotonic agent can be contained in the range of 0.1% to 25%, more preferably 1% to 5%, considering the relative amounts with other components. Preferred isotonic agents include polyhydric sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol, preferably sugar alcohols with three or more hydroxyl groups.

[0084] Nonionic surfactants or detergents (also known as "wetting agents") are used not only to assist in solubilizing therapeutic agents but also to protect active therapeutic proteins and antibodies from denaturation caused by aggregation during agitation to which the formulation is exposed to shear stress. Nonionic surfactants are used in the range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0085] Suitable nonionic surfactants include polysorbates (such as 20, 40, 60, 65, 80, etc.). Poloxamers (such as 184, 188, etc.), pluronics (登録商標) polyols, Triton (登録商標) , polyoxyethylene sorbitan monoethers (Tween (登録商標) -20, Tween (登録商標) -80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose, carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0086] Pharmaceutical carriers, excipients, diluents can be selected taking into account the intended route of administration and standard pharmaceutical practices. The pharmaceutical composition may further contain any suitable binder, lubricant, suspending agent, coating agent, solubilizing agent.

[0087] Depending on the various delivery systems, various composition / formulation elements may be present. For example, the pharmaceutical compositions useful in the present invention can be formulated to be administered using a minipump, formulated to be administered via a mucosal route, such as a nasal spray or aerosol for inhalation, as an ingestible solution, or formulated in the form of an injection for delivery via a parenteral route, such as intravenous, intramuscular, subcutaneous, etc.

[0088] In certain embodiments, the pharmaceutical composition of the invention is a lyophilized protein formulation. In other embodiments, the pharmaceutical composition can be an aqueous liquid formulation.

[0089] Treatment method The IL-2 fusion molecule can be used for the treatment of inflammatory and autoimmune diseases. In certain embodiments, a method of treating a disease (such as an autoimmune disease) in a subject comprises administering to the subject an effective amount of the isolated IL-2 fusion molecule disclosed herein. In certain embodiments, the inflammatory or autoimmune disease is asthma, diabetes (e.g., type I diabetes or latent autoimmune diabetes), lupus (e.g., systemic lupus erythematosus), arthritis (e.g., rheumatoid arthritis), allergy, organ transplant rejection, GVHD, Addison's disease, ankylosing spondylitis, anti-glomerular basement membrane disease, autoimmune hepatitis, dermatitis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), juvenile myositis, Kawasaki disease, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, PANDAS, psoriasis, psoriatic arthritis, Sjögren's syndrome, systemic sclerosis, systemic scleroderma, thrombocytopenic purpura, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and is selected from the group consisting of.

[0090] Generally, the dosage and route of administration of the pharmaceutical composition of the invention are determined according to standard pharmaceutical practice, depending on the subject's physique and condition. In certain embodiments, the pharmaceutical composition is administered to the subject by any route including oral, transdermal, inhalation, intravenous, intraarterial, intramuscular, direct application to the wound site, application to the surgical site, intraperitoneal, suppository, subcutaneous, intradermal, transdermal, spray, intrathoracic, intracardiac, intraarticular, intraocular, intracranial or intranasal. In certain embodiments, the composition is administered intravenously to the subject.

[0091] In certain embodiments, the dosage form of the pharmaceutical composition is single-dose or repeated dosing. In certain embodiments, the administration is performed on the subject once a day, twice a day, three times a day, or four or more times a day. In certain embodiments, about one or more (such as about two, three, four, five, six, or seven or more) administrations are performed in one week. In certain embodiments, the pharmaceutical composition is administered once a week, once every two weeks, once every three weeks, once every four weeks, once a week for two out of three weeks, or once a week for three out of four weeks. In certain embodiments, it is administered multiple times over several days, weeks, months, or years. In certain embodiments, the number of administrations is one or more (such as about two, three, four, five, seven, ten, fifteen, or twenty or more).

[0092] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings as commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. In case of conflict, the description herein (including definitions) shall control. Generally, the nomenclature used in connection with the technologies such as cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicine and pharmaceutical chemistry, protein and nucleic acid chemistry, hybridization, etc. described herein is well-known and commonly used in the art. Enzyme reactions and purification methods are carried out according to the manufacturer's specifications, by methods commonly practiced in the art or by the methods described herein. Further, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Throughout this specification and the embodiments, the expressions "comprising" and "including" and variations such as "comprises", "comprising", "includes" or "including" are to be understood as including the recited integer or group of integers but not excluding other integers or groups of integers. It should be understood that the embodiments and variations of the present invention described herein include embodiments and variations "consisting of" and / or "consisting essentially of". All publications and other documents cited herein are hereby incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not admit that these documents form part of the common general knowledge in the art.

[0093] Exemplary embodiments Further specific embodiments of the present invention are described below. These embodiments are intended to illustrate the compositions and methods described in the present invention and are not intended to limit the scope of the present invention. 1. A mutant of IL-2Rβ-ECD comprising one or more point mutations, wherein the mutant of IL-2Rβ-ECD has improved thermal stability compared to the wild-type one. 2. An IL-2Rβ-ECD variant of embodiment 1 having one or more mutations at positions selected from Δ1-5 (deletion of the first 5 amino acids), F11, V21, L28, W38, L51, P52, V53, I63, P67, I77, V88, V92, M93, I95, M107, I110, V115, P156, L157, Q162, Q164, W166, P174, L187, F191, P196, P200, P207, W90, H150, W152, W166, W194 and W197 (numbering according to SEQ ID NO: 3). 3. The following: a. F11 and F191, b. L51, P52 and V53, c. V92, M93, I95, d. M107, P196, I110 and e. P156 and L157 An IL-2Rβ-ECD variant of embodiment 1 comprising a mutation at a position selected from the group (numbering according to SEQ ID NO: 3). 4. The IL-2Rβ-ECD variant according to embodiment 2 or 3, wherein the hydrophobic amino acid or amino acid is mutated to a hydrophilic amino acid or amino acid selected from S, G, N, T and Q. 5. The following: a. F11S and F191G, b. L51S, P52G and V53S, c. V92S, M93G, I95G, d. M107G, P196S, I110G, and e. P156S and L157G, f. W166N, g. Q164E. W166N, V115S i. W152N, j. W152N, W166N k. V92S l. W166N, V92S m. L157S, n. W165N, W157S An IL-2Rβ-ECD variant of embodiment 3, comprising a mutation selected from the group (numbering follows SEQ ID NO: 3). 6. An IL-2Rβ-ECD variant of embodiment 1, comprising an amino acid sequence selected from SEQ ID NOs: 47, 48, and 49. 7. An isolated IL-2 fusion molecule useful for treating inflammatory and autoimmune diseases, comprising a cytokine portion and a masking portion, wherein the cytokine portion comprises an IL-2 polypeptide or mutein, and the masking portion comprises the extracellular domain (ECD) of IL-2Rβ or a functional analog or variant thereof; and the fusion molecule preferentially stimulates regulatory T cells compared to other T cells or NK cells in an in vitro assay. 8. The isolated IL-2 fusion molecule according to embodiment 7, wherein the fusion molecule has an EC 50 value of less than about 1 nM in a CTLL-2 cell proliferation assay. 9. The isolated IL-2 fusion molecule according to embodiment 7, wherein the fusion molecule has an EC 50 value of less than about 0.1 nM in a CTLL-2 cell proliferation assay. 10. The isolated fusion molecule according to any one of embodiments 7 to 9, wherein the masking portion comprises a variant of the IL-2Rβ-ECD according to any one of embodiments 1 to 6. 11. The isolated fusion molecule according to any one of embodiments 7 to 10, further comprising the extracellular domain (ECD) of IL-2Rγ or a functional analog thereof. 12. The isolated fusion molecule according to any one of embodiments 7 to 11, further comprising a carrier. 13. The isolated fusion molecule according to embodiment 12, wherein the masking portion is linked to the carrier portion via a cleavable or non-cleavable peptide linker. 14. The isolated fusion molecule according to any one of embodiments 7 to 13, wherein the IL-2 polypeptide or mutein has an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, and the IL-2Rβ ECD or a functional analog or variant thereof has an amino acid sequence that is at least 95% identical to SEQ ID NO: 3. 15. The isolated fusion molecule according to any one of embodiments 7 to 13, wherein the IL-2 mutein has the amino acid sequence shown in SEQ ID NO: 2. 16. The isolated fusion molecule according to any one of embodiments 7 to 13, wherein the IL-2 mutein has at least one mutation selected from L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88G, N88M, N88R, N88S, N88V, N88W, N90T, N90S, V91D, V91E, V91G, V91S, I92K, I92R, I92T, I92S, E95G, Q126E, Q126F, Q126G, Q126I, Q126L, Q126M, Q126N, Q126R, Q126V, and Q126Y (numbering according to SEQ ID NO: 1). 17. The isolated fusion molecule according to any one of embodiments 7 to 16, wherein the carrier moiety is selected from a PEG molecule, albumin, an albumin fragment, an antibody Fc domain, or an antibody or an antigen-binding fragment thereof. 18. The isolated fusion molecule according to embodiment 17, wherein the carrier moiety comprises an antibody Fc domain having a mutation at N297 and / or the mutations L234A and L235A ("LALA") (EU numbering). 19. The isolated fusion molecule according to embodiment 17 or 18, wherein the carrier moiety comprises an antibody Fc domain containing a knob-into-hole mutation, and the cytokine moiety and the masking moiety are fused to different polypeptide chains of the antibody Fc domain. 20. The isolated fusion molecule according to embodiment 19, wherein the cytokine moiety and the masking moiety are fused to the C-terminus of two different polypeptide chains of the Fc domain or the C-terminus of two different heavy chains of the antibody. 21. The isolated fusion molecule according to claim 19, wherein the carrier is the Fc domain of an antibody, and the cytokine portion and the masking portion are fused to the N-termini of two different polypeptide chains of the Fc domain. 22. The isolated fusion molecule according to embodiment 12, wherein the carrier portion is the Fc domain of an antibody, and comprises a first polypeptide chain comprising a molecular formula selected from F1-L1-E1, F1-L1-E1-L2-E2, and F1-L1-E2-L2-E1, and a second polypeptide chain comprising the molecular formula F2-L3-C, wherein the F1 and F2 are subunits of the Fc domain that form a heterodimer, L1, L2, and L3 are peptide linkers, E1 is the IL-2Rβ ECD or a functional analog thereof, E2 is the IL-2Rγ ECD or a functional analog thereof, and C is the cytokine portion. 23. The isolated fusion molecule according to embodiment 12, wherein the carrier portion is the Fc domain of an antibody, and comprises a first polypeptide chain comprising a molecular formula selected from E1-L1-F1, E1-L1-E2-L2-F1, and E2-L1-E1-L2-F1, and a second polypeptide chain comprising the molecular formula C-L3-F2, wherein the F1 and F2 are subunits of the Fc domain that form a heterodimer, L1, L2, and L3 are peptide linkers, E1 is the IL-2Rβ ECD or a functional analog thereof, E2 is the IL-2Rγ ECD or a functional analog thereof, and C is the cytokine portion. 24. The carrier portion is the antibody Fc domain, and the following: a. F1-L1-E1 and F2-L2-C-L3-E2, b. F1-L1-E1 and F2-L2-E2-L3-C, c. F1-L1-E2 and F2-L2-C-L3-E1, d. F1-L1-E2 and F2-L2-E1-L3-C, e. E1-L1-F1 and E2-L2-C-L3-F2, f. E1-L1-F1 and C-L2-E2-L3-F2, g. E2-L1-F1 and E2-L2-C-L3-F2, and h. E2-L1-F1 and C-L2-E1-L3-F2; A first polypeptide chain and a second polypeptide chain comprising a molecular formula selected from the group, wherein F1 and F2 are subunits of an Fc domain that form a heterodimer, L1, L2 and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, E2 is an IL-2Rγ ECD or a functional analog thereof, and C is a cytokine moiety, the isolated fusion molecule according to embodiment 12. 25. The isolated fusion molecule according to any one of embodiments 22 to 24, comprising an IL-2 mutein, wherein the IL-2Rβ ECD has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3, the IL-2Rγ ECD has the amino acid sequence shown in SEQ ID NO: 6, and the cytokine moiety has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2. 26. An isolated fusion molecule according to any one of embodiments 21 to 25, wherein the Fc domain comprises a knob-in-hole mutation. 27. The isolated fusion molecule according to any one of embodiments 18 to 26, wherein the knob-into-hole mutation comprises a T366Y "knob" mutation on the polypeptide chain of the Fc domain and a Y407T "hole" mutation in the other polypeptide of the Fc domain (EU numbering). 28. The isolated fusion molecule according to any one of embodiments 18 to 21 and 26, wherein the knob-into-hole mutation comprises mutations of Y349C and / or T366W in the CH3 domain of the "knob chain" and mutations of E356C, T366S, L368A and / or Y407V (EU numbering) in the CH3 domain of the "hole chain". 29. The carrier moiety is an Fc domain of an antibody, and the fusion molecule comprises a first polypeptide chain having an amino acid sequence that is at least 99% identical to one selected from SEQ ID NOs: 8 to 11, 28, 29 and 30 and a second polypeptide chain having an amino acid sequence that is at least 99% identical to one selected from SEQ ID NOs: 16 to 21, the isolated fusion molecule according to embodiment 12. 30. An isolated fusion molecule according to embodiment 12, wherein the carrier part is an antibody Fc domain, and the fusion molecule comprises a first polypeptide chain having an amino acid sequence that is at least 99% identical to one selected from SEQ ID NOs: 12 to 15, 31, 32, and 33, and a second polypeptide chain having an amino acid sequence that is at least 99% identical to one selected from SEQ ID NOs: 22 to 27. 31. An isolated fusion molecule according to embodiment 29 or 30, wherein the Fc domain further comprises a mutation of N297A or N297G (EU numbering). 32. An isolated fusion molecule according to embodiment 12, wherein the carrier is IgG4 Fc and also comprises a knob-into-hole mutation. 33. An isolated fusion molecule according to embodiment 32, comprising a first polypeptide chain having an amino acid sequence that is at least 99% identical or 100% identical to one selected from SEQ ID NOs: 50, 51, and 52, and a second polypeptide chain having an amino acid sequence that is at least 99% identical or 100% identical to one selected from SEQ ID NOs: 53 and 54. 34. An isolated fusion molecule according to any one of embodiments 22 to 24, wherein the peptide linkers L1, L2, and L3 independently have an amino acid sequence selected from SEQ ID NOs: 40 to 46, 55 to 57, 59, and 60. 35. An isolated fusion molecule according to any one of embodiments 22 to 24, wherein at least one of the peptide linkers L1, L2, and L3 has an amino acid sequence containing 20 to 44 amino acids. 36. An isolated fusion molecule according to any one of embodiments 7 to 11, which binds to the high-affinity IL-2 receptor (IL-2Rαβγ) having α, β, and γ subunits with an affinity that is at least 100-fold stronger than binding to the intermediate-affinity IL-2 receptor (IL-2Rβγ) formed by the β and γ subunits. 37. An isolated fusion molecule according to any one of embodiments 7 to 11, which binds to IL-2Rβγ with a binding dissociation equilibrium constant (K D ) of about 5 nM or more as measured by a surface plasmon resonance assay at 37°C. 38. An isolated fusion molecule according to any one of embodiments 7-37 that promotes the growth or survival of FOXP3-positive regulatory T cells in vitro. 39. An isolated fusion molecule according to any one of embodiments 7-37 that induces phosphorylation of STAT5 in ex vivo FOXP3-positive T cells that constitute a functional IL-2 receptor complex, but has a reduced ability to induce phosphorylation of STAT5 in FOXP3-negative T cells. 40. Further comprising an extracellular domain (ECD) of IL-2Rα or a functional analog thereof; the IL-2Rα ECD or a functional analog thereof is linked to the fusion molecule via a cleavable peptide linker. A fusion molecule according to any one of embodiments 7-11. 41. The fusion molecule according to embodiment 40, wherein the IL-2Rα ECD or a functional analog thereof comprises an amino acid sequence that is at least 95% identical to that shown in SEQ ID NO: 7. 42. One or more polynucleotides encoding the fusion molecule according to any one of embodiments 7-41 or the IL-2Rβ-ECD variant according to any one of embodiments 1-6. 43. One or more expression vectors comprising the one or more polynucleotides of embodiment 42. 44. A host cell comprising the vector of embodiment 43. 45. A method for producing an isolated fusion molecule according to any one of embodiments 7-41, comprising culturing the host cell according to claim 44 under conditions that allow expression of the fusion molecule and isolating the fusion molecule. 46. A pharmaceutical composition comprising a chimeric molecule according to any one of embodiments 7-41 and a pharmaceutically acceptable excipient. 47. A method for treating an inflammatory disease or an autoimmune disease in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a chimeric molecule according to any one of embodiments 7-41. 48. A method for treating an inflammatory disease or an autoimmune disease in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition of embodiment 46. 49. The method according to embodiment 48, wherein the inflammatory or autoimmune disease is selected from the group consisting of asthma, diabetes, arthritis, allergies, organ transplant rejection, and graft-versus-host disease.

Example

[0094] Transient transfection In transient transfection using HEK293 cells, an expression plasmid was co-introduced at 2.5 - 3 μg / mL into free-style HEK293 cells at 3×10 6 cells / mL using PEI (polyethyleneimine). For the Fc-based isolated IL-2 fusion molecule, the ratio of the Fc-IL-2 mutein fusion polypeptide to the Fc masking moiety fusion polypeptide was 1:2. For the antibody-based isolated IL-2 fusion molecule, the ratio of the knob heavy chain (including the IL-2 agonist polypeptide) and the whole heavy chain (including the masking moiety) to the light chain DNA was 2:1:2 molar ratio. On the 6th day after transfection, after centrifugation at 9,000 rpm for 45 minutes, the cell culture was recovered by 0.22 μM filtration.

[0095] For transient transfection using ExpiCHO cells, using Expifectamine CHO Reagent, 6×10 6 cells / mL ExpiCHO-S cells were co-transfected with the expression plasmid at 1 - 2 μg / mL. For 982 D1, the ratio of the knob heavy chain IL-2 mutein fusion polypeptide to the whole heavy chain (including the β masking moiety polypeptide) was 1:4. Similarly, for 982 D2, the ratio of the knob heavy chain IL-2E mutein polypeptide to the whole heavy chain (including the β masking moiety polypeptide) was 1:4. After centrifugation at 12,000 rpm for 40 minutes, 0.2 μM or 0.45 μM filtration was performed, and the cell culture was collected approximately 7 days after transfection.

[0096] Protein purification Purification of the protein IL-2 fusion molecule (protein) was performed using Protein A affinity chromatography CaptivA® resin (Repligen, Waltham, MA). For samples 982 Ref, 982 C1, and 982 C2, anion exchange chromatography using Sepharose® Q FF resin or Sepharose® Q HP resin was carried out in flow-through mode, followed by Capto TM A third column step using MMC ImpRes resin was performed for further purification. For samples 982 D1 and 982 D2, anion exchange chromatography using Sepharose® Q HP resin was carried out in flow-through mode, followed by Capto TM A third column step using SP ImpRes resin was performed for further purification. Sepharose® and Capto TM All resins were obtained from GE Healthcare Life Sciences (now Cytiva, Marlborough, MA). This sample was purified to a purity of over 98% by SEC-HPLC analysis prior to in vivo testing. The sample was formulated with 20 mM histidine, 7% sucrose, and 0.03% polysorbate-20. The sample was stored in an -80 °C freezer until use. Protease treatment

[0097] 0.1 μg / μL of Human MMP2 (Sino Biological #10082-HNAH) was activated with 1 mM p-aminophenylmercury acetate (APMA, Sigma #A-9563). 200 μg of the IL-2 fusion molecule was incubated with 0.5 μg of human MMP2 in HBS buffer (20 mM HEPES, 150 mM NaCl 2 and 10 μM ZnCl 2 containing) at 37 °C for 16 hours (overnight). 2 pH 7.4) for 16 hours (overnight) at 37 °C.

[0098] SDS-PAGE analysis 10 μL of the culture supernatant or 20 μg of the purified protein sample was mixed with Bolt TM LDS Sample Buffer (Novex) with or without a reducing reagent. After heating at 70 °C for 3 minutes, it was loaded onto a NuPAGE TM 4–12% BisTris Gel (Invitrogen). The gel was run at 200 V for 40 minutes in NuPAGE TM MOPS SDS Running buffer (Invitrogen) and then stained with Coomassie blue.

[0099] Figure 8 shows the results of SDS-PAGE analysis of the isolated IL-2 fusion molecule JR3.116.5 before activation (non-reduced and reduced) and after activation by the above protease treatment. JR3.116.5 contains two polypeptide chains with the amino acid sequences shown in SEQ ID NOs: 12 and 23, respectively, and has the structure shown in Figure 1B. The data indicated that the majority of the pool from the protein A column was the intended heterodimeric molecule of JR3.116.5. There appeared to be a small band of the homodimer of the heavy chain (SEQ ID NO: 23). Surprisingly, there were no obvious bands of the homodimers of the unpaired chain or any knob chain. The interaction between the cytokine portion and the mask site may have promoted the correct heterodimerization of the knob chain (SEQ ID NO: 12) and the heavy chain (SEQ ID NO: 23).

[0100] CTLL-2 assay CTLL-2 cells were cultured in RPMI 1640 medium supplemented with L-glutamine, 10% fetal bovine serum, 10% non-essential amino acids, 10% sodium pyruvate, and 55 μM β-mercaptoethanol. CTLL-2 cells are non-adherent and were maintained at 5×10 4 ~1×10 6 cells / mL in medium supplemented with 100 ng / mL of IL-2. Generally, the cells were split twice a week. It was optimal to use cells more than 48 hours after passage in the bioassay.

[0101] Samples were diluted to double concentration at 50 μL / well in a 96-well plate. The IL-2 standard solution was serially diluted threefold from 20 ng / mL (double concentration) and the titers were measured in 12 wells. The titer of the samples was tested as appropriate. After washing CTLL-2 cells five times to remove IL-2, 5000 cells / well were dispensed into 50 μL and cultured overnight or at least 18 hours using the samples. Then, 100 μL / well of Cell Titer Glo reagent (Promega) was added and luminescence was measured. The analysis results of CTLL-2 are shown in Figure 9. This data indicates that the masked portion decreased the activity of JR3.116.5 by approximately 20-fold. Furthermore, this masking effect is reversible, and when the masked portion is cleaved and activated with protease, the activity of the fusion molecule is restored.

[0102] NK92 cell proliferation assay The NK92 cell line is a factor-dependent cell line that requires IL-2 for growth and survival. Before the assay, NK92 cells were washed to remove IL-2 and cultured overnight without growth factors. The cells were harvested, washed again to remove residual growth factors, and resuspended at 4,000,000 cells / mL. Then, the cells (20,000 / well) were added to a 96-well plate. Basiliximab, an anti-CD25 antibody, was added at 10 μg / mL to half of the plate (48 wells). The cells were incubated for 15 minutes. Serial titrations of the IL-2 fusion molecule were added to each well at 50 μL / well. The plate was incubated overnight and Cell Titer Glo (Promega) was added before measuring luminescence. This allowed the measurement of the ATP level, which is an indicator of cell viability. Figure 11 shows the NK92 proliferation assays of 982 D1, 982 D1, and 982 Ref in the presence and absence of the anti-CD25 neutralizing antibody. The control molecule (982 Ref) is an IL-2 fusion molecule with IL-2 having substitution mutations V91K and C125A (the numbering of the IL-2 portion follows SEQ ID NO: 1). In the assay using the anti-CD25 antibody, the anti-CD25 antibody was added to the cells at 10 μg / mL.

[0103] From this data, it was found that 982 Ref has an activity to promote the proliferation of NK92 cells stronger than 982 D1 and 982 D2. All the fusion molecules tested showed minimal activity when a neutralizing anti-CD25 antibody was added to the assay.

[0104] Binding assay: Rat CD4 + T cells Blood collected from Sprague-Dawley male rats via jugular cannula was hemolyzed to remove red blood cells. The remaining cells were incubated with test articles 982 D1 or 982 D2 at various concentrations on ice for approximately 60 minutes. Goat anti-human IgG Fcγ-APC (Jackson ImmunoResearch Lab. Cat#109-135-170), the detection antibody, was added to each well. After incubation and subsequent washing, an anti-rat CD4 antibody (BD Bioscience, cat#554866) was added to stain rat CD4 + T cells to detect the IL-2 fusion molecule that binds to them.

[0105] Figure 12 shows the binding activity of 982 D1 and 982 D2 to rat CD4 + T cells. N.C. represents an irrelevant Ab control. Surprisingly, 982 D1 had slightly stronger binding to rat CD4 + T cells than 982 D2, but the difference was not significant.

[0106] Binding assay: Human CD4 + CD25 + T cells Human peripheral blood mononuclear cells (hPBMCs) were isolated from buffy coat blood (BioIVT and RBC) and cultured overnight in complete medium RPMI 1640 (Life Technologies, cat# 12633-020) containing 10% FBS (Life Technologies, cat# 10099141). The next day, human PBMCs were treated with anti-human CD3 antibody (Biolegend cat#317302) for 2 days, washed 3 times with complete medium RPMI 1640, and then left standing for 3 days. After adjusting the concentration to 4 - 5×10 6 cells / mL, 50 μL of cells (200 - 250K cells / well), and then 50 μL of IL-2 fusion molecules 982 C1, 982 D1, 982 D2, 982 Ref at various concentrations were loaded into the corresponding wells of a 96-well plate. As a negative control, irrelevant Ab was added in the same various concentration ranges. After incubating on ice for approximately 60 minutes, the cells were washed and the detection antibody, goat anti-human IgG Fcγ-APC (Jackson ImmunoResearch Lab. Cat#109-135-170), was added. After removing free IgG Fcγ-APC by extensive washing, FITC-labeled mouse anti-human CD4 Ab (BD bioscience, cat#555346) and PE-labeled mouse anti-human CD25 Ab (BD bioscience, cat#555432) were added to the wells for cell staining. Finally, to detect the binding of IL-2 fusion molecules to Treg (CD4 + CD25 + ) and T eff (CD4 + CD25 - ) cells, the stained samples were analyzed by flow cytometry.

[0107] Figures 13A and 13B show the binding of 982 C1, 982 D1, 982 Ref to human CD4 + / CD25 + T cells and CD4 + / CD25 - T cells. The results show that 982 Ref binds to CD4 + / CD25 +It showed strong binding affinity for T cells. In this assay, PBMCs were treated with anti-CD3 antibody for 2 days, rested for 3 days, and then incubated with various concentrations of IL-2 fusion molecules 982 C1, 982 D1, 982 Ref, and buffer control (N.C.) at room temperature for approximately 40 minutes. Anti-hFc secondary antibody was added, followed by staining with anti-CD4 antibody and anti-CD25 antibody. The stained samples were analyzed by flow cytometry to detect the binding of IL-2 fusion molecules to Treg (CD4 + CD25 + ) and T eff (CD4 + CD25 - ) cells, respectively. Subsequently, when 982 C1, 982 D1, and 982 D2 were compared, it was shown that the binding ability was high in the order of 982 D2 > 982 D1 > 982 C1. The IL-2 portion of 982 D2 has two point mutations that enhance the binding to CD25. From these results, it was suggested that the binding of 982 D1 decreased when masked with IL-2Rβ-ECD, and the binding of the masked IL-2 fusion molecule 982 C1 further decreased with double masking of IL-2Rβ-ECD and IL-2Rγ-ECD. CD4 + CD25 - T cells also showed the same ranking of binding activity, but the MFIs of 982 Ref, 982 D1, 982 Ref, 82 D2, and 982 C1 were lower than those of CD4 + CD25 + T cells, indicating that the IL-2 fusion molecule preferentially binds to CD4 + CD25 + T cells.

[0108] T cell proliferation assay Human PBMCs (BioIVT and RBC) separated from buffy coat blood were treated with anti-CD3 antibody (Biolegend cat#317302) for 2 days and then left standing for 3 days. The indicated various concentrations of IL-2 fusion molecules 982 C1, 982 D1, 982 D2, 982 Ref, or IL-2 were incubated at 37°C, 5% CO 2Incubated for 3 days in an incubator. Thereafter, the cells were lysed / fixed / permeabilized and antibody staining was performed with mouse anti-human CD4-FITC (BD bioscience, cat#555346), mouse anti-human CD25 - PE (BD bioscience, cat#555432), and mouse anti-human Ki67 Alex-647 (BD bioscience, cat#558615). After washing, the stained cells were analyzed by flow cytometry to identify Ki67+ (proliferation marker) cells of Treg (CD4 + CD25 + ) and T eff (CD4 + CD25 - ).

[0109] Figure 14 shows the concentration-dependent proliferation of CD4 + CD25 + T cells and CD4 + CD25 - T cells induced by 982 D1, 982 C1, 982 D2, and 982 Ref IL-2 fusion molecules. In this assay, PBMCs were treated with anti-CD3 antibody for 2 days and then rested for 3 days. Thereafter, the PBMCs were incubated with various concentrations of IL-2 fusion molecules 982 C1, 982 D1, 982 D2, and 982 Ref in an incubator at 37°C, 5% CO 2 for 3 days. Thereafter, the cells were lysed / fixed / permeabilized and stained with anti-CD4, CD25, and Ki67 antibodies. After washing, the stained cells were analyzed by flow cytometry to identify Ki67 + CD25 + cells of Treg (CD4 eff (CD4 + CD25 - ) and T + (proliferation marker) cells of (CD4

[0110] The in vitro activities of the IL-2 fusion molecules were in the following order, from strong to weak: 982 Ref, 982 D2, 982 D1, 982 C1. For CD4 + CD25 + T cells, they were more than CD4+ CD25 - Overall, much greater proliferation was observed than in T cells. As a result, in all three in vitro assays, 982 Ref showed the strongest activity, followed by 982 D2, 982 D1, and 982 C1 in that order. These results are consistent with the binding activity results shown in Figure 13.

[0111] Rat PK and PD studies Male Sprague-Dawley rats equipped with jugular vein cannulas were administered the IL-2 fusion molecule subcutaneously at 1 mg / kg or 3 mg / kg. Blood was collected at various time points from 0 to 144 hours.

[0112] For PK analysis, serum samples were assayed for the test article by ELISA. Briefly, ELISA plates were coated with 100 μL / well of 2 μg / mL F(ab’) in PBS 2 goat anti-human IgG Fcγ (Jackson ImmunoResearch, Cat.# 109-006-170). The plates were incubated overnight at 4°C. The plates were blocked with 100 μL / well of PBS containing 10% goat serum. After 1 hour of incubation and subsequent washing (4 times with pure water), 100 μL of serum samples diluted with PBS / 10% goat serum or standard solution were added to each well. After incubation (1 hour) and washing (6 times with pure water), the secondary antibody (anti-IL2-biotin (RandD Systems BAF202)) was added to each well at 0.5 μg / mL in 100 μL of PBS / 10% goat serum. After incubation (1 hour) and washing (6 times with pure water), 100 μL of Streptavidin-HRP (Jackson ImmunoResearch, Cat.#016-30-84, 1:1000) dissolved in PBS / 10% goat serum was added to each well. After incubation (1 hour), washing (8 times with DI water), 100 μL of TMB substrate was added to each well to initiate the color reaction. 1N H 2 SO 4 solution was added at 100 μL / well to stop the reaction. Then, OD450 was measured.

[0113] Figure 15 shows the changes over time in the serum and plasma concentrations of 982 C1, 982 D1, and 982 Ref IL-2 fusion molecules in a rat PK test. In this assay, male Sprague-Dawley rats equipped with jugular vein cannulas were subcutaneously administered with IL-2 fusion molecules 982 C1, 982 D1, and 982 Ref at 1 mg / kg. Blood was collected at 0 h, 1 h, 3 h, 6 h, 10 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h. Serum samples were measured for IL-2 fusion molecules by ELISA using goat anti-human IgG Fcγ capture and anti-human IL-2 biotin as detection reagents. 982 C1 had a larger AUC (0-t) (area under the concentration-time curve to the last measurable concentration) than 982 D1. On the other hand, both of them had a significantly larger AUC (0-t) than 982 Ref.

[0114] Figure 16 shows the changes over time in the serum and plasma concentrations of 982 D1, 982 Ref, and 982 D2 IL-2 fusion molecules in the second rat test. In this assay, male Sprague-Dawley rats equipped with jugular vein cannulas were subcutaneously administered with IL-2 fusion molecules 982 D1, 982 D2, and 982 Ref at 1 mg / kg and 982 D1 at 3 mg / kg as indicated. Blood was collected at 0 h, 1 h, 3 h, 6 h, 10 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h. Serum specimens were measured for the test articles by ELISA using goat anti-human IgG Fcγ capture and anti-human IL-2 biotin as detection reagents. 982 D2 had a larger AUC(0-t) than 982 D1, and both of them had a significantly larger AUC(0-t) than 982 Ref. Also, the measurement of plasma concentration over time showed that the PK profile of the masked IL-2 fusion molecule was better than that of 982 Ref with the V91K mutation in the IL-2 portion.

[0115] In the PD analysis, 982 molecules were subcutaneously injected, and blood was collected at various time points between 0 and 144 hours. Blood samples collected in K2 EDTA collection tubes from rats administered with 982 IL-2 fusion molecules were mixed with 1 volume of pre-warmed BD Phosflow TM lyse / fix buffer (1×, BD Biosciences cat# 558049) for lysis and fixation according to the manufacturer's recommendation. After washing the blood samples twice with PBS containing 2% FBS, permeabilization was performed on ice for 30 minutes using cold permeabilization buffer II (BD Biosciences cat#558052, -20°C) according to the manufacturer's instructions. Then, the samples were extensively washed four times with PBS containing 2% FBS, and the cell pellet was stored at 4°C or resuspended in staining buffer.

[0116] For the measurement of FOXP3 and Ki67, the above-fixed / permeabilized rat blood cells (300k - 400K cells / well) from each sampling were added to a 96-well working plate in 50 μL / well aliquots. Next, 50 μL of an Ab mixture containing mouse anti-rat CD4-FITC (Biolegend, cat#201505), mouse anti-rat CD25 - PE (BD Bioscience, cat#554866), mouse anti-rat FOXP3-APC (Biolegend, cat#320014); or mouse anti-Ki67-APC (Biolegend, cat#320514) was added to each well, and the cells in the plate were incubated at room temperature for 1 hour. After washing the plate twice with FACS buffer, flow cytometry analysis was performed to determine the percentage change of Treg (CD4 + FOXP3 + ) cells and T eff (CD4 + FOXP3 - ) cells over time. Also, flow cytometry was used to examine the time-course change of Ki67 + (proliferation marker) cells.

[0117] Figures 17A and 17B show the changes induced by 982 C1, 982 D1, and 982 Ref IL-2 fusion molecules in CD4 + / FOXP3 + and CD4 + / FOXP3 - cells (rat). Figures 18A and 18B show the CD4 induced by 982 C1, 982 D1, and 982 Ref IL-2 fusion molecules in rats in the first test + CD25 + and CD4 + CD25 - cell proliferation. Male Sprague-Dawley rats equipped with jugular cannulas were subcutaneously administered 1 mg / kg of IL-2 fusion molecules 982 C1, 982 D1, and 982 Ref. Blood was collected at 0, 24, 48, 96, and 144 hours, and Ab staining was performed after lysing / fixing / permeabilizing the blood samples. Subsequently, the percentage changes in Treg (CD4 + FOXP3 + ) and T eff (CD4 + FOXP3 - ) cells were analyzed respectively. Also, the time-course changes of Treg (CD4 + CD25 + ) and T eff (CD4 + CD25 - ) were measured respectively (Figures 17A, 17B) or the time-course changes of Ki67+ (proliferation marker) cells were measured (Figures 18A, 18B). Figures 19A and 19B show the results of the changes in CD4 + / FOXP3 + and CD4 + / FOXP3 - cells induced by 982 IL-2 fusion molecules in rats. Figures 20A and 20B show that the 982-IL-2 fusion molecule affects CD4 + / CD25 + and CD4 + / CD25 -Shows the results of inducing cell proliferation. Male Sprague-Dawley rats equipped with jugular cannulas were subcutaneously administered 1 mg / kg and 3 mg / kg of IL-2 fusion molecule 982 D1, 1 mg / kg of 982 D2, and 1 mg / kg of 982 Ref. Blood was collected at 0 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours, and Ab staining was performed after lysing / fixing / permeabilizing the blood samples. Subsequently, the time-course changes of Treg (CD4 + FOXP3 + ) and T eff (CD4 + FOXP3 - ) cells were analyzed respectively. Also, when examining the time-course changes of Ki67+ (proliferation marker) cells using flow cytometry, time-course changes were observed in gated Treg (CD4 + CD25 + ) cells and T eff (CD4 + CD25 - ) cells respectively (Figures 20A and 20B).

[0118] The results showed that 982 D1 showed a greater and longer effect on CD4 + FOXP3 + T cells and CD4 + CD25 + T cells compared to 982 Ref. This is surprising considering that the in vitro activity of 982 Ref was significantly higher. Similar results were obtained in the second rat in vivo test (Figures 19A, 19B, 20A, 20B). Surprisingly, 982 D1 compared to 982 D2 (Figures 19A, 19B, 20A, 20B), the CD4 + / FOXP3 + T cells, CD4 + / CD25 + and CD4 + / CD25 -By stimulating cell proliferation, a higher effect was exerted. This is in good agreement with the fact that in the binding assay for rat CD4 T cells, 982 D1 showed slightly higher binding activity than 982 D2 (Figure 12). However, the difference in activity observed between 982 D1 and 982 D2 was more apparent in vivo than in vitro (Figures 19A and 20A).

[0119] 982 D1 and 982 Ref showed selectivity for preferentially stimulating Treg cells rather than T cells, but the stimulating activity of T cells by 982 D1 was hardly observed compared to 982 Ref, indicating that 982 D1 has better selectivity than 982 Ref (Figures 18B and 20B). eff cells, but the stimulating activity of T cells by 982 D1 was hardly observed compared to 982 Ref, indicating that 982 D1 has better selectivity than 982 Ref (Figures 18B and 20B). eff +

[0120] Body weight Also, to evaluate the safety of the IL-2 fusion molecule, the body weights of animals were measured during a 6-day test period. The animals were administered 982 D1 at 1 mg / kg and 3 mg / kg, 982 D2 at 1 mg / kg, and 982 Ref at 1 mg / kg, respectively, by single subcutaneous injection. Body weight (BW) was measured daily for each animal from day 0 (administration) to day 6. The results are shown in Figure 21. Rats administered 982 D1 at 1 mg / kg and 3 mg / kg showed an increase in body weight compared to rats administered 982 Ref at 1 mg / kg.

[0121] As described above, in in vitro and in vivo tests, it was revealed that the masked IL-2 fusion molecule 982 D1 has a surprisingly superior PK profile compared to 982 Ref, which is a homodimeric IL-2 fusion molecule containing mutations V91K and C125A in the IL-2 portion. Surprisingly, 982 D1, compared to 982 Ref, for rat CD4 + CD25 + T cells and CD4 + FOXP3 +It had potent in vivo activity that stimulated the proliferation of T cells (Figs. 17A, 17B, 18A, 18B, 19A, 19B, 20A, 20B). Furthermore, all three masked IL-2 fusion molecules (982 C1, 982 D1, 982 D2) had longer PK than 982 Ref (Figs. 15 and 16). Also, it was surprising that 982 D1 had stronger in vivo activity in rats than 982 D2. Despite 982 D1 having relatively low activity in vitro compared to 982 D2 and 982 Ref., it had excellent in vivo activity compared to other molecules tested in the same rat assay. Furthermore, body weight data (Fig. 21) suggested that 982 D1 might be safer than 982 Ref. Also, the potent and selective in vivo activity of the masked IL-2 fusion molecule 982 D1 was surprising in that it could be achieved without the need for protease-dependent cleavage or removal of the masking moiety, as 982 D1 was not composed of any cleavable peptide linker. Such a novel mechanism of action is desirable as the distribution of protease(s) at the disease site may not be uniform, and non-specific cleavage and removal (or "leakage") of the masking moiety may occur in circulation or outside of other normal tissues and the disease site.

[0122] Although not bound by theory, differences in PK profiles may contribute in part to explaining the superior in vivo activity of 982 D1 compared to 982 Ref. Also, the observed differences in in vivo activity between 982 D1 and D2 may be explained by cross-reactivity between chemical species. Although not bound by theory, when the fusion molecule binds to CD25, the long linker between the masking portion of 982 D1 and the carrier may also facilitate competition between the endogenous IL-2Rβ ECD and the masking portion when both endogenous IL-2Rα and IL-2Rγ are present. For 982 D1 to promote the proliferation of Treg cells, it is necessary for the cytokine portion to bind to both endogenous IL-2Rβ and IL-2Rγ. The long linker between the masking portion, IL-2Rβ-ECD, and the carrier is thought to provide the flexibility necessary for the cytokine portion to form a tetrameric complex with endogenous IL-2Rα, IL-2Rβ, and IL-2Rγ. If the linker between the masking portion, IL-2Rβ-ECD, and the carrier is short, especially if the linker between the cytokine portion and the carrier is also short, the masking portion may pose a special constraint for the formation of the four-molecule complex. Figure 11 shows that although 982 D2 has stronger in vitro activity with human T cells than 982 D1, 982 D2 has slightly weaker binding affinity to rat CD4 + T cells. However, the difference in binding affinity to rat CD4 + cells is relatively small and may not explain the significant difference in in vivo activity between D1 and D2 (19A, 19B, 20A, 20B). The present invention further encompasses the following aspects. 1. An isolated IL-2 fusion molecule comprising a carrier moiety, a cytokine moiety, and one or more masking moieties, wherein the cytokine moiety is fused to the carrier moiety or the masking moiety, one or more masking moieties are fused to the carrier moiety or the cytokine moiety, the cytokine moiety comprises an IL-2 polypeptide comprising an IL-2 amino acid sequence comprising either a C125A or C125S substitution or one or more substitutions selected from T3A, C125S, V69A, and Q74P (numbering according to SEQ ID NO: 1), one or more masking moieties bind to the cytokine moiety and inhibit binding of the cytokine moiety to IL-2Rβ and / or IL-2Rγ but not to IL-2Rα on immune cells, isolated IL-2 fusion molecule. 2. A method of treating an inflammatory condition or an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of an isolated IL-2 fusion molecule comprising a carrier moiety, a cytokine moiety, and one or more masking moieties, wherein the cytokine moiety is fused to the carrier moiety or the masking moiety, one or more masking moieties are fused to the carrier moiety or the cytokine moiety, the cytokine moiety comprises an IL-2 polypeptide, and one or more masking moieties bind to the cytokine moiety and inhibit binding of the cytokine moiety to IL-2Rβ and / or IL-2Rγ on immune cells but do not inhibit binding to IL-2Rα, method. 3. The method of item 2, wherein the inflammatory condition or autoimmune disease is selected from the group consisting of asthma, type I diabetes, rheumatoid arthritis, allergy, systemic lupus erythematosus, multiple sclerosis, organ transplant rejection, and graft-versus-host disease. 4. The IL-2 fusion molecule has the following characteristics: (a) Binds to the high-affinity IL-2 receptor (IL-2Rαβγ) having α, β, and γ subunits with an affinity more than 100-fold that of the intermediate IL-2 receptor (IL-2Rβγ) having β and γ subunits, (b) K measured by surface plasmon resonance assay at 37°C D that binds to IL-2Rβγ at about 5 nM or more or 10 nM or more, (c) in a CTLL-2 cell proliferation assay, an EC of less than about 1 nM, 0.01 nM, 0.25 nM or 0.05 nM or more 50 having a value of, (d) in a proliferation assay of NK92 cells, an EC greater than about 0.05 nM, 0.1 nM, 0.25 nM or 0.5 nM 50 having a value of, (e) in a proliferation assay of NK92 cells in the presence of a neutralizing CD25 antibody, the Emax value is at least 5-fold or at least 10-fold lower compared to the absence of the neutralizing CD25 antibody, (f) preferentially stimulates FOXP3 + regulatory T cells compared to T effector cells and NK cells, (g) promotes the growth or survival of FOXP3 + regulatory T cells, and (h) induces phosphorylation of STAT5 in FOXP3 + T cells but has a reduced inducing ability in FOXP3 - T cells, The IL-2 fusion molecule of claim 1 or the method of claim 2 or 3 having one or more of the above. 5. The IL-2 fusion molecule according to any one of claims 1 to 4, or the method, wherein the IL-2 fusion molecule comprises a masking portion comprising an extracellular domain (ECD) of IL-2Rβ or IL-2Rγ or a functional analog thereof, and the masking portion is fused to the carrier portion with or without using a peptide linker. 6. The IL-2 fusion molecule comprises the following portions: A first masking portion comprising an extracellular domain (ECD) of IL-2Rβ or IL-2Rγ or a functional analog thereof, the first masking portion being fused to the carrier portion with or without using a peptide linker, and A second masking portion comprising an ECD of IL-2Rγ or IL-2Rβ or a functional analog thereof, the second masking portion being fused to the cytokine portion or the first masking portion with or without using a peptide linker The IL-2 fusion molecule according to any one of claims 1 to 4, or the method, comprising the above. 7. The IL-2 fusion molecule according to claim 5 or 6, or the method, wherein the IL-2Rβ ECD or a functional analog thereof has an amino acid sequence that is at least 95% identical to SEQ ID NO: 3. 8. The IL-2 fusion molecule according to any one of claims 5 to 7, or the method, wherein the IL-2Rγ ECD or a functional analog thereof has an amino acid sequence that is at least 95% identical to SEQ ID NO: 6. 9. An IL-2 fusion molecule or method according to any one of items 1 to 8, wherein the IL-2 polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, and optionally, the amino acid sequence is SEQ ID NO: 2. 10. An IL-2 fusion molecule or method according to any one of items 1 to 9, wherein the carrier moiety is selected from a PEG molecule, albumin, an albumin fragment, an antibody Fc domain, an antibody or an antigen-binding fragment thereof. 11. An IL-2 fusion molecule or method according to any one of items 1 to 10, wherein the cytokine moiety is fused to the carrier moiety or the masking moiety via a non-cleavable peptide linker, and the masking moiety is fused to the carrier moiety or the cytokine moiety via a non-cleavable peptide linker. 12. An IL-2 fusion molecule or method according to item 11, wherein the masking moiety is fused to the carrier moiety or the cytokine moiety via a peptide linker comprising at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 22 amino acids, at least 25 amino acids, at least 30 amino acids or up to 44 amino acids. 13. The carrier moiety is an antibody Fc domain, and the fusion molecule is: a first polypeptide chain composed of a molecular formula selected from F1-L1-E1, F1-L1-E1-L2-E2, and F1-L1-E2-L2-E1 in the direction from the N-terminus to the C-terminus and a second polypeptide chain composed of the molecular formula F2-L3-C in the direction from the N-terminus to the C-terminus a heterodimer comprising, wherein F1 and F2 are subunits of the Fc domain, L1, L2, and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, E2 is an IL-2Rγ ECD or a functional analog thereof, C is a cytokine moiety, An IL2 fusion molecule or method according to any one of items 1 to 12. 14. The carrier moiety is an antibody Fc domain, and the fusion molecule is: a first polypeptide chain composed of a molecular formula selected from E1-L1-F1, E1-L1-E2-L2-F1, and E2-L1-E1-L2-F1 in the direction from the N-terminus to the C-terminus and a second polypeptide chain composed of the molecular formula C-L3-F2 in the direction from the N-terminus to the C-terminus a heterodimer comprising, wherein F1 and F2 are subunits of the Fc domain, L1, L2, and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, and E2 is an IL-2Rγ ECD or a functional analog thereof, C is a cytokine moiety, An IL-2 fusion molecule or method according to any one of items 1 to 12. 15. The carrier moiety is an antibody Fc domain, and the fusion molecule has, in the N-terminal to C-terminal direction, respectively, the following pairs: F1-L1-E1 and F2-L2-C-L3-E2, F1-L1-E1 and F2-L2-E2-L3-C, F1-L1-E2 and F2-L2-C-L3-E1, F1-L1-E2 and F2-L2-E1-L3-C, E1-L1-F1 and E2-L2-C-L3-F2, E1-L1-F1 and C-L2-E2-L3-F2, E2-L1-F1 and E2-L2-C-L3-F2 and E2-L1-F1 and C-L2-E1-L3-F2 A heterodimer comprising a first polypeptide chain and a second polypeptide chain composed of a molecular formula selected from, wherein F1 and F2 are subunits of the Fc domain, L1, L2 and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, and E2 is an IL-2Rγ ECD or a functional analog thereof, C is a cytokine moiety, An IL-2 fusion molecule or method according to any one of items 1 to 12. 16. An IL-2 fusion molecule or method according to any one of items 13 to 15, wherein the peptide linkers L1, L2 and L3 are not cleavable. 17. An IL-2 fusion molecule or method according to any one of items 13 to 16, wherein L1, L2 and L3 each independently have an amino acid sequence selected from SEQ ID NOs: 40 to 46, 55 to 57 and 59. 18. An IL-2 fusion molecule or method according to any one of items 13 to 17, wherein at least one of L1, L2 and L3 has an amino acid sequence containing 20 to 44 amino acids. 19. The IL-2 fusion molecule is: A first polypeptide chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 50, 51 or 52 and A second polypeptide chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 53 or 54 An IL-2 fusion molecule or method according to any one of items 13 to 18. 20. The IL-2 fusion molecule is: (a) A first polypeptide chain comprising an amino acid sequence that is 99% or more identical to SEQ ID NO: 50 and a second polypeptide chain comprising an amino acid sequence that is 99% or more identical to SEQ ID NO: 53 or (b) A first polypeptide chain comprising SEQ ID NO: 50 and a second polypeptide chain comprising SEQ ID NO: 53 The IL-2 fusion molecule or method of claim 19, comprising. 21. The IL-2 fusion molecule or method of any one of claims 1-20, wherein the fusion molecule comprises at least two masking moieties, one of which is the ECD of IL-2Rα or a functional analog thereof, and the ECD masking moiety of IL-2Rα is fused to a cytokine moiety, a carrier moiety, or another masking moiety via a cleavable peptide linker. 22. The IL-2 fusion molecule or method of claim 21, wherein the IL-2Rα ECD moiety comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 7. 23. A polynucleotide encoding the IL-2 fusion molecule of any one of claims 1 and 4-22. 24. An expression vector comprising the polynucleotide of claim 23. 25. A host cell comprising the expression vector of claim 24. 26. A pharmaceutical composition comprising the IL-2 fusion molecule of any one of claims 1 and 4-22 and a pharmaceutically acceptable excipient. 27. The IL-2 fusion molecule of any one of claims 1 and 4-22 or the pharmaceutical composition of claim 26 for use in treating a subject by the method of claim 2 or 3. 28. Use of the IL-2 fusion molecule of any one of claims 1 and 4-22 for the manufacture of a medicament for treating a subject in the method of claim 2 or 3.

[0123] Array In the following array, the residues within the box indicate mutations. The underline of the cleavable linker indicates the protease substrate sequence. [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12]

Claims

**Claim 1** An isolated IL-2 fusion molecule comprising a carrier portion, a cytokine portion, and one or more masking portions, wherein the cytokine portion is fused to the carrier portion, one or more masking portions are fused to the carrier portion, the cytokine portion comprises an IL-2 polypeptide comprising an IL-2 amino acid sequence comprising (i) a C125A or C125S substitution or (ii) T3A, C125S, V69A, and Q74P substitutions, wherein the numbering follows SEQ ID NO: 1, the carrier portion is an antibody or an antibody Fc domain, one or more masking portions comprise the extracellular domain (ECD) of IL-2Rβ and / or IL-2Rγ or a functional analog thereof, one or more masking portions bind to the cytokine portion and inhibit binding of the cytokine portion to IL-2Rβ and / or IL-2Rγ but not to IL-2Rα on immune cells, An isolated IL-2 fusion molecule. **Claim 2** The IL-2 fusion molecule has the following properties: (a) Binds to the high-affinity IL-2 receptor (IL-2Rαβγ) having α, β, and γ subunits with an affinity that is at least 100-fold greater than that of the intermediate IL-2 receptor (IL-2Rβγ) having β and γ subunits, (b) K measured by surface plasmon resonance assay at 37 °C Ｄ that binds to IL-2Rβγ at about 5 nM or more or 10 nM or more, (c) In the CTLL-2 cell proliferation assay, having an EC value of less than about 1 nM, 0.01 nM, 0.25 nM or 0.05 nM or more ５０ value (d) In the NK92 cell proliferation assay, an EC value greater than about 0.05 nM, 0.1 nM, 0.25 nM or 0.5 nM ５０ having (e) In a proliferation assay of NK92 cells in the presence of a neutralizing CD25 antibody, has an Emax value that is at least 5-fold or at least 10-fold lower compared to the absence of the neutralizing CD25 antibody, (f) Compared to T effector cells and NK cells, preferentially stimulates FOXP3 ＋ regulatory T cells, (g) FOXP3 ＋ promote the growth or survival of regulatory T cells, and (h) FOXP3 ＋ In T cells, it induces phosphorylation of STAT5 but FOXP3 － In T cells, its inducing ability is reduced, The IL-2 fusion molecule of claim 1 having one or more of the above. **Claim 3** The IL-2 fusion molecule of claim 1 or 2, wherein one or more masking portions are fused to the carrier portion with or without using a peptide linker. **Claim 4** The IL-2 fusion molecule comprises the following portions: A first masking portion comprising the extracellular domain (ECD) of IL-2Rβ or IL-2Rγ or a functional analog thereof, the first masking portion being fused to the carrier portion with or without using a peptide linker, and A second masking portion comprising the ECD of IL-2Rγ or IL-2Rβ or a functional analog thereof, the second masking portion being fused to the first masking portion with or without using a peptide linker The IL-2 fusion molecule according to any one of claims 1 to 3, comprising. **Claim 5** The IL-2 fusion molecule of claim 3 or 4, wherein the IL-2Rβ ECD or a functional analog thereof has an amino acid sequence that is at least 95% identical to SEQ ID NO:

3. **Claim 6** An IL-2 fusion molecule according to any one of claims 3 to 5, wherein the IL-2Rγ ECD or a functional analog thereof has an amino acid sequence that is at least 95% identical to SEQ ID NO:

6. **Claim 7** An IL-2 fusion molecule according to any one of claims 1 to 6, wherein the IL-2 polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, and optionally, the amino acid sequence is SEQ ID NO:

2. **Claim 8** An IL-2 fusion molecule according to any one of claims 1 to 7, wherein the cytokine moiety is fused to the carrier moiety via a non-cleavable peptide linker, and the masking moiety is fused to the carrier moiety via a non-cleavable peptide linker. **Claim 9** An IL-2 fusion molecule according to claim 8, wherein the masking moiety is fused to the carrier moiety via a peptide linker comprising at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 22 amino acids, at least 25 amino acids, at least 30 amino acids or up to 44 amino acids. **Claim 10** The carrier moiety is an antibody Fc domain, and the fusion molecule is: A first polypeptide chain having a molecular formula selected from F1-L1-E1, F1-L1-E1-L2-E2, and F1-L1-E2-L2-E1 in the N-terminal to C-terminal direction and A second polypeptide chain having a molecular formula F2-L3-C in the N-terminal to C-terminal direction A heterodimer comprising, wherein F1 and F2 are subunits of the Fc domain, L1, L2 and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, E2 is an IL-2Rγ ECD or a functional analog thereof, C is the cytokine moiety, An IL-2 fusion molecule according to any one of claims 1 to 9. **Claim 11** The carrier moiety is an antibody Fc domain, and the fusion molecule is: A first polypeptide chain having a molecular formula selected from E1-L1-F1, E1-L1-E2-L2-F1, and E2-L1-E1-L2-F1 in the N-terminal to C-terminal direction and A second polypeptide chain having a molecular formula C-L3-F2 in the N-terminal to C-terminal direction A heterodimer comprising, wherein F1 and F2 are subunits of the Fc domain, L1, L2 and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, E2 is an IL-2Rγ ECD or a functional analog thereof, C is the cytokine moiety, An IL-2 fusion molecule according to any one of claims 1 to 9.

12. The carrier moiety is an antibody Fc domain, and the fusion molecule comprises, in the N-terminal to C-terminal direction, respectively, the following pairs: F1-L1-E1 and F2-L2-C-L3-E2, F1-L1-E1 and F2-L2-E2-L3-C, F1-L1-E2 and F2-L2-C-L3-E1, F1-L1-E2 and F2-L2-E1-L3-C, E1-L1-F1 and E2-L2-C-L3-F2, E1-L1-F1 and C-L2-E2-L3-F2, E2-L1-F1 and E2-L2-C-L3-F2 and E2-L1-F1 and C-L2-E1-L3-F2 A heterodimer comprising a first polypeptide chain and a second polypeptide chain composed of a molecular formula selected from, wherein F1 and F2 are subunits of the Fc domain, L1, L2 and L3 are peptide linkers, E1 is an IL-2Rβ ECD or a functional analog thereof, E2 is an IL-2Rγ ECD or a functional analog thereof, C is a cytokine moiety, An IL-2 fusion molecule according to any one of claims 1 to 11.

13. An IL-2 fusion molecule according to any one of claims 10 to 12, wherein the peptide linkers L1, L2 and L3 are not cleavable.

14. An IL-2 fusion molecule according to any one of claims 10 to 13, wherein L1, L2 and L3 each independently have an amino acid sequence selected from SEQ ID NOs: 40-46, 55-57 and 59.

15. An IL-2 fusion molecule according to any one of claims 10 to 14, wherein at least one of L1, L2 and L3 has an amino acid sequence comprising 20 to 44 amino acids.

16. The IL-2 fusion molecule is: A first polypeptide chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 50, 51 or 52 and A second polypeptide chain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 53 or 54 An IL-2 fusion molecule according to any one of claims 10 to 15.

17. The IL-2 fusion molecule is: (a) A first polypeptide chain comprising an amino acid sequence that is 99% or more identical to SEQ ID NO: 50 and a second polypeptide chain comprising an amino acid sequence that is 99% or more identical to SEQ ID NO: 53 or (b) A first polypeptide chain comprising SEQ ID NO: 50 and a second polypeptide chain comprising SEQ ID NO: 53 An IL-2 fusion molecule according to claim 16.

18. A polynucleotide encoding an IL-2 fusion molecule according to any one of claims 1 to 17.

19. An expression vector comprising the polynucleotide of claim 18.

20. A host cell comprising the expression vector of claim 19.

21. A pharmaceutical composition comprising an IL-2 fusion molecule according to any one of claims 1 to 17 and a pharmaceutically acceptable excipient.

22. The pharmaceutical composition of claim 21 for treating an inflammatory condition or an autoimmune disease.

23. The pharmaceutical composition of claim 22, wherein the inflammatory condition or autoimmune disease is selected from the group consisting of asthma, type I diabetes, rheumatoid arthritis, allergy, systemic lupus erythematosus, multiple sclerosis, organ transplant rejection, and graft-versus-host disease.

Images