Interleukin-2 (il-2) mutants and uses thereof

Abstract

This invention relates to the therapeutic use of IL-2 mutants, fusion proteins containing IL-2 mutants, nucleic acids encoding IL-2 mutants or their fusion proteins, vectors and host cells containing such nucleic acids, or pharmaceutical compositions containing them. It also includes methods for preparing IL-2 mutants and their fusion proteins.

Description

[0001] The contents of the sequence list submitted below are incorporated into this document by reference in their entirety. File name: IL-2-120230516.xml, record date: 2023.05.16, size: 145KB. Technical Field

[0002] This application relates to interleukin-2 (IL-2) mutants, and also includes fusion proteins of said IL-2 mutants, nucleic acid molecules encoding IL-2 mutants or fusion proteins, vectors and host cells containing nucleic acid molecules, pharmaceutical compositions containing the same and therapeutic uses, and further provides methods for preparing IL-2 mutants and their fusion proteins. Background of the Invention

[0004] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a bundle-like type I cell growth factor composed of four α-helices (A, B, C, D). It is mainly produced by antigen-activated T cells and promotes the proliferation, differentiation, and survival of mature T cells and B cells, as well as the cytolytic activity of natural killer (NK) cells in innate immune defense (KASmith, Science 240, 1169 (1988); BHNelson, DMWillerford, Adv. Immunol. 70, 1 (1998)).

[0005] IL-2 functions by binding to IL-2 receptors on the surface of target cells. IL-2 has three receptor subunits: the α chain (IL-2Rα or CD25), the β chain (IL-2Rβ or CD122), and the universal cytokine receptor γ chain (γc or IL-2Rγ or CD132) (WJ Leonard et al., Nature 311, 626 (1984); T. Nikaido et al., Nature 311, 631 (1984); D. Cosman et al., Nature 312, 768 (1984); M. Hatakeyama et al., Science 244, 551 (1989); T. Takeshita et al., Science 257, 379 (1992)).

[0006] Based on the different binding subunits and their affinity for IL-2, IL-2 receptors can be classified into the following types: The first type is the low-affinity receptor, composed of a single IL-2Rα subunit (Kd value approximately 10 nM). Since IL-2Rα lacks an intracellular signal transduction region, IL-2 binding to this type of receptor cannot transmit signals or regulate cells (HMWang, KASmith, J. Exp. Med. 166, 1055 (1987)). The second type is the intermediate-affinity receptor, composed of IL-2Rβ and γc subunits (Kd value approximately 1 nM). This type of receptor is mainly expressed on natural killer cells, macrophages, and resting T cells. At higher IL-2 concentrations, it can activate signaling pathways (BHNelson, DMWillerford, Adv. Immunol. 70, 1 (1998)). Although IL-2Rβ alone has a very low affinity for IL-2 (Kd value approximately 100 nM), and γc alone is almost undetectable in binding to IL-2 (M. Rickert, MJ Boulanger, N. Goriatcheva, K. C. Garcia, J. Mol. Biol. 339, 1115 (2004)), the complex of IL-2Rβ and γc (IL-2Rβγ) is essential for effective IL-2 signaling (Y. Nakamura, et al., Nature 369, 330 (1994); BH Nelson, et al., Nature 369, 333 (1994)). The third type is a high-affinity receptor composed of three subunits: IL-2Rα, IL-2Rβ, and γc (Kd value approximately 10 pM), which is typically found in activated lymphocytes and CD4+ cells. + CD25 + FoxP3 + It is expressed on regulatory T cells (T. Takeshita et al., Science 257, 379 (1992); Boyman O, et al., Nat Rev Immunol. 2012 Feb 17; 12(3):180-90).

[0007] Because IL-2 can enhance the function of effector T cells and natural killer cells, it can be used as an immune activator to treat lymphocytic proliferation-related diseases. Recent studies have found that IL-2 is also a key cytokine for the differentiation, function, and survival of Treg cells, playing a crucial role in the realization of Treg cell function, development, and the induction of their suppressive characteristics. Treg cells are generally defined by expressing CD4, CD25, and the transcription factor forkhead P3 (Foxp3). They can suppress autoreactive lymphocytes and regulate innate and adaptive immune responses, leading to immunosuppression. As one of the key effector cells in immunosuppression, Treg cells play a vital role in inducing and maintaining peripheral self-tolerance to antigens (including those expressed by tumors), and are of great significance in the development and progression of autoimmune diseases. (Malek TR, et al., Nat Rev Immunol 2004; 4:665-74; Nelson BH. J Immunol 2004; 172:3983-8; Piccirillo CA, et al., Semin Immunol 2004; 16:81-8).

[0008] Based on the broad biological effects of IL-2, by altering the selectivity or preference of IL-2 for different receptors, IL-2 can selectively activate target cells, thereby reducing the toxicity of IL-2 therapy and / or improving its efficacy, and increasing the safety window of IL-2 therapy for specific diseases. This is of great significance for improving the safety and effectiveness of IL-2 therapy for diseases, and there is still a need in this field to develop new IL-2 molecules with improved properties.

[0009] All publications, patents, patent applications, and published patent applications mentioned herein are incorporated herein by reference. Invention Overview

[0011] This application relates, in one aspect, to an IL-2 mutant comprising the deletion or mutation of amino acid residues RPRDL at positions 81-85 of the amino acid sequence of human wild-type IL-2. In some embodiments, the amino acid sequence of said human wild-type IL-2 is shown in SEQ ID NO:1.

[0012] In some embodiments, the IL-2 mutant described in this application comprises a complete deletion of amino acid residues RPRDL at positions 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises mutations at E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations at E67 and N71 are E67K and N71S, respectively. In some embodiments, the IL-2 mutant described in this application comprises a complete deletion of amino acid residues RPRDL at positions 81-85 of the human wild-type IL-2 amino acid sequence, and mutations at E67K and N71S relative to the human wild-type IL-2 amino acid sequence.

[0013] In some embodiments, the IL-2 mutant of this application comprises a mutation of RPRDL to RPL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant of this application further comprises a mutation at E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 mutation is E67A. In some embodiments, the IL-2 mutant of this application comprises a mutation of RPRDL to RPL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and an E67A mutation relative to the human wild-type IL-2 amino acid sequence.

[0014] In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RPL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises mutations at N71 and S75 of the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations at N71 and S75 are N71W and S75I, respectively. In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RPL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations at N71W and S75I of the human wild-type IL-2 amino acid sequence.

[0015] In some embodiments, the IL-2 mutant of this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant of this application further comprises mutations in E67 and / or V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation in E67 is E67T. In some embodiments, the mutation in V91 is V91K. In some embodiments, the IL-2 mutant of this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations in E67T and / or V91K relative to the human wild-type IL-2 amino acid sequence.

[0016] In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises mutations at L70 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations at L70 and S75 are L70I and S75F, respectively. In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations at L70I and S75F relative to the human wild-type IL-2 amino acid sequence.

[0017] In some embodiments, the IL-2 mutant of this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises a mutation of N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the N71 mutation is N71I. In some embodiments, the IL-2 mutant of this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and a mutation of N71I relative to the human wild-type IL-2 amino acid sequence.

[0018] In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to SPL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises mutations at E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations at E67 and N71 are E67M and N71S, respectively. In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to SPL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations at E67M and N71S relative to the human wild-type IL-2 amino acid sequence.

[0019] In some embodiments, the IL-2 mutant described in this application, in addition to the mutations described above, further comprises a mutation at C125 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the C125 mutation is C125S or C125A.

[0020] In some embodiments, the IL-2 mutant described in this application comprises any of the amino acid sequences shown in SEQ ID NOs:5-18 or variants thereof, wherein the variants have at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-18.

[0021] This application, in another aspect, relates to another IL-2 mutant comprising mutations at amino acid residue S (S75) and amino acid residue R (R83) at position 75 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the amino acid sequence of said human wild-type IL-2 is as shown in SEQ ID NO:1.

[0022] In some embodiments, the IL-2 mutant described in this application is wherein S75 is mutated to an uncharged amino acid residue. In some embodiments, the mutation of S75 is S75P, S75F, S75Y, S75G, S75A, S75V, S75T, S75I, or S75L. In some embodiments, the mutation of R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K, or R83E.

[0023] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75P and a mutation of R83 into R83Y. In some embodiments, the IL-2 mutant described in this application further includes mutations in E67 and D84 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E67 and D84 are E67A and D84N, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations in S75P, R83Y, E67A, and D84N relative to the human wild-type IL-2 amino acid sequence.

[0024] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75P and a mutation of R83 into R83A. In some embodiments, the IL-2 mutant described in this application further includes mutations in H16, L70, and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in H16, L70, and N71 are H16D, L70I, and N71G, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations in S75P, R83A, H16D, L70I, and N71G relative to the human wild-type IL-2 amino acid sequence.

[0025] In some embodiments, the IL-2 mutant of this application includes a mutation of S75 into S75F and a mutation of R83 into R83P. In some embodiments, the IL-2 mutant of this application further includes mutations in H16 and / or N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation of H16 is H16Q or H16N. In some embodiments, the mutation of N71 is N71S. In some embodiments, the IL-2 mutant of this application includes mutations in S75F, R83P, and H16Q relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant of this application includes mutations in S75F, R83P, H16N, and N71S relative to the human wild-type IL-2 amino acid sequence.

[0026] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75F and a mutation of R83 into R83L. In some embodiments, the IL-2 mutant described in this application further includes mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E67 and L70 are E67R and L70V, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations in S75F, R83L, E67R, and L70V relative to the human wild-type IL-2 amino acid sequence.

[0027] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75I and a mutation of R83 into R83L. In some embodiments, the IL-2 mutant described in this application further includes mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E67 and L70 are E67G and L70V, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations in S75I, R83L, E67G, and L70V relative to the human wild-type IL-2 amino acid sequence.

[0028] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75I and a mutation of R83 into R83K. In some embodiments, the IL-2 mutant described in this application further includes mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E67 and L70 are E67S and L70V, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations in S75I, R83K, E67S, and L70V relative to the human wild-type IL-2 amino acid sequence.

[0029] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75T and a mutation of R83 into R83A or R83F. In some embodiments, the IL-2 mutant described in this application further includes mutations at H16 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations at H16 and E67 are H16N and E67K, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations at S75T, R83A, H16N, and E67K relative to the human wild-type IL-2 amino acid sequence.

[0030] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75V and a mutation of R83 into R83F. In some embodiments, the IL-2 mutant described in this application further includes mutations in E15 and H16 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E15 and H16 are E15K and H16Q, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations in S75V, R83F, E15K, and H16Q relative to the human wild-type IL-2 amino acid sequence.

[0031] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75V and a mutation of R83 into R83N. In some embodiments, the IL-2 mutant described in this application further includes mutations in L70 and V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in L70 and V91 are L70F and V91K, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations in S75V, R83N, L70F, and V91K relative to the human wild-type IL-2 amino acid sequence.

[0032] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75G and a mutation of R83 into R83L. In some embodiments, the IL-2 mutant described in this application further includes mutations in L17, V91, and I92 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in L17, V91, and I92 are L17I, V91E, and I92L, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations in S75G, R83L, L17I, V91E, and I92L relative to the human wild-type IL-2 amino acid sequence.

[0033] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75Y and a mutation of R83 into R83N. In some embodiments, the IL-2 mutant described in this application further includes mutations in L17 and V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in L17 and V91 are L17I and V91K, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations in S75Y, R83N, L17I, and V91K relative to the human wild-type IL-2 amino acid sequence.

[0034] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75A and a mutation of R83 into R83K. In some embodiments, the IL-2 mutant described in this application further includes a mutation of L17 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation of L17 is L17I. In some embodiments, the IL-2 mutant described in this application includes mutations of S75A, R83K, and L17I relative to the human wild-type IL-2 amino acid sequence.

[0035] In some embodiments, the IL-2 mutant described in this application includes a mutation of S75 into S75L and a mutation of R83 into R83E. In some embodiments, the IL-2 mutant described in this application further includes mutations of L63 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of L63 and E67 are L63V and E67F, respectively. In some embodiments, the IL-2 mutant described in this application includes mutations of S75L, R83E, L63V, and E67F relative to the human wild-type IL-2 amino acid sequence.

[0036] In some embodiments, the IL-2 mutant described in this application, in addition to the mutations described above, further includes a mutation relative to the C125 amino acid sequence of human wild-type IL-2. In some embodiments, the C125 mutation is C125S or C125A.

[0037] In some embodiments, the IL-2 mutant described in this application comprises any of the amino acid sequences shown in SEQ ID NOs:19-34 or variants thereof, wherein the variants have at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:19-34.

[0038] In some embodiments, compared with wild-type IL-2, the IL-2 mutant described in this application has a reduced binding affinity to IL-2Rβγ, but is able to maintain binding with IL-2Rα.

[0039] This application, in another aspect, relates to fusion proteins comprising IL-2 mutants. In some embodiments, the fusion protein comprises an Fc. In some embodiments, the Fc comprises a mutation capable of altering effector function, and / or a mutation capable of prolonging half-life, and / or a mutation capable of promoting the formation of dimers from heteropeptides. In some embodiments, the Fc is derived from human IgG. In other embodiments, the Fc is derived from human IgG, including IgG1, IgG2, IgG3, or IgG4, preferably IgG1 or IgG4.

[0040] In some embodiments, the Fc is derived from human IgG1, which includes mutations L234A and L235A; and / or N297G; and / or N297A; and / or L234A, L235A and P331S; and / or L234A, L235E, G237A, A330S and P331S mutations, wherein the numbering is in the EU numbering system.

[0041] In some embodiments, the Fc is derived from human IgG4 and contains mutations S228P, F234A, and L235A, wherein the numbering is in the EU numbering system.

[0042] In some embodiments, the Fc further includes S354C and T366W mutations; and / or Y349C, T366S, L368A, and Y407V mutations, wherein the numbering is in the EU numbering system.

[0043] In some embodiments, the fusion protein as described in this application, wherein the IL-2 mutant is linked to the Fc. In some embodiments, the IL-2 mutant and the Fc are linked via a linker peptide. In some embodiments, the linker peptide comprises any of the amino acid sequences shown in SEQ ID NOs:43-74. In some embodiments, the linker peptide comprises the amino acid sequence GGGGS (SEQ ID NO:52).

[0044] In some embodiments, the fusion protein of the IL-2 mutant and Fc described in this application, wherein the IL-2 mutant is located at the N-terminus and / or C-terminus of Fc.

[0045] In some embodiments, the fusion protein described in this application comprises any of the amino acid sequences shown in SEQ ID NOs:76-105 or variants thereof, wherein the variants have at least about 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:76-105.

[0046] Additionally, this application also relates to isolated nucleic acids encoding any IL-2 mutant or fusion protein containing an IL-2 mutant described herein, vectors containing said nucleic acids, host cells (e.g., CHO cells, HEK293 cells, HeLa cells, or COS cells) containing said nucleic acids or vectors, compositions (e.g., pharmaceutical compositions), kits, and articles containing any IL-2 mutant or fusion protein containing an IL-2 mutant described herein. It also relates to methods of using any IL-2 mutant or its fusion protein described herein, or pharmaceutical compositions comprising the thereof, for the treatment in individuals (e.g., humans) of inflammatory or autoimmune diseases (e.g., lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, malnutrition-related epidermolysis bullosa, and Behçet's disease). Attached Figure Description

[0047] Figure 1A The diagram shows an exemplary structure of a bivalent fusion protein of IL-2 mutant and Fc, which contains two identical monomers, in which the IL-2 mutant molecule in each monomer is linked to the N-terminus of Fc. Figure 1B The diagram shows an exemplary structure of a bivalent fusion protein of IL-2 mutant and Fc, which contains two identical monomers, in which the IL-2 mutant molecule in each monomer is linked to the C-terminus of Fc.

[0048] Figure 2 The diagram shows an exemplary structure of a monovalent fusion protein of an IL-2 mutant and Fc, which contains two different monomers, one of which contains an IL-2 mutant molecule attached to the C-terminus of the Fc knob, and the other monomer is an Fc hole molecule.

[0049] Figure 3A , 3C Figures 3E and 3G show the results of STAT5 phosphorylation induced in Treg cells by the first group of IL-2 mutants and Fc bivalent fusion proteins: Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-(81-85)-mut2, Fc-(81-85)-mut3, Fc-(81-85)-mut9 and Fc-(81-85)-mut14. Figure 3B , 3D Figures 3F and 3H show the bivalent fusion proteins of the first group of IL-2 mutants and Fc: Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-(81-85)-mut2, Fc-(81-85)-mut3, Fc-(81-85)-mut9, and Fc-(81-85)-mut14 in CD8. + The result of inducing STAT5 phosphorylation in T lymphocytes.

[0050] Figure 3I The image shows the results of STAT5 phosphorylation induced in Treg cells by the second group of IL-2 mutants and Fc bivalent fusion proteins: Fc-S75R83-mut7 and Fc-S75R83-mut16. Figure 3J The image shows the second group of bivalent fusion proteins of IL-2 mutants and Fc: Fc-S75R83-mut7 and Fc-S75R83-mut16 on CD8. + The result of inducing STAT5 phosphorylation in T lymphocytes.

[0051] Figure 4AThe first group of IL-2 mutants and Fc bivalent fusion proteins, namely Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12, stimulated the proliferation of Treg cells in mice at a dose of 1 mg / kg.

[0052] Figure 4B The image shows the bivalent fusion proteins of the first group of IL-2 mutants and Fc: Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12, at a dose of 1 mg / kg, and their effects on CD8+ in mice. + The effect of T lymphocyte proliferation.

[0053] Figure 4C The image shows how the second group of IL-2 mutants and Fc bivalent fusion proteins Fc-S75R83-mut5, Fc-S75R83-mut9, and Fc-S75R83-mut16 stimulated the proliferation of Treg cells in mice at a dose of 1 mg / kg.

[0054] Figure 4D The image shows the effects of the second group of IL-2 mutants on CD8+ in mice at a dose of 1 mg / kg, representing the bivalent fusion proteins Fc-S75R83-mut5, Fc-S75R83-mut9, and Fc-S75R83-mut16. + The effect of T lymphocyte proliferation.

[0055] Figure 5A The image shows how the first group of IL-2 mutants and the bivalent fusion proteins Fc-(81-85)-mut8 and Fc-(81-85)-mut10 stimulated the proliferation of Treg cells in cynomolgus monkeys at a dose of 0.3 mg / kg.

[0056] Figure 5B The image shows the effects of the first group of IL-2 mutants on CD8 in cynomolgus monkeys at a dose of 0.3 mg / kg, representing the bivalent fusion proteins Fc-(81-85)-mut8 and Fc-(81-85)-mut10. + The effect of T lymphocyte proliferation.

[0057] Figure 5C The image shows how the second group of IL-2 mutants and the bivalent fusion protein Fc-S75R83-mut5 stimulated the proliferation of Treg cells in cynomolgus monkeys at doses of 0.3 mg / kg and Fc-S75R83-mut16 at doses of 0.3 mg / kg and 1.5 mg / kg, respectively.

[0058] Figure 5D The figures show the effects of the second group of IL-2 mutants and the bivalent fusion protein Fc-S75R83-mut5 on CD8 in cynomolgus monkeys at doses of 0.3 mg / kg and Fc-S75R83-mut16 at doses of 0.3 mg / kg and 1.5 mg / kg, respectively. + The effect of T lymphocyte proliferation.

[0059] Figure 6 The figure shows the PK curves in mice of exemplary IL-2 mutant and Fc bivalent fusion proteins Fc-(81-85)-mut10 and Fc-S75R83-mut16 at a dose of 1 mg / kg.

[0060] Detailed description of this application

[0061] This application relates to IL-2 mutants and fusion proteins containing IL-2 mutants. Compared to wild-type IL-2, the IL-2 mutants described in this application can selectively activate Treg cells, stimulate Treg cell proliferation, and have a higher safety window in the treatment of certain specific diseases (e.g., inflammatory diseases or autoimmune diseases). (For example, in HEK-BlueTMIL-2 cell line screening experiments, the ratio between the EC50 value of IL-2 mutant binding to IL-2Rβγ detected by HEK-BlueTMCD122 / CD132 cell line and the EC50 value of IL-2 mutant binding to IL-2Rαβγ detected by HEK-BlueTMIL-2 cell line was higher than that of wild-type IL-2.) Furthermore, the fusion protein of the IL-2 mutant and Fc described in this application reduces or minimizes antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) by modifying the Fc fragment, thereby avoiding unwanted immune responses during treatment and increasing the in vivo circulating half-life of the IL-2 mutant, thereby reducing the frequency and total number of administrations, providing convenience for patients and reducing treatment costs.

[0062] This application also relates to nucleic acids encoding the IL-2 mutant or fusion proteins containing the IL-2 mutant, vectors containing the nucleic acids, host cells containing the nucleic acids or vectors, and methods for producing the IL-2 mutant or fusion proteins, pharmaceutical compositions and articles thereof, as well as methods for treating diseases (e.g., lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, or inflammatory bowel disease) using the IL-2 mutant or fusion proteins containing the IL-2 mutant or pharmaceutical compositions.

[0063] I. Definition

[0064] Unless the context clearly indicates otherwise, implementation of this application will employ conventional methods within the scope of the art, including virology, immunology, microbiology, molecular biology, and recombinant DNA techniques, many of which will be detailed below. These techniques are well explained in the literature. See Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubelet al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: APractical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal CellCulture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0065] As described in this application, the terms "wild-type IL-2" or "WT IL-2" refer to naturally occurring IL-2, which may be derived from any vertebrate source of natural IL-2, such as mammals, including but not limited to livestock (e.g., cattle, sheep, goats, cats, dogs, donkeys, and horses), primates (e.g., human and non-human primates such as monkeys or chimpanzees), rabbits, and rodents (e.g., mice, rats, gerbils, and hamsters). This term includes unprocessed forms of IL-2 (e.g., containing a signal peptide) and processed, mature forms of IL-2 (e.g., not containing a signal peptide). This term also includes naturally occurring IL-2 allelic variants and splice variants, isotypes, homologs, and species homologs. This term also includes naturally occurring IL-2 variants, etc. For example, said naturally occurring IL-2 variant has at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) sequence homology compared to natural IL-2. In some embodiments, an exemplary human wild-type IL-2 amino acid sequence is shown in SEQ ID NO:1. In some embodiments, unprocessed human wild-type IL-2 additionally comprises a signal peptide with an N-terminal 20 amino acids, an exemplary amino acid sequence of which is shown in SEQ ID NO:4.

[0066] In some embodiments, wild-type IL-2 may further comprise one or more amino acid mutations that do not alter its binding to the IL-2 receptor. For example, introducing a serine mutation (C125S) at amino acid residue 125 of wild-type human IL-2 can prevent mismatches or aggregation caused by cysteine, and its exemplary amino acid sequence is shown in SEQ ID NO:2; introducing an alanine mutation (C125A) at position 125 can promote efficient folding and improve expression, and its exemplary amino acid sequence is shown in SEQ ID NO:3.

[0067] The "mutation" of an amino acid as described in this application includes substitution, deletion, and insertion. Any combination of substitution, deletion, and insertion can be performed to obtain constructs with desired properties (e.g., reduced binding affinity to IL-2Rβγ). In some embodiments, the deletion or insertion of an amino acid includes deletion or insertion within a polypeptide sequence. In some embodiments, the substitution of an amino acid can be a conserved amino acid substitution. In other embodiments, the substitution of an amino acid can be a non-conserved amino acid substitution, i.e., replacing one amino acid with another amino acid with different structure and / or chemical properties. Amino acid substitution also includes the application of non-naturally occurring amino acids or substitutions by naturally occurring amino acid derivatives of 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). In some embodiments, the mutation of an amino acid includes a combination of amino acid substitution and deletion, for example, simultaneously including substitution and deletion within a region of the polypeptide sequence (e.g., amino acid residues 81-85, RPRDL). Amino acid mutations can be generated using genetic or chemical methods known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, etc.

[0068] The “IL-2 mutant” described in this application includes one or more substitutions, deletions, insertions, or combinations thereof in the wild-type IL-2 amino acid sequence (e.g., SEQ ID NO. 1). In some embodiments, the “IL-2 mutant” described in this application is capable of reducing its binding affinity to IL-2Rβγ while maintaining binding to IL-2Rα.

[0069] In IL-2 mutants, the location of the amino acid mutation is determined based on the amino acid position in wild-type IL-2. In some embodiments, the amino acid sequence of wild-type IL-2 is shown in SEQ ID NO:1, and the location corresponding to the amino acid mutation in the IL-2 mutant can be identified by comparing the amino acid sequence with SEQ ID NO:1 (e.g., using BLAST). In some embodiments, when describing, for example, S75, it represents that the 75th amino acid residue relative to the wild-type IL-2 amino acid sequence is serine (S). "Mutation of S75" represents a mutation of the serine residue (S) at position 75. In some embodiments, when describing IL-2 mutants, they are described as follows. For example, "amino acid substitution" is expressed as "original amino acid residue / substituted amino acid position / substituted amino acid residue", such as "S75G" representing that the serine residue (S) at position 75 relative to the wild-type IL-2 amino acid sequence (e.g., SEQ ID NO.1) is substituted by a glycine residue (G). In this application, the "+" in the combination scheme of mutation sites indicates that mutations occur simultaneously at multiple specific positions. For example, "S75V+R83N" represents the substitution of the 75th serine residue (S) with a valine residue (V) and the 83rd arginine residue (R) with an asparagine residue (N) relative to the wild-type IL-2 amino acid sequence (e.g., SEQ ID NO.1).

[0070] As described in this application, the IL-2 mutant "selectively activates Treg cells" refers to the presence of other T cell subsets (such as CD4+). + T cells, CD8 + In the case of T cells or NK cells, they can more specifically target Treg cells and activate them. The ability of IL-2 mutants to "activate Treg cells" can be determined by methods known in the art or by methods disclosed in the embodiments of this application, including but not limited to: IL-2 mutants inducing IL-2 receptor signal transduction or inducing Treg cell proliferation in Treg cells. In some embodiments, IL-2 receptor signal transduction can be defined by the level of STAT5 phosphorylation. STAT5 phosphorylation is a necessary step in the IL-2 signal transduction pathway, therefore the phosphorylation level of STAT5 in Treg cells (pSTAT5) is considered a reflection of IL-2 activation of Treg cells. Induced proliferation can be defined by changes in the number of Treg cells after IL-2 stimulation, for example by measuring an increase in the number of Treg cells in a mixed cell population by flow cytometry or by measuring an increase in the expression of proliferation-related cell cycle proteins (e.g., Ki-67) in Treg cells.

[0071] As described in this application, “autoimmune disease” refers to a non-malignant disease or condition originating from and targeting an individual’s own tissues, in which the immune system attacks its own proteins, cells, and tissues. A list and review of autoimmune diseases can be found in The Autoimmune Diseases (Noel Rose, Ian Mackay, 2014, Academic Press). In some embodiments, examples of autoimmune diseases include, but are not limited to, lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, malnutrition-related epidermolysis bullosa, and Behçet's disease.

[0072] As described in this application, "treatment" or "treating" is a method for obtaining a beneficial or desired outcome, including clinical outcomes. For the purposes of this application, the beneficial or desired outcome includes, but is not limited to, one or more of the following: relieving one or more symptoms caused by a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying disease spread, preventing or delaying disease recurrence, delaying or slowing disease progression, improving disease status, relieving disease (partially or completely), reducing the dosage of one or more other medications required to treat the disease, delaying disease progression, improving quality of life, and / or prolonging survival. "Treatment" also includes reducing the pathological outcomes of the disease. The methods of this application consider any one or more of these treatments. For example, if one or more symptoms associated with the disease are relieved or eliminated, including but not limited to reducing symptoms caused by the disease, improving the quality of life of the patient with the disease, reducing the dosage of other medications required to treat the disease, and / or prolonging the individual's survival, the patient is considered to have been successfully "treated".

[0073] The term "prevent" and similar words such as "prevented" and "preventing" refer to methods of preventing, suppressing, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition. As stated in this application, "prevention" and similar words also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition, or reducing the likelihood of its occurrence or recurrence, before it occurs or recurs.

[0074] As described in this application, "delaying" disease progression means postponing, hindering, slowing, stabilizing, and / or delaying the development of a disease. The duration of delay may vary depending on the individual's medical history and / or the treatment received. A method for "delaying" disease progression refers to a method that reduces the probability of disease development and / or the severity of disease within a given time frame compared to not using the method. In some embodiments, such comparisons may be based on animal studies, involving observation and / or statistics in individual animals. In other embodiments, such comparisons may be based on clinical studies, using a statistically significant number of individuals.

[0075] As described in this application, the term "effective amount" refers to a dose of a drug or pharmaceutical composition sufficient to treat a specific disorder, symptom, or disease, such as improving, alleviating, reducing, and / or delaying one or more symptoms. In some embodiments, an effective amount is an amount sufficient to delay disease progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay the onset or recurrence of disease. An effective amount may be administered once or multiple times. In some embodiments, an effective amount of a drug or pharmaceutical composition refers to an amount capable of increasing the number of Treg cells in the patient's diseased tissue.

[0076] As described in this application, "individual" or "subject" refers to mammals, including but not limited to humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the individual refers to a human.

[0077] The term "constant region" refers to a portion of an immunoglobulin molecule (Ig) as opposed to the "variable region," which contains antigen-binding sites. The constant region has a more conserved amino acid sequence and includes the heavy chain constant region (C1). H ), which contains C H 1. C H 2 and C H 3. Structural domains and light chain constant regions (C L According to the immunoglobulin heavy chain constant region (C... H Immunoglobulins can be classified into different classes or subtypes based on their amino acid sequences. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains α, δ, ε, γ, and μ, respectively. According to C... H Relatively small differences in sequence and function further subdivide γ and α into subclasses, for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0078] As described in this application, the term IgG “subtype” or “subclass” refers to any subclass of immunoglobulins defined by the chemical and antigenic properties of their constant regions. Immunoglobulins are mainly classified into five classes: IgA, IgD, IgE, IgG, and IgM, and many of these can be further subdivided into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chains corresponding to different immunoglobulin classes are named using the Greek letters α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different immunoglobulin classes are well known and detailed in the fourth edition of *Cellular and Molecular Immunology* by Abbas et al. (WBSaunders, Co., 2000).

[0079] As described in this application, the terms "Fc," "Fc domain," "Fc fragment," "Fc region," or "crystallizable fragment" are used to define the C-terminal region of the immunoglobulin heavy chain, including native Fc and variant Fc. An exemplary Fc includes the C-terminal region of an immunoglobulin. H 2 and C H 3. Structural Domains. Although the boundaries of the immunoglobulin heavy chain Fc may vary, the human IgG heavy chain Fc is generally defined as extending from the amino acid residue at Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc (residue 447 according to the EU numbering system) may be removed, for example, during protein production or purification, or by recombinantly engineering the nucleic acid encoding the protein. Suitable native sequence Fc regions used for the constructs described herein are derived from, but are not limited to, human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0080] The term "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of a molecule containing an Fc (e.g., an antibody or an Fc-containing fusion protein). The preferred FcR is the natural human FcR sequence. Additionally, an exemplary FcR is an FcR that binds to an IgG antibody (a type of γ-receptor), including receptor subclasses such as FcγRI, FcγRII, and FcγRIII, as well as allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine inhibition motif (ITIM) in its cytoplasmic domain (see M). Annu.Rev.Immunol.15:203-234(1997)). FcRs are reviewed in Ravetch and Kinet, Annu.Rev.Immunol 9:457-92(1991); Capel et al., Immunomethods4:25-34(1994); and de Haas et al., J.Lab.Clin.Med.126:330-41(1995). In this paper, the term "FcR" encompasses other FcRs, including those to be identified in the future.

[0081] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for transporting maternal IgG to the fetus. Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994). Methods for determining binding to FcRn are known in the art (e.g., see Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6 (2004); WO 2004 / 92219 (Hinton et al.)). The half-life of human FcRn-binding peptides with high affinity can be determined in vivo and in serum, for example, in transgenic mice expressing human FcRn or in transfected human cell lines, or in primates administered peptides with variant Fc regions. WO2004 / 42072 (Presta) details antibody variants that enhance or weaken binding to FcRs. For information on FcRn, see Shieldet al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0082] "Fc effector function" refers to the biological activity induced by the Fc region (natural sequence Fc region or Fc region containing amino acid sequence mutations) in an Fc-containing molecule (e.g., an antibody or an Fc-containing fusion protein), which varies depending on the immunoglobulin subtype from which the Fc originates. Examples of effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors) and B cell activation. "Reduced or minimized" effector function means a reduction of at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) in effector function compared to wild-type Fc or an unmodified Fc-containing molecule (e.g., an antibody). Effector function can be readily determined and measured by those skilled in the art. In a preferred embodiment, effector functions such as complement binding, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity are all affected. In some embodiments, effector function is eliminated by removing glycosylation through mutations in the constant region, for example, a "no-effect function mutation." In some embodiments, the no-effect function mutant is a C H For N297A or DANA mutations (D265A+N297A) in region 2, see Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). Other mutations leading to reduced or eliminated effector function include K322A and L234A / L235A (LALA). Furthermore, effector function can be reduced or eliminated by altering production techniques, such as expression in host cells that do not undergo glycosylation (e.g., E. coli) or in host cells that alter the glycosylation pattern, which is ineffective or less effective in promoting effector function (e.g., Shinkaw et al., J. Biol. Chem. 278(5):3466-3473 (2003)).

[0083] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a form of cytotoxicity in which secretory Ig (or ligand-Fc structures) binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens (or carrying ligand receptors) and subsequently kill the target cells with cytotoxins. Antibodies (or other Fc-containing molecules) "arm" cytotoxic cells, which is necessary for killing target cells through this mechanism. The main cells mediating ADCC include NK cells and monocytes, with NK cells expressing only FcγRIII and monocytes expressing FcγRI, FcγRII, and FcγRIII. The expression of Fc in hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, in vitro ADCC assays can be performed, as detailed in US Pat. No. 5,500,362 or 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a target molecule can also be assessed in vivo, for example in animal models as disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0084] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component of the complement system (C1q) to an Fc-containing molecule (of an appropriate subclass), which binds to its homologous receptor via an Fc-fused ligand. To assess complement activation, CDC assays can be performed, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Antibody variants that increase or decrease C1q binding capacity by altering the amino acid sequence of the Fc region are detailed in USPat. No. 6,194,551B1 and WO99 / 51642. The contents of these patent publications are incorporated herein by reference. See Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0085] As described in this application, "regulatory T cells" or "Tregs" are a type of CD4 cells with significant immunosuppressive effects. +T cell subsets can suppress the immune responses of other cells. Tregs are characterized by the expression of the α subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3), and play an important role in maintaining immune homeostasis and preventing autoimmune diseases and transplant rejection. Tregs require IL-2 to achieve their function, development, and the induction of their inhibitory characteristics.

[0086] As stated in this application, "effect cells" refers to a population of lymphocytes that mediate IL-2-induced cytotoxic effects. Effector cells primarily include effector T cells such as CD8+. + Cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages, etc.

[0087] As described in this application, the terms "specific binding," "specific recognition," or "specifically used" refer to measurable and reproducible interactions, such as binding between a ligand and a receptor, where the presence of the ligand can be determined in the presence of a heterogeneous group of molecules, including biomolecules. For example, a ligand that specifically binds to a receptor exhibits greater affinity, strength, ease of binding, and / or longer duration of binding to the target receptor compared to binding to other receptors. In some embodiments, the degree of binding of the ligand to an irrelevant receptor is less than 10% of the degree of binding of the ligand to the target receptor, as determined by, for example, radioimmunoassay (RIA). In some embodiments, the equilibrium dissociation constant (Kd) of the ligand that specifically binds to the target receptor is ≤10. - 5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 M. In some embodiments, the ligand may specifically bind to receptors conserved in different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding. The binding specificity of the ligand can be determined experimentally using methods known in the art, such as, but not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, and BIACORE. TM - Testing and peptide scanning.

[0088] "Affinity" or "binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a ligand) and its binding partner (e.g., a receptor). Unless otherwise stated, "binding affinity" as used in this application refers to intrinsic binding affinity, which can reflect a 1:1 interaction between members of a binding pair. Binding affinity can be expressed as Kd, Koff, Kon, or Ka. As used in this application, the term "Koff" refers to the rate constant for the ligand to dissociate from the ligand / receptor complex, which can be determined by a kinetic selection device in s-1. -1 The term "Kon" is expressed in units of M. As described in this application, the term "Kon" refers to the binding rate constant of the ligand and receptor to form a ligand / receptor complex, expressed in megohms (M). -1 s -1 The terms "equilibrium dissociation constant" (Kd) are expressed in units of M (mol / L). As described in this application, the term "Kd" refers to the dissociation constant of a specific ligand-receptor interaction, which is the concentration of ligand required in the receptor solution for the ligand to occupy half of all receptor binding sites and reach equilibrium, equal to Koff / Kon. The dissociation constant (Kd) can serve as an indicator of the affinity between the ligand and the receptor. Kd values ​​obtained using the method described are expressed in units of M (mol / L). The determination of Kd assumes that all bound molecules are in solution. When the receptor is on a cell membrane, the corresponding dissociation rate constant is expressed as the EC50 value. The EC50 value is a good approximation of Kd. The affinity constant Ka is the reciprocal of the dissociation constant Kd, expressed in units of M. -1 It is expressed in units. The dissociation constant (Kd) can be used as an indicator of the affinity between the ligand and the receptor.

[0089] The half-maximal inhibitory concentration (IC50) is a measure of the effectiveness of a substance (e.g., a ligand) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, e.g., a ligand) is required to inhibit a given biological process by half. IC50 values ​​are typically expressed as molar concentrations. IC50 is equivalent to the “EC50” of an agonist drug or other substance (e.g., a ligand). EC50 also represents the plasma concentration required to achieve 50% of the maximum effect in vivo. As described in this application, “IC50” is used to represent the effective concentration of a ligand required to neutralize 50% of the receptor's biological activity in vitro. IC50 or EC50 can be determined by bioassays, such as ligand binding inhibition assays via FACS (competitive binding assays), cell-based cytokine release assays, or amplified luminescent homogeneous enzyme-linked immunosorbent assays (AlphaLISA).

[0090] As described in this application, "fusion" refers to the linking of components by direct peptide bonds or through one or more linker peptides (or "connectors"). The sequence of a "linker peptide (or connector)" can be a single amino acid or a polypeptide sequence. In some embodiments, the linker peptide (or connector) comprises or is composed of a glycine-serine connector. As described in this application, the term "glycine-serine connector" refers to a peptide composed of glycine and serine residues. An exemplary glycine-serine connector comprises an amino acid sequence of the general formula (Gly4Ser)n, where n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). A preferred glycine-serine connector is GGGGS.

[0091] As described in this application, a "covalent bond" is a stable bond formed between two atoms by sharing one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As described in this application, a "peptide bond" is a covalent bond formed between the carboxyl group of an amino acid and the amino group of an adjacent amino acid. As described in this application, a "disulfide bond" is a covalent bond formed between two sulfur atoms, such as two Fc segments linked by one or more disulfide bonds. One or more disulfide bonds between two segments may be formed by connecting thiol groups in the two segments. In some embodiments, one or more disulfide bonds may be formed between one or more cysteine ​​residues of two Fc segments. Oxidation of two thiol groups can form a disulfide bond. In some embodiments, covalent linkage is formed by direct covalent bonding. In some embodiments, covalent linkage is directly formed by peptide bonds or disulfide bonds.

[0092] As described in this application, the term "derivative" refers to a molecule having a protein amino acid sequence or an analogue thereof, but with additional modifications at one or more amino acid groups, α-carbon atoms, amino terms, or carboxyl terms. As described in this application, modifications include, but are not limited to, chemical modifications, amino acid side group modifications, amino terminal modifications, and carboxyl terminal modifications. Chemical modifications include, but are not limited to, adding chemical groups, forming new chemical bonds, and removing chemical groups. Amino acid side group modifications include, but are not limited to, ε-aminoacylation of lysine, N-alkylation of arginine, histidine, or lysine, carboxylalkylation of glutamic acid or aspartic acid, and deamination of glutamine or asparagine. Amino terminal modifications include, but are not limited to, deamination, N-low alkyl, N-dilow alkyl, and N-acyl modifications. Carboxyl terminal modifications include, but are not limited to, amide, low alkyl acyl, dialkylamide, and low alkyl ester modifications. In some embodiments, the low alkyl group is a C1-C4 alkyl group. Furthermore, one or more side groups or terminal groups may be protected by a person skilled in the chemical art using known protecting groups. The α-carbon of the amino acid may be monomethylated or dimethylated.

[0093] The "percentage of amino acid sequence identity (%)" or "homology" of a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a specific polypeptide or polypeptide sequence. This percentage of sequence identity is maximized after sequence alignment and the introduction of gaps (if necessary), without considering any conserved substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, various alignment methods within the scope of the art can be used, such as publicly available computer software like BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0094] As described in this application, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide, the amino group of which forms a peptide bond with the carboxyl group of its adjacent amino acid residue. As described in this application, the "N-terminus" of a polypeptide refers to the first amino acid residue of the polypeptide, the carboxyl group of which forms a peptide bond with the amino group of its adjacent amino acid residue.

[0095] "Isolated" polypeptide refers to a polypeptide that has been identified, isolated, and / or recovered from components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is not associated with any other components in its production environment. Contaminants in the production environment, such as those generated by recombinant transfected cells, can often interfere with the study, diagnosis, or treatment of the polypeptide and may include enzymes, hormones, and other proteolysinic or non-proteolysinic components. In some embodiments, the polypeptide will be purified to: (1) a polypeptide content greater than 95% by weight, as determined by the Lowry method, and in some embodiments, a polypeptide content greater than 99% by weight; (2) sufficient to obtain at least 15 N-terminal residues or an internal amino acid sequence by using a spin-cup sequencer; or (3) homogeneity achieved by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. Isolated polypeptides include in situ polypeptides within recombinant cells, since at least one element of the polypeptide's natural environment is absent. However, typically, an isolated polypeptide undergoes at least one purification step.

[0096] The term "amino acid" is used herein in its broadest sense, encompassing both naturally occurring amino acids and non-naturally occurring amino acids, including amino acid analogs and derivatives. The latter includes molecules containing an amino acid moiety. Based on this broad definition, those skilled in the art will find that the amino acids described herein include, for example, naturally occurring L-amino acids that form proteins; D-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; naturally occurring amino acids that do not form proteins, such as leucine, β-alanine, ornithine, GABA, etc.; and chemically synthesized compounds known in the art to possess the characteristics of amino acids. As described in this application, the term "protein-forming" refers to amino acids that can be used to synthesize cellular peptides, polypeptides, or proteins through metabolic pathways.

[0097] An "isolated" nucleic acid molecule encoding a protein or polypeptide (e.g., the IL-2 mutant or fusion protein containing the IL-2 mutant described herein) is a nucleic acid molecule identified and isolated from a nucleic acid molecule containing at least one impurity, which is generally associated with the environment in which it was produced. Preferably, the isolated nucleic acid is not associated with any components of its production environment. An isolated nucleic acid molecule encoding a polypeptide as described herein exists in a form or morphology that is not found in nature. Therefore, the isolated nucleic acid molecule differs from the nucleic acid naturally present in cells encoding the polypeptide described herein. An isolated nucleic acid includes nucleic acid molecules contained in cells containing that nucleic acid molecule, but which are located extrachromosomally or at a chromosomal location different from their natural chromosomal location.

[0098] The term "control sequence" refers to the DNA sequence necessary for the expression of an operable linker-coding sequence in a specific host organism. For example, control sequences suitable for prokaryotes include promoters, optional operable sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0099] When a nucleic acid establishes a functional relationship with another nucleic acid sequence, the connection between the two sequences is termed "operably ligated." For example, if DNA encoding a pre-sequence or secretory leader sequence is involved in the secretion of a polypeptide expressed as a precursor protein, then it is operably ligated to the DNA encoding that polypeptide molecule; if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably ligated to the coding sequence; or if the ribosome binding site is located in a position conducive to translation, then the ribosome binding site is operably ligated to the coding sequence. Generally, "operably ligated" means that the ligated DNA sequences are contiguous, and, for secretory leaders, not only contiguous but also in the reading phase. However, enhancers do not need to be contiguous. Ligation is accomplished by ligating at suitable restriction sites. If such sites are not present, synthetic oligonucleotide aptamers or adapters are used as a standard practice.

[0100] As described in this application, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid molecule linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors introduced into the genome of a known host cell. Some vectors are capable of directing the expression of the nucleic acid linked to them. Such vectors are referred to herein as "expression vectors."

[0101] As described in this application, the terms "transfection," "transformation," or "transduction" refer to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected," "transformed," or "transduced" cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary test cells and their progeny.

[0102] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably to refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and their resulting progeny, regardless of passage number. Progeny may not be identical to the parent cell in terms of nucleic acids; for example, they may contain mutations. This article includes screening or selecting mutant progeny that have the same function or biological activity as the original transformed cells.

[0103] The terms "pharmaceutical formulation" or "pharmaceutical composition" refer to a formulation that is a form in which the biological activity of the active ingredient is effective and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the formulation. Such a formulation is sterile. A "sterile" formulation is sterile or free of any live microorganisms or their spores.

[0104] The embodiments described in this application should be understood to include embodiments that are “composed of” and / or “substantially composed of”.

[0105] In this application, the reference to “about” as a numerical value or parameter includes (and describes) variations of that numerical value or parameter itself. For example, a description relating to “about X” includes a description of “X”.

[0106] As stated in this application, the reference to "not" a numerical value or parameter generally indicates and describes something other than a certain numerical value or parameter. For example, "this method cannot be used to treat type X cancer" means that this method is generally used to treat other types of diseases besides type X cancer.

[0107] As stated in this application, the term "about XY" has the same meaning as "about X to about Y".

[0108] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein and in the claims include plural objects.

[0109] II. IL-2 mutant

[0110] The IL-2 mutants described in this application exhibit a preference for IL-2 receptor selection and lymphocyte activation / proliferation, as well as biological characteristics. nature:

[0111] In some embodiments, the IL-2 mutant provided in this application has reduced affinity for IL-2Rβ and / or IL-2Rβγ compared to human wild-type IL-2 (e.g., SEQ ID NO:1). In some embodiments, the IL-2 mutant has reduced affinity for IL-2Rβ and / or IL-2Rβγ by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) compared to human wild-type IL-2.

[0112] In some embodiments, the IL-2 mutant provided in this application has increased affinity for IL-2Rα compared to human wild-type IL-2 (e.g., SEQ ID NO:1). In some embodiments, the IL-2 mutant has at least a 1-fold increase in affinity for IL-2Rα compared to human wild-type IL-2 (e.g., at least 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold).

[0113] In some embodiments, compared with human wild-type IL-2 (e.g., SEQ ID NO:1), the IL-2 mutant provided in this application has the same or no significantly weakened affinity for IL-2Rα or an increased affinity for IL-2Rα.

[0114] In some embodiments, compared to human wild-type IL-2 (e.g., SEQ ID NO:1), the IL-2 mutant provided in this application exhibits reduced affinity for IL-2Rβ and / or IL-2Rβγ, while maintaining or showing no significant decrease in affinity for IL-2Rα, or exhibiting increased affinity for IL-2Rα. The affinity between the IL-2 mutant and the receptor can be determined using conventional methods known in the art, such as combined ELISA assays, HEK-Blue™ IL-2 cell line assays, or surface plasmon resonance (SPR) assays.

[0115] In some embodiments, the IL-2 mutant provided in this application has higher safety in treating certain specific diseases compared with human wild-type IL-2 (e.g., SEQ ID NO:1).

[0116] For example, in experiments using the HEK-BlueTM IL-2 cell line (InvivoGen, cat#hkb-il2), compared to wild-type human IL-2, the ratio between the EC50 values ​​of IL-2 mutant binding to IL-2Rβγ (hereinafter referred to as the dual receptor) detected by the HEK-BlueTM CD122 / CD132 cell line and the EC50 values ​​of IL-2 mutant binding to IL-2Rαβγ (hereinafter referred to as the triple receptor) detected by the HEK-BlueTM IL-2 cell line was higher, indicating a longer therapeutic safety window and therefore higher safety. In some embodiments, the EC50 values ​​of IL-2 or IL-2 mutant binding to IL-2Rβγ are determined by the HEK-BlueTM CD122 / CD132 cell line assay, and the EC50 values ​​of IL-2 or IL-2 mutant binding to IL-2Rαβγ are determined by the HEK-BlueTM IL-2 cell line assay. In some embodiments, the EC50 of the IL-2 mutant... 二受体 / EC50 三受体 The ratio is the EC50 of human wild-type IL-2. 二受体 / EC50 三受体 The ratio must be at least twice that of other ratios, such as 2, 5, 10, 50, 100, 200, 500, 700, 1000, 2000, 10000, 20000, 30000 or higher.

[0117] In some embodiments, compared with wild-type human IL-2 (e.g., SEQ ID NO:1), the IL-2 mutant provided in this application can selectively activate Treg cells and reduce activation of other T cells, such as reducing activation of CD8 cells. + T cell activation; reduced NK cell activation.

[0118] In some embodiments, the IL-2 mutant provided in this application can selectively activate Treg cells compared to human wild-type IL-2 (e.g., SEQ ID NO:1). For example, in a STAT5 phosphorylation assay, the activation ability of the IL-2 mutant on Treg cells is reflected by detecting the activation of STAT5 phosphorylation signaling by IL-2 or the IL-2 mutant in Treg cells. Flow cytometry was used for detection, and GraphPad Prism software was used to analyze and calculate the EC50 value of IL-2 or the IL-2 mutant activating STAT5 phosphorylation in Treg cells. This EC50 value indirectly reflects the activation ability of IL-2 or the IL-2 mutant on Treg cells, indicating that the IL-2 mutant can effectively activate Treg cells.

[0119] In some embodiments, the IL-2 mutant provided in this application, compared with human wild-type IL-2 (e.g., SEQ ID NO:1), can reduce CD8 inhibition. + T cell activation. For example, in STAT5 phosphorylation assays, by detecting IL-2 or IL-2 mutants on CD8... + Activation of STAT5 phosphorylation signaling in T lymphocytes reflects the effect of IL-2 mutants on CD8. + The activation capacity of T lymphocytes was detected by flow cytometry, and the IL-2 mutant was analyzed and calculated using GraphPad Prism software. + The EC50 value for STAT5 phosphorylation activation in T lymphocytes, which indirectly reflects the effect of IL-2 or IL-2 mutants on CD8. + T lymphocyte activation capacity. In some embodiments, the IL-2 mutant, compared to wild-type human IL-2, showed increased activity against CD8+. + The activation capacity of T lymphocytes is reduced by at least 10%. For example: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0120] In some embodiments, the IL-2 mutant provided in this application can effectively expand Treg cells in vivo or in vitro. In some embodiments, the IL-2 mutant can increase CD4 in the subject. + The proportion of Treg cells in the T cell population (e.g., CD4+) + CD4 in the T cell population + CD25 + FoxP3 + (The proportion of cells increased).

[0121] In some embodiments, the IL-2 mutant can increase the ratio of Treg cells to non-Treg cells (e.g., CD4+) in a subject. + CD25 + FoxP3 + Cells and CD8 + (Increased cell ratio). For example, in the embodiments of this application, stimulation with an IL-2 mutant resulted in an increase in the number of Treg cells in the peripheral blood of cynomolgus monkeys over a certain period of time, while the number of CD8 cells increased. + The number of T lymphocytes was not significantly affected. In some embodiments, stimulation with an IL-2 mutant increased the number of Treg cells in CD4+ cells in the test animals. + The proportion of T lymphocytes increased by at least 1 time. For example: 1 time, 2 times, 3 times, 5 times, 7 times, 10 times, 15 times, 20 times or more.

[0122] The IL-2 mutant in this application:

[0123] In some embodiments, the IL-2 mutant described in this application has at least one amino acid mutation, such as substitution, deletion, insertion, or any combination thereof, compared to wild-type IL-2 (e.g., human wild-type IL-2).

[0124] Group 1 IL-2 mutants:

[0125] In some embodiments, the IL-2 mutant described in this application comprises a deletion or mutation of amino acid residues RPRDL at positions 81-85 of the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1). In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations of E67, L70, N71, S75, and V91. In some embodiments, the mutation of E67 is E67K, E67A, E67M, or E67T. In some embodiments, the mutation of L70 is L70I. In some embodiments, the mutation of N71 is N71I, N71S, or N71W. In some embodiments, the mutation of S75 is S75I or S75F. In some embodiments, the mutation of V91 is V91K.

[0126] In some embodiments, the IL-2 mutant described in this application has 1, 2, 3, 4, or all 5 amino acids missing from amino acid residues RPRDL at positions 81-85.

[0127] In some embodiments, the IL-2 mutant described in this application comprises the complete deletion of amino acid residues RPRDL at positions 81-85 of the amino acid sequence of human wild-type IL-2.

[0128] In some embodiments, the IL-2 mutant described in this application comprises the complete deletion of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence, and further comprises one or more mutations in E67 and N71. In some embodiments, the mutations in E67 and N71 are E67K and N71S, respectively.

[0129] In some embodiments, the IL-2 mutant described in this application comprises the complete deletion of amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2, and E67K and N71S mutations relative to the amino acid sequence of human wild-type IL-2.

[0130] In some embodiments, some or all of the amino acid residues in the RPRDL at positions 81-85 of the amino acid sequence of the wild-type human IL-2 mutant described in this application are substituted.

[0131] In some embodiments, the IL-2 mutant described in this application comprises an RPRDL mutation to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence.

[0132] In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RPL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises a mutation of E67. In some embodiments, the E67 mutation is E67A.

[0133] In some embodiments, the IL-2 mutant described in this application comprises an RPRDL mutation to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and an E67A mutation relative to the human wild-type IL-2 amino acid sequence.

[0134] In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RPL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises mutations at N71 and S75 of the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations at N71 and S75 are N71W and S75I, respectively. In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RPL at amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations at N71W and S75I of the human wild-type IL-2 amino acid sequence.

[0135] In some embodiments, the IL-2 mutant described in this application comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence.

[0136] In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and further comprises mutations in E67 and / or V91. In some embodiments, the E67 mutation is E67T. In some embodiments, the V91 mutation is V91K.

[0137] In some embodiments, the IL-2 mutant described in this application comprises an RPRDL mutation to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and an E67T mutation relative to the human wild-type IL-2 amino acid sequence.

[0138] In some embodiments, the IL-2 mutant described in this application comprises an RPRDL mutation to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and a V91K mutation relative to the human wild-type IL-2 amino acid sequence.

[0139] In some embodiments, the IL-2 mutant described in this application comprises an RPRDL mutation to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and E67T and V91K mutations relative to the human wild-type IL-2 amino acid sequence.

[0140] In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and further comprises mutations at L70 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations at L70 and S75 are L70I and S75F, respectively. In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and mutations at L70I and S75F relative to the human wild-type IL-2 amino acid sequence.

[0141] In some embodiments, the IL-2 mutant of this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and further comprises a mutation of N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the N71 mutation is N71I. In some embodiments, the IL-2 mutant of this application comprises a mutation of RPRDL to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and a mutation of N71I relative to the human wild-type IL-2 amino acid sequence.

[0142] In some embodiments, the IL-2 mutant described in this application comprises an RPRDL mutation to SPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence.

[0143] In some embodiments, the IL-2 mutant described in this application comprises a mutation of RPRDL to SPL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and further comprises one or more mutations in E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E67 and N71 are E67M and N71S, respectively.

[0144] In some embodiments, the IL-2 mutant described in this application comprises an RPRDL mutation to SPL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and E67M and N71S mutations relative to the human wild-type IL-2 amino acid sequence.

[0145] Group 2 IL-2 mutants:

[0146] In some embodiments, the IL-2 mutant described in this application comprises mutations at amino acid residue S (S75) and amino acid residue R (R83) at position 75 relative to the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1). In some embodiments, the mutation at S75 is S75P, S75F, S75Y, S75G, S75A, S75V, S75T, S75I, or S75L. In some embodiments, the mutation at R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K, or R83E. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations selected from E15, H16, L17, L63, E67, L70, N71, D84, V91, and I92. In some embodiments, the mutation at E15 is E15K. In some embodiments, the mutation of H16 is H16D, H16Q, or H16N. In some embodiments, the mutation of L17 is L17I. In some embodiments, the mutation of L63 is L63V. In some embodiments, the mutation of E67 is E67A, E67R, E67G, E67S, E67K, or E67F. In some embodiments, the mutation of L70 is L70I, L70V, or L70F. In some embodiments, the mutation of N71 is N71G or N71S. In some embodiments, the mutation of D84 is D84N. In some embodiments, the mutation of V91 is V91E or V91K. In some embodiments, the mutation of I92 is I92L.

[0147] In some embodiments, the IL-2 mutant described in this application comprises S75P and R83Y mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in E67 and D84 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E67 and D84 are E67A and D84N, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75P, R83Y, E67A, and D84N mutations relative to the human wild-type IL-2 amino acid sequence.

[0148] In some embodiments, the IL-2 mutant described in this application comprises S75P and R83A mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations of H16, L70, and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of H16, L70, and N71 are H16D, L70I, and N71G, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75P, R83A, H16D, L70I, and N71G mutations relative to the human wild-type IL-2 amino acid sequence.

[0149] In some embodiments, the IL-2 mutant described in this application comprises S75F and R83P mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in H16 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the H16 mutation is H16Q or H16N. In some embodiments, the N71 mutation is N71S. In some embodiments, the IL-2 mutant described in this application comprises S75F, R83P, and H16Q mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application comprises S75F, R83P, H16N, and N71S mutations relative to the human wild-type IL-2 amino acid sequence.

[0150] In some embodiments, the IL-2 mutant described in this application comprises S75F and R83L mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E67 and L70 are E67R and L70V, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75F, R83L, E67R, and L70V mutations relative to the human wild-type IL-2 amino acid sequence.

[0151] In some embodiments, the IL-2 mutant described in this application, relative to human wild-type IL-2, comprises S75I and R83L mutations relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in E67 and L70 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations in E67 and L70 are E67G and L70V, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75I, R83L, E67G, and L70V mutations relative to the amino acid sequence of human wild-type IL-2.

[0152] In some embodiments, the IL-2 mutant described in this application comprises S75I and R83K mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E67 and L70 are E67S and L70V, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75I, R83K, E67S, and L70V mutations relative to the human wild-type IL-2 amino acid sequence.

[0153] In some embodiments, the IL-2 mutant described in this application comprises S75T and R83F mutations relative to the amino acid sequence of human wild-type IL-2.

[0154] In some embodiments, the IL-2 mutant described in this application comprises S75T and R83A mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in H16 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in H16 and E67 are H16N and E67K, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75T, R83A, H16N, and E67K mutations relative to the human wild-type IL-2 amino acid sequence.

[0155] In some embodiments, the IL-2 mutant described in this application comprises S75V and R83F mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in E15 and H16 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in E15 and H16 are E15K and H16Q, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75V, R83F, E15K, and H16Q mutations relative to the human wild-type IL-2 amino acid sequence.

[0156] In some embodiments, the IL-2 mutant described in this application comprises S75V and R83N mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in L70 and V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in L70 and V91 are L70F and V91K, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75V, R83N, L70F, and V91K mutations relative to the human wild-type IL-2 amino acid sequence.

[0157] In some embodiments, the IL-2 mutant described in this application comprises S75G and R83L mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations among L17, V91, and I92 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in L17, V91, and I92 are L17I, V91E, and I92L, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75G, R83L, L17I, V91E, and I92L mutations relative to the human wild-type IL-2 amino acid sequence.

[0158] In some embodiments, the IL-2 mutant described in this application comprises S75Y and R83N mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in L17 and V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in L17 and V91 are L17I and V91K, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75Y, R83N, L17I, and V91K mutations relative to the human wild-type IL-2 amino acid sequence.

[0159] In some embodiments, the IL-2 mutant described in this application comprises S75A and R83K mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises a mutation at L17 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L17 mutation is L17I. In some embodiments, the IL-2 mutant described in this application comprises S75A, R83K, and L17I mutations relative to the human wild-type IL-2 amino acid sequence.

[0160] In some embodiments, the IL-2 mutant described in this application comprises S75L and R83E mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant described in this application further comprises one or more mutations in L63 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in L63 and E67 are L63V and E67F, respectively. In some embodiments, the IL-2 mutant described in this application comprises S75L, R83E, L63V, and E67F mutations relative to the human wild-type IL-2 amino acid sequence. Mutations at other sites:

[0161] In some embodiments, the IL-2 mutant of this application may further have one or more mutations at other positions or regions relative to the human wild-type IL-2 amino acid sequence, as long as it satisfies the reduced selectivity of the IL-2 mutant for the receptor IL-2Rβγ and / or one or more beneficial properties described in this invention. In some embodiments, the IL-2 mutant of this application also includes mutations at other positions in the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1) that do not alter the affinity for the IL-2 receptor. In some embodiments, the IL-2 mutant of this application has an amino acid mutation at position 125 relative to the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1), such as C125S, C125A, C125T, or C125V (see U.S. Patent No. 4,518,584 for details). Those skilled in the art will know how to add other additional mutations besides those described in this application.

[0162] In some embodiments, the IL-2 mutant described in this application further comprises a C125S mutation relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the IL-2 mutant described in this application further comprises a C125A mutation relative to the amino acid sequence of human wild-type IL-2.

[0163] In some embodiments, the IL-2 mutant described in this application comprises any of the amino acid sequences shown in SEQ ID NOs:5-18 or variants thereof, wherein the variants have at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-18.

[0164] In some embodiments, the IL-2 mutant described in this application comprises any of the amino acid sequences shown in SEQ ID NOs:19-34 or variants thereof, wherein the variants have at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:19-34.

[0165] In some embodiments, one or more mutations at the other positions or regions comprise one or more conserved amino acid substitutions. “Conservative substitution” means substitution with another amino acid that has the same net charge and is substantially the same size and shape as the substituted amino acid. Amino acids with aliphatic or substituted aliphatic amino acid side chains are substantially the same size when the total number of carbon atoms and heteroatoms on their side chains differs by no more than four. Amino acids with a side chain branch number that differs by no more than one are substantially the same shape. Amino acids having a phenyl or substituted phenyl on their side chains can be considered substantially the same in size and shape. Unless otherwise stated, conserved substitution is preferably applied to native amino acids. Exemplary conserved substitutions are shown in Table 1.

[0166] More substantial substitutions are provided under the heading “Substitution Examples” in Table 1, as detailed below in the section on amino acid side chain categories. Amino acids can be classified according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral-hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conserved substitutions require replacing one member of these categories with a member of another category. Amino acid substitutions can be introduced into protein constructs, and products that meet the desired activities described above can be screened.

[0167] Table 1 Amino Acid Substitution

[0168]

[0169]

[0170] In some embodiments, inserting non-natural amino acids, including synthetic non-natural amino acids or one or more D-amino acids, into the IL-2 mutant or IL-2 mutant fusion protein of this application may have several benefits. Peptides containing D-amino acids, etc., exhibit higher stability in vitro and in vivo compared to peptides containing L-amino acids. Therefore, constructing peptides by incorporating D-amino acids is particularly useful when better intracellular stability is required. In particular, D-peptides and their analogues are resistant to endogenous peptidases and proteases, thereby increasing the bioavailability of the molecule and prolonging its lifespan in vivo when needed. Furthermore, D-peptides and their analogues cannot be efficiently processed to be presented to helper T cells in a limited manner via the type II major histocompatibility complex (MHC), and therefore are not readily induced in subjects by humoral immune responses.

[0171] In some embodiments, the IL-2 mutant described in this application is a modified IL-2 mutant, such as a polyethylene glycol-modified IL-2 mutant, a covalently modified IL-2 mutant, or a glycosylated IL-2 mutant.

[0172] In some embodiments, the IL-2 mutant described in this application is a full-length sequence. In other embodiments, the N-terminus of the IL-2 mutant includes a signal peptide, which may be derived from a different molecule or from wild-type IL-2.

[0173] In some embodiments, the IL-2 mutant described in this application comprises any of the amino acid sequences shown in SEQ ID NOs:5-34 or variants thereof, wherein the variants have at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-34.

[0174] The amino acid sequences of exemplary wild-type IL-2 and its C125 mutants are shown in Table 2, the amino acid sequences of exemplary first group of IL-2 mutants are shown in Table 3, and the amino acid sequences of exemplary second group of IL-2 mutants are shown in Table 4.

[0175] Table 2. Exemplary mutants of wild-type IL-2 and C125

[0176]

[0177] Table 3. Exemplary Group 1 IL-2 mutants

[0178]

[0179]

[0180] Table 4. Exemplary second group of IL-2 mutant sequences

[0181]

[0182]

[0183] Table 5. Exemplary Fc mutant sequences

[0184]

[0185]

[0186]

[0187] Table 6. Exemplary linker peptide sequences

[0188]

[0189]

[0190] Table 7. Exemplary bivalent fusion protein sequences of the first group of IL-2 mutants and Fc.

[0191]

[0192]

[0193]

[0194] Table 8. Exemplary bivalent fusion protein sequences of the second group of IL-2 mutants and Fc.

[0195]

[0196]

[0197]

[0198] III. Fusion protein containing IL-2 mutant

[0199] This application also provides fusion proteins comprising IL-2 mutants. The IL-2 mutants described herein can be fused with other proteins to increase the circulating half-life by increasing molecular size and / or decreasing renal clearance. In some preferred embodiments, the IL-2 mutants described herein are fused with Fc. In other preferred embodiments, the IL-2 mutants described herein are fused with human serum albumin (HSA). In still other preferred embodiments, the IL-2 mutants described herein are fused with short peptides (e.g., XTEN).

[0200] IL-2 mutant fusion protein with Fc:

[0201] Fc fusion proteins can be generated using recombinant DNA technology, in which the translation reading frame of the Fc domain of a mammalian immunoglobulin (e.g., IgG) is fused to another protein to produce a new single recombinant polypeptide. Fc fusion proteins can typically be generated as disulfide-linked dimers, which are held together by disulfide bonds located in the hinge region of the Fc domain.

[0202] The IL-2 mutant can be fused to the N-terminus and / or C-terminus of the Fc. Preferably, the IL-2 mutant is fused to the C-terminus of the Fc. In some embodiments, the IL-2 mutant is directly fused to the Fc via a peptide bond. In some embodiments, a linker peptide is included between the IL-2 mutant and the Fc.

[0203] In some embodiments, the Fc described herein is derived from any one of IgA, IgD, IgE, IgG, and IgM and their subclasses. Of all immunoglobulins, IgG has the highest content in serum and the longest half-life. Unlike other immunoglobulins, IgG can be effectively recovered after binding to Fc receptors (FcRs). In some preferred embodiments, the Fc is derived from IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc is derived from human IgG. In some embodiments, the Fc contains C H 2 and C H 3. Structural Domains. In some embodiments, Fc further comprises all or part of a hinge region. In some embodiments, Fc is derived from human IgG1 or human IgG4. In some embodiments, the two subunits of Fc dimerize via one or more (e.g., 1, 2, 3, 4, or more) disulfide bonds. In some embodiments, each subunit of Fc comprises a full-length Fc sequence. In some embodiments, each subunit of Fc comprises an N-terminal truncated Fc sequence, such as a truncated Fc containing fewer N-terminal cysteine ​​residues to reduce disulfide bond mismatches during dimerization. In some embodiments, Fc is truncated at the N-terminus, for example, by deleting the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or even more amino acids of the complete immunoglobulin Fc. In some embodiments, Fc contains one or more mutations, such as insertions, deletions, and / or substitutions.

[0204] In some embodiments, the Fc described in this application has a reduced or eliminated Fc effect function. In some embodiments, the Fc has a reduced Fc-mediated effect function, such as a reduced ADCC, CDC, and / or ADCP effect function.

[0205] Fc-containing fusion proteins can activate complement and interact with Fc receptors (FcRs). In some cases, this inherent immunoglobulin property has been considered disadvantageous because such fusion proteins may target cells expressing Fc receptors instead of the preferred IL-2 receptors, potentially causing undesirable cytotoxicity. Furthermore, the long half-life of Fc fusion proteins makes them difficult to use for therapeutic purposes, or limits their therapeutic application, due to systemic toxicity. Therefore, in some embodiments, Fc is modified (e.g., by incorporating one or more amino acid mutations) to alter its binding to FcRs, particularly its binding to Fcγ receptors (responsible for ADCC) and / or to alter effector functions, such as altering antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). Preferably, such amino acid mutations do not reduce binding to FcRn receptors (related to half-life).

[0206] In some embodiments, the Fc (e.g., the Fc of human IgG1) is mutated to remove one or more effector functions, such as ADCC, ADCP, and / or CDC, hereinafter referred to as a "no-effect" or "near-no-effect" Fc. For example, in some embodiments, the Fc is a no-effect human IgG1 Fc containing the mutations L234A and L235A. In some embodiments, the human IgG1 Fc contains one or more of the following mutations (as in each Fc subunit): L234A, L235E, G237A, A330S, and P331S. As is known to those skilled in the art, the combination of K322A, L234A, and L235A in IgG1 Fc is sufficient to completely eliminate the binding of FcγR and C1q (Hezareh et al., J Virol 75, 12161–12168, 2001). MedImmune found that Fc containing a set of three mutations, L234F / L235E / P331S, exhibit very similar effects (Oganesyan et al., Acta Crystallographica 64, 700–704, 2008). In some embodiments, the Fc contains a glycosylation modification on the N297 region of the IgG1 Fc region, which is known to be necessary for generating optimal FcR interactions. The modification of the Fc can be any suitable engineered IgG Fc mentioned by Wang et al. (“IgG Fc engineering to modulate antibody effector functions,” Protein Cell. 2018 Jan; 9(1):63–73), the entire contents of which are incorporated herein by reference.

[0207] In some embodiments, as described in this application, the fusion protein of the IL-2 mutant and Fc does not have ADCC and / or CDC, or does not have detectable ADCC and / or CDC. In some embodiments, as described in this application, the fusion protein of the IL-2 mutant and Fc produces ADCC and / or CDC reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) compared to a fusion protein containing the same IL-2 mutant moiety but fused to wild-type or unmodified Fc.

[0208] Glycosylation variants

[0209] In some embodiments, the degree of glycosylation of the construct can be increased or decreased by altering the fusion protein of Fc or IL-2 mutant with Fc. Glycosylation sites can be added or deleted in Fc by changing the amino acid sequence to create or remove one or more glycosylation sites.

[0210] Naturally occurring Fc-containing proteins produced by mammalian cells typically contain a branched chain of biantennary oligosaccharides, which are usually linked to FcC via N-bonds. H On Asn297 of the 2-domain. See Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in Fc can be modified to produce certain improved properties.

[0211] In some embodiments, the fusion protein of Fc or IL-2 mutant with Fc as described in this application has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, in such a fusion protein of Fc or IL-2 mutant with Fc, the fucose content may range from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. As described in WO 2008 / 077546, the fucose content is determined by MALDI-TOF mass spectrometry measurement of the average intra-glycan fucose content attached to Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures). Asn297 refers to the asparagine residue located at position 297 of the Fc region (Fc region residue EU numbering system); however, due to minor sequence variations in the Fc region, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may possess enhanced ADCC function. See US Patent Publication Nos. US2003 / 0157108 (Presta, L.), US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to antibody variants that are “defucosylated” or “fucosylated” include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated Fc protein include Lec13 CHO cells lacking protein fucosylation function (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells with α-1,6-fucosylation gene and FUT8 gene knockout (see Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0212] Effect function variants

[0213] In some embodiments, this application contemplates an Fc possessing some, but not all, of its effector functions, making it an ideal candidate for an application where the in vivo half-life of the fusion protein of the IL-2 mutant and Fc is important, but certain effector functions (such as CDC and ADCC) are non-essential or detrimental. Cytotoxicity assays can be performed in vitro or in vivo to determine the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the Fc or the fusion protein containing this Fc and IL-2 mutant lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC, natural killer (NK) cells, express only FcγRIII; however, monocytes express FcγRI, FcγRII, and FcγRIII. Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991) summarizes the expression of FcR on hematopoietic cells. Non-limiting examples of in vitro assays for assessing the ADCC activity of target molecules are detailed in US Patent No. 5,500,362 (see Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be used (see ACTITM non-radioactive toxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA) and CytoTox). Non-radiotoxicity assays (Promega, Madison, WI). Effector cells suitable for this assay include peripheral blood mononuclear cells (PBMCs) and NK cells. Alternatively, ADCC activity of the target molecule can be assessed in vivo, for example, in animal models disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to determine that the fusion protein of the IL-2 mutant and Fc cannot bind to C1q and therefore lacks CDC activity. See C1q and C3c binding enzyme-linked immunosorbent assays in WO 2006 / 029879 and WO2005 / 100402. The CDC assay can be performed to assess complement activity (see Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can be performed using methods known in the art (see Petkova, S B et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0214] Reduced effector function Fc contains substitutions of one or more residues at positions 238, 265, 269, 270, 297, 327, and 329 in the Fc region (US Patent No. 6,737,056). Such Fc mutants include substitutions at two or more of the amino acid sites 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, which replaces residues 265 and 297 with alanine (US Patent No. 7,332,581). Certain antibody variants that enhance or reduce binding to FcRs are described in detail (see US Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001). In some embodiments, the Fc region is modified to alter (i.e., increase or decrease) C1q binding and / or CDC, as described in, for example, US Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4 178-4184 (2000).

[0215] In some embodiments, the Fc region contains one or more amino acid substitutions, which increase the half-life and / or enhance binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and enhanced binding to the neonatal FcRn are responsible for transporting maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and are detailed in US2005 / 0014934A1 (Hinton et al.). Antibodies containing an Fc region with one or more substitutions thus increase the binding of the Fc region to the FcRn. Such Fc variants include those with one or more Fc region residue substitutions, for example, a substitution of Fc region residue 434 (US Patent No. 7,371,826).

[0216] See Duncan and Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc variants.

[0217] Cysteine ​​engineered variants

[0218] In some embodiments, it may be necessary to create a cysteine-engineered Fc or a fusion protein comprising this Fc and an IL-2 mutant, wherein one or more residues of the Fc are replaced by cysteine ​​residues. In some embodiments, the substituted residues appear at accessible sites on the Fc or the fusion protein of the IL-2 mutant and Fc. By replacing these residues with cysteine, the active thiol group is thus located at an accessible site on the Fc or the fusion protein of the IL-2 mutant and Fc and can be used to conjugate the molecule with other parts, such as a drug moiety or a linker-drug moiety, to create a conjugate of the IL-2 mutant and Fc fusion protein. In some embodiments, any one or more of the following residues may be replaced by cysteine: heavy chain A118 (EU numbering system) and heavy chain Fc domain S400 (EU numbering system). Cysteine-engineered molecules can be produced as described in US Patent No. 7,521,541.

[0219] In some embodiments, Fc is derived from IgG1 Fc. In some embodiments, Fc is derived from human IgG1 Fc. In some embodiments, Fc is derived from human wild-type IgG1 Fc. In some embodiments, Fc does not contain the hinge region of IgG1 Fc. In some embodiments, Fc contains one or more effector mutations and / or deglycosylation mutations.

[0220] In some embodiments, Fc contains one or more mutations relative to amino acid residues L234 (L234) and L235 (L235) of the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc contains mutations L234 and L235 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc contains mutations L234A and L235A relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc contains (or is substantially composed of, or is composed of) the amino acid sequence SEQ ID NO:35 or a variant thereof; said variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:35.

[0221] In some embodiments, Fc contains a mutation at amino acid residue N (N297) at position 297 of the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc contains the mutation N297G or N297A relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc contains (or is substantially composed of, or is composed of) the amino acid sequence SEQ ID NO:36 or a variant thereof; said variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:36. In some embodiments, Fc contains (or is substantially composed of, or is composed of) the amino acid sequence SEQ ID NO:37 or a variant thereof; said variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:37.

[0222] In some embodiments, Fc contains one or more mutations relative to amino acid residues L (L234), L (L235), and P (P331) at position 234 of the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc contains mutations at L234, L235, and P331 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc contains mutations L234A, L235A, and P331S relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc contains (or is substantially composed of, or is composed of) the amino acid sequence SEQ ID NO:38 or a variant thereof; said variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:38.

[0223] In some embodiments, Fc comprises one or more mutations relative to amino acid residues L (L234), L (L235), G (G237), A (A330), and P (P331) at position 234 of the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc comprises mutations in L234, L235, G237, A330, and P331 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc comprises mutations L234A, L235E, G237A, A330S, and P331S relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, Fc comprises (or is substantially composed of, or is composed of) the amino acid sequence SEQ ID NO:39 or a variant thereof; the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:39.

[0224] In some embodiments, Fc is derived from IgG4 Fc. In some embodiments, Fc is derived from human IgG4 Fc. In some embodiments, Fc is derived from human wild-type IgG4 Fc. In some embodiments, Fc does not contain the hinge region of IgG4. In some embodiments, Fc contains one or more effector mutations and / or deglycosylation mutations.

[0225] In some embodiments, Fc contains one or more mutations relative to amino acid residue S (S228), amino acid residue F (F234), and amino acid residue L (L235) of the human wild-type IgG4 Fc amino acid sequence. In some embodiments, Fc contains mutations S228, F234, and L235 relative to the human wild-type IgG4 Fc amino acid sequence. In some embodiments, Fc contains mutations S228P, F234A, and L235A relative to the human wild-type IgG4 Fc amino acid sequence. In some embodiments, the Fc portion contains (or is substantially composed of, or is composed of) the amino acid sequence SEQ ID NO:40 or a variant thereof; said variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:40.

[0226] KIH (Knob-into-Hole) technology:

[0227] Additional mutations that can be introduced into Fc include those that promote the formation of heterodimers between Fc peptides. In some embodiments, the assembly of heterodimers is addressed using KIH (Knob-into-Hole) technology, which involves... H 3. The structural domain introduces an asymmetric mutation structure ("knob" mutation refers to a mutation in C...). H In the 3 domain, a larger amino acid residue replaces a smaller residue, while a "hole" mutation refers to replacing a larger residue with a smaller amino acid residue. Modified Fc molecules, due to steric hindrance, are more prone to heterodimerization than homodimerization (Ridgway JB, et al. "Knobs-into-holes" engineering of antibody C). H 3 domains for heavy chainheterodimerization[J]. Protein Eng. 1996, 9(7):617-621). (This refers to a C...) H The threonine residue (T) at position 366 of the 3rd domain is replaced with a tryptophan residue (W) to form a "knob" structure, and the other paired C is replaced with a tryptophan residue (W). HThe 366th threonine residue (T) of the 3 domain is replaced with a serine residue (S), the 368th leucine residue (L) is replaced with an alanine residue (A), and the 407th tyrosine residue (Y) is replaced with a valine residue (V) to form a "holes" structure. At the same time, two cysteine ​​residue (C) mutations are introduced to form a stabilizing disulfide bridge (S354C on the "knob" side and Y349C on the "hole" side), which enhances the stability of the KIH. The numbering follows the EU index as described in Kabat (Kuglstatter A, et al. Structural differences between glycosylated, disulfide-linked heterodimeric knob-into-hole Fc fragment and its homodimeric knob-knob and hole-hole side products[J]. Protein Eng Des Sel., 2017, 30(9): 649-656).

[0228] Exemplary Fc mutant sequences are shown in Table 5.

[0229] In some embodiments, the IL-2 mutant fusion protein of this application with Fc is in a divalent form, comprising two IL-2 mutant molecules. For example, a homodimer composed of two of the aforementioned fusion polypeptide chains. In some embodiments, its exemplary structure is as follows: Figure 1A and Figure 1B As shown.

[0230] In some embodiments, the IL-2 mutant fusion protein of this application is in a monovalent form, comprising only one IL-2 mutant molecule. For example, it can be a heterodimer composed of one of the aforementioned fusion polypeptide chains and one Fc subunit. In some embodiments, its exemplary structure is as follows: Figure 2 As shown. In other embodiments, the IL-2 mutant fusion protein of this application with Fc is in monovalent form, consisting of one of the above-described fusion polypeptide chains.

[0231] connector

[0232] In some embodiments, the IL-2 mutant and Fc are linked via a linker (e.g., a linker peptide, a non-linker peptide). In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a stable linker. Generally, an ideal linker does not affect or does not significantly affect the correct folding and conformation of the IL-2 mutant-Fc fusion protein described herein. Preferably, the linker imparts flexibility to the IL-2 mutant-Fc fusion protein, preserves or enhances the biological function of the IL-2 mutant, and / or does not affect or significantly affect the half-life and / or stability of the IL-2 mutant-Fc fusion protein in vivo. In some embodiments, the linker is a stable linker (e.g., not cleaved by proteases, particularly MMPs).

[0233] In some embodiments, the linker is a linker peptide. The linker peptide can be of any length. In some embodiments, the linker peptide length is 1 to 10 amino acids, 3 to 18 amino acids, 1 to 20 amino acids, 10 to 20 amino acids, 21 to 30 amino acids, 1 to 30 amino acids, 10 to 30 amino acids, 1 to 50 amino acids, 5 to 40 amino acids, 12 to 18 amino acids, or 4 to 25 amino acids. In some embodiments, the linker peptide length is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the linker peptide length is 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the linker peptide is 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some embodiments, the linker peptide length does not exceed the length necessary to prevent undesirable domain interactions and / or optimize biological function and / or stability. In some embodiments, the linker peptide length is up to 30 amino acids, for example up to 20 amino acids, or up to 15 amino acids. In some embodiments, the linker peptide length is 5 to 30 amino acids, or 5 to 18 amino acids.

[0234] Linker peptides can have naturally occurring sequences or non-naturally occurring sequences. For example, sequences from the hinge region of an antibody heavy chain can be used as linkers. See, for example, WO1996 / 34103. In some embodiments, the linker peptide is a hinge region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the linker peptide is a mutated hinge region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the linker is a flexible linker. Typical flexible linkers include, but are not limited to, glycine polymers (G)n (SEQ ID NO:43), glycine-serine polymers (including, for example, (GS)n (SEQ ID NO:44), (GGS)n (SEQ ID NO:45), (GGGS)n (SEQ ID NO:46), (GGGGS)n (SEQ ID NO:47) or (GGS)m(GGGS)n (SEQ ID NO:48), where m and n are integers at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore can serve as a neutral chain between components. Glycine has more phi-psi space than alanine and is less restricted compared to residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Examples of flexible linkers include, but are not limited to, the amino acid sequences shown in Table 6. Generally, those skilled in the art will recognize that the designed IL-2 mutant fusion protein with Fc may include all or part of a flexible linker, such that the linker may include a flexible linker portion and one or more portions providing less flexible structure to provide an ideal structure and function for the IL-2 mutant fusion protein with Fc. In some embodiments, the linker peptide comprises any of the amino acid sequences shown in SEQ ID NOs:43-74 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:43-74.

[0235] Exemplary linker peptide sequences are shown in Table 6.

[0236] Other considerations regarding the adapter include its impact on the physical or pharmacokinetic properties of the resulting IL-2 mutant fusion protein with Fc, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stability and planned degradation), rigidity, flexibility, immunogenicity, binding of the IL-2 mutant to the IL-2 receptor, and binding ability to colloids or liposomes.

[0237] In some embodiments, the IL-2 mutant fusion protein of this application comprises a bivalent form and a monovalent form. In other embodiments, the bivalent form comprises two IL-2 mutant molecules, which may be composed of two identical or different monomers, for example, the monomer is a fusion polypeptide chain of the IL-2 mutant and Fc. In other embodiments, the monovalent form comprises one IL-2 mutant molecule, which may be composed of two different monomers, for example, one monomer is a fusion polypeptide chain of the IL-2 mutant and an Fc subunit, and the other monomer is an Fc subunit. In other embodiments, the monovalent form comprises one IL-2 mutant molecule, composed of only one monomer, for example, the monomer is a fusion polypeptide chain of the IL-2 mutant and an Fc subunit.

[0238] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises a deletion or mutation of amino acid residue RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the amino acid sequence of the human wild-type IL-2 is as shown in SEQ ID NO:1.

[0239] In some embodiments, the IL-2 mutant fusion protein of the present application and Fc (e.g., in the form of a bivalent or monovalent fusion protein) comprises a complete deletion of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant further comprises mutations at E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 and N71 mutations are E67K and N71S, respectively. In some embodiments, the IL-2 mutant comprises a complete deletion of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence, and mutations at E67K and N71S relative to the human wild-type IL-2 amino acid sequence.

[0240] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises a mutation of RPRDL to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant further comprises a mutation of E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 mutation is E67A. In some embodiments, the IL-2 mutant comprises a mutation of RPRDL to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and an E67A mutation relative to the human wild-type IL-2 amino acid sequence.

[0241] In some embodiments, the IL-2 mutant fusion protein of the present application and Fc (e.g., in the form of a bivalent or monovalent fusion protein) comprises an RPRDL mutation to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises mutations at N71 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the N71 and S75 mutations are N71W and S75I, respectively. In some embodiments, the IL-2 mutant comprises an RPRDL mutation to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations at N71W and S75I relative to the human wild-type IL-2 amino acid sequence.

[0242] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant further comprises mutations in E67 and / or V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 mutation is E67T. In some embodiments, the V91 mutation is V91K. In some embodiments, the IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and E67T and / or V91K mutations relative to the human wild-type IL-2 amino acid sequence.

[0243] In some embodiments, the IL-2 mutant fusion protein of the present application and Fc (e.g., in the form of a bivalent or monovalent fusion protein) comprises an RPRDL mutation to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and further comprises mutations at L70 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L70 and S75 mutations are L70I and S75F, respectively. In some embodiments, the IL-2 mutant comprises an RPRDL mutation to RHL at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, and mutations at L70I and S75F relative to the human wild-type IL-2 amino acid sequence.

[0244] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises an N71 mutation relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the N71 mutation is N71I. In some embodiments, the IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and an N71I mutation relative to the human wild-type IL-2 amino acid sequence.

[0245] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises an RPRDL mutation to SPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant further comprises mutations at E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 and N71 mutations are E67M and N71S, respectively. In some embodiments, the IL-2 mutant comprises an RPRDL mutation to SPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations at E67M and N71S relative to the human wild-type IL-2 amino acid sequence.

[0246] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises a mutation at amino acid residue S (S75) at position 75 and amino acid residue R (R83) at position 83 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the amino acid sequence of the human wild-type IL-2 is as shown in SEQ ID NO:1.

[0247] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein S75 is mutated to an uncharged amino acid residue. In some embodiments, the mutation of S75 is S75P, S75F, S75Y, S75G, S75A, S75V, S75T, S75I, or S75L. In some embodiments, the mutation of R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K, or R83E.

[0248] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75P and the R83 mutation is R83Y. In some embodiments, the IL-2 mutant further comprises mutations in E67 and D84 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 and D84 mutations are E67A and D84N, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75P, R83Y, E67A, and D84N relative to the human wild-type IL-2 amino acid sequence.

[0249] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75P and the R83 mutation is R83A. In some embodiments, the IL-2 mutant further comprises mutations in H16, L70, and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in H16, L70, and N71 are H16D, L70I, and N71G, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75P, R83A, H16D, L70I, and N71G relative to the human wild-type IL-2 amino acid sequence.

[0250] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75F and the R83 mutation is R83P. In some embodiments, the IL-2 mutant further comprises mutations in H16 and / or N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the H16 mutation is H16Q or H16N. In some embodiments, the N71 mutation is N71S. In some embodiments, the IL-2 mutant comprises mutations in S75F, R83P, and H16Q relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant comprises mutations in S75F, R83P, H16N, and N71S relative to the human wild-type IL-2 amino acid sequence.

[0251] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75F and the R83 mutation is R83L. In some embodiments, the IL-2 mutant further comprises mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 and L70 mutations are E67R and L70V, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75F, R83L, E67R, and L70V relative to the human wild-type IL-2 amino acid sequence.

[0252] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75I and the R83 mutation is R83L. In some embodiments, the IL-2 mutant further comprises mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 and L70 mutations are E67G and L70V, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75I, R83L, E67G, and L70V relative to the human wild-type IL-2 amino acid sequence.

[0253] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75I and the R83 mutation is R83K. In some embodiments, the IL-2 mutant further comprises mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 and L70 mutations are E67S and L70V, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75I, R83K, E67S, and L70V relative to the human wild-type IL-2 amino acid sequence.

[0254] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75T and the R83 mutation is R83A or R83F. In some embodiments, the IL-2 mutant further comprises mutations at H16 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the H16 and E67 mutations are H16N and E67K, respectively. In some embodiments, the IL-2 mutant comprises mutations at S75T, R83A, H16N, and E67K relative to the human wild-type IL-2 amino acid sequence.

[0255] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75V and the R83 mutation is R83F. In some embodiments, the IL-2 mutant further comprises mutations in E15 and H16 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E15 and H16 mutations are E15K and H16Q, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75V, R83F, E15K, and H16Q relative to the human wild-type IL-2 amino acid sequence.

[0256] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75V and the R83 mutation is R83N. In some embodiments, the IL-2 mutant further comprises mutations in L70 and V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L70 and V91 mutations are L70F and V91K, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75V, R83N, L70F, and V91K relative to the human wild-type IL-2 amino acid sequence.

[0257] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75G and the R83 mutation is R83L. In some embodiments, the IL-2 mutant further comprises mutations in L17, V91, and I92 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations in L17, V91, and I92 are L17I, V91E, and I92L, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75G, R83L, L17I, V91E, and I92L relative to the human wild-type IL-2 amino acid sequence.

[0258] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75Y and the R83 mutation is R83N. In some embodiments, the IL-2 mutant further comprises mutations in L17 and V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L17 and V91 mutations are L17I and V91K, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75Y, R83N, L17I, and V91K relative to the human wild-type IL-2 amino acid sequence.

[0259] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75A and the R83 mutation is R83K. In some embodiments, the IL-2 mutant further comprises a mutation at L17 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L17 mutation is L17I. In some embodiments, the IL-2 mutant comprises mutations at S75A, R83K, and L17I relative to the human wild-type IL-2 amino acid sequence.

[0260] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in this application (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein the S75 mutation is S75L and the R83 mutation is R83E. In some embodiments, the IL-2 mutant further comprises mutations at L63 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L63 and E67 mutations are L63V and E67F, respectively. In some embodiments, the IL-2 mutant comprises mutations at S75L, R83E, L63V, and E67F relative to the human wild-type IL-2 amino acid sequence.

[0261] In some embodiments, the IL-2 mutant fusion protein of this application and Fc (e.g., in the form of a bivalent or monovalent fusion protein) further comprises, in addition to the above-described mutations, a C125 mutation relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the C125 mutation is C125S or C125A.

[0262] In some embodiments, the IL-2 mutant fusion protein of the present application and Fc (e.g., in the form of a bivalent or monovalent fusion protein) comprises an IL-2 mutant containing any of the amino acid sequences shown in SEQ ID NOs:5-34 or variants thereof, wherein the variants have at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-34.

[0263] The amino acid sequences of the first group of IL-2 mutants and Fc bivalent fusion proteins are shown in Table 7, and the amino acid sequences of the second group of IL-2 mutants and Fc bivalent fusion proteins are shown in Table 8.

[0264] In some embodiments, in the bivalent fusion protein form of IL-2 mutant and Fc described in this application, each monomer comprises, from the N-terminus to the C-terminus, or from the C-terminus to the N-terminus, (i) IL-2 mutant and (ii) Fc.

[0265] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described in this application, each monomer comprises, from the N-terminus to the C-terminus, or from the C-terminus to the N-terminus, the following: (i) the IL-2 mutant, (ii) the linker peptide, and (iii) the Fc.

[0266] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described in this application, the Fc is IgG1 Fc, and the Fc contains L234A and L235A (LALA) mutations. In some embodiments, the Fc contains the amino acid sequence SEQ ID NO:35.

[0267] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described in this application, the Fc is IgG1 Fc, and the Fc contains the N297G mutation. In some embodiments, the Fc contains the amino acid sequence SEQ ID NO:36.

[0268] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described in this application, the Fc is IgG1 Fc, and the Fc contains the N297A mutation. In some embodiments, the Fc contains the amino acid sequence SEQ ID NO:37.

[0269] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described in this application, the Fc is IgG1 Fc, and the Fc contains L234A, L235A, and P331S mutations. In some embodiments, the Fc contains the amino acid sequence SEQ ID NO:38.

[0270] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described in this application, the Fc is IgG1 Fc, and the Fc contains L234A, L235E, G237A, A330S, and P331S mutations. In some embodiments, the Fc contains the amino acid sequence SEQ ID NO:39.

[0271] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described in this application, the Fc is IgG4 Fc, and the Fc contains S228P, F234A, and L235A mutations. In some embodiments, the Fc contains the amino acid sequence SEQ ID NO:40.

[0272] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described in this application, each monomer comprises, from the N-terminus to the C-terminus, or from the C-terminus to the N-terminus: (i) an IL-2 mutant comprising any of the amino acid sequences shown in SEQ ID NOs:5-34 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-34 and (ii) an Fc comprising any of the amino acid sequences shown in SEQ ID NOs:35-40 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:35-40.

[0273] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described in this application, each monomer comprises, from the N-terminus to the C-terminus, or from the C-terminus to the N-terminus: (i) an IL-2 mutant comprising any of the amino acid sequences shown in SEQ ID NOs:5-34 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-34; (ii) a linker peptide comprising any of the amino acid sequences shown in SEQ ID NOs:43-74 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:43-74; and (iii) an Fc comprising, as in SEQ ID NOs:43-74. The amino acid sequence shown in any of SEQ ID NOs:35-40 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:35-40.

[0274] In some embodiments, the IL-2 mutant and Fc bivalent fusion protein form described in this application comprises any of the amino acid sequences shown in SEQ ID NOs:76-105 or variants thereof, wherein the variants have at least about 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:76-105.

[0275] In some embodiments, in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described in this application, Fc is IgG1 Fc. In some embodiments, the Fc contains a KIH mutation (referred to as Fc knob and Fchole, respectively). In some preferred embodiments, the Fc contains a LALA mutation and a KIH mutation (referred to as Fc LALA knob and Fc LALA hole, respectively). In some more preferred embodiments, the Fc LALA knob may contain mutations L234A, L235A, T366W, and S354C, and the Fc LALA hole may contain mutations L234A, L235A, T366S, L368A, Y407V, and Y349C.

[0276] In some embodiments, the IgG1 Fc LALA knob contains the amino acid sequence SEQ ID NO:41, and the Fc LALA hole contains the amino acid sequence SEQ ID NO:42.

[0277] In some embodiments, in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described in this application, one monomer comprises, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: (i) the IL-2 mutant and (ii) the Fc knob, and another monomer comprises: the Fc hole.

[0278] In some embodiments, in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described in this application, one monomer comprises, from the N-terminus to the C-terminus, or from the C-terminus to the N-terminus,: (i) an IL-2 mutant comprising any of the amino acid sequences shown in SEQ ID NOs:5-34 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-34; and (ii) an Fc LALA knob comprising the amino acid sequence SEQ ID NO:41 or a variant thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:41; and another monomer is an Fc LALA hole comprising the amino acid sequence SEQ ID NO:41. NO:42 or a variant thereof, said variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO:42.

[0279] In some embodiments, in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described in this application, one monomer comprises, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: (i) the IL-2 mutant and (ii) the Fc hole; and the other monomer comprises: the Fc knob.

[0280] In some embodiments, in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described in this application, one monomer comprises, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: (i) an IL-2 mutant comprising any of the amino acid sequences shown in SEQ ID NOs:5-34 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-34 and (ii) an Fc LALA hole comprising the amino acid sequence SEQ ID NO:42 or a variant thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:42; and another monomer is an Fc LALA knob comprising the amino acid sequence SEQ ID NO:42. NO:41 or a variant thereof, said variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology with SEQ ID NO:41.

[0281] In some embodiments, in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described in this application, one monomer comprises, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: (i) the IL-2 mutant, (ii) the linker peptide and (iii) Fcknob; and the other monomer comprises: Fc hole.

[0282] In some embodiments, in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described in this application, one of the monomers comprises, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: (i) an IL-2 mutant comprising any of the amino acid sequences shown in SEQ ID NOs:5-34 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-34; (ii) a linker peptide comprising any of the amino acid sequences shown in SEQ ID NOs:43-74 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:43-74; and (iii) Fc. LALAknob, comprising the amino acid sequence SEQ ID NO:41 or a variant thereof, said variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:41; and another monomer, Fc LALA hole, comprising the amino acid sequence SEQ ID NO:42 or a variant thereof, said variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:42.

[0283] In some embodiments, in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described in this application, one monomer comprises, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: (i) the IL-2 mutant, (ii) a linker peptide and (iii) Fchole, and the other monomer comprises: Fc knob.

[0284] In some embodiments, in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described in this application, one of the monomers comprises, from the N-terminus to the C-terminus or from the C-terminus to the N-terminus: (i) an IL-2 mutant comprising any of the amino acid sequences shown in SEQ ID NOs:5-34 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:5-34; (ii) a linker peptide comprising any of the amino acid sequences shown in SEQ ID NOs:43-74 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:43-74; and (iii) Fc. LALAhole, comprising the amino acid sequence SEQ ID NO:42 or a variant thereof, said variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:42; and another monomer, Fc LALA knob, comprising the amino acid sequence SEQ ID NO:41 or a variant thereof, said variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:41.

[0285] In some embodiments, when administered to an individual (e.g., a human) via intravenous, intramuscular, or subcutaneous injection, the half-life of the IL-2 mutant fusion protein of this application with Fc is at least 10 hours (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 hours or longer).

[0286] Combining affinity

[0287] The binding affinity of a molecule (e.g., an IL-2 mutant or a fusion protein containing an IL-2 mutant) to its binding target (e.g., an IL-2 receptor, such as IL-2Rα, IL-2Rβ, IL-2Rγ, IL-2Rβγ, or IL-2Rαβγ) can be determined by any suitable ligand binding assay known in the art, such as Western blot, enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence, bead-based multiplex immunoassay (MIA), RIA, surface plasmon resonance (SPR), ECL, IRMA, EIA, Biacore assay, Octet assay, peptide scanning, etc. For example, simple analyses can be performed using various labeling reagents to label IL-2 mutants or fusion proteins containing IL-2 mutants or their receptors (e.g., IL-2Rα, IL-2Rβ, IL-2Rγ, IL-2Rβγ, or IL-2Rαβγ), or their subunits, using BiacoreX (Amersham Biosciences), an over-the-counter assay kit or similar kit, which can be operated according to the user manual and experimental procedures included with the kit.

[0288] In some embodiments, protein microarrays are used for large-scale analysis of the interaction, function, and activity of the IL-2 mutant or fusion protein containing the IL-2 mutant described in this application with its receptor. The protein microarray has a supporting surface that binds to a series of capture proteins (e.g., the IL-2 receptor or its subunits). Fluorescently labeled probe molecules (e.g., the IL-2 mutant or fusion protein containing the IL-2 mutant described in this application) are then added to the array and interact with the bound capture proteins, emitting fluorescent signals that are read by a laser scanner.

[0289] Binding affinity can also be measured using Biacore. For example, IL-2Rβγ or IL-2Rαβγ was coupled to the surface of the CM-5 sensor chip as a ligand using an EDC / NHS chemistry method. Then, a series of dilutions of the IL-2 mutant or the IL-2 mutant-Fc fusion protein described herein were used as analytes to bind to the ligands coupled to the chip, and the binding and dissociation of IL-2 with IL-2Rβγ or IL-2Rαβγ could be monitored in real time. Affinity (Kd) can be determined by kinetic analysis using BIA evaluation software.

[0290] In some embodiments, the Biacore assay can be applied to determine the Kd value of the IL-2 mutant or the IL-2 mutant-Fc fusion protein provided in this application with IL-2Rβγ to determine binding affinity. In some embodiments, the IL-2 mutant-Fc fusion protein of this application has a lower binding affinity for IL-2Rβγ compared to the wild-type IL-2-Fc fusion protein. In some embodiments, the affinity of the IL-2 mutant-Fc fusion protein with IL-2Rβ and / or IL-2Rβγ is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) compared to the wild-type IL-2-Fc fusion protein. In some embodiments, the IL-2 mutant fusion protein of this application, compared to the wild-type IL-2 fusion protein of Fc (e.g., human wild-type IL-2), exhibits increased, unchanged, or no significant decrease in affinity for IL-2Rα. In some embodiments, compared to the wild-type IL-2 fusion protein of Fc, the IL-2 mutant fusion protein of this application exhibits increased, unchanged, or no significant decrease in binding affinity for IL-2Rαβγ.

[0291] In some embodiments, the Kd value of the binding between the IL-2 mutant described herein or the fusion protein of the IL-2 mutant and Fc and the intermediate affinity receptor (IL-2Rβγ) is greater than the Kd value of the binding between wild-type IL-2 or the fusion protein of wild-type IL-2 and Fc and its same receptor. In some embodiments, the Kd value of the binding between the IL-2 mutant described herein or the fusion protein of the IL-2 mutant and Fc and its high affinity receptor (IL-2Rαβγ) is close to (e.g., equal to, less than, or slightly greater than) the Kd value of the binding between wild-type and the same receptor (IL-2Rαβγ).

[0292] Pharmacokinetics (PK)

[0293] Pharmacokinetics (PK) refers to the absorption, distribution, metabolism, and excretion of a drug (e.g., the IL-2 mutant or fusion protein containing the IL-2 mutant described herein) after administration to a subject. Pharmacokinetic parameters that can be used to determine clinical efficacy include, but are not limited to, serum / plasma concentration, serum / plasma concentration over time, and peak serum / plasma concentration (Cmax). max ), time to reach maximum concentration (T) max ), half-life (T) 1 / 2 The area under the concentration-time curve within the dosing interval (AUC) τ )wait.

[0294] Techniques for obtaining PK curves of drugs (e.g., the IL-2 mutant or fusion protein containing the IL-2 mutant described herein) are known in the art. See Heller et al., Annu Rev Anal Chem, 11, 2018; and Ghandforoush Sattari et al., J Amino Acids, Article ID 346237, Volume 2010. In some embodiments, PK curves of the IL-2 mutant or fusion protein containing the IL-2 mutant as described herein are measured in a blood, plasma, or serum sample of an individual. In some embodiments, mass spectrometry techniques (e.g., LC-MS / MS or ELISA) are used to measure the PK curves of the IL-2 mutant or fusion protein containing the IL-2 mutant as described herein in an individual. PK analysis can be performed on PK curves using any method known in the art, such as non-interlaboratory analysis using PKSolver V2 software (Zhang Y. et al., “PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel,” Comput Methods Programs Biomed. 2010; 99(3):306-1). See Example 8 for an example method.

[0295] “C” represents the concentration of the drug (e.g., an IL-2 mutant or a fusion protein containing an IL-2 mutant) in the subject’s plasma, serum, or any suitable body fluid or tissue, typically expressed as mass per unit volume, such as nanograms per milliliter. For convenience, the drug concentration in serum or plasma is referred to herein as “serum concentration” or “plasma concentration.” The serum / plasma concentration at any time following administration (e.g., IL-2 mutant or a fusion protein polypeptide containing an IL-2 mutant via intravenous, intraperitoneal, or subcutaneous injection) is referred to as C. time Or C t The maximum serum / plasma drug concentration during administration is called C. max C min This refers to the minimum serum / plasma drug concentration at the end of the dosing interval; C ave This refers to the average concentration during the dosing interval.

[0296] The term "bioavailability" refers to the extent or rate at which a drug (e.g., an IL-2 mutant or a fusion protein containing an IL-2 mutant) reaches its site of action via systemic circulation.

[0297] "AUC" is the area under the serum / plasma concentration-time curve and is considered the most reliable measure of bioavailability, such as the area under the concentration-time curve within the dosing interval (AUCτ), "total exposure," or "total drug exposure over a period of time" (AUC). 0-∞ The area under the concentration-time curve (AUC) at time t after drug administration. 0-t )wait.

[0298] Time to peak serum / plasma concentration (T) max ) is the serum / plasma concentration (C) reached after administration (e.g., IL-2 mutant or fusion protein containing IL-2 mutant). max The peak time.

[0299] Half-life (T) 1 / 2 Elimination half-life refers to the time required for the concentration of a drug (e.g., an IL-2 mutant or a fusion protein containing an IL-2 mutant) in plasma or serum (or other biological matrix) to decrease to half its concentration or amount at a specific time point. For example, after intravenous administration, the concentration of the drug in plasma or serum decreases due to distribution and elimination. In the curve of plasma or serum drug concentration versus time after intravenous administration, the first phase, or rapid decline phase, is considered to be primarily due to distribution, while the later decline is typically slower and primarily due to elimination, although both processes occur in both phases. Distribution is considered to be completed after sufficient time. Generally, the elimination half-life is determined by the terminal phase or elimination (primary) phase of the plasma / serum concentration-time curve. See Michael Schrag and Kelly Regal, "Comprehensive Guide to Toxicology in Preclinical Drug Development," Chapter 3 – Pharmacokinetics and Toxicokinetics, 2013.

[0300] In some embodiments, the fusion protein comprising an IL-2 mutant described herein (e.g., a bivalent fusion protein of an IL-2 mutant and Fc) has a half-life of at least 10 hours (e.g., for intravenous, subcutaneous, or intramuscular injection, such as for human injection), such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 hours, or longer.

[0301] IL-2 mutants or derivatives of fusion proteins containing IL-2 mutants

[0302] In some embodiments, the IL-2 mutant or fusion protein containing the IL-2 mutant described herein may be further modified to include additional non-protein portions known in the art and readily available. Exemplary non-protein portions include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxolane, ethylene / maleic anhydride copolymers, polyamic acid (homomers or random copolymers), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the IL-2 mutant or the fusion protein containing the IL-2 mutant may vary, and if multiple polymers are attached, they may be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the IL-2 mutant or the fusion protein containing the IL-2 mutant to be improved, and whether the derivative of the IL-2 mutant or the fusion protein containing the IL-2 mutant will be used for treatment under specific conditions.

[0303] In some embodiments, the IL-2 mutant or fusion protein containing the IL-2 mutant described herein further comprises a tag selected from chromophores, fluorophores (e.g., coumarin, xanthracene, cyanine, pyrene, boron polybenzozaindole, oxazine and their derivatives), fluorescent proteins (e.g., GFP, phycobiliproteins and their derivatives), phosphorescent dyes (e.g., dioxane, xanthracene or carbocyanine dyes, lanthanide chelates), tandem dyes (e.g., cyanine-phycobiliprotein derivatives and xanthracene-phycobiliprotein derivatives), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), haptens, enzymes (e.g., peroxidase, phosphatase, glycosidase, luciferase), and radioisotopes (e.g., 125 I, 3 H, 14 C 32 P).

[0304] In some embodiments, IL-2 mutants or fusion proteins containing IL-2 mutants may be further modified to include one or more other bioactive proteins, peptides, or fragments thereof. As described herein, “bioactive” or “biologically active” is used interchangeably and refers to exhibiting biological activity in vivo to perform a specific function. For example, it may mean binding to a specific biomolecule, such as a protein, DNA, etc., and then promoting or inhibiting the activity of that biomolecule. In some embodiments, bioactive proteins or fragments thereof include proteins and peptides administered to patients as active pharmaceutical ingredients for the prevention or treatment of diseases or symptoms, proteins and peptides for diagnostic purposes, such as enzymes for diagnostic tests or in vitro detection, and proteins and peptides administered to patients to prevent disease, such as vaccines. In some embodiments, bioactive proteins or fragments thereof have immunostimulatory / immunomodulatory, membrane transport, or enzymatic activity. In some embodiments, bioactive proteins, peptides, or fragments thereof are enzymes, hormones, growth factors, cytokines, or mixtures thereof. In some embodiments, bioactive proteins, peptides, or fragments may specifically recognize target peptides (e.g., antigens or other proteins).

[0305] In some embodiments, the bioactive protein or fragment thereof that may be included in the IL-2 mutant or fusion protein containing the IL-2 mutant described herein is an antigen-binding protein (e.g., an antibody). In some embodiments, the bioactive protein or fragment thereof that may be included in the IL-2 mutant or fusion protein containing the IL-2 mutant as described herein is an antibody mimic, which is a small engineered protein containing an antigen-binding domain reminiscent of an antibody (GGeering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and consist of a single polypeptide. Examples of antibody mimics that may be included in IL-2 mutants or fusion proteins containing IL-2 mutants as described herein may be, but are not limited to, designed ankyrin repeat proteins (DARPin; comprising 3-5 fully synthetic ankyrin repeat sequences flanked by N-terminal and C-terminal cap domains), an affinity multimer (avimer; a high-affinity protein comprising multiple A domains, each with low affinity for the target), or an anticoagulant (lipid-based scaffold with four accessible loops, the sequence of each loop may be randomized). In some embodiments, the bioactive protein or fragment thereof that may be included in IL-2 mutants or fusion proteins containing IL-2 mutants as described herein is an armadillo repeat protein (e.g., β-catenin, α-introducing protein, globin, colorectal adenomatous polyposis (APC)) comprising armadillo repeat units (characteristics, the repeat amino acid sequence is approximately 40 residues in length). Each armadillo repeat unit consists of a pair of α-helices forming a hairpin structure. Multiple repeat copies form the α-solenoid structure described herein. Armadillo repeat proteins can bind to different types of peptides, relying on a constant binding mode of the peptide backbone without requiring specific conserved side chains or interactions with the free N- or C-terminus of the peptide. The possibility of recognizing peptide residues through residue recognition, coupled with the inherent modularity of repeat proteins, makes armadillo repeat proteins promising candidates for universal peptide-binding scaffolds.

[0306] IV. Nucleic acids encoding IL-2 mutants or fusion proteins containing IL-2 mutants

[0307] This application also relates to isolated nucleic acids encoding any IL-2 mutant or fusion protein containing an IL-2 mutant described herein, and vectors containing nucleic acids encoding any IL-2 mutant or fusion protein containing an IL-2 mutant described herein. It also relates to isolated host cells (e.g., CHO cells, HEK 293 cells, HeLa cells, or COS cells) containing the aforementioned nucleic acids or vectors. In some embodiments, the isolated nucleic acid further comprises a nucleic acid sequence encoding an N-terminal signal peptide of an IL-2 mutant or fusion protein containing an IL-2 mutant.

[0308] In some embodiments, a vector containing nucleic acid encoding any of the IL-2 mutants described herein or a fusion protein containing an IL-2 mutant is adapted to replicate and integrate in eukaryotic cells, such as mammalian cells (e.g., CHO cells, HEK 293 cells, HeLa cells, COS cells). In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector, such as pTT5.

[0309] Numerous virus-based systems have been developed for transferring genes into mammalian cells. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retrovirus vectors, herpes simplex virus vectors, and their derivatives. Viral vector technology is well-known in the art and is detailed, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral microparticles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or under in vitro conditions. Many retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Many adenovirus vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying construct protein-coding sequences can be packaged using experimental methods known in the art. The resulting lentiviral vectors can be used to transduce mammalian cells using methods known in the art. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for long-term gene transduction because they allow transgenes to integrate stably and continuously into daughter cells. Lentiviral vectors also exhibit low immunogenicity and can transduce non-proliferating cells.

[0310] In some embodiments, the vector is a nonviral vector. In some embodiments, the vector is a pTT5 vector. In some embodiments, the vector is a transposon, such as the Sleeping Beauty (SB) transposon system or the PiggyBac transposon system. In some embodiments, the vector is a polymer-based nonviral vector, including, for example, poly(lactic-co-glycolic acid copolymer) (PLGA) and polylactic acid (PLA), poly(ethyleneimine) (PEI), and dendritic macromolecules. In some embodiments, the vector is a cationic lipid-based nonviral vector, such as cationic liposomes, lipid nanoemulsions, and solid lipid nanoparticles (SLN). In some embodiments, the vector is a peptide-based nonviral gene vector, such as poly-L-lysine. Any known nonviral vector suitable for genome editing can be used to introduce nucleic acids encoding IL-2 mutants or fusion proteins containing IL-2 mutants into host cells. See Yin H. et al., Nature Rev. Genetics (2014) 15:521-555; Aronovich EL et al., “The Sleeping Beauty transposon system: a non-viral vector for gene therapy.” Hum. Mol. Genet. (2011) R1: R14-20; and Zhao S. et al., “PiggyBac transposon vectors: the tools of the human gene editing.” Transl. Lung Cancer Res. (2016) 5(1):120-125, which are incorporated herein by reference. In some embodiments, any one or more nucleic acids or vectors encoding the IL-2 mutant described herein or containing a fusion protein of the IL-2 mutant are introduced into host cells by physical methods (e.g., CHO, HEK 293, HeLa, or COS), including but not limited to electroporation, acoustic perforation, photoperforation, magnetic transfection, and hydroperforation.

[0311] In some embodiments, the vector contains an optional marker gene or reporter gene for selecting cells expressing the IL-2 mutant or a fusion protein containing the IL-2 mutant from a host cell population transfected with the vector (e.g., a lentiviral vector, pTT5 vector). Both the optional marker and reporter genes may be surrounded by appropriate regulatory sequences to enable their expression in host cells. For example, the vector may contain transcription and translation terminators, a start sequence, and a promoter for regulating nucleic acid sequence expression.

[0312] Any molecular cloning method known in the art can be used, including, for example, cloning nucleic acids into a vector using restriction endonuclease sites and one or more optional markers. In some embodiments, the nucleic acid is operatively linked to a promoter. A variety of promoters for gene expression in prokaryotic or eukaryotic cells (e.g., mammalian cells) have been explored, and any promoter known in the art can be used in this application. Promoters can be broadly classified into constitutive promoters or regulatory promoters, such as inducible promoters.

[0313] In some embodiments, nucleic acids encoding the IL-2 mutant or fusion protein containing the IL-2 mutant described herein are operatively linked to a constitutive promoter. Constitutive promoters allow heterologous genes (also known as transgenes) to be constitutively expressed in host cells. Examples of promoters considered herein include, but are not limited to, the CMV promoter (CMV), human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), glycerol phosphokinase promoter (PGK), simian virus 40 early promoter (SV40), chicken β-actin promoter coupled with CMV early enhancer (CAGG), Roche sarcoma virus (RSV) promoter, polyomavirus enhancer / herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, and the "myeloproliferative sarcoma virus enhancer, negative control region deletion, d1587rev primer binding site substitution (MND)" promoter. The efficiency of these constitutive promoters in driving transgene expression has been extensively compared in numerous studies. In some embodiments, a nucleic acid encoding the IL-2 mutant described herein or a fusion containing the IL-2 mutant is operatively linked to a CMV promoter.

[0314] In some embodiments, a nucleic acid encoding the IL-2 mutant or a fusion protein containing the IL-2 mutant described herein is operatively linked to an inducible promoter. Inducible promoters fall under the category of regulatory promoters. An inducible promoter can be induced by one or more conditions, such as physical conditions, the host cell microenvironment or physiological state of the host cell, an inducer (i.e., an inducing agent), or a combination thereof. In some embodiments, the inducing conditions do not induce the expression of endogenous genes in the host cell. In some embodiments, the inducing conditions are selected from: inducers, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, and the activation state of the host cell. In some embodiments, the inducible promoter may be an NFAT promoter, Promoter or NFκB promoter.

[0315] V. Preparation Method

[0316] This application also relates to methods for preparing any IL-2 mutant or fusion protein containing an IL-2 mutant as described herein. Therefore, in some embodiments, a method for preparing an IL-2 mutant or fusion protein containing an IL-2 mutant is provided, comprising: (a) culturing host cells (e.g., CHO cells, HEK 293 cells, HeLa cells, or COS cells) containing a nucleic acid or vector encoding any IL-2 mutant or fusion protein containing an IL-2 mutant as described herein, under conditions that allow for efficient expression of the encoded IL-2 mutant or fusion protein containing an IL-2 mutant; and (b) obtaining the expressed IL-2 mutant or fusion protein containing an IL-2 mutant from the host cells. In some embodiments, step (a) further comprises generating host cells containing a nucleic acid or vector encoding an IL-2 mutant or fusion protein containing an IL-2 mutant as described herein. The IL-2 mutant or fusion protein containing an IL-2 mutant as described herein may be prepared using any method known in the art or described herein.

[0317] In some embodiments, the IL-2 mutant or fusion protein containing the IL-2 mutant described herein may be expressed in eukaryotic cells, such as mammalian cells. In some embodiments, the IL-2 mutant or fusion protein containing the IL-2 mutant described herein may be expressed in prokaryotic cells.

[0318] 1. Recombinant products of prokaryotic cells

[0319] a) Carrier construction

[0320] The polynucleotide sequence encoding the protein construct described in this application can be obtained using standard recombinant techniques. Polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once the sequence encoding the polypeptide is obtained, it is inserted into a recombinant vector capable of replicating and expressing the heteropolynucleotide in a prokaryotic host. Many vectors known and usable in the art can be used in this application. The selection of a suitable vector depends primarily on the size of the nucleic acid to be inserted and the specific host cell to which the vector is transformed. Each vector contains various components depending on its function (amplification or expression of the heteropolynucleotide, or both) and its compatibility with the specific host cell in which it resides. Vector components typically include, but are not limited to: a replication initiation site, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, heteronucleotide insertion, and a transcription termination sequence.

[0321] Generally, plasmid vectors contain replicons and control sequences derived from host cell-compatible species and are used in conjunction with these host cells. The vector typically carries a replication site and a marker sequence that provides phenotypic selection in transformed cells. For example, *E. coli* is often transformed using pBR322, a plasmid derived from *E. coli*. pBR322 contains genes encoding resistance to ampicillin (Amp) and tetracycline (Tet), thus providing a simple way to identify transformed cells. pBR322, its derivatives, or other bacterial plasmids or phages may also contain, or be modified to contain, promoters that can be used by microorganisms to express endogenous proteins. Carter et al., US Pat. No. 5, 648, 237 details examples of pBR322 derivatives used to express specific antibodies.

[0322] Furthermore, phage vectors containing replicons and control sequences compatible with the host microorganism can be used as transformation vectors with these host cells. For example, phages such as GEM... TM -11 can be used to prepare recombinant vectors, which can be used to transform susceptible host cells, such as Escherichia coli LE392.

[0323] A promoter is a non-translational regulatory sequence located upstream (5′) of a cistron that regulates the expression of downstream genes. Prokaryotic promoters are generally classified into two types: inducible and constitutive. An inducible promoter is a promoter that can initiate and enhance cistron transcription in response to changes in culture conditions (e.g., the presence or absence of nutrients or changes in temperature).

[0324] Many promoters that can be recognized by potential host cells are well known. The promoter is removed from the source DNA by restriction endonuclease and the isolated promoter sequence is inserted into the vector of this application, and the selected promoter is operatively linked to the cis-trans DNA encoding the polypeptide. Natural promoter sequences and many heterologous promoters can be used to guide the amplification and / or expression of target genes. In some embodiments, heterologous promoters are used because they generally allow for greater transcription and higher yields of target gene expression compared to natural target polypeptide promoters.

[0325] Promoters suitable for prokaryotic hosts include the PhoA promoter, β-galactosidase and lactose promoter systems, tryptophan (trp) promoter systems, and hybrid promoters such as the tac or trc promoters. However, other promoters that function in bacteria (such as other known bacterial or bacteriophage promoters) are also suitable. Their nucleic acid sequences have been published, allowing technicians to use adapters or aptamers to provide any desired restriction sites to ligate them to the cistrons encoding the target light and heavy chains (Siebenlist et al., (1980) Cell 20:269).

[0326] In some embodiments, each cistron within the recombinant vector contains a secretory signal sequence component that directly guides the transmembrane transfer of the expressed polypeptide. Generally, the signal sequence can be a component of the vector or a portion of the target polypeptide DNA inserted into the vector. The signal sequence selected for this application should be a sequence that can be recognized and processed by the host cell (i.e., cleaved by a signal peptidase). For prokaryotic host cells that cannot recognize and process the intrinsic signal sequence of the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp or a thermostable enterotoxin II (STII) lead, LamP, PhoE, PelB, OmpA, and MBP.

[0327] In some embodiments, the production of the IL-2 mutant of this application or a fusion protein containing the IL-2 mutant can occur in the cytoplasm of a host cell, thus eliminating the need for a secretory signal sequence within each cistron. In some embodiments, the polypeptide component is expressed, folded, and assembled to form a protein construct in the cytoplasm. Certain host strains (e.g., *E. coli* trxB- strain) provide cytoplasmic conditions favorable for disulfide bond formation, thereby allowing the expressed protein subunits to fold and assemble properly. See Proba and Pullthun, Gene, 159:203 (1995).

[0328] b) Prokaryotic host cells

[0329] Suitable prokaryotic host cells for expressing the proteins of this application include archaea and eubacteria, such as Gram-negative or Gram-positive bacteria. Examples of usable bacteria include *Escherichia coli* (e.g., *Escherichia coli*), bacilli (e.g., *Bacillus subtilis*), *Enterobacter*, *Pseudomonas* (e.g., *Pseudomonas aeruginosa*), *Salmonella typhimurium*, *Serratia marcescens*, *Klebsiella*, *Proteus*, *Shigella*, rhizobia, *Vibrio hygroscopicus*, or *Paragonimella*. In some embodiments, Gram-negative cells are used. In some embodiments, *Escherichia coli* cells serve as the host for this application. Examples of *E. coli* strains include strain W3110 (Bachmann, *Cellular and Molecular Biology*, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27, 325) and its derivatives, including strain 33D3 (US Pat. No. 5, 639, 635) with the genotype W3110 AfhuA(AtonA)ptr3 lac Iq lacL8AompT A(nmpc fepE)degP41 kanR). Other strains and their derivatives, such as *E. coli* 294 (ATCC 31446), *E. coli* B, *E. coli* 1776 (ATCC 31537), and *E. coli* RV308 (ATCC 31608), are also applicable. These examples are illustrative and not restrictive. Methods for constructing bacterial derivatives of any of the aforementioned known genotypes are known in the art and are detailed, for example, in Bass et al., Proteins, 8:309-314 (1990). Given the reproducibility of replicons in bacterial cells, it is generally necessary to select suitable bacteria. For example, *Escherichia coli*, *Serratia marcescens*, or *Salmonella* are suitable hosts when using known plasmids such as pBR322, pBR325, pACYC177, or pKN410 to provide replicons.

[0330] Typically, host cells should secrete a minimum amount of proteolytic enzymes, and additional protease inhibitors need to be added appropriately to the cell culture.

[0331] c) Protein production

[0332] The aforementioned expression vectors are used to transform host cells, which are then cultured in appropriately modified conventional nutrient media to induce promoters, select transformants, or amplify genes encoding desired sequences. Transformation refers to the introduction of DNA into a prokaryotic host, enabling the DNA to replicate as an extrachromosomal element or through chromosomal integration. Depending on the host cell type used, standard techniques suitable for that cell type are employed for transformation. Calcium treatment with calcium chloride is commonly used for bacterial cells containing significant cell wall barriers. Another transformation method uses polyethylene glycol / dimethyl sulfoxide. Another technique is electroporation.

[0333] Prokaryotic cells used to produce the protein constructs of this application are grown in a culture medium known in the art and suitable for culturing selected host cells. Suitable culture media include luria broth (LB) and necessary nutrient supplements. In some embodiments, the culture medium also contains a selector chosen based on the structure of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium for the growth of cells expressing an ampicillin resistance gene.

[0334] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may be added alone or as a mixture with other supplements or media (such as compound nitrogen sources) at appropriate concentrations. Optionally, the culture medium may contain one or more reducing agents selected from glutathione, cysteine, cystamine, thioglycine, dithioerythritol, and dithiothreitol. Prokaryotic host cells are cultured at a suitable temperature. For example, for the growth of *Escherichia coli*, a preferred temperature range is 20°C to 39°C, more preferably 25°C to 37°C, and even more preferably 30°C. The pH of the culture medium can be any pH between 5 and 9, depending primarily on the host organism. For *Escherichia coli*, a pH of 6.8 to 7.4 is preferred, and more preferably 7.0.

[0335] If an inducible promoter is used in the expression vector of this application, protein expression is induced under conditions suitable for promoter activation. In one aspect of this application, the PhoA promoter is used to control the transcription of the polypeptide. Therefore, transformed host cells are cultured in a phosphate-restricted medium for induction. Preferably, the phosphate-restricted medium is CRAP medium (see Simmons et al., J. Immunol. Methods (2002), 263:133-147). Depending on the vector structure used, a variety of other inducers known in the art can be used.

[0336] The protein construct expressed in this application is secreted into and recovered from the periplasm of host cells. Protein recovery typically involves disrupting the microorganism, usually through methods such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. For example, the protein can be further purified by affinity resin chromatography. Alternatively, the protein can be transported to a culture medium and isolated therein. Cells can be removed from the culture medium, and the supernatant can be filtered and concentrated for further purification of the resulting protein. The expressed peptides can be further separated and identified using common methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0337] Alternatively, proteins can be produced on a large scale through fermentation. Various large-scale fed-batch fermentation programs can be used to produce recombinant proteins. Large-scale fermentations have a capacity of at least 1000 liters, preferably between 1000 and 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, particularly glucose (preferably carbon / energy). Small-scale fermentation typically refers to fermentation in fermenters with a volume not exceeding 100 liters, ranging from 1 liter to 100 liters.

[0338] During fermentation, protein expression is typically induced after cells have grown to the desired density under suitable conditions, for example, when the OD550 is approximately 180-220, at which point the cells are in the early quiescent phase. Depending on the vector structure used, a variety of inducers known in the art and described above can be used. Cells can be grown for a relatively short period before induction. Induction typically lasts approximately 12-50 hours, although longer or shorter induction times may be possible.

[0339] To improve the yield and quality of the IL-2 mutant or fusion protein containing the IL-2 mutant of this application, various fermentation conditions can be modified. For example, to improve the correct assembly and folding of secreted peptides, host prokaryotic cells can be co-transformed using an additional vector overexpressing a chaperone protein, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, or DsbG) or FkpA (a peptide prolyl cis-trans isomerase with chaperone activity). Chaperone proteins have been shown to help promote the correct folding and dissolution of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem274:19601-19605; Georgiou et al., US Pat. No. 6,083,715; Georgiou et al. al., US Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275: 17106-17113; Arie et al. al., (2001) Mol. Microbiol. 39:199-210.

[0340] To minimize the hydrolysis of expressed heterologous proteins (especially proteolytically sensitive proteins), certain host strains lacking proteolytic enzymes can be used in this application. For example, the host cell strain can be modified to mutate genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Some *E. coli* protease-deficient strains can be used, as detailed in Joly et al., (1998), supra; Georgiou et al., US Pat. No. 5, 264, 365; Georgiou et al., US Pat. No. 5, 508, 192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996).

[0341] Escherichia coli strains lacking proteolytic enzymes and transformed with plasmids expressing one or more chaperone proteins can serve as host cells in expression systems encoding the IL-2 mutant described in this application or fusion proteins containing the IL-2 mutant.

[0342] d) Protein purification

[0343] Further purification of the protein constructs produced herein is necessary to obtain a substantially homogeneous formulation for further analysis and use. Standard protein purification methods known in the art may be employed. The following procedures are examples of suitable purification procedures: fractionation on an immunoaffinity column or ion exchange column, ethanol precipitation, reversed-phase liquid chromatography (HPLC), silica or cation exchange resin (such as DEAE) chromatography, chromatographic focusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, e.g., dextran gel G-75.

[0344] In some embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of a protein construct containing the Fc region described herein. Protein A is a 42 kDa surface protein from Staphylococcus aureus, which has a high binding affinity to Fc-containing structures, such as the IL-2 mutant-Fc fusion protein described herein. (Lindmark et al., (1983) J. Immunol. Meth. 62:1-1). The solid phase immobilizing protein A preferably comprises a column with a glass or silica surface, more preferably a glass column with controlled pore size or a silica column. In some applications, the column is coated with a reagent, such as glycerol, to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants that have nonspecifically bound to it. Finally, the target protein construct is recovered from the solid phase by elution.

[0345] 2. Recombinant products of eukaryotic cells

[0346] For eukaryotic expression, vector components typically include, but are not limited to, one or more of the following: signal sequence, replication start site, one or more marker genes, enhancer element, promoter, and transcription termination sequence.

[0347] a) Signal sequence elements

[0348] The vector for a eukaryotic host can also be an insert that encodes a signal sequence or another polypeptide having a specific cleavage site at the N-terminus of a mature protein or polypeptide. The chosen heterologous signal sequence is preferably a sequence that is recognized and processed by the host cell (i.e., cleaved by a signal peptidase). In mammalian cell expression, mammalian signal sequences, as well as viral secretion precursors, such as the herpes simplex gD signal, are useful. The DNA of this precursor region is linked within the reading frame to the DNA encoding the protein construct of this application.

[0349] b) Copying start point

[0350] Generally, mammalian expression vectors do not require replication start elements (SV40 starters are typically used only because they contain early promoters).

[0351] c) Selecting gene elements

[0352] Expression and cloning vectors may contain selection genes, also known as selection markers. Typical selection genes encode proteins that: (a) are resistant to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) are complement auxotrophic proteins; or (c) provide proteins that provide key nutrients that complex culture media cannot provide, such as the gene encoding Bacillus D-alanine racemic enzyme.

[0353] An example of a selection strategy is using drugs to inhibit the growth of host cells. Cells that are successfully transformed with a foreign gene produce a drug-resistant protein, thus surviving the selection process. Examples of this dominant selection include the use of drugs such as neomycin, mycophenolic acid, and hygromycin.

[0354] Another example of optional markers suitable for mammalian cells is optional markers that can identify cells capable of carrying nucleic acids encoding the protein constructs described in this application, such as DHFR, thymidine kinase, metallothionein-I and-II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0355] For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When using wild-type DHFR, suitable host cells are Chinese hamster ovary (CHO) cell lines lacking DHFR activity (e.g., ATCC CRL-9096).

[0356] Alternatively, host cells transformed or co-transformed with the DNA sequence encoding the polypeptide, the wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3′-phosphotransferase (APH), can be selected by cell growth in a medium containing the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See US Pat. No. 4,965,199.

[0357] d) Starter element

[0358] Expression and cloning vectors typically contain a promoter that is recognized by the host and operatively linked to a nucleic acid encoding the desired polypeptide sequence. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 80 bases upstream of the transcription start site in many genes is the CNCAAT region, where N can be any nucleotide. Most eukaryotes have an AATAAA sequence at the 3′ end, which may be a signal for the addition of a poly-A tail to the 3′ end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0359] Peptide transcription in mammalian host cell vectors is controlled by promoters, such as promoters derived from viral genomes, such as polyomavirus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40), promoters from heterologous mammals, such as actin promoters or immunoglobulin promoters, and heat shock promoters, provided that such promoters are compatible with the host cell system.

[0360] The early and late promoters of SV40 virus are readily available as SV40 restriction fragments, which also contain the SV40 virus replication initiation site. The immediate early promoter of human cytomegalovirus is readily available as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papillomavirus as a vector is disclosed in USPat. No. 4,419,446. Modifications to this system are detailed in USPat. No. 4,601,978. See Reyes et al., Nature 297:598-601 (1982), on the expression of human interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter. Alternatively, a long terminal repeat sequence of Rous sarcoma virus can be used as a promoter.

[0361] e) Enhancement sub-element

[0362] Transcription of DNA encoding the protein construct described herein in higher eukaryotes is typically enhanced by inserting enhancer sequences into the vector. Numerous enhancer sequences have been identified in mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic viruses are commonly used. Examples include the SV40 enhancer at the end of the replication origin (100–270 bp), the early promoter enhancer of cytomegalovirus, the polyomavirus enhancer at the end of the replication origin, and the adenovirus enhancer. See Yaniv, Nature 297:17–18 (1982), on enhancing elements that activate eukaryotic promoters. The enhancer can be spliced ​​into the vector at the 5′ or 3′ position of the polypeptide coding sequence, but is preferably located at the 5′ position of the promoter.

[0363] f) Transcription termination element

[0364] Expression vectors used in eukaryotic host cells (nucleated cells of yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also contain sequences necessary for transcription termination and stabilization of mRNA. These sequences are typically obtained from the 5′ untranslated region of eukaryotic or viral DNA or cDNA, and occasionally from the 3′ end. These regions contain nucleic acid fragments that are transcribed as polyadenylate fragments in the untranslated portion of mRNA encoding a polypeptide. A suitable transcription termination element is the polyadenylate region of bovine growth hormone. See WO94 / 11026 and the expression vectors disclosed therein.

[0365] g) Host cell selection and transformation

[0366] Suitable host cells for cloning or expressing DNA in the vectors described herein include higher eukaryotic cells, including vertebrate host cells. The culture and propagation of vertebrate cells (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); the COS fibroblast-like cell line derived from monkey kidney tissue; the human embryonic kidney line (293 or 293 cell subclones, used for suspension culture growth, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Seltorius cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); and African green monkey kidney cells (VERO-76, ATCC). CRL-1587); human cervical cancer cells (HELA, ATCC-ccl2); canine kidney cells (MDCK, ATCC-ccl34); buffalo-rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells and human hepatocellular carcinoma cell line (Hep G2).

[0367] The host cells were transformed using the above expression vectors or cloning vectors to produce protein structures, and then cultured in a properly modified conventional nutrient medium to induce promoters, select transformants, or amplify genes encoding the desired sequences.

[0368] h) Culture host cells

[0369] The host cells used to produce the protein constructs of this application can be cultured in a variety of media. Commercial media such as Ham's F10 (Sigma), Minimal Basis Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655 or 5,122,469, WO 90 / 03430, WO 87 / 00195 or US Pat. Re. 30,985 can be used as the culture medium for host cells. Any of these culture media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPE), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin™), trace elements (defined as inorganic compounds typically present in micromolar concentrations), and glucose or equivalent energy. Any other necessary supplements may also be added at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, are those previously used for host cell expression and are readily apparent to those skilled in the art.

[0370] VI. Pharmaceutical Composition

[0371] This application further relates to pharmaceutical compositions comprising any IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein, or pharmaceutical compositions comprising nucleic acids encoding any IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein, or pharmaceutical compositions comprising a vector encoding any IL-2 mutant or fusion protein comprising an IL-2 mutant as described herein. These compositions may optionally further comprise pharmaceutically acceptable vectors and / or excipients. In some embodiments, the excipients include excipients and / or stabilizers. Pharmaceutical compositions may be prepared as lyophilized formulations or aqueous solutions by mixing an IL-2 mutant or fusion protein comprising an IL-2 mutant of desired purity as described herein with optional pharmaceutically acceptable vectors, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)).

[0372] VII. Treatment methods for diseases

[0373] The IL-2 mutants, fusion proteins containing IL-2 mutants, or compositions containing the same (e.g., pharmaceutical compositions) described in this application can be used for a variety of purposes, such as diagnostics, molecular detection, and therapy. In some embodiments, this application relates to a method of treating a disease in an individual (e.g., a human), comprising administering to the individual an effective dose of any IL-2 mutant as described herein, a fusion protein containing an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a vector and host cell containing such nucleic acid, or a pharmaceutical composition containing the same. In some preferred embodiments, the fusion protein containing an IL-2 mutant is a fusion protein of an IL-2 mutant and an Fc. In some embodiments, the disease is associated with the regulation of Treg cells in the subject. In some embodiments, the disease includes inflammatory diseases or autoimmune diseases. In some embodiments, the IL-2 mutants, fusion proteins containing IL-2 mutants, or pharmaceutical compositions containing IL-2 mutants or fusion proteins thereof are administered via intravenous, intramuscular, or subcutaneous injection.

[0374] In some implementations, the diseases mentioned include lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, malnutrition-related epidermolysis bullosa, and Behçet's disease.

[0375] For example, in some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises a deletion or mutation of amino acid residues RPRDL relative to positions 81-85 of the amino acid sequence of human wild-type IL-2. In some embodiments, the amino acid sequence of the human wild-type IL-2 is as shown in SEQ ID NO:1.

[0376] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant of this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises a complete deletion of amino acid residues RPRDL at positions 81-85 of the amino acid sequence of human wild-type IL-2. In some embodiments, the IL-2 mutant further comprises mutations in E67 and N71 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations in E67 and N71 are E67K and N71S, respectively. In some embodiments, the IL-2 mutant comprises a complete deletion of amino acid residues RPRDL at positions 81-85 of the amino acid sequence of human wild-type IL-2, and mutations in E67K and N71S relative to the amino acid sequence of human wild-type IL-2.

[0377] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant of this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises a mutation of RPRDL to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant further comprises a mutation of E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 mutation is E67A. In some embodiments, the IL-2 mutant comprises a mutation of RPRDL to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and an E67A mutation relative to the human wild-type IL-2 amino acid sequence.

[0378] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises a mutation of RPRDL to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises mutations of N71 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the N71 and S75 mutations are N71W and S75I, respectively. In some embodiments, the IL-2 mutant comprises a mutation of RPRDL to RPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations of N71W and S75I relative to the human wild-type IL-2 amino acid sequence.

[0379] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant of this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant further comprises mutations in E67 and / or V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 mutation is E67T. In some embodiments, the V91 mutation is V91K. In some embodiments, the IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and E67T and / or V91K mutations relative to the human wild-type IL-2 amino acid sequence.

[0380] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant of this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises mutations at L70 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L70 and S75 mutations are L70I and S75F, respectively. In some embodiments, the IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations at L70I and S75F relative to the human wild-type IL-2 amino acid sequence.

[0381] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant of this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and further comprises an N71 mutation relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the N71 mutation is N71I. In some embodiments, the IL-2 mutant comprises an RPRDL mutation to RHL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and an N71I mutation relative to the human wild-type IL-2 amino acid sequence.

[0382] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant of this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises an RPRDL mutation to SPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant further comprises mutations in E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 and N71 mutations are E67M and N71S, respectively. In some embodiments, the IL-2 mutant comprises an RPRDL mutation to SPL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence, and mutations in E67M and N71S relative to the human wild-type IL-2 amino acid sequence.

[0383] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises a mutation at amino acid residue S (S75) and amino acid residue R (R83) at position 75 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the amino acid sequence of the human wild-type IL-2 is as shown in SEQ ID NO:1.

[0384] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. The IL-2 mutant comprises a mutation at amino acid residue S (S75) and amino acid residue R (R83) at position 75 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, S75 in the IL-2 mutant is mutated to an uncharged amino acid residue. In some embodiments, the mutation at S75 is S75P, S75F, S75Y, S75G, S75A, S75V, S75T, S75I, or S75L. In some embodiments, the mutation of R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K, or R83E.

[0385] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the mutation of S75 is S75P, and the mutation of R83 is R83Y. In some embodiments, the IL-2 mutant further comprises mutations of E67 and D84 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of E67 and D84 are E67A and D84N, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75P, R83Y, E67A, and D84N relative to the amino acid sequence of human wild-type IL-2.

[0386] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the mutation of S75 is S75P, and the mutation of R83 is R83A. In some embodiments, the IL-2 mutant further comprises mutations of H16, L70, and N71 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of H16, L70, and N71 are H16D, L70I, and N71G, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75P, R83A, H16D, L70I, and N71G relative to the amino acid sequence of human wild-type IL-2.

[0387] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant of this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the S75 mutation is S75F, and the R83 mutation is R83P. In some embodiments, the IL-2 mutant further comprises mutations in H16 and / or N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the H16 mutation is H16Q or H16N. In some embodiments, the N71 mutation is N71S. In some embodiments, the IL-2 mutant comprises mutations in S75F, R83P, and H16Q relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant comprises S75F, R83P, H16N, and N71S mutations relative to the amino acid sequence of human wild-type IL-2.

[0388] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the mutation of S75 is S75F, and the mutation of R83 is R83L. In some embodiments, the IL-2 mutant further comprises mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of E67 and L70 are E67R and L70V, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75F, R83L, E67R, and L70V relative to the amino acid sequence of human wild-type IL-2.

[0389] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the mutation at S75 is S75I, and the mutation at R83 is R83L. In some embodiments, the IL-2 mutant further comprises mutations at E67 and L70 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations at E67 and L70 are E67G and L70V, respectively. In some embodiments, the IL-2 mutant comprises mutations at S75I, R83L, E67G, and L70V relative to the amino acid sequence of human wild-type IL-2.

[0390] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the S75 mutation is S75I, and the R83 mutation is R83K. In some embodiments, the IL-2 mutant further comprises mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E67 and L70 mutations are E67S and L70V, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75I, R83K, E67S, and L70V relative to the human wild-type IL-2 amino acid sequence.

[0391] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the S75 mutation is S75T, and the R83 mutation is R83A or R83F. In some embodiments, the IL-2 mutant further comprises mutations in H16 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the H16 and E67 mutations are H16N and E67K, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75T, R83A, H16N, and E67K relative to the human wild-type IL-2 amino acid sequence.

[0392] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant of this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the S75 mutation is S75V, and the R83 mutation is R83F. In some embodiments, the IL-2 mutant further comprises mutations in E15 and H16 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the E15 and H16 mutations are E15K and H16Q, respectively. In some embodiments, the IL-2 mutant or its fusion protein comprises mutations in S75V, R83F, E15K, and H16Q relative to the human wild-type IL-2 amino acid sequence.

[0393] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the mutation of S75 is S75V, and the mutation of R83 is R83N. In some embodiments, the IL-2 mutant further comprises mutations of L70 and V91 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of L70 and V91 are L70F and V91K, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75V, R83N, L70F, and V91K relative to the amino acid sequence of human wild-type IL-2.

[0394] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the mutation of S75 is S75G, and the mutation of R83 is R83L. In some embodiments, the IL-2 mutant further comprises mutations of L17, V91, and I92 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of L17, V91, and I92 are L17I, V91E, and I92L, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75G, R83L, L17I, V91E, and I92L relative to the amino acid sequence of human wild-type IL-2.

[0395] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the S75 mutation is S75Y, and the R83 mutation is R83N. In some embodiments, the IL-2 mutant described in this application further comprises mutations in L17 and V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L17 and V91 mutations are L17I and V91K, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75Y, R83N, L17I, and V91K relative to the human wild-type IL-2 amino acid sequence.

[0396] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the mutation at S75 is S75A, and the mutation at R83 is R83K. In some embodiments, the IL-2 mutant further comprises a mutation at L17 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation at L17 is L17I. In some embodiments, the IL-2 mutant comprises mutations at S75A, R83K, and L17I relative to the amino acid sequence of human wild-type IL-2.

[0397] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the thereof. In the IL-2 mutant, the S75 mutation is S75L, and the R83 mutation is R83E. In some embodiments, the IL-2 mutant described in this application further comprises mutations in L63 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L63 and E67 mutations are L63V and E67F, respectively. In some embodiments, the IL-2 mutant comprises mutations in S75L, R83E, L63V, and E67F relative to the human wild-type IL-2 amino acid sequence.

[0398] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, or a fusion protein containing an IL-2 mutant. The IL-2 mutant or its fusion protein, in addition to the mutations described above, further comprises a mutation relative to the C125 amino acid sequence of human wild-type IL-2. In some embodiments, the C125 mutation is C125S or C125A.

[0399] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, or a fusion protein comprising the IL-2 mutant. The IL-2 mutant comprises any of the amino acid sequences shown in SEQ ID NOs:5-34 or variants thereof, the variants having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to any of the amino acid sequences shown in SEQ ID NOs:5-34. In some embodiments, the fusion protein comprising the IL-2 mutant is a fusion protein of the IL-2 mutant and Fc. In some embodiments, the IL-2 mutant and Fc fusion protein is in a divalent form. In some embodiments, the IL-2 mutant-Fc bivalent fusion protein comprises any of the amino acid sequences shown in SEQ ID NOs:76-105 or variants thereof, wherein the variants have at least about 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with any of the amino acid sequences shown in SEQ ID NOs:76-105. In some embodiments, the IL-2 mutant-Fc fusion protein is in monovalent form. In some embodiments, the disease or condition is selected from lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, malnutrition-related epidermolysis bullosa, and Behçet's disease.

[0400] In some embodiments, this application relates to a method of treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of the IL-2 mutant described in this application, a fusion protein containing the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell containing the nucleic acid, or a pharmaceutical composition containing the thereof, and further comprising administering to the individual one or more other active ingredients required for a specific indication of treatment, preferably having active ingredients that do not affect the therapeutic effects of each other. For example, when used to treat autoimmune diseases or to alleviate or prevent autoimmune responses after organ transplantation, it can be used in combination with immunosuppressants. In some embodiments, the immunosuppressant is selected from: glucocorticoids; azathioprine; cyclosporine A; methotrexate; anti-CD3 antibodies; or anti-TNF-α antibodies, etc.

[0401] The administration routes of the IL-2 mutants, fusion proteins containing IL-2 mutants, nucleic acids encoding IL-2 mutants or their fusion proteins, vectors and host cells containing such nucleic acids, or pharmaceutical compositions containing them, described herein can be any known and convenient manner, including by injection or infusion. The route of administration shall follow known and accepted methods, such as by single or multiple bolus injections or by prolonged infusion in an appropriate manner.

[0402] The dosage and required drug concentration of the pharmaceutical composition of this application may vary depending on the specific application. Determining the appropriate dosage or route of administration is entirely within the scope of the skill of a person skilled in the art. Animal studies have provided reliable guidance for determining effective dosages for human treatment. Interspecies analogies for effective dosages can be made based on the principles outlined in Mordenti, J. and Chappell, W., “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobie et al., Eds, Pergamon Press, New York 1989, pp. 42-46.

[0403] When administered in vivo using an IL-2 mutant, a fusion protein containing an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and host cell containing such nucleic acid, or a pharmaceutical composition containing such a substance, the therapeutically effective dose may depend, for example, on the treatment context and target. Those skilled in the art will understand that the appropriate dose level for treatment will vary depending on the molecule delivered, the indication for treatment with the IL-2 mutant, the route of administration, and the patient's body size (weight, size of body surface area or organs) and / or condition (age and general health status). Within the scope of this application, different formulations will be effective for different treatments and different diseases, and the route of administration intended to treat a specific organ or tissue may differ from that intended for another organ or tissue. Furthermore, the dose may be administered by single or multiple administrations or by continuous infusion. For repeated administration over several days or longer, treatment continues until the desired level of disease symptom suppression is achieved, depending on the condition. Alternatively, other dosing regimens may be useful. Progression of such treatment can be easily monitored using conventional techniques and analyses.

[0404] VIII. Products and Reagent Kits

[0405] This application further relates to kits, unit doses, and articles comprising any IL-2 mutant described herein, fusion proteins comprising IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells comprising such nucleic acids, or pharmaceutical compositions comprising them. In some embodiments, kits comprising any of the pharmaceutical compositions described herein are involved, and preferably, instructions for use are provided, such as for treating the diseases described herein (e.g., autoimmune diseases).

[0406] The kits of this application comprise one or more containers containing the IL-2 mutant described herein, a fusion protein containing the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or its fusion protein, a vector and host cell containing such nucleic acid, or a pharmaceutical composition containing the thereof, for example, for treating a disease. For example, they may include instructions describing the administration of the IL-2 mutant, the fusion protein containing the IL-2 mutant, the nucleic acid encoding the IL-2 mutant or its fusion protein, the vector and host cell containing such nucleic acid, or a pharmaceutical composition containing the thereof to treat a disease (such as an autoimmune disease). The kit may further include descriptions of selecting suitable individuals (e.g., humans) for treatment based on identifying whether an individual has a disease and the stage of the disease. Instructions relating to the use of the IL-2 mutant, the fusion protein containing the IL-2 mutant, the nucleic acid encoding the IL-2 mutant or its fusion protein, the vector and host cell containing such nucleic acid, or the pharmaceutical composition containing the thereof typically include information about the intended therapeutic dose, dosing schedule, and route of administration. Containers may be unit doses, bulk packaging (e.g., multi-dose packaging), or subunit doses. The instructions provided in the kits of this application are typically written instructions on a label or instruction manual (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions stored on a disk or CD) are also acceptable. The kits of this application are packaged appropriately. Appropriate packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. Packaging for use with specific devices, such as infusion devices like micropumps, is also considered. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an IL-2 mutant as described herein, a fusion protein containing an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and host cell containing such nucleic acid, or a pharmaceutical composition containing such a substance. The container may further contain a second pharmaceutical active agent. The kit may optionally provide additional components, such as buffers and explanatory information. Generally, the kit includes a container and a label or instruction manual on or associated with the container.

[0407] For example, in some embodiments, the kit includes: a) an IL-2 mutant comprising any of the IL-2 mutants described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising thereof; and b) at least one effective amount of another agent capable of enhancing the effects (e.g., therapeutic effects) of the IL-2 mutant, the fusion protein comprising an IL-2 mutant, the nucleic acid encoding an IL-2 mutant or a fusion protein thereof, the vector and host cell comprising such nucleic acid, or the pharmaceutical composition comprising thereof.

[0408] In some embodiments, the kit includes: a) an IL-2 mutant comprising any of the IL-2 mutants described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising thereof; and b) instructions for use for administering to an individual an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising thereof for the treatment of an autoimmune disease (e.g., lupus).

[0409] In some embodiments, the kit includes: a) an IL-2 mutant comprising any of the IL-2 mutants described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a vector and host cell comprising such nucleic acid, or a pharmaceutical composition comprising thereof; and b) at least one effective amount of another agent capable of enhancing the effect (e.g., therapeutic effect) of the IL-2 mutant, the fusion protein comprising an IL-2 mutant, the nucleic acid encoding an IL-2 mutant or a fusion protein thereof, the vector and host cell comprising such nucleic acid, or the pharmaceutical composition comprising thereof; and c) instructions for use to administer to an individual the IL-2 mutant, the fusion protein comprising an IL-2 mutant, the nucleic acid encoding an IL-2 mutant or a fusion protein thereof, the vector comprising such nucleic acid, or the pharmaceutical composition comprising thereof, and other substances for the treatment of an autoimmune disease (e.g., lupus). The IL-2 mutant, the fusion protein comprising an IL-2 mutant, the nucleic acid encoding an IL-2 mutant or a fusion protein thereof, the vector and host cell comprising such nucleic acid, or the pharmaceutical composition comprising thereof, and other substances may be present in separate containers or in the same container. For example, the kit may contain one specific composition or two or more compositions, wherein one composition includes an IL-2 mutant, a fusion protein containing an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a vector and host cell containing the nucleic acid, or a pharmaceutical composition containing the thereof, and another composition includes another pharmaceutical agent.

[0410] In some embodiments, the kit comprises a nucleic acid (or set of nucleic acids) encoding an IL-2 mutant or a fusion protein thereof (e.g., a fusion protein of an IL-2 mutant and Fc). In some embodiments, the kit comprises: a) a nucleic acid (or set of nucleic acids) encoding an IL-2 mutant or a fusion protein thereof (e.g., a fusion protein of an IL-2 mutant and Fc), and b) a host cell expressing the nucleic acid (or set of nucleic acids). In some embodiments, the kit comprises: a) a nucleic acid (or set of nucleic acids) encoding an IL-2 mutant or a fusion protein thereof (e.g., a fusion protein of an IL-2 mutant and Fc), and b) instructions for use suitable for: i) expressing an IL-2 mutant or a fusion protein thereof in a host cell, ii) preparing a composition comprising an IL-2 mutant or a fusion protein thereof, and iii) administering a composition comprising an IL-2 mutant or a fusion protein thereof to an individual for the treatment of an autoimmune disease (e.g., lupus). In some embodiments, the kit includes: a) a nucleic acid (or a set of nucleic acids) encoding an IL-2 mutant or a fusion protein thereof (e.g., a fusion protein of an IL-2 mutant and Fc), b) a host cell expressing the nucleic acid (or set of nucleic acids), and c) instructions for use suitable for: i) expressing an IL-2 mutant or a fusion protein thereof in a host cell, ii) preparing a composition comprising an IL-2 mutant or a fusion protein thereof, and iii) administering a composition comprising an IL-2 mutant or a fusion protein thereof to an individual for the treatment of an autoimmune disease (e.g., lupus).

[0411] On the other hand, this application also relates to articles of manufacture, including vials (such as sealed vials), bottles, jars, flexible packaging, etc. These articles of manufacture include containers and labels or instructions for use on or associated with the containers. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be made of a variety of materials, such as glass or plastic. Generally, the composition contained in the container is effective in treating the diseases or disorders described herein (such as autoimmune diseases) and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The label or instructions for use indicate that the composition is intended to treat a specific condition in an individual. The label or instructions for use further include instructions for administering the composition to the individual. The label may indicate instructions for reconstitution and / or use. Containers containing pharmaceutical compositions may be reusable vials, allowing for repeated administration of the reconstituted formulation (e.g., 2-6 administrations). Instructions for use refer to the instructions typically included in the commercial packaging of therapeutic products, which contain information on the indications, usage, dosage, administration, contraindications, and / or warnings for using such therapeutic products. In some embodiments, the product description specifies that treatable diseases include lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, and Behçet's disease. Furthermore, the product may further comprise a second container containing pharmaceutically acceptable buffer solutions, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. From a commercial and user perspective, it may further include other necessary materials, including other buffer solutions, diluents, filters, needles, and syringes.

[0412] The kit or product contains a pharmaceutical composition in multiple unit doses and instructions for use, and is packaged in sufficient quantities for storage and use in a pharmacy, such as a hospital pharmacy or a multi-dose pharmacy. Detailed Implementation

[0413] Those skilled in the art will recognize several possible embodiments within the scope and spirit of this application. The application will now be described in more detail with reference to the following non-limiting embodiments. These embodiments further illustrate the application but should not be construed as limiting its scope in any way.

[0414] Example 1: Design and expression of human wild-type IL-2 and IL-2 mutants

[0415] Construction and expression of wild-type IL-2:

[0416] The nucleic acid sequence encoding human wild-type IL-2 (amino acid sequence as shown in SEQ ID NO:1) was synthesized by Nanjing Genscript Biotech Co., Ltd., and restriction enzyme sites were introduced at the 5' and 3' ends of the synthesized wild-type IL-2 nucleic acid sequence, respectively. After digestion with enzymes, the synthesized nucleic acid fragment was ligated with a vector fragment that had undergone the same double digestion to obtain the expression vector for wild-type IL-2. Furthermore, based on this expression vector, a C125S mutation was introduced at position 125 of wild-type IL-2 (amino acid sequence as shown in SEQ ID NO:2; this mutation can prevent cysteine ​​mismatch or aggregation), thus obtaining the expression vector for the IL-2 C125S mutant; a C125A mutation was introduced at position 125 of wild-type IL-2 (amino acid sequence as shown in SEQ ID NO:3; this mutation can promote efficient folding and improve expression), thus obtaining the expression vector for the IL-2 C125A mutant. Compared with wild-type IL-2, the above two IL-2 mutants did not alter their binding to the IL-2 receptor.

[0417] Design, screening, and expression of IL-2 mutants:

[0418] Through research, design, and FACS screening, a series of IL-2 mutants were obtained. The screening process is as follows:

[0419] First, biotinylated IL-2Rα and IL-2Rβγ required for the screening process are prepared:

[0420] Preparation of IL-2Rα The extracellular coding sequence of IL-2Rα (Uniprot ID: P01589) was codon-optimized and synthesized in its entirety by Nanjing Genscript Biotech Co., Ltd. The synthesized gene sequence was constructed into an expression vector carrying a His tag gene and expressed in 293F cells. IL-2Rα was purified using a (Ni) nickel column according to the manufacturer's instructions. In short, immobilized metal affinity chromatography (IMAC) was performed using Ni-NTA from QIAGEN. First, the nickel column was equilibrated with buffer A1 (50 mM Na3PO4, 0.15 M NaCl, pH 7.2) at a flow rate of 150 cm / h. The pH of the culture supernatant was adjusted to 7.2, and the sample was loaded at room temperature at a flow rate of 150 cm / h. Subsequently, the column was reequilibrated with 6 column volumes of A1 buffer at a flow rate of 150 cm / h. Finally, elution was performed using 10 column volumes of 50 mM PB solution (containing 0.15 M NaCl and 0.2 M imidazole, pH 7.2), and the eluent was collected.

[0421] Preparation of IL-2RβγThe IL-2Rβγ used in this application is an Fc heterodimer (IL-2Rβγ-Fc heterodimer) constructed using KIH (Knob-into-Hole) technology. The extracellular coding sequences of IL-2Rβ (Uniprot ID: P14784) and IL-2Rγ (Uniprot ID: P31785) were cloned and fused into the N-terminus of Fc hole (SEQ ID NO: 109) and Fc knob (SEQ ID NO: 108), respectively, to prepare the IL-2Rβγ-Fc heterodimer. The IL-2Rβ-Fc hole and IL-2Rγ-Fc knob were constructed into the pcDNA3.1 eukaryotic expression vector, and then co-transfected into 293F cells. The cell culture medium was collected. The IL-2Rβγ-Fc heterodimer was purified sequentially using protein A affinity chromatography and Superdex 200 column chromatography with gel filtration. In short, the culture medium of 293F cells was purified using a protein A affinity chromatography column. First, the protein A column was equilibrated with 50 mM PBS buffer containing 0.15 M NaCl (pH 7.2) at a flow rate of 150 cm / h, at a volume six times the column volume. The supernatant of the culture medium (pH adjusted to 7.2) was passed through the column at a flow rate of 150 cm / h. After further equilibration, the column was washed with 50 mM sodium citrate (pH 3.5), and the eluent was collected. Then, the IL-2Rβγ-Fc heterodimer was obtained after purification by gel filtration and Superdex 200 column chromatography.

[0422] Biotinylation labeling of IL-2Rα and IL-2Rβγ Following the instructions for the Biotin-Protein Ligase / BirAEnzyme (GeneCopoeia cat#BI001) kit, biotinylated IL-2Rα and IL-2Rβγ were biotinylated using biotinylated ligases. In short, the corresponding Biotin Ligase Buffer A, Buffer B, and BirA ligase were added to the IL-2Rα and IL-2Rβγ protein solutions, respectively, and incubated at 30°C for 2 hours. Biotinylation efficiency was assessed using ELISA to confirm successful biotinylation.

[0423] FACS Screening: The designed IL-2 mutant library was constructed into a yeast display system. Flow cytometry fluorescence sorting (FACS) was used to screen for mutants with decreased affinity for IL-2Rβγ but whose affinity for IL-2Rα remained essentially unchanged or not significantly weakened. The FACS screening method is well-known to those skilled in the art. First, four rounds of FACS screening were performed using biotinylated IL-2Rβγ (Biotin-IL-2Rβγ) to enrich the yeast positive population with decreased affinity for IL-2Rβγ. In short, yeast cells induced in SGCAA medium were precipitated, 1 mL of PBSM was added, and the mixture was centrifuged at 14000g for 30 seconds. The supernatant was discarded, and the cell pellet was washed. The yeast cells were resuspended in PBSM buffer containing Biotin-IL-2Rβγ and incubated at room temperature for 1 h. After washing, cells were stained with Streptavidin-PE (BD Biosciences, 554061) and anti-V5 tag antibody [iFluor647] (GenScript, A01805-100) for cell sorting. The enriched positive yeast population with weakened binding to Biotin-IL-2Rβγ was then further screened using FACS to identify single clones whose affinity for biotinylated IL-2Rα (Biotin-IL-2Rα) remained essentially unchanged or not significantly weakened. Finally, the selected single clones were sequenced, constructed into expression vectors carrying His tag genes, samples were prepared, and appropriate biological evaluations were performed.

[0424] Analysis of the sequencing results of single clones revealed that these mutants can be roughly divided into two groups:

[0425] The first group of IL-2 mutants involves mutations at amino acid residues 81-85 of the amino acid sequence relative to the human wild-type IL-2, including but not limited to deletions and / or substitutions. Specific information on each mutant in this group is shown in Table 9. In the table, “(81-85)RPL,” “(81-85)RHL,” or “(81-85)SPL” indicates that amino acid residues RPRDL at positions 81-85 of IL-2 have been replaced with amino acid residues RPL, RHL, or SPL, respectively. “(81-85) Complete deletion of RPRDL” indicates that all amino acid residues RPRDL at positions 81-85 of IL-2 have been deleted. “+” indicates simultaneous mutation.

[0426] Table 9

[0427]

[0428]

[0429] The second group of IL-2 mutants exhibits simultaneous amino acid mutations at S75 and R83 relative to the human wild-type IL-2 amino acid sequence. Specific information on each mutant in this group is shown in Table 10, with "+" indicating simultaneous mutation.

[0430] Table 10

[0431] mutant mutation site S75R83-mut1 S75P+R83Y+E67A+D84N+C125S S75R83-mut2 S75P+R83A+H16D+L70I+N71G+C125S S75R83-mut3 S75F+R83P+H16Q+C125S S75R83-mut4 S75F+R83P+H16N+N71S+C125S S75R83-mut5 S75F+R83L+E67R+L70V+C125S S75R83-mut6 S75G+R83L+L17I+V91E+I92L+C125S S75R83-mut7 S75Y+R83N+L17I+V91K+C125S S75R83-mut8 S75A+R83K+L17I+C125S S75R83-mut9 S75I+R83L+E67G+L70V+C125S S75R83-mut10 S75I+R83K+E67S+L70V+C125S S75R83-mut11 S75T+R83F+C125S S75R83-mut12 S75T+R83A+H16N+E67K+C125S S75R83-mut13 S75L+R83E+L63V+E67F+C125S S75R83-mut14 S75V+R83N+C125S S75R83-mut15 S75V+R83F+E15K+H16Q+C125S S75R83-mut16 S75V+R83N+L70F+V91K+C125S

[0432] Expression and purification of IL-2 mutants: The gene coding sequences of the above-mentioned IL-2 mutants were obtained by PCR, digested with enzymes, and ligated into expression vectors carrying the His tag gene that had also been double-digested, to obtain recombinant expression vectors for each IL-2 mutant. These recombinant expression vectors were transfected into 293F cells and cultured at 37°C, 5% CO2, and 120 rpm for 5 days. The cell culture supernatant was collected, and the IL-2 mutants were purified according to the (Ni) nickel column purification instructions. The specific steps are as described above.

[0433] The following experiments further examine whether the two groups of IL-2 mutants obtained can selectively activate Treg cells. The ability of wild-type IL-2 or IL-2 mutants to "activate Treg cells" can be determined by methods known in the art or by methods disclosed in the embodiments of this application, including but not limited to, for example, by comparing the EC50 values ​​of IL-2 mutants binding to IL-2Rβγ or IL-2Rαβγ. The greater the difference between the two (e.g., the greater the EC50 value of IL-2 mutants binding to IL-2Rβγ while maintaining binding to IL-2Rαβγ), the better the selectivity of IL-2 mutants for Treg cells. Alternatively, it can be determined by the change in the number of Treg cells after stimulation with IL-2 mutants, such as by measuring the change in the number of Treg cells in a mixed cell population by flow cytometry.

[0434] Example 2: Determination of the binding affinity of IL-2 mutants to IL-2Rαβγ and IL-2Rβγ

[0435] HEK-BLUE TM The experimental principle of cell line detection:

[0436] HEK-Blue TM The IL-2 cell line (InvivoGen, cat#hkb-il2) is a HEK293 cell line stably transfected with human IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132) genes. Another cell line, HEK-Blue... TMThe CD122 / CD132 (InvivoGen, cat#hkb-il2bg) cell line expresses only IL-2Rβ (CD122) and IL-2Rγ (CD132), but not IL-2Rα (CD25). Furthermore, both cell lines stably express human JAK3 and STAT5 genes, exhibiting a fully functional IL-2 signaling pathway. Additionally, STAT5-induced SEAP reporter gene expression was introduced, allowing for the use of QUANTI-Blue after IL-2 stimulation. TM The expression level of STAT5-induced SEAP was detected using a solution (InvivoGen, cat#rep-qbs). HEK-Blue was among the solutions used. TM IL-2 cell lines were used to detect the binding of IL-2 or its mutants to IL-2Rαβγ. HEK-Blue TM The CD122 / CD132 cell line was used to detect the binding of IL-2 or its mutants to IL-2Rβγ. When IL-2 or its mutants were added to these cell lines, IL-2 or its mutants could bind to the high-affinity receptor IL-2Rαβγ or the intermediate-affinity receptor IL-2Rβγ, activating the intracellular Janus family tyrosine kinase signaling cascade (JAK1 and JAK3), initiating the phosphorylation of signal transducer and activator of transcription 5 (STAT5), and subsequently inducing SEAP expression. The substrate QUANTI-Blue was used. TM SEAP expression levels were detected by reading OD values ​​at 630 nm and calculating corresponding EC50 values ​​to reflect the activation of STAT5 after IL-2 or its mutants bind to IL-2Rαβγ or IL-2Rβγ. Wild-type human IL-2 and AMG592 (AMGEN, SEQ ID NO:107) were used as controls in this experiment.

[0437] HEK-Blue TM The EC50 values ​​of IL-2 mutant binding to IL-2Rβγ were determined using CD122 / CD132 cell lines and HEK-Blue microarrays. TM The EC50 values ​​of IL-2 mutant binding to IL-2Rαβγ were measured using IL-2 cell lines. The ratio between the two, namely the EC50 of HEK-IL-2Rβγ / EC50 of HEK-IL-2Rαβγ, was defined as the therapeutic safety window for IL-2 selective activation of Treg cells. The safety window value reflects the selective targeting of Treg cells by the IL-2 mutant; the higher the value, the stronger the selective targeting of Treg cells by the IL-2 mutant.

[0438] HEK-BLUE TM Cell line detection experiments:

[0439] First, the IL-2 mutant to be tested was diluted to 800 μg / mL with the test culture medium, and then serially diluted 4-fold in 96-well Coring U-plates to obtain 12 concentrations. The digested cells (HEK-Blue) were then... TM IL-2 cell lines or HEK-Blue TM The CD122 / CD132 cell line suspension was adjusted to 2.8 × 10⁻⁶. 5 After reaching a cell count of 180 μL, seed 180 μL of cell suspension per well into a 96-well flat-bottom cell culture plate. Add 20 μL of diluted IL-2 or IL-2 mutant to each well, gently vortex to mix, and then incubate at 37°C, 5% CO2 for 24 h. Transfer 20 μL of cell culture supernatant per well from this cell culture plate to another new 96-well flat-bottom cell culture plate, and add 180 μL of QUANTI-Blue to each well. TM The test solution was mixed thoroughly and incubated at 37°C for 1 hour. The absorbance value at 620 nm was then read. The EC50 value of the test sample was statistically calculated using GraphPad Prism 8.0.1. The fitting method was to first convert the concentration value to logarithm, i.e., X = log(X), and then calculate the EC50 value of the test sample according to the log(agonist) vs. response-variable slope (four parameters) fitting curve.

[0440] HEK-BLUE TM Experimental results for cell line analysis:

[0441] The detection results of the first and second groups of IL-2 mutants are shown in Tables 11 and 12. The results showed that, compared with wild-type IL-2, both groups of IL-2 mutants were able to bind to IL-2Rαβγ, and their EC50 value for binding to IL-2Rβγ was increased, their affinity was decreased, and their therapeutic safety window was significantly improved, indicating that these mutants have excellent Treg cell selective targeting.

[0442] Table 11 Group 1 IL-2 mutant HEK-BLUE TM Test results

[0443]

[0444]

[0445] (Note: * indicates that these mutants have low activation ability of IL-2Rβγ cells, and accurate EC50 values ​​could not be obtained by fitting within the concentration range used in the experiment.)

[0446] Table 12 Group 2 IL-2 mutant HEK-BLUE TM Test results

[0447]

[0448] (Note: * indicates that these mutants have low activation ability of IL-2Rβγ cells, and accurate EC50 values ​​could not be obtained by fitting within the concentration range used in the experiment.)

[0449] Example 3: Expression and purification of IL-2 mutant and Fc fusion protein

[0450] The aforementioned IL-2 mutant was linked to IgG1 Fc LALA via a linker peptide, resulting in an IL-2-Fc fusion protein with two construction forms: a bivalent form and a monovalent form. IgG1 Fc LALA refers to IgG1 Fc with L234A and L235A mutations, and the numbering follows the EU Kabat numbering system (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991)).

[0451] The bivalent form of the IL-2 mutant fusion protein with Fc comprises two IL-2 mutant molecules: each containing two identical monomers, in which the IL-2 mutant is linked to either the N-terminus or C-terminus of the Fc. An exemplary structure of this form is shown below. Figure 1A The (IL-2-Fc) shown consists of two identical monomers, each containing, from the N-terminus to the C-terminus: an IL-2 mutant (SEQ ID NOs:5-34), a linker peptide (SEQ ID NO:52), and an IgG1 Fc LALA (SEQ ID NO:35). Another exemplary structure of this form is shown below. Figure 1B As shown (Fc-IL-2), it consists of two identical monomers, each of which contains from the N-terminus to the C-terminus: IgG1 Fc LALA (SEQ ID NO:35), a linker peptide (SEQ ID NO:52), and an IL-2 mutant (SEQ ID NOs:5-34).

[0452] The monovalent form of the IL-2 mutant fusion protein comprises a single IL-2 mutant molecule containing two distinct monomers. In one monomer, the IL-2 mutant is fused to the N-terminus or C-terminus of one Fc subunit, and the other monomer is the other Fc subunit. Pairing between the two asymmetric Fc subunits is promoted through knot and hole mutations. In a preferred form, it comprises two distinct monomers. In one monomer, the IL-2 mutant is fused to the C-terminus of one Fc subunit via a linker peptide. This monomer, from N-terminus to C-terminus, comprises an IgG1 Fc LALA knob (SEQ ID NO:41), a linker peptide (SEQ ID NO:52), and an IL-2 mutant (SEQ ID NOs:5-34); and the other monomer is an IgG1 Fc LALA hole (SEQ ID NO:42). An exemplary structure of the fusion protein is shown below. Figure 2 As shown.

[0453] The specific construction process of the IL-2 mutant fusion protein with Fc (taking the bivalent fusion protein form Fc-IL-2 of IL-2 mutant and Fc as an example): Recognition sites of restriction endonucleases HindIII and XhoI were introduced via PCR. The nucleic acid sequence encoding IgG1 FcLALA was linked to the nucleic acid sequence encoding the IL-2 mutant via an oligonucleotide sequence encoding GGGGS, and constructed into the pcDNA3.1 eukaryotic expression vector. This was transfected into 293F cells and cultured at 37℃, 5% CO2, and 120 rpm for 5 days. The culture medium was collected, and the Fc-IL-2 mutant fusion protein was purified by protein A affinity chromatography and gel filtration using a Superdex 200 column chromatography. Specific operational steps are as described in Example 1.

[0454] Activity assays of different forms of IL-2 mutant and Fc fusion protein:

[0455] HEK-BLUETM cell line experiments were used to detect and verify whether different fusion modalities affected the activity of IL-2 mutants. The specific experimental procedures for HEK-BLUETM are as described in Example 2.

[0456] The bivalent forms of the fusion proteins of Fc with (81-85)-mut8 and (81-85)-mut10 from the first group of IL-2 mutants and S75R83-mut16 from the second group of IL-2 mutants, namely Fc-(81-85)-mut8, Fc-(81-85)-mut10 and Fc-S75R83-mut16, were used as exemplary fusion proteins. Each fusion protein contains two identical monomers in which the IL-2 mutant is fused to the C-terminus of Fc LALA via the linker peptide GGGGS.

[0457] Meanwhile, the above mutant fusion proteins with Fc were tested in their monovalent forms: Fc knob-(81-85)-mut8 / Fc-hole, Fc knob-(81-85)-mut10 / Fc-hole, and Fc knob-S75R83-mut16 / Fc-hole. These fusion proteins contain two different monomers. One monomer contains an IL-2 mutant molecule fused to the C-terminus of Fc LALA knob via the linker peptide GGGGS. The other monomer is Fc LALA hole.

[0458] The specific test results are shown in Tables 13 and 14: The results demonstrate that the fusion protein of IL-2 mutant and Fc, whether in bivalent or monovalent form, does not affect the affinity of IL-2 mutant for IL-2Rβγ or IL-2Rαβγ, and the fusion protein of IL-2 mutant and Fc still has a preference for binding to different receptors.

[0459] Table 13

[0460]

[0461] (Note: * indicates that these mutants have low activation ability of IL-2Rβγ cells, and accurate EC50 values ​​could not be obtained by fitting within the concentration range used in the experiment.)

[0462] Table 14

[0463]

[0464]

[0465] Example 4: BIACORE Affinity Determination of IL-2 Mutant

[0466] The Biacore assay was used to determine the affinity of exemplary Fc-IL-2 mutant bivalent fusion proteins Fc-(81-85)-mut8 or Fc-S75R83-mut16 for human IL-2Rβγ and human IL-2Rαβγ, respectively. The IL-2Rβγ used was the IL-2Rβγ-Fc heterodimer prepared in Example 1, and the IL-2Rαβγ used was a heterodimeric fusion protein (IL-2Rαβγ-Fc) composed of IL-2Rαβγ-Fc knob monomer and IL-2Rβ-Fc hole monomer. The Fc-wild-type IL-2 bivalent fusion protein served as a control in this experiment. The specific operating steps are as follows: IL-2Rβγ-Fc or IL-2Rαβγ-Fc was captured using the Protein A sensor chip of a Biacore instrument (Biacore T200, GE). IL-2Rβγ-Fc or IL-2Rαβγ-Fc was diluted to 1 μg / mL and flowed at a rate of 10 μL / min for 30 seconds. Then, a series of concentration gradients of wild-type IL-2 or its mutant bivalent fusion protein were flowed onto the chip surface at a rate of 30 μL / min. Binding lasted for 120 seconds, and dissociation lasted for 360 seconds. The reaction signals were detected in real time using the Biacore instrument to obtain binding and dissociation curves. The Kon, Koff, and Kd values ​​are shown in Table 15.

[0467] Table 15

[0468]

[0469] Example 5: In vitro biological activity assay of IL-2 mutant

[0470] When IL-2 binds to its cell surface receptor, it activates the JAK-STAT signaling pathway, leading to STAT5 phosphorylation. STAT5 phosphorylation is a necessary step in the IL-2 signaling pathway; after phosphorylation, STAT5 enters the cell nucleus to participate in downstream signal transduction processes. Therefore, phosphorylated STAT5 signals in cells can reflect the activation status of IL-2.

[0471] CD8 + T lymphocytes express IL-2Rβγ but not IL-2Rα. Treg cells express both IL-2Rβγ and IL-2Rα. CD8+ was measured by flow cytometry. + The level of STAT5 phosphorylation (pSTAT5) in T lymphocytes and Treg cells indirectly reflects the activation of CD8 by IL-2 mutants. + The activity of T lymphocytes and Treg cells (see, for example, Peterson LB, et al. J Autoimmun. 2018 Dec; 95: 1-14).

[0472] The specific operating steps are as follows: PBMC cells (Shanghai Miaoshun Biotechnology Co., Ltd., cat#PB050C) were revived one day in advance, and PBMCs were collected overnight. Cells were washed with PBS and collected by centrifugation. After mixing with 2% complete culture medium, cells were counted, and 60 μL of cell suspension was added to each well, approximately 5–6 × 10⁶ cells / well. 5 Cells were seeded into cell culture plates and placed in a cell culture incubator for later use. The test samples included exemplary first group of IL-2 mutant and Fc bivalent fusion proteins Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-(81-85)-mut2, Fc-(81-85)-mut3, Fc-(81-85)-mut9 and Fc-(81-85)-mut14, and exemplary second group of IL-2 mutant and Fc bivalent fusion proteins Fc-S75R83-mut7 and Fc-S75R83-mut16. After serially diluting these samples, they were added to cells for stimulation for 30 min. Cell fixation medium was added, and the cells were washed. Then, anti-CD4 antibody (BD cat#562424), anti-CD25 antibody (Biolegend cat#356128), and anti-CD8 antibody (Biolegend cat#300932) were added, and the cells were incubated at 4°C in the dark for 30 min. After washing the cells, 150 μL / well of pre-chilled Phosflow was added. TM PermBuffer III (BD cat#558050) was incubated on ice for 30 min. After washing cells with FACS buffer, 100 μL / well of PE Mouse Anti-Stat5 (pY694) (BD cat#612567) and anti-Foxp3 antibody (Biolegend cat#320112) diluted in FACS buffer were added, and the cells were incubated at 4°C in the dark for 30 min. Cells were washed with FACS buffer, centrifuged, and the supernatant was discarded. Cells were resuspended in 100 μL / well of FACS buffer and analyzed. CD8 + The surface marker of T lymphocytes is CD8, and in this experiment, CD4 was used. - CD8 + The cells are defined as CD8 + T lymphocytes. Surface markers of Treg cells typically include CD4, CD25, and Foxp3; in this experiment, CD4... + CD25 + FoxP3 +The cells were defined as Treg cells. Data processing: The pSTAT5 fluorescence values ​​(MFI) of the two cell populations treated with IL-2 mutants at different concentrations were statistically analyzed using GraphPad Prism 8.0.1, and the EC50 values ​​were calculated. The fitting method was to first convert the concentration values ​​to logarithms, i.e., X = log(X), and then fit a curve according to log(agonist) vs. response-variable slope (four parameters) to calculate the Treg cells and CD8+. + EC50 value of T lymphocytes.

[0473] Figure 3A , 3C Figures 3E and 3G show the results of STAT5 phosphorylation induced in Treg cells by the exemplary first group of IL-2 mutants and Fc divalent fusion proteins; Figure 3B , 3D Figures 3F and 3H illustrate exemplary first-group IL-2 mutants and Fc bivalent fusion proteins in CD8. + The result of inducing STAT5 phosphorylation in T lymphocytes.

[0474] Figure 3I The image shows the results of STAT5 phosphorylation induced in Treg cells by an exemplary second group of IL-2 mutants and Fc divalent fusion proteins; Figure 3J The image shows an exemplary second group of IL-2 mutants and Fc bivalent fusion proteins in CD8. + The result of STAT5 phosphorylation induced in T cells.

[0475] The results above show that both the IL-2 mutant and the Fc bivalent fusion protein can induce STAT5 phosphorylation in Treg cells, while in CD8... + In T lymphocytes, the IL-2 mutant fusion protein with Fc did not significantly induce STAT5 phosphorylation. These results further demonstrate that the IL-2 mutant of this invention can selectively activate Treg cells without activating CD8. + T lymphocytes have good Treg cell targeting and are safer in human treatment compared to wild-type IL-2.

[0476] Example 6: Activity assay of IL-2 mutant in mice

[0477] To demonstrate the selectivity of the IL-2 mutant for Treg cells in rodents, Treg cells and CD8+ were measured in the blood of mice after a single administration of the Fc-IL-2 mutant bivalent fusion protein. + The proliferation of T lymphocytes.

[0478] The specific procedures were as follows: C57LB / 6 mice were purchased from Vital River and housed in a pathogen-free environment. All animal experiments were conducted in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC).

[0479] Animal treatment: 21 female SPF C57BL / 6 mice were divided into 7 groups of 3 mice each. Each group of mice was subcutaneously injected with exemplary IL-2 mutant and Fc bivalent fusion proteins: Fc-S75R83-mut5, Fc-S75R83-mut9, Fc-S75R83-mut16, Fc-(81-85)-mut8, Fc-(81-85)-mut10, or Fc-(81-85)-mut12. Fc-AMG592 (SEQ ID NO:106) served as the control sample in this experiment.

[0480] First, a single dose of 1 mg / kg was administered to each mouse on day 0. 100 μL of blood was collected intravenously before and 3 days after administration on day 0, and the animal number was marked on the EDTA anticoagulant blood collection tube.

[0481] The Treye-Nulear Transcription Factor Buffer Set (Biolegend CAT#424401) was used for detection. In short, 80 μL of blood sample was added to 1 mL of 1×Lyse / Fix buffer (BD CAT#558049), mixed well, and incubated at 37°C for 30 min. Then, the cells were centrifuged at 400 g for 8 min and the supernatant was discarded. Flow cytometry was used for detection: First, cells were fixed by adding 1000 μL / tube of 1×Transcription Factor Fix working solution and incubating at room temperature in the dark for 40 min. Then, cell membrane perforation was performed, followed by centrifugation at 400 g for 8 min and discarding the supernatant. 2 mL / tube of 1×Perm buffer was added for resuspending, followed by centrifugation at 400 g for 8 min and discarding the supernatant. This cell membrane perforation step was repeated once. The cells were then transferred to a V-type 96-well plate. Resuspend cells in 1×Perm buffer and add anti-CD4 antibody (Biolegend cat#317429), anti-CD25 antibody (Biolegend cat#356128), anti-Foxp3 antibody (Biolegend cat#320112), and anti-CD8 antibody (Biolegend cat#344742), respectively. Incubate at room temperature for 1 h, then centrifuge at 600 g × 8 min and discard the supernatant. Wash twice with 200 μL / well of 1% BSA, centrifuge, discard the supernatant, and resuspend in 100 μL of LACS buffer for analysis.

[0482] Statistical analysis was performed using the flow cytometry software CytExpert. The lymphocyte population (P1) was counted at 30,000. The percentage of Treg cells in CD4+ cells was calculated. + Percentage of cell population, and CD8 + The percentage of T lymphocytes in total lymphocytes. CD8 + The surface marker of T lymphocytes is CD8. + The cells are defined as CD8 + T lymphocytes. Treg cells are marked by surface markers CD4, CD25, and Foxp3. CD4+... + CD25 + FoxP3 + The cells are defined as Treg cells.

[0483] The results are as follows Figure 4A As shown, the first group of IL-2 mutant bivalent fusion proteins with Fc: Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12, at a dose of 1 mg / kg, all stimulated the proliferation of Treg cells in mice. Furthermore, compared to the control Fc-AMG592 (SEQ ID NO:106), Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12 exhibited Treg cell proliferation-stimulating activity comparable to or higher than that of Fc-AMG592. Additionally, as... Figure 4B As shown, Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12 did not stimulate the proliferation of CD8+ T lymphocytes. This indicates their specificity in promoting the proliferation of Treg cells in vivo.

[0484] The results are as follows Figure 4C As shown, the second group of IL-2 mutants and Fc bivalent fusion proteins Fc-S75R83-mut5, Fc-S75R83-mut9, and Fc-S75R83-mut16, at a dose of 1 mg / kg, were able to stimulate the proliferation of Treg cells in mice. Compared with the control Fc-AMG592, the proliferative activity of Fc-S75R83-mut5 and Fc-S75R83-mut9 in stimulating Treg cell proliferation was comparable to that of Fc-AMG592, while the proliferative activity of Fc-S75R83-mut16 in stimulating Treg cell proliferation was higher. Additionally, as... Figure 4DAs shown, Fc-S75R83-mut5, Fc-S75R83-mut9, and Fc-S75R83-mut16 stimulate Treg cell proliferation but do not stimulate CD8. + Proliferation of T lymphocytes.

[0485] The above results demonstrate that the IL-2 mutant molecule of the present invention can specifically activate Treg cells in rodents without affecting CD8. + T cells have an impact.

[0486] Example 7: Activity determination of IL-2 mutant in cynomolgus monkeys

[0487] To demonstrate the selectivity of the IL-2 mutant for Treg cells in primates, Treg cells and CD8+ were measured in the blood of cynomolgus monkeys after a single administration of the Fc-IL-2 mutant bivalent fusion protein. + The proliferation of T lymphocytes.

[0488] The specific procedures were as follows: Six cynomolgus monkeys, of average size, weighing 2.5-3 kg, were purchased from Beijing Zhongke Lingrui Biotechnology Co., Ltd., and housed in an environment free of specific pathogens. All animal experiments were conducted in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC).

[0489] First, a single subcutaneous injection was administered to each cynomolgus monkey on day 0. The injection consisted of an exemplary Fc-IL-2 mutant bivalent fusion protein: Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-S75R83-mut5, Fc-S75R83-mut16, or the control drug Fc-AMG592 (AMGEN, SEQ ID NO: 106). The injection dose was divided into a low-dose group (0.3 mg / kg) and a high-dose group (1.5 mg / kg). Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-S75R83-mut5, and the control drug Fc-AMG592 were only tested in the low-dose group, while Fc-S75R83-mut16 was tested in both the low-dose and high-dose groups. One mL of blood was collected intravenously on day 0 before administration and on day 7 after administration.

[0490] The Treye-Nulear Transcription Factor Buffer Set (Biolegend cat#424401) was used for detection, and the specific operation method is as described in Example 6.

[0491] The results are as follows Figures 5A-5BAs shown, the first group of IL-2 mutants and the bivalent fusion proteins Fc-(81-85)-mut8 and Fc-(81-85)-mut10, when administered at a low dose of 0.3 mg / kg, were able to stimulate the proliferation of Treg cells in cynomolgus monkeys. Figure 5A Furthermore, compared to the control drug Fc-AMG592, Fc-(81-85)-mut8 and Fc-(81-85)-mut10 showed higher activity in stimulating Treg cell proliferation. Simultaneously, they did not stimulate CD8+. + T lymphocytes proliferated, but their number did not change significantly. Figure 5B ).

[0492] The results are as follows Figures 5C-5D As shown, the second group of IL-2 mutants and the bivalent fusion protein Fc-S75R83-mut5, at a low dose of 0.3 mg / kg, stimulated the proliferation of Treg cells in cynomolgus monkeys. Figure 5C (At the same time, neither of them stimulated CD8 in cynomolgus monkeys.) + T lymphocytes proliferated, but their number did not change significantly. Figure 5D ).

[0493] Fc-S75R83-mut16, in both the low-dose group (0.3 mg / kg) and the high-dose group (1.5 mg / kg), stimulated the proliferation of Treg cells in cynomolgus monkeys, and its effect on promoting Treg cell proliferation increased with increasing dosage. Figure 5C Furthermore, regardless of whether the dose is low or high, Fc-S75R83-mut16 does not stimulate CD8 in cynomolgus monkeys. + T lymphocytes proliferated, but their number did not change significantly. Figure 5D ).

[0494] Furthermore, compared to the control Fc-AMG592, at the same dose, Fc-S75R83-mut5 and Fc-S75R83-mut16 exhibited significantly higher activity in stimulating Treg cell proliferation. Figure 5C ).

[0495] The above results demonstrate that the IL-2 mutant of this invention can specifically activate Treg cells in primates without affecting CD8 cells. + The T cells had an effect that was comparable to, or even better than, that of the control Fc-AMG592.

[0496] Example 8: PK experiment of IL-2 mutant in mice

[0497] C57LB / 6 mice were purchased from Vital River and housed in a specific pathogen-free environment. All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) protocols and guidelines.

[0498] The specific procedures for the in vivo pharmacokinetic (PK) experiment in mice were as follows: Twenty-four C57LB / 6 mice were randomly divided into four groups of six mice each, with half male and half female. Each group of mice received a single tail vein injection of an exemplary IL-2 mutant fusion protein with Fc, namely Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-S75R83-mut5, or Fc-S75R83-mut16, at a dose of 1 mg / kg. Anticoagulated whole blood was collected at 0 hours before administration, and at 15 minutes, 5 hours, 24 hours, 72 hours, 168 hours, 240 hours, 336 hours, 432 hours, and 600 hours after administration, and plasma was separated. Plasma drug concentrations in each sample were determined using ELISA. Pharmacokinetic parameters for each animal were calculated using a WinNonlin 6.0 non-compartmental (NCA) model.

[0499] In mice, following intravenous injection of 1 mg / kg of the exemplary IL-2 mutant with the Fc bivalent fusion proteins Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-S75R83-mut5, and Fc-S75R83-mut16, the plasma drug concentration gradually decreased over time. The results of the pharmacokinetic (PK) experiment are as follows... Figure 6 As shown, the bivalent fusion protein of IL-2 mutant and Fc of the present invention has good pharmacokinetic properties in mice (the PK curves of Fc-(81-85)-mut8 and Fc-S75R83-mut5 are not shown).

Claims

1. An IL-2 mutant, the amino acid sequence of which is the amino acid sequence obtained by mutating amino acid residues RPRDL at positions 81-85 of SEQ ID NO:1 to RHL.

2. The IL-2 mutant according to claim 1 also has an E67T mutation.

3. The IL-2 mutant according to claim 1 also has a V91K mutation.

4. The IL-2 mutant according to claim 1 also exhibits mutations in E67T and V91K.

5. The IL-2 mutant according to claim 1 also exhibits mutations in L70I and S75F.

6. The IL-2 mutant according to claim 1 also has an N71I mutation.

7. The IL-2 mutant according to any one of claims 1-6 also has a C125 mutation, wherein the C125 mutation is C125S or C125A.

8. The IL-2 mutant according to claim 1, compared with human wild-type IL-2, has a reduced binding affinity to IL-2Rβγ, but maintains binding to IL-2Rα.

9. An IL-2 mutant, characterized in that, The amino acid sequence of the mutant is shown in any of SEQ ID NOs: 12-18.

10. A fusion protein comprising the IL-2 mutant of any one of claims 1-9 and Fc.

11. The fusion protein of claim 10, wherein Fc comprises a mutation capable of altering effector function, and / or a mutation capable of prolonging half-life, and / or a mutation capable of promoting the formation of dimers from heteropeptides.

12. The fusion protein according to claim 10, wherein the Fc is derived from human IgG1 or human IgG4.

13. The fusion protein according to any one of claims 10-12, wherein the Fc comprises: (i) L234A and L235A mutations; or (ii) N297G mutation; or (iii) N297A mutation; or (iv) L234A, L235A, and P331S mutations; or (v) L234A, L235E, G237A, A330S and P331S mutations, wherein the mutations are numbered according to the EU numbering system.

14. The fusion protein according to any one of claims 10-12, wherein the Fc comprises S228P, F234A and L235A mutations, wherein the mutations are numbered according to the EU numbering system.

15. The fusion protein according to any one of claims 10-12, wherein the Fc further comprises: (i) S354C and T366W mutations; or (ii) Y349C, T366S, L368A and Y407V mutations, wherein the mutations are numbered according to the EU numbering system.

16. The fusion protein according to any one of claims 10-12, wherein the IL-2 mutant is linked to Fc via a linker peptide.

17. The fusion protein according to claim 16, wherein the amino acid sequence of the linker peptide is shown in any one of SEQ ID NOs:43-74.

18. The fusion protein according to any one of claims 10-12, wherein the IL-2 mutant is located at the N-terminus or C-terminus of Fc.

19. A fusion protein comprising an IL-2 mutant, characterized in that, The amino acid sequence of the fusion protein is shown in any of SEQ ID NOs: 83-89.

20. A nucleic acid encoding an IL-2 mutant of any one of claims 1-9 or a fusion protein of any one of claims 10-19.

21. A vector comprising the nucleic acid of claim 20.

22. A host cell comprising the vector of claim 21.

23. A method for preparing an IL-2 mutant or a fusion protein containing an IL-2 mutant, comprising: a) Culture the host cells of claim 22 under conditions that enable efficient expression of the IL-2 mutant or a fusion protein containing the IL-2 mutant; and b) Obtain the expressed IL-2 mutant or the fusion protein containing the IL-2 mutant from the host cell.

24. A pharmaceutical composition comprising the IL-2 mutant of any one of claims 1-9, the fusion protein of any one of claims 10-19, the nucleic acid of claim 20, the vector of claim 21, or the host cell of claim 22, and a pharmaceutically acceptable vector and / or excipient.

Images