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

Abstract

The present invention relates to an IL-2 mutant, a fusion protein comprising the IL-2 mutant, a nucleic acid encoding the IL-2 mutant or the fusion protein thereof, a vector and a host cell comprising the nucleic acid, or a pharmaceutical composition comprising the same, meanwhile, the invention also discloses an IL-2 mutant, a fusion protein containing the IL-2 mutant, a nucleic acid for coding the IL-2 mutant or the fusion protein thereof, a vector and a host cell containing the nucleic acid, or a therapeutic application of a pharmaceutical composition containing the nucleic acid. The invention also relates to a preparation method of the IL-2 mutant and the fusion protein thereof.

Description

Interleukin-2 (IL-2) mutants and their uses

[0001] The contents of the sequence listing submitted below are incorporated herein by reference in its entirety, file name: IL-2-1 20230516.xml, record date: 2023.05.16, size: 145 KB. Technical Field

[0002] The present application relates to interleukin-2 (IL-2) mutants, and also includes fusion proteins of the IL-2 mutants, nucleic acid molecules encoding IL-2 mutants or fusion proteins, vectors and host cells containing nucleic acid molecules, as well as pharmaceutical compositions and therapeutic uses thereof. It further provides methods for preparing IL-2 mutants and their fusion proteins.

[0003] Background of the Invention

[0004] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a bundled 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 cells (NK cells) in innate immune defense (KA Smith, Science 240, 1169 (1988); BH Nelson, D.M. Willerford, Adv. Immunol. 70, 1 (1998)).

[0005] IL-2 functions by binding to the IL-2 receptor 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] IL-2 receptors can be divided into the following types based on their binding subunits and affinity for IL-2: The first type is a low-affinity receptor, composed of a single IL-2Rα subunit (Kd value approximately 10 nM). Because IL-2Rα lacks an intracellular signaling domain, IL-2 binding to this type of receptor cannot transmit signals or regulate cells (HM Wang, KA Smith, J. Exp. Med. 166, 1055 (1987)). The second type is an intermediate-affinity receptor, composed of IL-2Rβ and γc subunits (Kd value approximately 1 nM). This type of receptor is primarily expressed on natural killer cells, macrophages, and resting T cells, and can activate signaling pathways at higher IL-2 concentrations (BH Nelson, D.M. Willerford, Adv. Immunol. 70, 1 (1998)). Although the affinity of IL-2Rβ alone for IL-2 is very low (Kd value of approximately 100 nM), and γc alone has almost no detectable binding to IL-2 (M. Rickert, MJ Boulanger, N. Goriatcheva, KC Garcia, J. Mol. Biol. 339, 1115 (2004)), the complex composed of IL-2Rβ and γc (IL-2Rβγ) is essential for effective IL-2 cell 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 of approximately 10 pM), which is usually expressed in activated lymphocytes and CD4 + CD25 + Foxp3 + It is expressed on regulatory T cells (Treg) (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 lymphoproliferative diseases. Recent studies have found that IL-2 is also a key cytokine for the differentiation, function, and survival of Treg cells, and plays an important role in achieving the function, development, and induction of Treg cell suppressive characteristics. Treg cells are generally defined by the expression of CD4, CD25, and the transcription factor forkhead P3 (Foxp3). They can inhibit autoreactive lymphocytes and regulate innate and adaptive immune responses, leading to immunosuppression. As one of the key effector cells of 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 to the occurrence and development 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 wide range of biological effects of IL-2, by changing the selectivity or preference of IL-2 for different receptors, IL-2 can selectively activate target cells to reduce the toxicity and / or improve the efficacy of IL-2 treatment, thereby increasing the safety window of IL-2 treatment for specific diseases. This is of great significance to improving the safety and effectiveness of IL-2 treatment of diseases. There is still a need in this field to develop new IL-2 molecules with improved properties.

[0009] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect, the present application relates to an IL-2 mutant comprising a deletion or mutation of amino acid residues 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 human wild-type IL-2 is as shown in SEQ ID NO: 1.

[0012] In some embodiments, the IL-2 mutants described herein comprise 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 mutants described herein further comprise 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 E67K and N71S, respectively. In some embodiments, the IL-2 mutants described herein comprise 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 in E67K and N71S relative to the human wild-type IL-2 amino acid sequence.

[0013] In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL, and an E67A mutation relative to the human wild-type IL-2 amino acid sequence.

[0014] In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL, and further comprise mutations of N71 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of N71 and S75 are N71W and S75I, respectively. In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL, and N71W and S75I mutations relative to the human wild-type IL-2 amino acid sequence.

[0015] In some embodiments, the IL-2 mutants described herein comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RHL. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RHL, and E67T and / or V91K mutations relative to the human wild-type IL-2 amino acid sequence.

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

[0017] In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and further comprise a mutation of N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation of N71 is N71I. In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and a mutation of N71I relative to the human wild-type IL-2 amino acid sequence.

[0018] In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to SPL. In some embodiments, the IL-2 mutants described herein further comprise mutations of E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of E67 and N71 are E67M and N71S, respectively. In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to SPL, as well as mutations of E67M and N71S relative to the human wild-type IL-2 amino acid sequence.

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

[0020] In some embodiments, the IL-2 mutant described herein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5-18, 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 to the amino acid sequence shown in any one of SEQ ID NOs: 5-18.

[0021] Another aspect of the present application relates to another IL-2 mutant comprising a mutation of amino acid residue S at position 75 (S75) and amino acid residue R at position 83 (R83) relative to the amino acid sequence of wild-type human IL-2. In some embodiments, the amino acid sequence of wild-type human IL-2 is as shown in SEQ ID NO: 1.

[0022] In some embodiments, the IL-2 mutants described herein are characterized in that 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 mutants described herein comprise S75 mutations to S75P and R83 mutations to R83Y. In some embodiments, the IL-2 mutants described herein further comprise E67 and D84 mutations 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 mutants described herein comprise S75P, R83Y, E67A, and D84N mutations relative to the human wild-type IL-2 amino acid sequence.

[0024] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75P and R83 mutations to R83A. In some embodiments, the IL-2 mutants described herein further comprise H16, L70, and N71 mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the H16, L70, and N71 mutations are H16D, L70I, and N71G, respectively. In some embodiments, the IL-2 mutants described herein comprise S75P, R83A, H16D, L70I, and N71G mutations relative to the human wild-type IL-2 amino acid sequence.

[0025] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75F and R83 mutations to R83P. In some embodiments, the IL-2 mutants described herein further comprise H16 and / or N71 mutations 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 mutants described herein comprise S75F, R83P, and H16Q mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein comprise S75F, R83P, H16N, and N71S mutations relative to the human wild-type IL-2 amino acid sequence.

[0026] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75F and R83 mutations to R83L. In some embodiments, the IL-2 mutants described herein further comprise E67 and L70 mutations 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 mutants described herein comprise S75F, R83L, E67R, and L70V mutations relative to the human wild-type IL-2 amino acid sequence.

[0027] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75I and R83 mutations to R83L. In some embodiments, the IL-2 mutants described herein further comprise E67 and L70 mutations 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 mutants described herein comprise S75I, R83L, E67G, and L70V mutations relative to the human wild-type IL-2 amino acid sequence.

[0028] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75I and R83 mutations to R83K. In some embodiments, the IL-2 mutants described herein further comprise E67 and L70 mutations 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 mutants described herein comprise S75I, R83K, E67S, and L70V mutations relative to the human wild-type IL-2 amino acid sequence.

[0029] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75T and R83 mutations to R83A or R83F. In some embodiments, the IL-2 mutants described herein further comprise H16 and E67 mutations 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 mutants described herein comprise S75T, R83A, H16N, and E67K mutations relative to the human wild-type IL-2 amino acid sequence.

[0030] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75V and R83 mutations to R83F. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise S75V, R83F, E15K, and H16Q mutations relative to the human wild-type IL-2 amino acid sequence.

[0031] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75V and R83 mutations to R83N. In some embodiments, the IL-2 mutants described herein further comprise L70 and V91 mutations 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 mutants described herein comprise S75V, R83N, L70F, and V91K mutations relative to the human wild-type IL-2 amino acid sequence.

[0032] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75G and R83 mutations to R83L. In some embodiments, the IL-2 mutants described herein further comprise L17, V91, and I92 mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the L17, V91, and I92 mutations are L17I, V91E, and I92L, respectively. In some embodiments, the IL-2 mutants described herein comprise S75G, R83L, L17I, V91E, and I92L mutations relative to the human wild-type IL-2 amino acid sequence.

[0033] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75Y and R83 mutations to R83N. In some embodiments, the IL-2 mutants described herein further comprise L17 and V91 mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, L17 and V91 mutations are L17I and V91K, respectively. In some embodiments, the IL-2 mutants described herein comprise S75Y, R83N, L17I, and V91K mutations relative to the human wild-type IL-2 amino acid sequence.

[0034] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75A and R83 mutations to R83K. In some embodiments, the IL-2 mutants described herein further comprise L17 mutations 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 mutants described herein comprise S75A, R83K, and L17I mutations relative to the human wild-type IL-2 amino acid sequence.

[0035] In some embodiments, the IL-2 mutants described herein comprise S75 mutations to S75L and R83 mutations to R83E. In some embodiments, the IL-2 mutants described herein further comprise L63 and E67 mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, L63 and E67 mutations are L63V and E67F, respectively. In some embodiments, the IL-2 mutants described herein comprise S75L, R83E, L63V, and E67F mutations relative to the human wild-type IL-2 amino acid sequence.

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

[0037] In some embodiments, the IL-2 mutant described herein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 19-34 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 to the amino acid sequence shown in any one of SEQ ID NOs: 19-34.

[0038] In some embodiments, the IL-2 mutants described herein have reduced binding affinity to IL-2Rβγ compared to wild-type IL-2, while maintaining binding to IL-2Rα.

[0039] Another aspect of the present application relates to fusion proteins comprising IL-2 mutants. In some embodiments, the fusion protein comprises an Fc. In some embodiments, the Fc comprises mutations that alter effector function, and / or mutations that extend half-life, and / or mutations that promote dimerization of heterologous polypeptides. In some embodiments, the Fc is derived from human. 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 comprising 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 according to the EU numbering system.

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

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

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

[0044] In some embodiments, the fusion protein of the IL-2 mutant described herein and Fc, 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 herein comprises an amino acid sequence as shown in any one of SEQ ID NOs:76-105 or a variant thereof, wherein the variant has 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 the amino acid sequence shown in any one of SEQ ID NOs:76-105.

[0046] At the same time, the present application also relates to isolated nucleic acids encoding any IL-2 mutant described herein or fusion proteins comprising IL-2 mutants, vectors comprising the nucleic acids, host cells comprising the nucleic acids or vectors (e.g., CHO cells, HEK 293 cells, Hela cells or COS cells), compositions (e.g., pharmaceutical compositions), kits, and products comprising any IL-2 mutant described herein or fusion proteins comprising IL-2 mutants. Also provided are methods of using any of the IL-2 mutants described herein, or fusion proteins thereof, or pharmaceutical compositions comprising the same, for treating inflammatory diseases or autoimmune diseases (e.g., 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) in individuals in need thereof (e.g., humans). BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0049] Figures 3A, 3C, 3E, and 3G show the results of 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 inducing STAT5 phosphorylation in Treg cells; Figures 3B, 3D, 3F, and 3H show the results of 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 in CD8 + Results of the induction of STAT5 phosphorylation in T lymphocytes.

[0050] FIG3I shows the results of the second group of IL-2 mutants and Fc bivalent fusion proteins: Fc-S75R83-mut7 and Fc-S75R83-mut16 inducing STAT5 phosphorylation in Treg cells; FIG3J shows the results of the second group of IL-2 mutants and Fc bivalent fusion proteins: Fc-S75R83-mut7 and Fc-S75R83-mut16 in CD8 + Results of the induction of STAT5 phosphorylation in T lymphocytes.

[0051] Figure 4A shows that the first group of bivalent fusion proteins of IL-2 mutants with Fc: 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 shows the first group of IL-2 mutants and Fc bivalent fusion proteins: Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12, which inhibited CD8 in mice at a dose of 1 mg / kg. + Effects on T lymphocyte proliferation.

[0053] Figure 4C shows that 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 shows the effects of the second group of IL-2 mutants and Fc bivalent fusion proteins Fc-S75R83-mut5, Fc-S75R83-mut9, and Fc-S75R83-mut16 on CD8 in mice at a dose of 1 mg / kg. + Effects on T lymphocyte proliferation.

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

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

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

[0058] FIG5D shows the effects of the second group of IL-2 mutants and Fc bivalent fusion proteins Fc-S75R83-mut5 at a dose of 0.3 mg / kg and Fc-S75R83-mut16 at a dose of 0.3 mg / kg and 1.5 mg / kg, respectively, on CD8 + Effects on T lymphocyte proliferation.

[0059] FIG6 shows the PK curves of exemplary bivalent fusion proteins of IL-2 mutants and Fc, Fc-(81-85)-mut10 and Fc-S75R83-mut16, in mice at a dose of 1 mg / kg.

[0060] Detailed description of this application

[0061] The present application relates to IL-2 mutants, and fusion proteins containing IL-2 mutants. Compared with wild-type IL-2, the IL-2 mutants described in the present application can selectively activate Treg cells, stimulate Treg cell proliferation, and have a higher safety window (for example, in HEK-Blue TM In the IL-2 cell line screening experiment, compared with wild-type IL-2, HEK-Blue TM EC50 values ​​of IL-2 mutants binding to IL-2Rβγ detected by CD122 / CD132 cell line were similar to those by HEK-Blue TM The ratio between the EC50 values ​​of the IL-2 mutant binding to IL-2Rαβγ detected by the IL-2 cell line is higher). Furthermore, the fusion protein of the IL-2 mutant and Fc described in the present application reduces or minimizes antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) by modifying the Fc fragment, thereby avoiding the generation of undesirable immune responses during treatment, and can increase the in vivo circulation half-life of the IL-2 mutant, thereby reducing the frequency of administration and the total number of administrations, providing convenience for patients and reducing treatment costs.

[0062] The present application also relates to nucleic acids encoding the IL-2 mutant or a fusion protein comprising the IL-2 mutant, vectors comprising the nucleic acid, host cells comprising the nucleic acid or vector, and methods for producing the above-mentioned IL-2 mutant or fusion protein, pharmaceutical compositions and manufactured products comprising the same, as well as methods for using the IL-2 mutant or a fusion protein or pharmaceutical composition comprising the IL-2 mutant to treat diseases (e.g., lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopic diseases, asthma or inflammatory bowel disease).

[0063] I. Definition

[0064] Unless the context clearly indicates otherwise, the practice of this application will employ conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of the art, many of which are described in detail below for illustration. Such techniques are fully explained in the literature. See Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et 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: A Practical 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 Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

[0065] As used herein, the term "wild-type IL-2" or "WT IL-2" refers to naturally occurring IL-2, which can be native IL-2 from any vertebrate source, such as mammals, including but not limited to livestock (e.g., cattle, sheep, goats, cats, dogs, donkeys, and horses), primates (e.g., humans and non-human primates such as monkeys or chimpanzees), rabbits, and rodents (e.g., mice, rats, gerbils, and hamsters). The term includes unprocessed IL-2 forms (e.g., containing a signal peptide) and processed mature IL-2 forms (e.g., not containing a signal peptide). The term also includes naturally occurring IL-2 allelic variants and splice variants, isoforms, homologs, and species homologs. The term also includes naturally occurring IL-2 variants, etc. For example, the naturally occurring IL-2 variant has at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) sequence homology compared to native 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 an N-terminal 20 amino acid signal peptide, 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 binding to the IL-2 receptor. For example, introduction of a serine mutation (C125S) at amino acid residue 125 of wild-type human IL-2 can prevent mispairing or aggregation caused by cysteine, and an exemplary amino acid sequence thereof is shown in SEQ ID NO: 2; introduction of an alanine mutation (C125A) at position 125 can promote efficient folding and improve expression, and an exemplary amino acid sequence thereof is shown in SEQ ID NO: 3.

[0067] As described herein, "mutations" of amino acids include substitutions, deletions, and insertions. Any combination of substitutions, deletions, and insertions can be performed to obtain a construct having desired properties (e.g., reduced binding affinity to IL-2Rβγ). In some embodiments, deletions or insertions of amino acids include deletions or insertions in the polypeptide sequence. In some embodiments, amino acid substitutions can be conservative amino acid substitutions. In other embodiments, amino acid substitutions can be non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties. Amino acid substitutions also include replacement with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). In some embodiments, amino acid mutations include a combination of amino acid substitutions and deletions, for example, within a certain region of the polypeptide sequence (e.g., amino acid residues 81-85 RPRDL), amino acid substitutions and deletions are simultaneously included. Amino acid mutations can be generated using genetic or chemical methods known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like.

[0068] As described herein, the "IL-2 mutants" include one or more substitutions, deletions, insertions, or any combination thereof in the wild-type IL-2 amino acid sequence (e.g., SEQ ID NO. 1). In some embodiments, the "IL-2 mutants" described herein can reduce their binding affinity to IL-2Rβγ while maintaining binding to IL-2Rα.

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

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

[0071] As described herein, "autoimmune disease" refers to a non-malignant disease or condition that originates from and is directed against 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, dystrophic epidermolysis bullosa, and Behçet's disease.

[0072] As described herein, "treatment" or "treating" is a method for obtaining a beneficial or desired result, including a clinical result. For the purposes of this application, the beneficial or desired result includes, but is not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, alleviating the disease (partially or completely), reducing the dosage of one or more other drugs required to treat the disease, delaying the progression of the disease, improving the quality of life and / or prolonging life. At the same time, "treatment" also includes reducing the pathological consequences of the disease. The methods of this application take into account any one or more aspects of these treatments. For example, if one or more symptoms associated with the disease are alleviated or eliminated, including but not limited to reducing the symptoms caused by the disease, improving the quality of life of patients with the disease, reducing the dosage of other drugs required to treat the disease, and / or prolonging the survival of the individual, the patient is considered to be successfully "treated."

[0073] The term "prevent" and similar words, such as "prevented," "preventing," and the like, refer to methods of preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. It also refers to delaying the occurrence or recurrence of a disease or condition. As used herein, "prevent" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before it occurs or recurs, or reducing the likelihood of the disease or condition occurring or recurring.

[0074] As described in the present application, "delaying" the development of a disease means postponing, hindering, slowing down, slowing down, stabilizing and / or postponing the development of a disease. Depending on the individuality of the disease history and / or treatment, the time of delay may be different. A method of "delaying" the development of a disease refers to a method that reduces the probability of disease development and / or reduces the degree of disease within a given time frame compared to not using the method. In certain embodiments, this comparison can be based on animal experiments, observed and / or counted in individual animals. In other embodiments, this comparison can be based on clinical studies, using statistically significant individual quantities.

[0075] As described herein, the term "effective amount" refers to a dosage of a drug or pharmaceutical composition sufficient to treat a specific disorder, condition, or disease, such as to improve, alleviate, weaken, and / or delay one or more symptoms. In some embodiments, an effective amount is an amount sufficient to delay the progression of a disease. In some embodiments, an effective amount is an amount sufficient to prevent or delay the occurrence or recurrence of a disease. An effective amount may be administered in a single dose or multiple doses. In some embodiments, an effective amount of a drug or pharmaceutical composition refers to an amount that can increase the number of Treg cells in a patient's diseased tissue.

[0076] As used herein, "individual" or "subject" refers to a mammal, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

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

[0078] As described herein, the term IgG "subtype" or "subclass" refers to any subclass of immunoglobulins defined by the chemical and antigenic properties of the constant region. Immunoglobulins are mainly divided into five major classes: IgA, IgD, IgE, IgG, and IgM, and many of them can be further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chains corresponding to the different immunoglobulin classes are named using the Greek letters α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and are described in detail in Abbas et al., Cellular and Molecular Immunology, 4th edition (WB Saunders, Co., 2000).

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

[0080] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an Fc-containing molecule (e.g., an antibody or an Fc-containing fusion protein). The preferred FcR is a native human FcR sequence. In addition, an exemplary FcR is an FcR that binds to an IgG antibody (a gamma receptor), including receptor subclasses such as FcγRI, FcγRII, and FcγRIII, as well as allelic variants and alternatively spliced ​​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-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see M. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). As used herein, the term "FcR" encompasses other FcRs, including those that will be identified in the future.

[0081] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transport of 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 high-affinity binding polypeptides binding to FcRn in vivo and in serum can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered polypeptides with variant Fc regions. WO 2004 / 42072 (Presta) details antibody variants that enhance or diminish binding to FcRs. Regarding FcRn, see Shield et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0082] "Fc effector function" refers to those biological activities caused by the Fc region (either a native sequence Fc region or an Fc region comprising amino acid sequence mutations) of an Fc-containing molecule (e.g., an antibody or an Fc-containing fusion protein), which vary depending on the immunoglobulin subtype from which the Fc is derived. Examples of effector function 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 receptor) and B cell activation. "Reducing or minimizing" effector function means that the effector function is reduced by at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) compared to wild-type Fc or an unmodified Fc-containing molecule (e.g., an antibody). Determining effector function can be readily determined and measured by one of ordinary skill in the art. In preferred embodiments, the effector functions of complement fixation, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity are all affected. In some embodiments, the effector function is eliminated by eliminating glycosylation by mutations in the constant region, e.g., "null effector function mutations." In some embodiments, the null effector function mutant is a C H For example, N297A or DANA mutation (D265A+N297A) in region 2 can be found in Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). In addition, other mutations that reduce or eliminate effector function include K322A and L234A / L235A (LALA). In addition, effector function can be reduced or eliminated by changes in production technology, such as expression in a host cell that does not glycosylate (e.g., E. coli) or in a host cell that alters 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" or "ADCC" refers to a form of cytotoxicity in which secreted Ig (or ligand-Fc structures) bind 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 bearing the antigen (or bearing the ligand receptor) and subsequently kill the target cells with cytotoxins. Antibodies (or other Fc-containing molecules) "arm" the cytotoxic cells and are necessary to kill the target cells by this mechanism. The primary cells that mediate ADCC include NK cells and monocytes, of which NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression 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, an in vitro ADCC assay can be performed, such as those described in detail in US Pat. No. 5,500,362 or 5,821,337. Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a target molecule can also be assessed in vivo, for example, in an animal model 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 is initiated by binding of the first component of the complement system (Clq) to an Fc-containing molecule (of the appropriate subclass) that binds to its cognate receptor via an Fc-fused ligand. To assess complement activation, a CDC assay can be performed, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Antibody variants with altered Fc region amino acid sequences that increase or decrease Clq binding capacity are described in detail in US Pat. No. 6,194,551 Bl and WO 99 / 51642. The contents of these patent publications are incorporated herein by reference. See Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0085] As used herein, “regulatory T cells” or “Tregs” are a type of CD4 T cells that have a significant immunosuppressive effect. +T cell subsets 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). They play a vital role in maintaining immune homeostasis and preventing autoimmune diseases and transplant rejection. Tregs require IL-2 for their function and development, as well as for the induction of their suppressive characteristics.

[0086] As used herein, "effector cells" refer to lymphocyte populations that mediate the cytotoxic effects induced by IL-2. 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 used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, such as binding between a ligand and a receptor, wherein the presence of the ligand can be determined in the presence of a heterogeneous population of molecules, including biomolecules. For example, a ligand that specifically binds to a receptor binds to the target receptor with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other receptors. In some embodiments, the extent of binding of the ligand to an unrelated receptor is less than 10% of the extent of binding of the ligand to the target receptor, as determined, for example, by radioimmunoassay (RIA). In some embodiments, the equilibrium dissociation constant (Kd) of a ligand that specifically binds to a 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 In some embodiments, the ligand can specifically bind to a receptor that is conserved across species. In some embodiments, specific binding can include but does not require exclusive binding. The binding specificity of the ligand can be determined experimentally using methods known in the art, including but not limited to Western blots, ELISA, RIA, ECL, IRMA, EIA, BIACORE, etc. 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 indicated, "binding affinity" as used herein 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 herein, the term "Koff" refers to the rate constant for the dissociation of a ligand from a ligand / receptor complex, which can be measured by a kinetic selection apparatus and expressed as s. -1 As used herein, the term "Kon" refers to the binding rate constant of a ligand to a receptor to form a ligand / receptor complex, expressed in M. -1 s -1 As described in this application, the term equilibrium dissociation constant "Kd" refers to the dissociation constant for a specific ligand-receptor interaction, which refers to the ligand concentration required when the ligand occupies half of all receptor binding sites in the receptor solution and reaches equilibrium, which is equal to Koff / Kon. The dissociation constant (Kd) can be used as an indicator reflecting the affinity between the ligand and the receptor. The Kd value obtained using the method is expressed in M ​​(mol / L). The premise for determining Kd is that all binding molecules are in solution. When the receptor is on the cell membrane, the corresponding dissociation rate constant is expressed by 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 M -1 The dissociation constant (Kd) can be used as an indicator to reflect the affinity between ligand and receptor.

[0089] The half 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 comparable 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 maximal effect in vivo. As described herein, "IC50" is used to represent the effective concentration of a ligand required to neutralize 50% of the biological activity of a receptor in vitro. IC50 or EC50 can be determined by biological measurements, such as inhibition of ligand binding by FACS analysis (competitive binding assay), cell-based cytokine release assays, or amplified luminescence homogeneous enzyme-linked immunosorbent assays (AlphaLISA).

[0090] As described in the present application, "fusion" refers to the connection of the various components by direct connection by peptide bonds or by one or more connecting peptides (or referred to as "linkers"). The sequence of a "connecting peptide (or linker)" can be a single amino acid or polypeptide sequence. In some embodiments, the connecting peptide (or linker) comprises a glycine-serine linker or consists of a glycine-serine linker. As described in the present application, the term "glycine-serine linker" refers to a peptide consisting of glycine and serine residues. Exemplary glycine-serine linkers include amino acid sequences 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 linker is GGGGS.

[0091] As used herein, a "covalent bond" refers to a stable bond between two atoms formed by sharing one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, a "peptide bond" refers to a covalent bond formed between the carboxyl group of an amino acid and the amine group of an adjacent amino acid. As used herein, a "disulfide bond" refers to a covalent bond formed between two sulfur atoms, such as two Fc fragments being bound by one or more disulfide bonds. The one or more disulfide bonds between the two fragments may be formed by linking thiol groups in the two fragments. In some embodiments, one or more disulfide bonds may be formed between one or more cysteines in the two Fc fragments. Oxidation of two thiol groups may form a disulfide bond. In some embodiments, the covalent linkage is formed by direct covalent bonding. In some embodiments, the covalent linkage is directly linked by a peptide bond or a disulfide bond.

[0092] As used herein, the term "derivative" refers to a molecule having the amino acid sequence of a protein or its analog, but with additional modifications at one or more amino acid groups, the alpha carbon atom, the amino terminus, or the carboxyl terminus. As used herein, modifications include, but are not limited to, chemical modifications, amino acid side group modifications, amino terminus modifications, and carboxyl terminus modifications. Chemical modifications include, but are not limited to, the addition of chemical groups, the creation of new chemical bonds, and the removal of chemical groups. Modifications of amino acid side groups include, but are not limited to, acylation of the epsilon amino group of lysine, N-alkylation of arginine, histidine, or lysine, carboxyl alkylation of glutamic acid or aspartic acid, and deamination of glutamine or asparagine. Modifications of the amino terminus include, but are not limited to, deamination, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the carboxyl terminus include, but are not limited to, amide, lower alkyl acyl, dialkylamide, and lower alkyl ester modifications. In some embodiments, the lower alkyl group is a C1-C4 alkyl group. In addition, one or more side groups or terminal groups may be protected using known protecting groups as used by those skilled in the art of chemistry. The alpha carbon of an amino acid may be monomethylated or dimethylated.

[0093] "Percentage (%) amino acid sequence identity" or "homology" of a peptide or polypeptide sequence is defined as the percentage of identical amino acid residues in a candidate sequence to a particular polypeptide or polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to maximize the percentage of sequence identity, and without considering any conservative substitutions as part of the sequence identity. To determine percentage amino acid sequence identity, various alignments within the skill of the art can be used, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Suitable parameters for measuring alignment can be determined by those skilled in the art, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0094] As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide whose amine group forms a peptide bond with the carboxyl group of the adjacent amino acid residue. As used herein, the "N-terminus" of a polypeptide refers to the first amino acid residue of the polypeptide whose carboxyl group forms a peptide bond with the amine group of the adjacent amino acid residue.

[0095] An "isolated" polypeptide is one that has been identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). Preferably, the isolated polypeptide is free from all other components of its production environment. Contaminating components of the production environment, such as those produced by recombinant transfected cells, often interfere with the research, diagnosis, or treatment of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide will be purified: (1) to greater than 95% polypeptide by weight as determined by the Lowry method, in some embodiments, greater than 99% polypeptide by weight; (2) to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated polypeptide includes the polypeptide in situ within recombinant cells because at least one element of the polypeptide's natural environment is absent. However, generally, an isolated polypeptide will be subjected to at least one purification step.

[0096] "Amino acid" is used herein in its broadest sense, encompassing naturally occurring amino acids as well as non-naturally occurring amino acids, including analogs and derivatives of amino acids. The latter include molecules containing amino acid moieties. Based on this broad definition, one skilled in the art will recognize 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 norleucine, β-alanine, ornithine, GABA, etc.; and chemically synthesized compounds with amino acid characteristics known in the art. As used herein, the term "protein-forming" refers to amino acids that can be synthesized into peptides, polypeptides, or proteins in a cell through metabolic pathways.

[0097] An "isolated" nucleic acid molecule encoding a protein or polypeptide (e.g., an IL-2 mutant or a fusion protein comprising an IL-2 mutant as described herein) is a nucleic acid molecule that has been identified and separated from at least one impurity nucleic acid molecule that is typically associated with the environment in which it was produced. Preferably, the isolated nucleic acid is free from all components of its production environment. An isolated nucleic acid molecule encoding a polypeptide as described herein is in a form or morphology that is not the same as it is found in nature. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid encoding a polypeptide as described herein that is naturally present in a cell. An isolated nucleic acid includes a nucleic acid molecule contained in a cell containing the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0098] The term "control sequence" refers to DNA sequences necessary for expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. 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 nucleic acid is "operably linked" to the other nucleic acid sequence. For example, the DNA encoding the presequence or secretory leader sequence participates in the secretion process of a polypeptide expressed as a precursor protein, and is operably linked to the DNA encoding the polypeptide molecule; if a promoter or enhancer affects the transcription of the coding sequence, the promoter or enhancer is operably linked to the coding sequence; or if the ribosome binding site is in a position that facilitates translation, the ribosome binding site is operably linked to the coding sequence. In general, "operably linked" means that the connected DNA sequences are continuous, and, for secretory leaders, they are not only continuous but also in the reading phase. However, enhancers do not have to be continuous. Connection is accomplished by ligation at appropriate restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0100] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of amplifying another nucleic acid molecule to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are introduced into the genome of a known host cell. Certain vectors are capable of directing the expression of nucleic acids to which they are linked. Such vectors are referred to herein as "expression vectors."

[0101] As used herein, the terms "transfection," "transformation," or "transduction" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0102] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny produced therefrom, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid and, for example, may contain mutations. Screening or selecting for mutant progeny that have the same function or biological activity as the original transformed cell is contemplated herein.

[0103] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that contains no additional ingredients that are unacceptably toxic to a subject to which the formulation is administered. Such formulations are sterile. A "sterile" formulation is sterile or free of all living microorganisms and their spores.

[0104] The embodiments described in this application should be understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0105] In this application, "about" is mentioned as a value or parameter, including (and describing) variations of the value or parameter itself. For example, the description of "about X" includes the description of "X".

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

[0107] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0108] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0109] II. IL-2 mutants

[0110] The IL-2 mutants of the present application have the following preferences and biological properties for IL-2 receptor selection and lymphocyte activation / proliferation:

[0111] In some embodiments, the IL-2 mutants provided herein have 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 mutants have 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 mutants provided herein have increased affinity for IL-2Rα compared to human wild-type IL-2 (e.g., SEQ ID NO: 1). In some embodiments, the affinity of the IL-2 mutants for IL-2Rα is increased by at least 1-fold (e.g., at least 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold) compared to human wild-type IL-2.

[0113] In some embodiments, compared to human wild-type IL-2 (e.g., SEQ ID NO: 1), the IL-2 mutants provided herein have the same or no significant reduction in affinity for IL-2Rα, or have 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 mutants provided herein have a reduced affinity for IL-2Rβ and / or IL-2Rβγ, while maintaining the same affinity for IL-2Rα or not significantly weakening or increasing the affinity for IL-2Rα. The affinity between the IL-2 mutant and the receptor can be determined by conventional methods known in the art, such as ELISA assays, HEK-Blue TM IL-2 cell line detection experiments or surface plasmon resonance (SPR) assays, etc.

[0115] In some embodiments, the IL-2 mutants provided herein have higher safety in treating certain specific diseases compared to human wild-type IL-2 (eg, SEQ ID NO: 1).

[0116] For example, in HEK-Blue TM In the IL-2 cell line (InvivoGen, cat#hkb-il2) experiment, compared with human wild-type IL-2, HEK-Blue TM The EC50 values ​​of IL-2 mutants binding to IL-2Rβγ (hereinafter referred to as the second receptor) detected by CD122 / CD132 cell line were similar to those of HEK-Blue TM The ratio of the EC50 values ​​of the IL-2 mutants to IL-2Rαβγ (hereinafter referred to as the three receptors) detected by the IL-2 cell line is higher, that is, it has a higher therapeutic safety window and thus has higher safety. TM CD122 / CD132 cell line assays were used to determine the EC50 values ​​of IL-2 or IL-2 mutants binding to IL-2Rβγ. TM IL-2 cell line assays are used to determine the EC50 value of IL-2 or IL-2 mutant binding to IL-2Rαβγ. In some embodiments, the EC50 of the IL-2 mutant is 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 A ratio of at least 2-fold, for example, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 700-fold, 1000-fold, 2000-fold, 10000-fold, 20000-fold, 30000-fold or more.

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

[0118] In certain embodiments, compared with human wild-type IL-2 (e.g., SEQ ID NO: 1), the IL-2 mutants provided herein can selectively activate Treg cells. For example, in a STAT5 phosphorylation experiment, the activation of STAT5 phosphorylation signals by detecting IL-2 or IL-2 mutants in Treg cells is used to reflect the activation ability of IL-2 mutants to Treg cells. Detected by flow cytometry, GraphPad Prism software analyzes and calculates the EC50 value of IL-2 or IL-2 mutants activating STAT5 phosphorylation in Treg cells. The EC50 value indirectly reflects the activation ability of IL-2 or IL-2 mutants to Treg cells, indicating that IL-2 mutants can effectively activate Treg cells.

[0119] In some embodiments, compared to human wild-type IL-2 (e.g., SEQ ID NO: 1), the IL-2 mutants provided herein can reduce the effect of IL-2 on CD8 + T cell activation. For example, in STAT5 phosphorylation experiments, by detecting IL-2 or IL-2 mutants in CD8 + The activation of STAT5 phosphorylation signal in T lymphocytes reflects the effect of IL-2 mutants on CD8 + The activation capacity of T lymphocytes was detected by flow cytometry and analyzed by GraphPad Prism software to calculate the activation capacity of IL-2 mutants in CD8 + The EC50 value of activating STAT5 phosphorylation in T lymphocytes indirectly reflects the effect of IL-2 or IL-2 mutants on CD8 + In some embodiments, the IL-2 mutant has a greater ability to activate CD8 T lymphocytes than human wild-type IL-2. + The activation capacity of T lymphocytes is decreased by at least 10%, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0120] In some embodiments, the IL-2 mutants provided herein can effectively expand Treg cells in vivo or in vitro. In some embodiments, the IL-2 mutants can increase CD4 + The proportion of Treg cells in the T cell population (e.g., CD4 +CD4 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 in a subject (e.g., CD4 + CD25 + Foxp3 + cells and CD8 + For example, in the present embodiment, after stimulation with IL-2 mutants, the number of Treg cells in the peripheral blood of the test animal cynomolgus monkey increased within a certain period of time, while the number of CD8 + There is no significant effect on the number of T lymphocytes. In some embodiments, the Treg cells in the test animals are stimulated by IL-2 mutants in the CD4 + The proportion of T lymphocytes increases by at least 1-fold. For example: 1-fold, 2-fold, 3-fold, 5-fold, 7-fold, 10-fold, 15-fold, 20-fold or more.

[0122] IL-2 mutants of the present application:

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

[0124] The first group of IL-2 mutants:

[0125] In some embodiments, the IL-2 mutants described herein comprise deletions or mutations of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1). In some embodiments, the IL-2 mutants described herein further comprise one or more mutations in 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, in the IL-2 mutant described herein, the amino acid residues RPRDL at positions 81-85 are deleted by 1, 2, 3, 4 or all 5 amino acids.

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

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

[0129] In some embodiments, the IL-2 mutant described herein 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 E67K and N71S mutations relative to the human wild-type IL-2 amino acid sequence.

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

[0131] In some embodiments, the IL-2 mutant described herein comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL.

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

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

[0134] In some embodiments, the IL-2 mutants described herein comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RPL, and further comprise mutations of N71 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of N71 and S75 are N71W and S75I, respectively. In some embodiments, the IL-2 mutants described herein comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RPL, and N71W and S75I mutations relative to the human wild-type IL-2 amino acid sequence.

[0135] In some embodiments, the IL-2 mutant described herein comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL.

[0136] In some embodiments, the IL-2 mutants described herein comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RHL, and further comprise 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 herein comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and an E67T mutation relative to the human wild-type IL-2 amino acid sequence.

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

[0139] In some embodiments, the IL-2 mutant described herein comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, as well as E67T and V91K mutations relative to the human wild-type IL-2 amino acid sequence.

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

[0141] In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and further comprise a mutation of N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation of N71 is N71I. In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, 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 herein comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to SPL.

[0143] In some embodiments, the IL-2 mutants described herein comprise a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to SPL, and further comprise one or more 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.

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

[0145] The second group of IL-2 mutants:

[0146] In some embodiments, the IL-2 mutants described herein comprise mutations of amino acid residue S (S75) at position 75 and amino acid residue R (R83) at position 83 relative to the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1). 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. In some embodiments, the IL-2 mutants described herein further comprise mutations of one or more of E15, H16, L17, L63, E67, L70, N71, D84, V91, and I92. In some embodiments, the mutation of 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 mutants described herein comprise S75P and R83Y mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise S75P, R83Y, E67A, and D84N mutations relative to the human wild-type IL-2 amino acid sequence.

[0148] In some embodiments, the IL-2 mutants described herein comprise S75P and R83A mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise one or more 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 mutants described herein comprise 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 mutants described herein comprise S75F and R83P mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise one or more mutations in H16 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation in H16 is H16Q or H16N. In some embodiments, the mutation in N71 is N71S. In some embodiments, the IL-2 mutants described herein comprise S75F, R83P, and H16Q mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein comprise S75F, R83P, H16N, and N71S mutations relative to the human wild-type IL-2 amino acid sequence.

[0150] In some embodiments, the IL-2 mutants described herein comprise S75F and R83L mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise S75F, R83L, E67R, and L70V mutations relative to the human wild-type IL-2 amino acid sequence.

[0151] In some embodiments, the IL-2 mutants described herein comprise S75I and R83L mutations relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise 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 mutants described herein comprise S75I and R83K mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise 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 herein comprises S75T and R83F mutations relative to the human wild-type IL-2 amino acid sequence.

[0154] In some embodiments, the IL-2 mutants described herein comprise S75T and R83A mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise S75T, R83A, H16N, and E67K mutations relative to the human wild-type IL-2 amino acid sequence.

[0155] In some embodiments, the IL-2 mutants described herein comprise S75V and R83F mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise S75V, R83F, E15K, and H16Q mutations relative to the human wild-type IL-2 amino acid sequence.

[0156] In some embodiments, the IL-2 mutants described herein comprise S75V and R83N mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise S75V, R83N, L70F, and V91K mutations relative to the human wild-type IL-2 amino acid sequence.

[0157] In some embodiments, the IL-2 mutants described herein comprise S75G and R83L mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise one or more 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 mutants described herein comprise 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 mutants described herein comprise S75Y and R83N mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise S75Y, R83N, L17I, and V91K mutations relative to the human wild-type IL-2 amino acid sequence.

[0159] In some embodiments, the IL-2 mutants described herein comprise S75A and R83K mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise L17 mutations 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 mutants described herein comprise S75A, R83K, and L17I mutations relative to the human wild-type IL-2 amino acid sequence.

[0160] In some embodiments, the IL-2 mutants described herein comprise S75L and R83E mutations relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise 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 mutants described herein comprise S75L, R83E, L63V, and E67F mutations relative to the human wild-type IL-2 amino acid sequence.

[0161] Mutations at other sites:

[0162] In some embodiments, the IL-2 mutants of the present 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 they satisfy the reduced selective preference of the IL-2 mutant for the receptor IL-2Rβγ and / or one or more beneficial properties described in the present invention. In some embodiments, the IL-2 mutants described in the present application further include mutations at other positions relative to 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 mutants described in the present application have 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 appreciate how to add additional mutations beyond those described herein.

[0163] In some embodiments, the IL-2 mutants described herein further comprise a C125S mutation relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutants described herein further comprise a C125A mutation relative to the human wild-type IL-2 amino acid sequence.

[0164] In some embodiments, the IL-2 mutant described herein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5-18, 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 to the amino acid sequence shown in any one of SEQ ID NOs: 5-18.

[0165] In some embodiments, the IL-2 mutant described herein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 19-34, 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 to the amino acid sequence shown in any one of SEQ ID NOs: 19-34.

[0166] In some embodiments, the one or more mutations in the other positions or regions comprise one or more conservative amino acid substitutions. "Conservative substitution" refers to substitution with another amino acid that has the same net charge and roughly the same size and shape as the substituted amino acid. When the total number of carbon atoms and heteroatoms in the side chain differs by no more than 4, the amino acids with aliphatic or substituted aliphatic amino acid side chains are roughly the same size. When the number of branches in their side chains differs by no more than 1, the shape of the amino acids is roughly the same. Amino acids with phenyl or substituted phenyl groups on the side chain can be considered to have roughly the same size and shape. Unless otherwise stated, conservative substitutions preferably use natural amino acids. Exemplary conservative substitutions are shown in Table 1.

[0167] More substantial substitutions are provided in Table 1 under the heading "Examples of Substitutions," as further detailed below in the section on amino acid side chain classes. Amino acids can be categorized 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 direction: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve replacing a member of one of these classes with a member of another. Amino acid substitutions can be introduced into protein constructs and screened for products that meet the desired activity described above.

[0168] Table 1 Amino acid substitutions

[0169] In some embodiments, the insertion of non-natural amino acids, including synthetic non-natural amino acids or one or more D-amino acids, into the IL-2 mutants or IL-2 mutant fusion proteins of the present application can have a variety of benefits. Compared with polypeptides containing L-amino acids, polypeptides containing D-amino acids, etc., show higher stability in vitro and in vivo. Therefore, when better intracellular stability is required, it is particularly useful to construct polypeptides such as by adding D-amino acids. In particular, D-peptides and their analogs are resistant to endogenous peptidases and proteases, thereby increasing the bioavailability of the molecule and extending its lifespan in vivo when needed. In addition, D-peptides and their analogs cannot be effectively processed to be presented to helper T cells in a limited manner by the type II major histocompatibility complex (MHC), and are therefore not easy to induce humoral immune responses in subjects.

[0170] In some embodiments, the IL-2 mutant described herein is a modified IL-2 mutant, such as a PEGylated IL-2 mutant, a covalently modified IL-2 mutant, or a glycosylation-modified IL-2 mutant.

[0171] In some embodiments, the IL-2 mutants described herein are full-length sequences. In other embodiments, the N-terminus of the IL-2 mutants comprises a signal peptide, which may be derived from a different molecule or from wild-type IL-2.

[0172] In some embodiments, the IL-2 mutant described herein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5-34, 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 to the amino acid sequence shown in any one of SEQ ID NOs: 5-34.

[0173] Exemplary amino acid sequences of wild-type IL-2 and its C125 mutant are shown in Table 2, exemplary amino acid sequences of the first group of IL-2 mutants are shown in Table 3, and exemplary amino acid sequences of the second group of IL-2 mutants are shown in Table 4.

[0174] Table 2 Exemplary wild-type IL-2 and C125 mutants

[0175] Table 3 Exemplary first group of IL-2 mutants

[0176] Table 4 Exemplary second group of IL-2 mutant sequences

[0177] Table 5 Exemplary Fc mutant sequences

[0178] Table 6 Exemplary connecting peptide sequences

[0179] Table 7 Exemplary first group of bivalent fusion protein sequences of IL-2 mutants and Fc

[0180] Table 8 Exemplary second group of IL-2 mutants and Fc bivalent fusion protein sequences

[0181] III. Fusion proteins containing IL-2 mutants

[0182] The present application also provides fusion proteins comprising IL-2 mutants. The IL-2 mutants described herein can be fused with other proteins to increase circulatory half-life by increasing molecular size and / or reducing renal clearance. In some preferred embodiments, the IL-2 mutants described herein are fused to Fc. In other preferred embodiments, the IL-2 mutants described herein are fused to human serum albumin (HSA). In other preferred embodiments, the IL-2 mutants described herein are fused to short peptides (e.g., XTEN).

[0183] Fusion protein of IL-2 mutant and Fc:

[0184] Fc fusion proteins can be produced by recombinant DNA technology, in which the translational 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 produced as disulfide-linked dimers, which are held together by disulfide bonds located in the hinge region of the Fc domain.

[0185] 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.

[0186] In some embodiments, the Fc described herein is derived from any of IgA, IgD, IgE, IgG, and IgM, and their subclasses. Of all immunoglobulins, IgG has the highest serum content and the longest half-life. Unlike other immunoglobulins, IgG can be effectively recovered after binding to Fc receptors (FcRs). In some preferred embodiments, Fc is derived from IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, Fc is derived from human IgG. In some embodiments, Fc comprises C H 2 and C H 3 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 are dimerized by 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 cysteines to reduce mispairing of disulfide bonds during dimerization. In some embodiments, Fc is truncated at the N-terminus, for example, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more amino acids of a complete immunoglobulin Fc are missing. In some embodiments, Fc comprises one or more mutations, such as insertions, deletions and / or substitutions.

[0187] In some embodiments, the Fc described herein has reduced or eliminated Fc effector functions. In some embodiments, the Fc has reduced Fc-mediated effector functions, such as reduced ADCC, CDC, and / or ADCP effector functions.

[0188] Fusion proteins containing Fc 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 rather than the preferred cells expressing IL-2 receptors, which may cause undesirable cytotoxicity. Furthermore, considering the long half-life of Fc fusion proteins, due to systemic toxicity, it is difficult to use them in treatment, or their therapeutic applications are limited. Therefore, in some embodiments, Fc is modified (e.g., comprising one or more amino acid mutations) to change its binding to FcR, in particular, to change its binding to Fcγ receptors (responsible for ADCC) and / or to change effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Preferably, such amino acid mutations do not reduce binding to FcRn receptors (related to half-life).

[0189] In some embodiments, an Fc (e.g., a human IgG1 Fc) is mutated to eliminate one or more effector functions, such as ADCC, ADCP, and / or CDC, hereinafter referred to as an "effector-free" or "nearly-effector-free" Fc. For example, in some embodiments, the Fc is an effector-free human IgG1 Fc comprising the mutations L234A and L235A. In some embodiments, the human IgG1 Fc comprises one or more of the following mutations (e.g., in each Fc subunit): L234A, L235E, G237A, A330S, and P331S. As known to those skilled in the art, the combination of K322A, L234A, and L235A in an IgG1 Fc is sufficient to completely abolish FcγR and C1q binding (Hezareh et al., J Virol 75, 12161–12168, 2001). MedImmune found that an Fc containing a set of three mutations, L234F / L235E / P331S, had very similar effects (Oganesyan et al., Acta Crystallographica 64, 700–704, 2008). In some embodiments, the Fc comprises a glycosylation modification on N297 of the IgG1 Fc region, which is known to be necessary for optimal FcR interaction. The Fc modification 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 contents of which are incorporated herein by reference in their entirety.

[0190] In some embodiments, the fusion protein of the IL-2 mutant and Fc, as described herein, does not have ADCC and / or CDC, or does not have detectable ADCC and / or CDC. In some embodiments, the fusion protein of the IL-2 mutant and Fc, as described herein, produces ADCC and / or CDC that is 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 comprising the same IL-2 mutant portion but fused to a wild-type or unmodified Fc.

[0191] Glycosylation variants

[0192] In some embodiments, the degree of glycosylation of the construct is increased or decreased by altering the Fc or fusion protein of the IL-2 mutant with the Fc. Glycosylation sites can be added or deleted in the Fc by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0193] Natural Fc-containing proteins produced by mammalian cells typically contain a branched chain of biantennary oligosaccharides, which are usually linked to the Fc C H The oligosaccharide is located at 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 attached to the GlcNAc on the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in Fc can be modified to produce certain improved properties.

[0194] In some embodiments, the Fc or IL-2 mutant and Fc fusion protein described herein has a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, in such an Fc or IL-2 mutant and Fc fusion protein, the fucose content may be 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 to measure the average fucose content within the sugar chains 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 at position 297 of the Fc region (EU numbering system for Fc region residues); however, due to minor sequence variations in the Fc region, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have enhanced ADCC function. See US Patent Publication Nos. US 2003 / 0157108 (Presta, L.) and US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 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-containing proteins include Lec13 CHO cells lacking protein fucosylation function (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); US Pat Appl No US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, is knocked out (see Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 615 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 616 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 617 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 618 (2004); Kanda, Y. et al., Biotech. Bioeng. 87: 619 ... al., Biotechnol. Bioeng., 94(4):680-688(2006); and WO2003 / 085107).

[0195] effector function variants

[0196] In some embodiments, the present application contemplates an Fc that has some, but not all, Fc 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 with the Fc is important, but certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. Cytotoxicity assays can be performed in vitro or in vivo to determine 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 a fusion protein comprising the Fc and IL-2 mutant lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. The primary cells that mediate ADCC, natural killer (NK) cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991), page 464. Non-limiting examples of in vitro assays for assessing ADCC activity of target molecules are described in detail 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, nonradioactive detection methods can be used (see ACTI for flow cytometry). TM Non-radioactive toxicity assay (CellTechnology, Inc. Mountain View, CA) and CytoTox Non-radioactive toxicity assay (Promega, Madison, WI). Suitable effector cells for this assay include peripheral blood mononuclear cells (PBMC) and NK cells. Furthermore, ADCC activity of the target molecule can also be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that IL-2 mutant Fc fusion proteins are unable to bind to C1q and therefore lack CDC activity. See WO 2006 / 029879 and WO 2005 / 100402 for C1q and C3c binding enzyme-linked immunosorbent assays. CDC assays 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, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0197] Fc with reduced effector function comprises substitutions of one or more residues at positions 238, 265, 269, 270, 297, 327, and 329 of the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants comprise substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are replaced with alanine (U.S. 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 engineered to alter (i.e., increase or decrease) C1q binding and / or CDC, for example, as described in US Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0198] In some embodiments, the Fc region comprises one or more amino acid substitutions that increase half-life and / or enhance binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-life and enhanced binding to neonatal FcRn are responsible for the transport of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and are described in detail in US 2005 / 0014934A1 (Hinton et al.). Antibodies comprising an Fc region with one or more substitutions thus enhance binding of the Fc region to FcRn. Such Fc variants include those with one or more Fc region residue substitutions, for example, substitutions of residue 434 in the Fc region (US Patent No. 7,371,826).

[0199] 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.

[0200] Cysteine ​​engineered variants

[0201] In some embodiments, it may be desirable to create a cysteine-engineered Fc or a fusion protein comprising such an Fc and an IL-2 mutant, wherein one or more residues of the Fc are substituted with cysteine ​​residues. In some embodiments, the substituted residues occur at accessible sites on the Fc or IL-2 mutant and Fc fusion protein. By replacing these residues with cysteine, active thiol groups are positioned at accessible sites on the Fc or IL-2 mutant and Fc fusion protein and can be used to conjugate the molecule to other moieties, such as drug moieties or linker-drug moieties, to create conjugates of the IL-2 mutant and Fc fusion protein. In some embodiments, any one or more of the following residues may be substituted with cysteine: heavy chain A118 (EU numbering system) and heavy chain Fc domain S400 (EU numbering system). Cysteine-engineered molecules can be generated as described in US Patent No. 7,521,541.

[0202] In some embodiments, the Fc is derived from an IgG1 Fc. In some embodiments, the Fc is derived from a human IgG1 Fc. In some embodiments, the Fc is derived from a wild-type human IgG1 Fc. In some embodiments, the Fc does not comprise the hinge region of an IgG1 Fc. In some embodiments, the Fc comprises one or more null mutations and / or aglycosylation mutations.

[0203] In some embodiments, the Fc comprises one or more mutations in amino acid residue L at position 234 (L234) and amino acid residue L at position 235 (L235) relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations in L234 and L235 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations L234A and L235A relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 35 or a variant thereof; the variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO: 35.

[0204] In some embodiments, the Fc comprises a mutation of amino acid residue N (N297) at position 297 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises a mutation N297G or N297A relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 36 or a variant thereof; the variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO: 36. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 37 or a variant thereof; the variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO: 37.

[0205] In some embodiments, the Fc comprises one or more mutations of amino acid residue L at position 234 (L234), amino acid residue L at position 235 (L235), and amino acid residue P at position 331 (P331) relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations of L234, L235, and P331 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations L234A, L235A, and P331S relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 38 or a variant thereof; the variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO: 38.

[0206] In some embodiments, the Fc comprises one or more mutations of amino acid residue L at position 234 (L234), amino acid residue L at position 235 (L235), amino acid residue G at position 237 (G237), amino acid residue A at position 330 (A330), and amino acid residue P at position 331 (P331) relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations of L234, L235, G237, A330, and P331 relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations L234A, L235E, G237A, A330S, and P331S relative to the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 39, 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 to SEQ ID NO: 39.

[0207] In some embodiments, the Fc is derived from an IgG4 Fc. In some embodiments, the Fc is derived from a human IgG4 Fc. In some embodiments, the Fc is derived from a wild-type human IgG4 Fc. In some embodiments, the Fc does not comprise the hinge region of an IgG4. In some embodiments, the Fc comprises one or more null mutations and / or aglycosylation mutations.

[0208] In some embodiments, the Fc comprises one or more mutations at amino acid residue S at position 228 (S228), amino acid residue F at position 234 (F234), and amino acid residue L at position 235 (L235) relative to the human wild-type IgG4 Fc amino acid sequence. In some embodiments, the Fc comprises mutations S228, F234, and L235 relative to the human wild-type IgG4 Fc amino acid sequence. In some embodiments, the Fc comprises mutations S228P, F234A, and L235A relative to the human wild-type IgG4 Fc amino acid sequence. In some embodiments, the Fc portion comprises (or consists essentially of, or consists of) the amino acid sequence of SEQ ID NO: 40 or a variant thereof; the variant having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO: 40.

[0209] KIH (Knob-into-Hole) technology:

[0210] Additional mutations that can be made to Fc include those that promote heterodimer formation between Fc polypeptides. In some embodiments, the problem of heterodimer assembly is solved by the KIH (Knob-into-Hole) technique, which is a technique that involves knocking out a C H 3 domains introduced asymmetric mutation structures (“knob” mutation refers to the mutation in C H In the 3 domain, a large amino acid residue is substituted for a smaller residue, while a "hole" mutation refers to the use of a small amino acid residue to replace a larger residue). The engineered Fc is more inclined to heterodimerization rather than homodimerization due to steric hindrance (Ridgway JB, et al. "Knobs-into-holes" engineering of antibody C H 3 domains for heavy chain heterodimerization[J].Protein Eng.1996,9(7):617-621). H The threonine residue (T) at position 366 of the 3 domain was replaced by a tryptophan residue (W) to form a "knobs" structure, and the other paired C HThe threonine residue (T) at position 366 of the 3-domain was replaced by a serine residue (S), the leucine residue (L) at position 368 was replaced by an alanine residue (A), and the tyrosine residue (Y) at position 407 was replaced by a valine residue (V) to form a "hole" structure. At the same time, two cysteine ​​residue (C) mutations (S354C on the "knob" side and Y349C on the "hole" side) that form a stabilizing disulfide bridge were introduced to enhance the stability of KIH. The numbering is based on the EU index of 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).

[0211] Exemplary Fc mutant sequences are shown in Table 5.

[0212] In some embodiments, the IL-2 mutant and Fc fusion protein of the present application is bivalent, comprising two IL-2 mutant molecules. For example, it may be a homodimer composed of two fusion polypeptide chain monomers. In some embodiments, exemplary structures thereof are shown in Figures 1A and 1B.

[0213] In some embodiments, the IL-2 mutant and Fc fusion proteins of the present application are monovalent, comprising only one IL-2 mutant molecule. For example, they may be in the form of a heterodimer composed of one fusion polypeptide chain monomer and one Fc subunit. In some embodiments, an exemplary structure thereof is shown in Figure 2. In other embodiments, the IL-2 mutant and Fc fusion proteins of the present application are monovalent, consisting of one fusion polypeptide chain monomer.

[0214] connector

[0215] In some embodiments, the IL-2 mutant and Fc can be connected by a linker (e.g., a connecting peptide, a non-connecting peptide). In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a stable linker. In general, an ideal linker does not affect or does not significantly affect the correct folding and conformation of the fusion protein of the IL-2 mutant and Fc described herein. Preferably, the linker imparts flexibility to the fusion protein of the IL-2 mutant and Fc, retains or improves the biological function of the IL-2 mutant, and / or does not affect or does not significantly affect the half-life and / or stability of the fusion protein of the IL-2 mutant and Fc in vivo. In some embodiments, the linker is a stable linker (e.g., cannot be cut by proteases, particularly MMPs).

[0216] In some embodiments, the linker is a connecting peptide. The connecting peptide can be of any length. In some embodiments, the connecting peptide 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, 4 to 25 amino acids in length. In some embodiments, the connecting peptide 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 length. In some embodiments, the connecting peptide is 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In certain embodiments, the connecting 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 certain embodiments, the connecting peptide is no more than the length necessary to prevent undesirable domain interactions and / or optimize biological function and / or stability. In certain embodiments, the length of the connecting peptide is at most 30 amino acids, for example at most 20 amino acids, or at most 15 amino acids. In certain embodiments, the connecting peptide is 5 to 30 amino acids in length, or 5 to 18 amino acids.

[0217] The connecting peptide can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence from the hinge region of an antibody heavy chain can be used as a linker. For example, see WO1996 / 34103. In some embodiments, the connecting peptide is a human IgG1, IgG2, IgG3 or IgG4 hinge region. In some embodiments, the connecting peptide is a mutated human IgG1, IgG2, IgG3 or IgG4 hinge region. 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), wherein m and n are integers of 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 act as neutral links between components. Glycine has more phi-psi space than alanine and is less constrained than 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, one skilled in the art will recognize that the designed IL-2 mutant and Fc fusion protein can include all or part of a flexible linker, such that the linker can include a flexible linker portion and one or more portions that provide less flexible structure to provide an ideal structure and function of the IL-2 mutant and Fc fusion protein. In some embodiments, the connecting peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs:43-74 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 to the amino acid sequence shown in any one of SEQ ID NOs:43-74.

[0218] Exemplary connecting peptide sequences are shown in Table 6.

[0219] Other considerations regarding the linker include the effect on the physical or pharmacokinetic properties of the resulting fusion protein of the IL-2 mutant and 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 the ability to bind to colloids or liposomes.

[0220] In some embodiments, the fusion protein of the IL-2 mutant and Fc described herein includes a bivalent form and a monovalent form. In other embodiments, the bivalent form, which comprises two IL-2 mutant molecules, can 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, which comprises one IL-2 mutant molecule, can 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, which comprises one IL-2 mutant molecule, is composed of only one monomer, for example, the monomer is a fusion polypeptide chain of the IL-2 mutant and an Fc subunit.

[0221] In some embodiments, the IL-2 mutant described herein in a fusion protein with Fc (e.g., a bivalent or monovalent fusion protein), comprises a deletion or mutation of amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of wild-type human IL-2. In some embodiments, the amino acid sequence of wild-type human IL-2 is as shown in SEQ ID NO: 1.

[0222] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises a complete deletion of the 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 of E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of E67 and N71 are E67K and N71S, respectively. In some embodiments, the IL-2 mutant comprises a complete deletion of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence, and mutations of E67K and N71S relative to the human wild-type IL-2 amino acid sequence.

[0223] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL. 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 mutation of E67 is E67A. In some embodiments, the IL-2 mutant comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL, and an E67A mutation relative to the human wild-type IL-2 amino acid sequence.

[0224] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein form), the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL, and further comprises a mutation of N71 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of N71 and S75 are N71W and S75I, respectively. In some embodiments, the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL, and a mutation of N71W and S75I relative to the human wild-type IL-2 amino acid sequence.

[0225] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described in the present application (for example, in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL. In some embodiments, the IL-2 mutant further comprises a mutation of E67 and / or V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation of E67 is E67T. In some embodiments, the mutation of V91 is V91K. In some embodiments, the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and a mutation of E67T and / or V91K relative to the human wild-type IL-2 amino acid sequence.

[0226] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein form), the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and further comprises a mutation of L70 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of L70 and S75 are L70I and S75F, respectively. In some embodiments, the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and a mutation of L70I and S75F relative to the human wild-type IL-2 amino acid sequence.

[0227] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein form), the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and further comprises a mutation of N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation of N71 is N71I. In some embodiments, the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and an N71I mutation relative to the human wild-type IL-2 amino acid sequence.

[0228] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to SPL. In some embodiments, the IL-2 mutant further comprises a mutation of E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of E67 and N71 are E67M and N71S, respectively. In some embodiments, the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to SPL, as well as E67M and N71S mutations relative to the human wild-type IL-2 amino acid sequence.

[0229] In some embodiments, the IL-2 mutant described herein in the fusion protein with Fc (e.g., a bivalent or monovalent fusion protein), comprises a mutation of amino acid residue S at position 75 (S75) and amino acid residue R at position 83 (R83) relative to the amino acid sequence of wild-type human IL-2. In some embodiments, the amino acid sequence of wild-type human IL-2 is as shown in SEQ ID NO: 1.

[0230] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., 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.

[0231] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein), the IL-2 mutant wherein 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.

[0232] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant, wherein 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, wherein the mutations of H16, L70, and N71 are H16D, L70I, and N71G, respectively. In some embodiments, the IL-2 mutant comprises S75P, R83A, H16D, L70I, and N71G mutations relative to the amino acid sequence of human wild-type IL-2.

[0233] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant, wherein the mutation of S75 is S75F, and the mutation of R83 is R83P. In some embodiments, the IL-2 mutant further comprises a mutation of 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 comprises S75F, R83P, and H16Q mutations 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 human wild-type IL-2 amino acid sequence.

[0234] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein), the IL-2 mutant wherein 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.

[0235] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., 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 of 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 S75I, R83L, E67G, and L70V mutations relative to the human wild-type IL-2 amino acid sequence.

[0236] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein), the IL-2 mutant comprises a mutation of S75 to S75I and a mutation of R83 to R83K. In some embodiments, the IL-2 mutant further comprises mutations of E67 and L70 relative to the amino acid sequence of wild-type human IL-2. In some embodiments, the mutations of E67 and L70 are E67S and L70V, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75I, R83K, E67S, and L70V relative to the amino acid sequence of wild-type human IL-2.

[0237] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein), the IL-2 mutant wherein the mutation of S75 is S75T and the mutation of R83 is R83A or R83F. In some embodiments, the IL-2 mutant further comprises mutations of H16 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of H16 and E67 are H16N and E67K, respectively. In some embodiments, the IL-2 mutant comprises S75T, R83A, H16N, and E67K mutations relative to the human wild-type IL-2 amino acid sequence.

[0238] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein), the IL-2 mutant wherein the mutation of S75 is S75V and the mutation of R83 is R83F. In some embodiments, the IL-2 mutant further comprises mutations of E15 and H16 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of E15 and H16 are E15K and H16Q, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75V, R83F, E15K, and H16Q relative to the amino acid sequence of human wild-type IL-2.

[0239] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant wherein 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 S75V, R83N, L70F, and V91K mutations relative to the amino acid sequence of human wild-type IL-2.

[0240] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., in the form of a bivalent or monovalent fusion protein), the IL-2 mutant comprises a mutation of S75 to S75G and a mutation of R83 to R83L. In some embodiments, the IL-2 mutant further comprises mutations of L17, V91, and I92 relative to the amino acid sequence of wild-type human 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 wild-type human IL-2.

[0241] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., 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 S75Y, R83N, L17I, and V91K mutations 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 herein (e.g., a bivalent or monovalent fusion protein), the IL-2 mutant comprises a mutation of S75 to S75A and a mutation of R83 to R83K. In some embodiments, the IL-2 mutant further comprises a mutation of L17 relative to the amino acid sequence of wild-type human IL-2. In some embodiments, the mutation of L17 is L17I. In some embodiments, the IL-2 mutant comprises S75A, R83K, and L17I mutations relative to the amino acid sequence of wild-type human IL-2.

[0243] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein), the IL-2 mutant wherein the mutation of S75 is S75L and the mutation of R83 is R83E. In some embodiments, the IL-2 mutant further comprises mutations of L63 and E67 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of L63 and E67 are L63V and E67F, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75L, R83E, L63V, and E67F relative to the amino acid sequence of human wild-type IL-2.

[0244] In some embodiments, the IL-2 mutant described herein in a fusion protein with Fc (e.g., a bivalent or monovalent fusion protein) further comprises, in addition to the aforementioned mutations, a mutation at C125 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the C125 mutation is C125S or C125A.

[0245] In some embodiments, in the fusion protein of the IL-2 mutant and Fc described herein (e.g., a bivalent or monovalent fusion protein), the IL-2 mutant comprises the amino acid sequence shown in any one of SEQ ID NOs: 5-34 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 to the amino acid sequence shown in any one of SEQ ID NOs: 5-34.

[0246] The amino acid sequences of the exemplary first group of bivalent fusion proteins of IL-2 mutants and Fc are shown in Table 7, and the amino acid sequences of the exemplary second group of bivalent fusion proteins of IL-2 mutants and Fc are shown in Table 8.

[0247] In some embodiments, in the bivalent fusion protein form of IL-2 mutant and Fc described herein, each monomer comprises from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) IL-2 mutant and (ii) Fc.

[0248] In some embodiments, in the bivalent fusion protein form of IL-2 mutant and Fc described herein, each monomer comprises, from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) IL-2 mutant, (ii) connecting peptide and (iii) Fc.

[0249] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described herein, the Fc is an IgG1 Fc, and the Fc comprises L234A and L235A (LALA) mutations. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 35.

[0250] In some embodiments, in the bivalent fusion protein format of the IL-2 mutant and Fc described herein, the Fc is an IgG1 Fc, and the Fc comprises an N297G mutation. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 36.

[0251] In some embodiments, in the bivalent fusion protein format of the IL-2 mutant and Fc described herein, the Fc is an IgG1 Fc, and the Fc comprises an N297A mutation. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 37.

[0252] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described herein, the Fc is an IgG1 Fc, and the Fc comprises L234A, L235A, and P331S mutations. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 38.

[0253] In some embodiments, in the bivalent fusion protein format of the IL-2 mutant and Fc described herein, the Fc is an IgG1 Fc, and the Fc comprises L234A, L235E, G237A, A330S, and P331S mutations. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 39.

[0254] In some embodiments, in the bivalent fusion protein form of the IL-2 mutant and Fc described herein, the Fc is an IgG4 Fc, and the Fc comprises S228P, F234A, and L235A mutations. In some embodiments, the Fc comprises the amino acid sequence of SEQ ID NO: 40.

[0255] In some embodiments, in the bivalent fusion protein form of an IL-2 mutant and Fc described herein, each monomer comprises, from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) an IL-2 mutant comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5-34 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 to the amino acid sequence shown in any one of SEQ ID NOs: 5-34 and (ii) an Fc comprising an amino acid sequence as shown in any one 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 to the amino acid sequence shown in any one of SEQ ID NOs: 35-40.

[0256] In some embodiments, in the bivalent fusion protein form of an IL-2 mutant and Fc described herein, each monomer comprises, from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) an IL-2 mutant comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5-34 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 to the amino acid sequence as shown in any one of SEQ ID NOs: 5-34, (ii) a connecting peptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 43-74 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 to the amino acid sequence as shown in any one of SEQ ID NOs: 43-74. The amino acid sequence of any one of SEQ ID NOs: 43-74 has at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity and (iii) Fc comprises an amino acid sequence as set forth in any one 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 identity to the amino acid sequence of any one of SEQ ID NOs: 35-40.

[0257] In some embodiments, the bivalent fusion protein of the IL-2 mutant and Fc described herein comprises the amino acid sequence shown in any one of SEQ ID NOs: 76-105 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology to the amino acid sequence shown in any one of SEQ ID NOs: 76-105.

[0258] In some embodiments, in one of the monovalent fusion protein forms of an IL-2 mutant and an Fc described herein, the Fc is an IgG1 Fc. In some embodiments, the Fc comprises a KIH mutation (referred to as an Fc knob and an Fc hole, respectively). In some preferred embodiments, the Fc comprises a LALA mutation and a KIH mutation (referred to as an Fc LALA knob and an Fc LALA hole, respectively). In some more preferred embodiments, the Fc LALA knob may comprise mutations L234A, L235A, T366W, and S354C, and the Fc LALA hole may comprise mutations L234A, L235A, T366S, L368A, Y407V, and Y349C.

[0259] In some embodiments, the IgG1 Fc LALA knob comprises the amino acid sequence of SEQ ID NO:41, and the Fc LALA hole comprises the amino acid sequence of SEQ ID NO:42.

[0260] In some embodiments, in one of the monovalent fusion protein forms of an IL-2 mutant and Fc described herein, one monomer comprises, from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant and (ii) an Fc knob, and one monomer comprises: an Fc hole.

[0261] In some embodiments, in one of the monovalent fusion protein forms of an IL-2 mutant and Fc described herein, one monomer comprises, from N-terminus to C-terminus, or from C-terminus to N-terminus: (i) an IL-2 mutant comprising the amino acid sequence of any one of SEQ ID NOs: 5-34 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 to the amino acid sequence of any one of SEQ ID NOs: 5-34 and (ii) an Fc LALA knob comprising the amino acid sequence of SEQ ID NO: 41 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 to SEQ ID NO: 41; and the other monomer is an Fc LALA hole comprising the amino acid sequence of SEQ ID NO: 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 to SEQ ID NO:42.

[0262] In some embodiments, in one of the monovalent fusion protein forms of IL-2 mutant and Fc described herein, one monomer comprises, from N-terminus to C-terminus or from C-terminus to N-terminus: (i) IL-2 mutant and (ii) Fc hole; and the other monomer comprises: Fc knob.

[0263] In some embodiments, in one of the monovalent fusion protein forms of an IL-2 mutant and Fc described herein, one monomer comprises, from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant comprising the amino acid sequence of any one of SEQ ID NOs: 5-34 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 to the amino acid sequence of any one of SEQ ID NOs: 5-34 and (ii) an Fc LALA hole comprising the amino acid sequence of SEQ ID NO: 42 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 to SEQ ID NO: 42; and the other monomer is an Fc LALA knob comprising the amino acid sequence of SEQ ID NO: 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 to SEQ ID NO:41.

[0264] In some embodiments, in one of the monovalent fusion protein forms of an IL-2 mutant and Fc described herein, one monomer comprises, from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant, (ii) a connecting peptide, and (iii) an Fc knob; and the other monomer comprises: an Fc hole.

[0265] In some embodiments, in one of the monovalent fusion protein forms of an IL-2 mutant and Fc described herein, one monomer comprises, from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5-34 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 to the amino acid sequence as shown in any one of SEQ ID NOs: 5-34; (ii) a connecting peptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 43-74 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 to the amino acid sequence as shown in any one of SEQ ID NOs: 43-74; and (iii) Fc LALA The present invention also provides a monomer comprising an Fc LALA hole comprising an amino acid sequence of SEQ ID NO:42 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 identity to SEQ ID NO:42; and an Fc LALA hole comprising an amino acid sequence of SEQ ID NO:42 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 identity to SEQ ID NO:42.

[0266] In some embodiments, in one of the monovalent fusion protein forms of an IL-2 mutant and Fc described herein, one monomer comprises, from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant, (ii) a connecting peptide, and (iii) an Fc hole, and the other monomer comprises: an Fc knob.

[0267] In some embodiments, in one of the monovalent fusion protein forms of an IL-2 mutant and Fc described herein, one monomer comprises, from N-terminus to C-terminus or from C-terminus to N-terminus: (i) an IL-2 mutant comprising an amino acid sequence as shown in any one of SEQ ID NOs: 5-34 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 to the amino acid sequence as shown in any one of SEQ ID NOs: 5-34; (ii) a connecting peptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 43-74 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 to the amino acid sequence as shown in any one of SEQ ID NOs: 43-74; and (iii) Fc LALA The invention also provides a monomer comprising an Fc LALA knob comprising an amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 41.

[0268] In some embodiments, the half-life of the fusion protein of an IL-2 mutant and Fc described herein 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 more) when administered intravenously, intramuscularly, or subcutaneously to a subject (e.g., a human).

[0269] Binding affinity

[0270] The binding affinity of a molecule (e.g., an IL-2 mutant or a fusion protein comprising an IL-2 mutant) to its binding partner (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, for example, 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 analysis, peptide scanning, and the like. For example, simple analysis can be performed by using 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 labeled with various labeling reagents. BiacoreX (Amersham Biosciences), an over-the-counter measurement kit or a similar kit can also be used and can be operated according to the user manual and experimental operation method included in the kit.

[0271] In some embodiments, protein microarrays are used to analyze the interaction, function, and activity of the IL-2 mutants or fusion proteins comprising the IL-2 mutants described herein with their receptors on a large scale. The protein microarray has a support surface bound to a series of capture proteins (e.g., IL-2 receptors or subunits thereof). Fluorescently labeled probe molecules (e.g., IL-2 mutants or fusion proteins comprising the IL-2 mutants described herein) are then added to the array and interact with the bound capture proteins, releasing fluorescent signals that are read by a laser scanner.

[0272] Binding affinity can also be measured using Biacore. For example, IL-2Rβγ or IL-2Rαβγ is coupled to the surface of a CM-5 sensor chip as a ligand using EDC / NHS chemistry. A series of dilutions of the IL-2 mutant or IL-2 mutant-Fc fusion protein described herein is then used as an analyte to bind to the ligand coupled to the chip, and the binding and dissociation of IL-2 to IL-2Rβγ or IL-2Rαβγ can be monitored in real time. Affinity (Kd) can be determined by kinetic analysis using BIA evaluation software.

[0273] In some embodiments, a Biacore assay can be used to determine the Kd value of the IL-2 mutants or IL-2 mutant-Fc fusion proteins provided herein for IL-2Rβγ to determine binding affinity. In some embodiments, the IL-2 mutant-Fc fusion proteins of the present application have lower binding affinity for IL-2Rβγ than the wild-type IL-2-Fc fusion protein. In some embodiments, the affinity of the IL-2 mutant-Fc fusion protein for IL-2Rβ and / or IL-2Rβγ is reduced by at least 5% (e.g., at least any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) relative to the wild-type IL-2-Fc fusion protein. In some embodiments, the IL-2 mutant and Fc fusion proteins described herein have an increased affinity for IL-2Rα, or the affinity remains unchanged, or the affinity is not significantly reduced relative to a wild-type IL-2 and Fc fusion protein (e.g., human wild-type IL-2). In some embodiments, the binding affinity of the IL-2 mutant and Fc fusion proteins of the present application to IL-2Rαβγ is increased, remains unchanged, or the affinity is not significantly reduced relative to a wild-type IL-2 and Fc fusion protein.

[0274] In some embodiments, the Kd value of the binding between the IL-2 mutant or the fusion protein of the IL-2 mutant and Fc described herein and the intermediate affinity receptor (IL-2Rβγ) is greater than the Kd value of the binding between the wild-type IL-2 or the fusion protein of the wild-type IL-2 and Fc and the same receptor. In some embodiments, the Kd value of the binding between the IL-2 mutant or the fusion protein of the IL-2 mutant and Fc described herein and the 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 the wild-type and the same receptor (IL-2Rαβγ).

[0275] Pharmacokinetics (PK)

[0276] Pharmacokinetics (PK) refers to the absorption, distribution, metabolism, and excretion of a drug (e.g., an IL-2 mutant or a fusion protein comprising an IL-2 mutant as 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, maximum serum / plasma concentration (C max ), time to reach maximum concentration (T max ), half-life (T 1 / 2 ), the area under the concentration-time curve during the dosing interval (AUC τ )wait.

[0277] Techniques for obtaining PK profiles of drugs (e.g., IL-2 mutants or fusion proteins comprising IL-2 mutants as 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, the PK profile of an IL-2 mutant or a fusion protein comprising an IL-2 mutant as described herein is measured in a blood, plasma, or serum sample of an individual. In some embodiments, the PK profile of an IL-2 mutant or a fusion protein comprising an IL-2 mutant as described herein in an individual is measured using mass spectrometry techniques (e.g., LC-MS / MS or ELISA). PK analysis can be performed on the PK curve by any method known in the art, for example, non-compartmental 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). For an exemplary method, see Example 8.

[0278] "C" represents the concentration of the drug (e.g., IL-2 mutant or fusion protein comprising 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 concentration of the drug in serum or plasma is referred to herein as "serum concentration" or "plasma concentration." The serum / plasma concentration at any time after administration (e.g., IL-2 mutant or fusion protein polypeptide comprising IL-2 mutant by intravenous, intraperitoneal, or subcutaneous injection) is referred to as C. time or C t The maximum serum / plasma drug concentration during drug administration is called C max ; C min Refers to the minimum serum / plasma drug concentration at the end of the dosing interval; C ave Refers to the average concentration during the dosing interval.

[0279] The term "bioavailability" refers to the extent or rate at which a drug (eg, an IL-2 mutant or a fusion protein comprising an IL-2 mutant) passes through the systemic circulation and thereby reaches the site of action.

[0280] “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 during the dosing interval (AUCτ), “total exposure” or “total drug exposure over time” (AUC 0-∞ ), the area under the concentration-time curve at time t after administration (AUC 0-t )wait.

[0281] Time to peak serum / plasma concentration (T max ) is the serum / plasma concentration (C max ) peak time.

[0282] Half-life (T 1 / 2 ) refers to the time required for the concentration of a drug (e.g., an IL-2 mutant or a fusion protein comprising an IL-2 mutant) measured in plasma or serum (or other biological matrix) to drop to half of its concentration or amount at a specific time point. For example, after intravenous administration, the drug concentration in plasma or serum decreases due to the distribution and elimination of the drug. In the curve of plasma or serum drug concentration over time after intravenous administration, the first phase or rapid decline is considered to be mainly due to distribution, while the later decline is usually slower and mainly due to elimination, although both processes occur in both phases. Distribution is considered to be complete after sufficient time. Generally speaking, 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, "A Comprehensive Guide to Toxicology in Preclinical Drug Development," "Chapter 3 - Pharmacokinetics and Toxicokinetics," 2013.

[0283] 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., intravenous, subcutaneous, or intramuscular injection, such as in humans), 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.

[0284] Derivatives of IL-2 mutants or fusion proteins containing IL-2 mutants

[0285] In some embodiments, the IL-2 mutants or fusion proteins comprising IL-2 mutants as described herein may be further modified to include additional non-protein moieties known in the art and readily available. Exemplary non-protein moieties 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, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxolane, ethylene / maleic anhydride copolymers, polyamic acid (homopolymer or random copolymer), dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Due to the stability of polyethylene glycol propionaldehyde in water, it may have advantages in manufacturing. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the IL-2 mutant or fusion protein comprising the IL-2 mutant may vary; 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 fusion protein comprising the IL-2 mutant to be modified, whether the derivative of the IL-2 mutant or fusion protein comprising the IL-2 mutant will be useful therapeutically for a specific condition, and the like.

[0286] In some embodiments, the IL-2 mutant or fusion protein comprising the IL-2 mutant described herein further comprises a tag selected from the group consisting of a chromophore, a fluorophore (e.g., coumarin, xanthene, cyanine, pyrene, boron polybenzazaindole, oxazine, and derivatives thereof), a fluorescent protein (e.g., GFP, phycobiliprotein, and derivatives thereof), a phosphorescent dye (e.g., dioxetanes, xanthene or carbocyanine dyes, lanthanide chelates), a tandem dye (e.g., cyanine-phycobiliprotein derivatives and xanthene-phycobiliprotein derivatives), a particle (e.g., gold clusters, colloidal gold, microspheres, quantum dots), a hapten, an enzyme (e.g., peroxidase, phosphatase, glycosidase, luciferase), and a radioisotope (e.g., 125 I. 3 H. 14 C. 32 P).

[0287] In some embodiments, the IL-2 mutant or the fusion protein comprising the IL-2 mutant may be further modified to comprise one or more other biologically active proteins, polypeptides or fragments thereof. As described herein, "biological activity" or "biologically active" are used interchangeably and refer to the display of biological activity in vivo to perform a specific function. For example, it may mean binding to a specific biological molecule, such as a protein, DNA, etc., and then promoting or inhibiting the activity of the biological molecule. In some embodiments, biologically active proteins or fragments thereof include proteins and polypeptides administered to patients as active drugs to prevent or treat a disease or symptom, as well as proteins and polypeptides for diagnostic purposes, such as enzymes for diagnostic tests or in vitro detection, and proteins and polypeptides administered to patients to prevent a disease, such as vaccines. In some embodiments, the biologically active protein or fragment thereof has immunostimulatory / immunomodulatory, membrane transport or enzymatic activity. In some embodiments, the biologically active protein, polypeptide or fragment thereof is an enzyme, hormone, growth factor, cytokine or a mixture thereof. In some embodiments, the biologically active protein, polypeptide or fragment can specifically recognize a target peptide (e.g., an antigen or other protein).

[0288] In some embodiments, the biologically active protein or fragment thereof that can be included in the IL-2 mutants described herein or in fusion proteins comprising the IL-2 mutants is an antigen binding protein (e.g., an antibody). In some embodiments, the biologically active protein or fragment thereof that can be included in the IL-2 mutants described herein or in fusion proteins comprising the IL-2 mutants is an antibody mimetic, which is a small engineered protein that contains an antigen binding domain reminiscent of an antibody (G Geering 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 mimetics that can be included in the IL-2 mutants or fusion proteins comprising the IL-2 mutants described herein include, but are not limited to, designed ankyrin repeat proteins (DARPins; comprising 3-5 fully synthetic ankyrin repeat sequences flanked by N-terminal and C-terminal cap domains), an avimer (a high-affinity protein comprising multiple A domains, each with a low affinity for the target), or an anticoagulant (based on a lipid scaffold with four accessible loops, each with a randomized sequence). In some embodiments, a biologically active protein or fragment thereof that can be included in the IL-2 mutants or fusion proteins comprising the IL-2 mutants described herein is an armadillo repeat protein (e.g., β-catenin, α-importin, plakoglobin, adenomatous polyposis coli (APC)), comprising an armadillo repeat unit (characteristically, a repeated amino acid sequence of approximately 40 residues in length). Each armadillo repeat unit consists of a pair of α-helices that form a hairpin structure. Multiple copies of the repeats form the α-solenoid structure. Armadillo repeat proteins are able to bind different types of peptides, relying on a constant binding pattern to the peptide backbone without requiring specific conserved side chains or interactions with the free N- or C-termini of the peptide. The potential for residue-by-residue recognition of peptides, combined with the intrinsic modularity of the repeat proteins, makes armadillo repeat proteins promising candidates for universal scaffolds for peptide binding.

[0289] IV. Nucleic Acids Encoding IL-2 Mutants or Fusion Proteins Containing IL-2 Mutants

[0290] The present application also relates to isolated nucleic acids encoding any of the IL-2 mutants described herein or fusion proteins comprising IL-2 mutants, and vectors comprising nucleic acids encoding any of the IL-2 mutants described herein or fusion proteins comprising IL-2 mutants. It also relates to isolated host cells (e.g., CHO cells, HEK293 cells, HeLa cells, or COS cells) comprising 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 the IL-2 mutant or fusion protein comprising the IL-2 mutant.

[0291] In some embodiments, the vector comprising a nucleic acid encoding any of the IL-2 mutants described herein or a fusion protein comprising an IL-2 mutant is suitable for replication and integration 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.

[0292] Many virus-based systems have been developed for transferring genes into mammalian cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, herpes simplex viral vectors, and their derivatives. Viral vector technology is well known in the art and is described in detail, 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 in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral 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 for transduction into mammalian cells using methods known in the art. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transduction because they allow for long-term, stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0293] In some embodiments, the vector is a non-viral 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 non-viral vector, including, for example, poly (lactic-co-glycolic acid) (PLGA) and polylactic acid (PLA), poly (ethyleneimine) (PEI) and dendrimers. In some embodiments, the vector is a cationic lipid-based non-viral vector, such as cationic liposomes, lipid nanoemulsions and solid lipid nanoparticles (SLN). In some embodiments, the vector is a peptide-based non-viral gene vector, such as poly-L-lysine. Any known non-viral vector suitable for genome editing can be used to introduce nucleic acids encoding IL-2 mutants or fusion proteins comprising 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, incorporated herein by reference. In some embodiments, any one or more nucleic acids or vectors encoding the IL-2 mutants or fusion proteins comprising the IL-2 mutants described herein are introduced into host cells (e.g., CHO, HEK 293, Hela or COS) by physical methods, including but not limited to electroporation, sonoporation, photoporation, magnetofection, and hydroporation.

[0294] In some embodiments, the vector comprises a selectable marker gene or a reporter gene for selecting cells expressing an IL-2 mutant described herein or a fusion protein comprising an IL-2 mutant from a population of host cells transfected with a vector (e.g., a lentiviral vector, a pTT5 vector). Both the selectable marker and the reporter gene may be surrounded by appropriate regulatory sequences to enable expression in the host cell. For example, the vector may comprise a transcriptional and translational terminator, an initiation sequence, and a promoter for regulating expression of the nucleic acid sequence.

[0295] Can use any molecular cloning method known in the art, comprise, for example, use restriction endonuclease site and one or more selectable markers that nucleic acid is cloned in vector.In certain embodiments, nucleic acid is operably connected on the promoter.Explore out multiple promoters that are used for gene expression in prokaryotic cell or eukaryotic cell (for example, mammalian cell), and any promoter known in the art can be used for the application.Promoter can be roughly divided into constitutive promoter or regulatable promoter, such as inducible promoter.

[0296] In some embodiments, the nucleic acid encoding the IL-2 mutant described herein or the fusion protein comprising the IL-2 mutant is operably linked to a constitutive promoter. A constitutive promoter allows a heterologous gene (also referred to as a transgene) to be constitutively expressed in a host cell. Examples of promoters contemplated herein include, but are not limited to, CMV promoter (CMV), human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), simian virus 40 early promoter (SV40), chicken β-actin promoter coupled to CMV early enhancer (CAGG), Rosenberg's sarcoma virus (RSV) promoter, polyoma virus enhancer / herpes simplex thymidine kinase (MC1) promoter, β-actin (β-ACT) promoter, "myeloproliferative sarcoma virus enhancer, negative control region deletion, d1587rev primer binding site substitution (MND)" promoter. The efficiency of these constitutive promoters in driving transgenic expression has been extensively compared in a large number of studies. In some embodiments, the nucleic acid encoding an IL-2 mutant or a fusion comprising an IL-2 mutant described herein is operably linked to a CMV promoter.

[0297] In some embodiments, the nucleic acid encoding the IL-2 mutant described herein or the fusion protein comprising the IL-2 mutant is operably linked to an inducible promoter. Inducible promoters belong to the category of regulatable promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the host cell or the 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 (such as ionizing radiation, light), temperature (such as heat), redox state, and activation state of the host cell. In some embodiments, the inducible promoter can be a NFAT promoter, promoter or NFκB promoter.

[0298] V. Preparation Method

[0299] The present application also relates to a method for preparing any IL-2 mutant described herein or a fusion protein comprising an IL-2 mutant. Therefore, in some embodiments, it relates to a method for preparing an IL-2 mutant or a fusion protein comprising an IL-2 mutant, comprising: (a) culturing a host cell (e.g., CHO cells, HEK 293 cells, Hela cells, or COS cells) containing a nucleic acid or vector encoding any IL-2 mutant or a fusion protein comprising an IL-2 mutant described herein under conditions that effectively express the encoded IL-2 mutant or the fusion protein comprising an IL-2 mutant; and (b) obtaining the expressed IL-2 mutant or the fusion protein comprising an IL-2 mutant from the host cell. In some embodiments, the method of step (a) further comprises producing a host cell comprising a nucleic acid or vector encoding an IL-2 mutant or a fusion protein comprising an IL-2 mutant described herein. The IL-2 mutant or the fusion protein comprising an IL-2 mutant described herein can be prepared using any method known in the art or described herein.

[0300] In some embodiments, the IL-2 mutants or fusion proteins comprising IL-2 mutants described herein can be expressed in eukaryotic cells, such as mammalian cells. In some embodiments, the IL-2 mutants or fusion proteins comprising IL-2 mutants described herein can be expressed in prokaryotic cells.

[0301] 1. Recombinant products of prokaryotic cells

[0302] a) Vector construction

[0303] The polynucleotide sequences encoding the protein constructs described herein 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 heterologous polynucleotide in a prokaryotic host. Many vectors known and available in the art can be used for this application. Selecting a suitable vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell transformed by the vector. Each vector comprises various components, which depends on the function of the vector (amplification or expression of heterologous polynucleotides, or both) and the compatibility between the vector and the specific host cell in which it is located. Vector components typically include, but are not limited to: an initiation of replication site, a selectable marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, heterologous nucleic acid insertion, and a transcription termination sequence.

[0304] In general, plasmid vectors contain replicons and control sequences from species compatible with the host cells, and are used with these host cells. The vector typically carries a replication site and a marker sequence that can provide phenotypic selection in the transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from E. coli. pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, and therefore provides a simple method for identifying 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 for expressing specific antibodies.

[0305] In addition, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transformation vectors with these host cells. For example, phage such as GEM TM -11 can be used to prepare a recombinant vector, which can be used to transform susceptible host cells, such as Escherichia coli LE392.

[0306] A promoter is an untranslated regulatory sequence located upstream (5') of a cistron that regulates downstream gene expression. Prokaryotic promoters are generally divided into two categories: inducible and constitutive. An inducible promoter is one that responds to changes in culture conditions (e.g., the presence or absence of nutrients or temperature) to initiate and increase transcription from the cistron.

[0307] Many promoters that can be recognized by potential host cells are all known.Through restriction endonuclease, promoter is shifted out from source DNA and the isolating promoter sequence is inserted in the application's carrier, selected promoter is operably connected on the cistron DNA of coded polypeptide.Natural promoter sequence and many heterologous promoters can be used for instructing amplification and / or expression of target gene.In certain embodiments, utilize heterologous promoter, because compared with natural target polypeptide promoter, heterologous promoter allows larger transcription usually, and the output of expressing target gene is higher.

[0308] Promoters suitable for prokaryotic hosts include the PhoA promoter, the β-galactosidase and lactose promoter systems, the tryptophan (trp) promoter system, and hybrid promoters such as the tac or trc promoters. However, other promoters that are functional in bacteria (e.g., other known bacterial or phage promoters) are also suitable. Their nucleic acid sequences are publicly available, allowing the skilled artisan to link them to the cistrons encoding the light and heavy chains of interest using linkers or adapters to provide any desired restriction sites (Siebenlist et al., (1980) Cell 20:269).

[0309] In certain embodiments, each cistron in the recombinant vector comprises a secretion signal sequence component, which directly guides the transmembrane transfer of the expressed polypeptide. In general, the signal sequence can be an integral part of the vector or a part 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 (i.e., cut by a signal peptidase) by the host cell. 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 heat-stable enterotoxin II (STII) leader, LamP, PhoE, PelB, OmpA and MBP.

[0310] In some embodiments, the production of the IL-2 mutants or fusion proteins comprising the IL-2 mutants of the present application can occur in the cytoplasm of the host cell, thus eliminating the need for a secretion signal sequence to be present in each cistron. In some embodiments, the polypeptide components are expressed, folded, and assembled to form a protein construct in the cytoplasm. Certain host strains (e.g., E. coli trxB- strains) provide cytoplasmic conditions that are favorable for disulfide bond formation, thereby allowing the expressed protein subunits to fold and assemble appropriately. See Proba and Pullthun, Gene, 159:203 (1995).

[0311] b) Prokaryotic host cells

[0312] Prokaryotic host cells suitable for expressing the protein of the present application include archaebacteria and eubacteria, such as gram-negative bacteria or gram-positive bacteria. Examples of available bacteria include Escherichia coli (e.g., Escherichia coli), bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreoscilla or Paracoccus. In certain embodiments, gram-negative cells are used. In certain embodiments, Escherichia coli cells are used as hosts of the present 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) having the genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT 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 suitable. These examples are illustrative and not limiting. Methods for constructing bacterial derivatives of any of the above-mentioned known genotypes are known in the art and are described in detail, for example, in Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select suitable bacteria, taking into account the replicability of the replicon in bacterial cells. For example, when using known plasmids such as pBR322, pBR325, pACYC177, or pKN410 to provide the replicon, Escherichia coli, Serratia marcescens, or Salmonella typhimurium are suitable hosts.

[0313] Generally, the host cells should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors should be appropriately included in the cell culture.

[0314] c) Protein production

[0315] Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified appropriately to induce promoters, select transformants, or amplify the gene encoding the desired sequence. Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA can replicate as an extrachromosomal element or through chromosomal integration. Depending on the host cell used, transformation is performed using standard techniques appropriate for that cell type. Calcium treatment with calcium chloride is commonly used for bacterial cells with a substantial cell wall barrier. Another transformation method uses polyethylene glycol / dimethyl sulfoxide. Another technique is electroporation.

[0316] Prokaryotic cells used to produce the protein constructs of the present application are grown in a culture medium known in the art and suitable for culturing the selected host cells. Suitable culture medium includes Luria broth (LB) and necessary nutritional supplements. In some embodiments, the culture medium also contains a selective agent selected 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 to grow cells expressing an ampicillin resistance gene.

[0317] In addition to the carbon source, nitrogen source and inorganic phosphate source, any necessary supplements may also be added at appropriate concentrations, either alone or as a mixture with other supplements or media (such as a complex nitrogen source). 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, the preferred temperature range is 20°C to 39°C, more preferably 25°C to 37°C, and even more preferably 30°C. The pH value of the culture medium can be any pH value between 5 and 9, depending primarily on the host organism. For Escherichia coli, the pH value is preferably 6.8 to 7.4, and more preferably 7.0.

[0318] If an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation. In one aspect of the present application, the PhoA promoter is used to control transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-limited medium for induction. Preferably, the phosphate-limited 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, which are known in the art.

[0319] The protein construct expressed by the application is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery generally involves destroying microorganisms, usually by modes such as osmotic shock, ultrasonic treatment or cracking. Once the cells are destroyed, 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 the culture medium and isolated therein. Cells can be removed from the culture medium, filtered and concentrated, and the culture supernatant is used to further purify the protein produced. The expressed polypeptide can be further separated and identified by common methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot tests.

[0320] Alternatively, proteins can be produced on a large scale through fermentation. Various large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. Large-scale fermentations have a capacity of at least 1,000 liters, preferably 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, particularly glucose (the preferred carbon / energy source). Small-scale fermentation typically refers to fermentation in fermenters with a capacity of no more than 100 liters, ranging from 1 liter to 100 liters.

[0321] During the fermentation process, induction of protein expression is typically initiated after the cells have grown to a desired density under suitable conditions, for example, at an OD550 of approximately 180-220, when the cells are in the early stationary phase. Depending on the vector structure employed, a variety of inducers known in the art and described above may be used. Cells may be grown for a shorter period of time before induction. Cells are typically induced for approximately 12-50 hours, although longer or shorter induction times may be used.

[0322] To improve the yield and quality of the IL-2 mutants or fusion proteins comprising the IL-2 mutants of the present application, various fermentation conditions can be modified. For example, to improve the correct assembly and folding of the secreted polypeptide, an additional vector that overexpresses a chaperone protein, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, or DsbG) or FkpA (a peptide prolyl cis-trans isomerase with chaperone activity), can be used to co-transform the host prokaryotic cells. Chaperone proteins have been shown to help promote the correct folding and solubilization of heterologous proteins produced in bacterial host cells. Chen et al., (1999) J Bio Chem 274:19601-19605; 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.

[0323] To minimize hydrolysis of expressed heterologous proteins, particularly 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 cause mutations in 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 described in detail 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).

[0324] E. coli strains lacking proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins can be used as host cells in expression systems encoding the IL-2 mutants or fusion proteins comprising the IL-2 mutants described herein.

[0325] d) Protein purification

[0326] The protein constructs produced herein are further purified to obtain substantially homogeneous preparations for further analysis and use. Standard protein purification methods known in the art can be used. The following procedures are exemplary of suitable purification procedures: fractionation on an immunoaffinity column or an ion exchange column, ethanol precipitation, reversed-phase liquid chromatography HPLC, chromatography on silica or a cation exchange resin (such as DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, for example, Sephadex G-75.

[0327] In some embodiments, protein A immobilized on a solid phase is used for immunoaffinity purification of protein constructs comprising an Fc region as described herein. Protein A is a 42 kDa surface protein from Staphylococcus aureus that has a high binding affinity for Fc-containing structures, such as, for example, the IL-2 mutant-Fc fusion protein described herein. Lindmark et al., (1983) J. Immunol. Meth. 62:1-1. The solid phase to which protein A is immobilized preferably comprises a column having a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some applications, the chromatographic 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 are nonspecifically bound to the solid phase. Finally, the target protein construct is recovered from the solid phase by elution.

[0328] 2. Recombinant products of eukaryotic cells

[0329] For eukaryotic expression, vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0330] a) Signal sequence elements

[0331] The carrier that is used for eukaryotic hosts can also be an inset, and this inset encoding signal sequence or has other polypeptides of specific cleavage site at the N-end of mature protein or polypeptide.The heterologous signal sequence of selection is preferably the sequence by host cell recognition and processing (that is, cut by signal peptidase).In mammalian cell expression, can obtain mammalian signal sequence and virus secretion leader, for example, herpes simplex gD signal, all is useful.The DNA in this precursor region is connected with the DNA of coding protein construct of the application in reading frame.

[0332] b) Origin of replication

[0333] Generally, mammalian expression vectors do not require an origin of replication element (the SV40 origin is usually used only because it contains the early promoter).

[0334] c) Select genetic elements

[0335] Expression and cloning vectors may contain a selection gene, also called a selectable marker. Typical selection genes encode proteins such as (a) proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) proteins that complement auxotrophic deficiencies, or (c) proteins that provide critical nutrients that are not available in complex culture media, such as the gene encoding Bacillus D-alanine racemase.

[0336] An example of a selective strategy involves using a drug to prevent host cell growth. Cells successfully transformed with the heterologous gene produce a protein that confers drug resistance, allowing them to survive the selection. Examples of this type of dominant selection include the drugs neomycin, mycophenolic acid, and hygromycin.

[0337] Another example of a selectable marker suitable for mammalian cells is one that can identify cells that are competent to carry the nucleic acid encoding the protein construct described in the present application, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[0338] For example, cells transformed with the DHFR selection gene are first cultured in a medium containing methotrexate (Mtx) for identification. Methotrexate is a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity (e.g., ATCC CRL-9096).

[0339] Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3′-phosphotransferase (APH), can be selected by growing the cells in a medium containing a selectable marker, such as an aminoglycoside antibiotic, for example, kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

[0340] d) Promoter element

[0341] Expression and cloning vectors typically contain a promoter that is recognized by the host and operably linked to the 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 of many genes is the CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic organisms, there is an AATAAA sequence, which may be the signal for adding a poly A tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

[0342] Transcription of the polypeptide in a mammalian host cell vector is controlled by a promoter, for example, by promoters obtained from viral genomes, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably, Simian Virus 40 (SV40), from heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, from heat shock promoters, provided that such promoters are compatible with the host cell systems.

[0343] The SV40 virus early and late promoters are readily available as SV40 restriction fragments that also contain the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is readily available as a HindIII E restriction fragment. US Pat. No. 4,419,446 discloses a system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector. Improvements to this system are detailed in US Pat. No. 4,601,978. See Reyes et al., Nature 297:598-601 (1982) for expression of human interferon cDNA in mouse cells under the control of the herpes simplex virus thymidine kinase promoter. Alternatively, the Rous sarcoma virus long terminal repeat sequence can be used as a promoter.

[0344] e) Enhancer elements

[0345] The transcription of the DNA encoding the protein construct of the present application by higher eukaryotes is usually increased by inserting an enhancer sequence into the vector. Many enhancer sequences have been found in mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). However, enhancers from eukaryotic cell viruses are commonly used. Examples include the SV40 enhancer at the end of the replication origin (100-270bp), the cytomegalovirus early promoter enhancer, the polyoma enhancer at the end of the replication origin, and the adenovirus enhancer. See Yaniv, Nature 297:17-18 (1982), for enhancing elements that activate eukaryotic promoters. The enhancer can be spliced ​​into the vector at 5' or 3' of the polypeptide coding sequence, but is preferably located at 5' of the promoter.

[0346] f) Transcription termination element

[0347] 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 mRNA stabilization. These sequences are typically obtained from the 5′ untranslated region, and occasionally the 3′ untranslated region, of eukaryotic or viral DNA or cDNA. These regions contain nucleic acid segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. One suitable transcription termination element is the bovine growth hormone polyadenylation region. See WO 94 / 11026 and the expression vectors disclosed therein.

[0348] g) Host cell selection and transformation

[0349] Suitable host cells for cloning or expressing the DNA in the vectors described herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) is a routine procedure. Examples of useful mammalian host cell lines are 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 a subclone of 293 cells for growth in suspension culture, 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 Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); 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 liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells and a human hepatoma cell line (Hep G2).

[0350] Host cells are transformed with the above-described expression vectors or cloning vectors to produce the protein construct and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify genes encoding the desired sequences.

[0351] h) Cultivating host cells

[0352] Host cells used to produce the protein constructs of the present application can be cultured in a variety of culture media. Commercial culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US Pat. Nos. 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 culture media for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPE), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), and / or thiazolinone. TM Drugs), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be added at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used for host cell expression and will be apparent to those of ordinary skill in the art.

[0353] VI. Pharmaceutical Compositions

[0354] The present application further relates to pharmaceutical compositions comprising any of the IL-2 mutants described herein or fusion proteins comprising IL-2 mutants, or pharmaceutical compositions comprising nucleic acids encoding any of the IL-2 mutants described herein or fusion proteins comprising IL-2 mutants, or pharmaceutical compositions comprising vectors encoding nucleic acids encoding any of the IL-2 mutants described herein or fusion proteins comprising IL-2 mutants. These compositions may optionally further comprise a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the excipient comprises an excipient and / or a stabilizer. The pharmaceutical composition can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing an IL-2 mutant or fusion protein comprising an IL-2 mutant having the desired purity as described herein with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0355] VII. Methods of treating the disease

[0356] The IL-2 mutants described herein or fusion proteins comprising IL-2 mutants or compositions comprising them (e.g., pharmaceutical compositions) can be used for a variety of purposes, such as diagnosis, molecular detection, and treatment. In some embodiments, the present application relates to a method for treating an individual (e.g., human) disease, comprising administering to the individual an effective dose of any IL-2 mutant as described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising it. In some preferred embodiments, the fusion protein comprising 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 mutant or a fusion protein comprising an IL-2 mutant or a pharmaceutical composition comprising an IL-2 mutant or its fusion protein is administered by intravenous injection, intramuscular injection, or subcutaneous injection.

[0357] In some embodiments, the disease comprises lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopic disease, 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.

[0358] For example, in some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising the nucleic acid, or a pharmaceutical composition comprising the same. Wherein the IL-2 mutant comprises a deletion or mutation of amino acid residues 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 human wild-type IL-2 is as shown in SEQ ID NO: 1.

[0359] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein the IL-2 mutant comprises a complete deletion of the amino acid residues RPRDL relative to the 81st to 85th positions of the human wild-type IL-2 amino acid sequence. In some embodiments, the IL-2 mutant further comprises mutations of E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of E67 and N71 are E67K and N71S, respectively. In some embodiments, the IL-2 mutant comprises a complete deletion of the amino acid residues RPRDL relative to the 81st to 85th positions of the human wild-type IL-2 amino acid sequence, and E67K and N71S mutations relative to the human wild-type IL-2 amino acid sequence.

[0360] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein the IL-2 mutant comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL. 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 mutation of E67 is E67A. In some embodiments, the IL-2 mutant comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RPL, and an E67A mutation relative to the human wild-type IL-2 amino acid sequence.

[0361] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant as described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein the IL-2 mutant comprises an amino acid residue RPRDL mutated to RPL relative to the 81st-85th position of the human wild-type IL-2 amino acid sequence, and further comprises a mutation of N71 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of N71 and S75 are N71W and S75I, respectively. In some embodiments, the IL-2 mutant comprises an amino acid residue RPRDL mutated to RPL relative to the 81st-85th position of the human wild-type IL-2 amino acid sequence, and N71W and S75I mutations relative to the human wild-type IL-2 amino acid sequence.

[0362] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL. In some embodiments, the IL-2 mutant further comprises a mutation of E67 and / or V91 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation of E67 is E67T. In some embodiments, the mutation of V91 is V91K. In some embodiments, the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to RHL, and a mutation of E67T and / or V91K relative to the human wild-type IL-2 amino acid sequence.

[0363] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant as described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein the IL-2 mutant comprises an amino acid residue RPRDL mutation relative to the 81st to 85th position of the human wild-type IL-2 amino acid sequence to RHL, and further comprises mutations of L70 and S75 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of L70 and S75 are L70I and S75F, respectively. In some embodiments, the IL-2 mutant comprises an amino acid residue RPRDL mutation relative to the 81st to 85th position of the human wild-type IL-2 amino acid sequence to RHL, and L70I and S75F mutations relative to the human wild-type IL-2 amino acid sequence.

[0364] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant as described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein the IL-2 mutant comprises an amino acid residue RPRDL mutation relative to the 81st to 85th position of the human wild-type IL-2 amino acid sequence to RHL, and further comprises a mutation of N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutation of N71 is N71I. In some embodiments, the IL-2 mutant comprises an amino acid residue RPRDL mutation relative to the 81st to 85th position of the human wild-type IL-2 amino acid sequence to RHL, and an N71I mutation relative to the human wild-type IL-2 amino acid sequence.

[0365] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to SPL. In some embodiments, the IL-2 mutant further comprises mutations of E67 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of E67 and N71 are E67M and N71S, respectively. In some embodiments, the IL-2 mutant comprises a mutation of the amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence to SPL, and E67M and N71S mutations relative to the human wild-type IL-2 amino acid sequence.

[0366] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising the nucleic acid, or a pharmaceutical composition comprising the same as described herein. The IL-2 mutant comprises a mutation of 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 human wild-type IL-2 is as shown in SEQ ID NO: 1.

[0367] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant as described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein the IL-2 mutant comprises a mutation of the 75th amino acid residue S (S75) and the 83rd amino acid residue R (R83) relative to the human wild-type IL-2 amino acid sequence. In some embodiments, S75 in the IL-2 mutant 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 in R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K, or R83E.

[0368] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein 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.

[0369] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising such a nucleic acid, or a pharmaceutical composition comprising the same. 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 in H16, L70, and N71 relative to the amino acid sequence of human wild-type IL-2. 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 amino acid sequence of human wild-type IL-2.

[0370] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. In the IL-2 mutant, the mutation of S75 is S75F, and the mutation of R83 is R83P. In some embodiments, the IL-2 mutant further comprises a mutation of H16 and / or N71 relative to the amino acid sequence of human wild-type IL-2. 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 comprises S75F, R83P, and H16Q mutations relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the IL-2 mutant comprises S75F, R83P, H16N and N71S mutations relative to the human wild-type IL-2 amino acid sequence.

[0371] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein 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.

[0372] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein in the IL-2 mutant, the mutation of S75 is S75I, 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 E67G and L70V, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75I, R83L, E67G, and L70V relative to the amino acid sequence of human wild-type IL-2.

[0373] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein in the IL-2 mutant, the mutation of S75 is S75I, and the mutation of R83 is R83K. 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 E67S and L70V, respectively. In some embodiments, the IL-2 mutant comprises mutations of S75I, R83K, E67S, and L70V relative to the amino acid sequence of human wild-type IL-2.

[0374] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein in the IL-2 mutant, the mutation of S75 is S75T, and the mutation of R83 is R83A or R83F. In some embodiments, the IL-2 mutant further comprises mutations in H16 and E67 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations in H16 and E67 are H16N and E67K, respectively. In some embodiments, the IL-2 mutant comprises S75T, R83A, H16N, and E67K mutations relative to the amino acid sequence of human wild-type IL-2.

[0375] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein in the IL-2 mutant, the mutation of S75 is S75V, and the mutation of R83 is R83F. In some embodiments, the IL-2 mutant further comprises mutations in E15 and H16 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations in E15 and H16 are E15K and H16Q, respectively. In some embodiments, the IL-2 mutant or its fusion protein comprises S75V, R83F, E15K and H16Q mutations relative to the amino acid sequence of human wild-type IL-2.

[0376] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein 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 S75V, R83N, L70F, and V91K mutations relative to the amino acid sequence of human wild-type IL-2.

[0377] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein 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.

[0378] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein in the IL-2 mutant, the mutation of S75 is S75Y, and the mutation of R83 is R83N. In some embodiments, the IL-2 mutant described herein further comprises mutations in L17 and V91 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations in L17 and V91 are L17I and V91K, respectively. In some embodiments, the IL-2 mutant comprises S75Y, R83N, L17I, and V91K mutations relative to the amino acid sequence of human wild-type IL-2.

[0379] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. In the IL-2 mutant, the mutation of S75 is S75A, and the mutation of R83 is R83K. In some embodiments, the IL-2 mutant further comprises a mutation of L17 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of L17 is L17I. In some embodiments, the IL-2 mutant comprises S75A, R83K, and L17I mutations relative to the amino acid sequence of human wild-type IL-2.

[0380] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising this nucleic acid, or a pharmaceutical composition comprising the same. Wherein in the IL-2 mutant, the mutation of S75 is S75L, and the mutation of R83 is R83E. In some embodiments, the IL-2 mutant described herein further comprises mutations of L63 and E67 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of L63 and E67 are L63V and E67F, respectively. In some embodiments, the IL-2 mutant comprises S75L, R83E, L63V, and E67F mutations relative to the amino acid sequence of human wild-type IL-2.

[0381] In some embodiments, the present application relates to a method for treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, or a fusion protein comprising an IL-2 mutant. The IL-2 mutant or fusion protein thereof, in addition to the above-mentioned mutations, further comprises a mutation relative to the human wild-type IL-2 amino acid sequence C125. In some embodiments, the C125 mutation is C125S or C125A.

[0382] In some embodiments, the present application relates to a method for treating an individual suffering from an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant described herein, or a fusion protein comprising an IL-2 mutant. The IL-2 mutant comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 5-34, 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 to the amino acid sequence as set forth in any one of 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 fusion protein of the IL-2 mutant and Fc is in a bivalent form. In some embodiments, the bivalent fusion protein of the IL-2 mutant and Fc comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 76-105, or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to the amino acid sequence as set forth in any one of SEQ ID NOs: 76-105. In some embodiments, the fusion protein of the IL-2 mutant and Fc is monovalent. In some embodiments, the disease or condition is selected from lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopic disease, 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.

[0383] In some embodiments, the present application relates to a method for treating an individual with an inflammatory disease or an autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising the nucleic acid, or a pharmaceutical composition comprising the same, as described herein, and further comprising administering to the individual one or more other active ingredients, the active ingredients being required for the specific indications 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 reactions after organ transplantation, it can be used in combination with an immunosuppressant. In some embodiments, the immunosuppressant is selected from: glucocorticoids; azathioprine; cyclosporine A; methotrexate; anti-CD3 antibodies; or anti-TNF-α antibodies, etc.

[0384] Administration of the IL-2 mutants, fusion proteins comprising the IL-2 mutants, nucleic acids encoding the IL-2 mutants or their fusion proteins, vectors and host cells comprising the nucleic acids, or pharmaceutical compositions comprising the same as described herein can be carried out in any known and convenient manner, including by injection or infusion. The route of administration is in accordance with known and recognized methods, such as by single or multiple boluses or by prolonged infusion in an appropriate manner.

[0385] The dosage and desired drug concentration of the pharmaceutical compositions of the present application may vary depending on the specific application. Determining the appropriate dosage or route of administration is well within the skill of those of ordinary skill. Animal studies provide reliable guidance for determining effective dosages for human therapy. Interspecies analogies of effective dosages can be made based on the principles of Mordenti, J. and Chappell, W., "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-46.

[0386] When an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and host cell comprising such a nucleic acid, or a pharmaceutical composition comprising such a nucleic acid is administered in vivo, the therapeutically effective amount may depend, for example, on the therapeutic context and the goal. One of ordinary skill in the art will appreciate that the appropriate dosage level for treatment will vary depending on the molecule being delivered, the indication for treatment with the IL-2 mutant, the route of administration, and the patient's size (weight, body surface or organ size) and / or condition (age and general health). Within the scope of this application, different formulations will be effective for different treatments and different diseases, and the administration regimen intended to treat a specific organ or tissue may differ from that for another organ or tissue. Furthermore, the dosage may be administered by one or more single administrations or by continuous infusion. For repeated administration over several days or longer, depending on the condition, treatment is continued until the desired degree of suppression of disease symptoms is achieved. Alternatively, other dosage regimens may be useful. The progress of such treatment is readily monitored by conventional techniques and assays.

[0387] VIII. Products and Kits

[0388] The present application further relates to kits, unit doses, and articles of manufacture comprising any of the IL-2 mutants 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 the same. In some embodiments, the present application relates to kits comprising any of the pharmaceutical compositions described herein, and preferably provides instructions for their use, such as for treating a disease described herein (e.g., an autoimmune disease).

[0389] The kit of the present application includes one or more containers containing the IL-2 mutants described herein, 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 such containers, for example, for treating a disease. For example, instructions describing the administration 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 such containers to treat a disease (such as an autoimmune disease) are included. The kit may further include instructions for selecting individuals (e.g., humans) suitable for treatment based on identifying whether the individual has the disease and the stage of the disease. Instructions for use 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 such containers typically include information about the dose, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions provided in the kits of the present application are typically written instructions on a label or package insert (e.g., a paper included in the kit), but machine-readable instructions (e.g., instructions stored on a disk or CD) are also acceptable. The kits of the present application are packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. Packaging used in conjunction with specific devices, such as infusion devices such as micropumps, is also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an IL-2 mutant as described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and host cell comprising the nucleic acid, or a pharmaceutical composition comprising the same. The container may further contain a second pharmaceutically active agent. The kit may optionally provide additional components, such as a buffer and explanatory information. Generally, the kit comprises a container and a label or package insert on or associated with the container.

[0390] For example, in some embodiments, the kit includes: a) any one of the IL-2 mutants described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising such a nucleic acid, or a pharmaceutical composition comprising such a pharmaceutical composition, and b) at least one other agent in an effective amount that can enhance the effect (such as a therapeutic effect) of the IL-2 mutant, the fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising such a nucleic acid, or a pharmaceutical composition comprising such a pharmaceutical composition.

[0391] In some embodiments, the kit comprises: a) any one of the IL-2 mutants described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising such a nucleic acid, or a pharmaceutical composition comprising such a composition, and b) instructions for administering the IL-2 mutant, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or its fusion protein, a vector and a host cell comprising such a nucleic acid, or a pharmaceutical composition comprising such a composition to an individual for treating an autoimmune disease (e.g., lupus).

[0392] In some embodiments, the kit comprises: a) 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 its fusion protein, a vector and host cell comprising such a nucleic acid, or a pharmaceutical composition comprising such a pharmaceutical composition, and b) at least one other agent in an effective amount that enhances 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 its fusion protein, the vector and host cell comprising such a nucleic acid, or a pharmaceutical composition comprising such a pharmaceutical composition, and c) instructions for administering the IL-2 mutant, the fusion protein comprising an IL-2 mutant, the nucleic acid encoding an IL-2 mutant or its fusion protein, the vector and host cell comprising such a nucleic acid, or a pharmaceutical composition comprising such a pharmaceutical composition, and other substances to an individual for treating 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 its fusion protein, the vector and host cell comprising such a nucleic acid, or the pharmaceutical composition comprising such a pharmaceutical composition, and other substances, can be present in separate containers or in the same container. For example, the kit may comprise one specific composition or two or more compositions, wherein one composition comprises 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 a host cell comprising the nucleic acid, or a pharmaceutical composition comprising the same, and the other composition comprises another pharmaceutical agent.

[0393] 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 an 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 an 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 an Fc), and b) instructions for use for: i) expressing the IL-2 mutant or fusion protein thereof in a host cell, ii) preparing a composition comprising the IL-2 mutant or fusion protein thereof, and iii) administering a composition comprising the IL-2 mutant or fusion protein thereof to a subject to treat an autoimmune disease (e.g., lupus). In some embodiments, the kit comprises: 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 nucleic acid set), and c) instructions for use, suitable for: i) expressing the IL-2 mutant or a fusion protein thereof in the host cell, ii) preparing a composition comprising the IL-2 mutant or a fusion protein thereof, and iii) administering the composition comprising the IL-2 mutant or a fusion protein thereof to an individual to treat an autoimmune disease (e.g., lupus).

[0394] On the other hand, the present application also relates to articles, including vials (such as sealed vials), bottles, cans, flexible packaging, etc. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be made of a variety of materials, such as glass or plastic. In general, the composition contained in the container can effectively treat the diseases or disorders described herein (such as autoimmune diseases) and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition of an individual. The label or package insert further includes instructions for administering the composition to an individual. The label may indicate instructions for reconstitution and / or use. The container holding the pharmaceutical composition can be a reusable vial that allows for repeated administration of the reconstituted formulation (for example, 2-6 administrations). Package insert refers to instructions typically included in the commercial packaging of a therapeutic product, which contains information on the indications, usage, dosage, administration, contraindications, and / or warnings for the use of such therapeutic products. In some embodiments, the instructions indicate that the treatable diseases include 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, dystrophic epidermolysis bullosa, and Behçet's disease. In addition, the product may further include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. From a commercial and user perspective, other required materials may be further included, including other buffers, diluents, filters, needles, and syringes.

[0395] The kit or article of manufacture includes multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies. DETAILED DESCRIPTION

[0396] Those skilled in the art will recognize that several embodiments are possible within the scope and purpose of this application. The application will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the application, but should not be construed as limiting its scope in any way.

[0397] Example 1: Design and expression of human wild-type IL-2 and IL-2 mutants

[0398] Construction and expression of wild-type IL-2:

[0399] 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 Biotechnology Co., Ltd., and restriction sites were introduced at the 5' and 3' ends of the synthesized wild-type IL-2 nucleic acid sequence. After enzyme digestion, the synthesized nucleic acid fragment was ligated with a vector fragment that had undergone the same double enzyme digestion to obtain an expression vector for wild-type IL-2. In addition, 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, which can prevent cysteine ​​mispairing or aggregation), thereby obtaining an expression vector for the IL-2 C125S mutant; and a C125A mutation was introduced at position 125 of wild-type IL-2 (amino acid sequence as shown in SEQ ID NO: 3, which can promote efficient folding and improve expression), thereby obtaining an expression vector for the IL-2 C125A mutant. Compared with wild-type IL-2, the above two IL-2 mutants did not change their binding to the IL-2 receptor.

[0400] Design, screening and expression of IL-2 mutants:

[0401] Through research, design, and FACS screening, a series of IL-2 mutants were finally obtained. The screening process is as follows:

[0402] First, prepare the biotinylated IL-2Rα and IL-2Rβγ required for the screening process:

[0403] Preparation of IL-2Rα: The extracellular domain coding sequence of IL-2Rα (Uniprot ID: P01589) was codon-optimized and fully synthesized by Nanjing GenScript Biotechnology 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 nickel column according to the operating instructions. Briefly, immobilized metal affinity chromatography (IMAC) was performed using Ni-NTA from QIAGEN. The nickel column was first 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 buffer A1 at a flow rate of 150 cm / h. Finally, elution was performed with 10 column volumes of 50 mM PB solution (containing 0.15 M NaCl and 0.2 M imidazole, pH 7.2), and the eluate was collected.

[0404] Preparation of IL-2Rβγ: The IL-2Rβγ used in the examples of this application is an Fc heterodimer (IL-2Rβγ-Fc heterodimer) constructed using the KIH (Knob-into-Hole) technique. The extracellular domain coding sequences of IL-2Rβ (Uniprot ID: P14784) and IL-2Rγ (Uniprot ID: P31785) were cloned and fused to the N-termini of the Fc hole molecule (SEQ ID NO: 109) and the Fc knob molecule (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, co-transfected into 293F cells, and the cell culture fluid was collected. The IL-2Rβγ-Fc heterodimer was purified using protein A affinity chromatography followed by gel filtration on a Superdex 200 column. Briefly, 293F cell culture medium was purified using a protein A affinity column. The column was first equilibrated with 50 mM PBS buffer containing 0.15 M NaCl (pH 7.2) at a flow rate of 150 cm / h and a volume of six times the column volume. The culture supernatant (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 eluate was collected. The IL-2Rβγ-Fc heterodimer was then purified by gel filtration on a Superdex 200 column to obtain the IL-2Rβγ-Fc heterodimer.

[0405] Biotinylation of IL-2Rα and IL-2Rβγ: IL-2Rα and IL-2Rβγ were biotinylated using a biotinylation ligase according to the instructions of the Biotin-Protein Ligase / BirA Enzyme (GeneCopoeia cat#BI001) kit. Briefly, 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 by ELISA to confirm successful biotin labeling.

[0406] FACS screening: A library of designed IL-2 mutants was constructed and displayed in a yeast display system. Flow cytometry (FACS) was used to screen for mutants with reduced affinity for IL-2Rβγ, while maintaining or not significantly reducing affinity for IL-2Rα. FACS screening methods are well known to those skilled in the art. Four rounds of FACS screening were performed using biotinylated IL-2Rβγ (Biotin-IL-2Rβγ) to enrich for yeast positive for reduced affinity for IL-2Rβγ. Briefly, yeast cells induced in SGCAA medium were pelleted, 1 mL of PBSM was added, and the cells were centrifuged at 14,000 g for 30 seconds. The supernatant was discarded and the cell pellet washed. The yeast cells were resuspended in PBSM buffer containing Biotin-IL-2Rβγ and incubated at room temperature for 1 hour. After washing, cells were stained with Streptavidin-PE (BD Biosciences, 554061) and an anti-V5 tag antibody [iFluor 647] (GenScript, A01805-100) and sorted. The enriched yeast population with reduced binding to Biotin-IL-2Rβγ was then screened using FACS for single clones whose affinity for biotinylated IL-2Rα (Biotin-IL-2Rα) remained essentially unchanged or not significantly reduced. Finally, the selected single clones were sequenced, constructed into an expression vector carrying the His-tag gene, and samples were prepared and subjected to appropriate biological evaluations.

[0407] By analyzing the sequencing results of single clones, it was found that these mutants can be roughly divided into two groups:

[0408] The first group of IL-2 mutants has undergone mutations at amino acid residues 81-85 relative to the human wild-type IL-2 amino acid sequence, including but not limited to deletions and / or substitutions. Detailed information on each mutant in this group is shown in Table 9. "(81-85)RPL", "(81-85)RHL" or "(81-85)SPL" in the table means that the amino acid residues RPRDL at positions 81-85 of IL-2 are replaced with amino acid residues RPL, RHL or SPL, respectively. "(81-85)RPRDL is completely deleted" means that the amino acid residues RPRDL at positions 81-85 of IL-2 are completely deleted. "+" represents simultaneous mutations.

[0409] Table 9

[0410] The second group of IL-2 mutants had simultaneous amino acid mutations at S75 and R83 relative to the human wild-type IL-2 amino acid sequence. Detailed information for each mutant in this group is shown in Table 10, where "+" represents simultaneous mutations.

[0411] Table 10

[0412] Expression and Purification of IL-2 Mutants: The gene coding sequences of the aforementioned IL-2 mutants were obtained by PCR, digested with enzymes, and ligated into expression vectors carrying the His-tag gene that had been double-digested with enzymes to generate recombinant expression vectors for each IL-2 mutant. 293F cells were transfected with these recombinant expression vectors and cultured for 5 days at 37°C, 5% CO2, and 120 rpm. The cell culture supernatant was collected and purified using a nickel column according to the manufacturer's instructions. The specific steps are as described above.

[0413] The following experiments were conducted to further examine whether the two groups of IL-2 mutants finally 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 examples 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 (for example, while the IL-2 mutant remains bound to IL-2Rαβγ, the greater the EC50 value of its binding to IL-2Rβγ), the better the selectivity of the IL-2 mutant for Treg cells; for another example, it can also be judged by the changes in the number of Treg cells after stimulation with the IL-2 mutant, such as by measuring the changes in the number of Treg cells in a mixed cell population by flow cytometry.

[0414] Example 2: Determination of Binding Affinity of IL-2 Mutants to IL-2Rαβγ and IL-2Rβγ

[0415] HEK-BLUE TM Experimental principle of cell line detection:

[0416] 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 is HEK-Blue TMCD122 / CD132 (InvivoGen, cat#hkb-il2bg) only expresses IL-2Rβ (CD122) and IL-2Rγ (CD132), but not IL-2Rα (CD25). In addition, both cell lines stably express human JAK3 and STAT5 genes, and have a fully functional IL-2 signaling pathway; in addition, the expression of the SEAP reporter gene induced by STAT5 is introduced. After IL-2 stimulation, the QUANTI-Blue TM Solution (InvivoGen, cat# rep-qbs) was used to detect the expression level of SEAP induced by STAT5. TM IL-2 cell line is used to detect the binding of IL-2 or its mutants to IL-2Rαβγ. TM The CD122 / CD132 cell line is used to detect the binding of IL-2 or its mutants to IL-2Rβγ. When IL-2 or its mutants are added to the above cell lines, IL-2 or its mutants can bind to the IL-2 high-affinity receptor IL-2Rαβγ or the medium-affinity receptor IL-2Rβγ, activating the intracellular Janus family tyrosine kinase signaling cascade (JAK1 and JAK3), initiating phosphorylation of signal transducer and activator of transcription 5 (STAT5), and thereby inducing SEAP expression. The substrate QUANTI-Blue was used. TM SEAP expression levels were measured by reading the OD at 630 nm and calculating the corresponding EC50 value, which reflects STAT5 activation 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.

[0417] Using HEK-Blue TM The EC50 values ​​of IL-2 mutants binding to IL-2Rβγ were determined using the CD122 / CD132 cell line and HEK-Blue TM The EC50 values ​​of IL-2 mutants binding to IL-2Rαβγ were determined using an IL-2 cell line. The ratio of the two values ​​(EC50 of HEK-IL-2Rβγ / EC50 of HEK-IL-2Rαβγ) was defined as the therapeutic safety window for IL-2 selectively activating Treg cells. This safety window reflects the selective targeting of IL-2 mutants for Treg cells; a higher value indicates a stronger targeting of IL-2 mutants for Treg cells.

[0418] HEK-BLUE TM Cell line testing experiments:

[0419] First, dilute the IL-2 mutant to be tested to 800 μg / mL with the test culture medium, and then dilute it 4-fold in a series to 12 concentrations in a 96-well U-shaped plate (Coring) for later use. TM IL-2 cell line or HEK-Blue TM CD122 / CD132 cell line) suspension was adjusted to 2.8×10 5 After 180 μL of cell suspension / well was seeded into a 96-well flat-bottom cell culture plate, 20 μL of diluted IL-2 or IL-2 mutant was added to each well, and the cell suspension was gently shaken to mix, and then placed in a 37°C, 5% CO2 incubator for 24 hours. 20 μL / well of cell culture supernatant was transferred from the cell plate to another new 96-well flat-bottom cell culture plate, and 180 μL of QUANTI-Blue was added to each well. TM The test solution was mixed and incubated at 37°C for 1 hour. The absorbance was read at 620 nm. GraphPad Prism 8.0.1 was used to calculate the EC50 value of the test sample. The concentration value was first converted to a logarithm (X = log(X)). The EC50 value of the test sample was then calculated using a curve fitting method based on the log(agonist) vs. response-variable slope (four parameters).

[0420] HEK-BLUE TM Experimental results of cell line testing:

[0421] 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 values ​​for binding to IL-2Rβγ were increased, their affinity was reduced, and their therapeutic safety window was significantly improved, indicating that these mutants have excellent Treg cell selection and targeting.

[0422] Table 11 The first group of IL-2 mutants HEK-BLUE TM Test results

[0423] (Note: The values ​​marked with * are due to the low activation ability of these mutants on IL-2Rβγ cells, and accurate EC50 values ​​cannot be obtained through fitting within the concentration range used in the experiment)

[0424] Table 12 The second group of IL-2 mutants HEK-BLUE TM Test results

[0425] (Note: The values ​​marked with * are due to the low activation ability of these mutants on IL-2Rβγ cells, and accurate EC50 values ​​cannot be obtained through fitting within the concentration range used in the experiment)

[0426] Example 3: Expression and purification of IL-2 mutant and Fc fusion protein

[0427] The above-mentioned IL-2 mutant was linked to IgG1 Fc LALA via a linker peptide to form a fusion protein of IL-2 and Fc, comprising two constructs: a bivalent form and a monovalent form. IgG1 Fc LALA refers to an IgG1 Fc with L234A and L235A mutations, and the numbering is based on the EU Kabat numbering system (Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., US Pat. of Health and Human Services, "Sequences of proteins of immunological interest" (1991)).

[0428] A bivalent form of an IL-2 mutant-Fc fusion protein comprises two IL-2 mutant molecules: it comprises two identical monomers, in each of which the IL-2 mutant is linked to the N-terminus or C-terminus of the Fc. An exemplary structure of one such form is shown in Figure 1A (IL-2-Fc), which consists of two identical monomers, each of which comprises, from the N-terminus to the C-terminus, an IL-2 mutant (SEQ ID NOs: 5-34), a connecting peptide (SEQ ID NO: 52), and an IgG1 Fc LALA (SEQ ID NO: 35). Another exemplary structure of this form is shown in Figure 1B (Fc-IL-2), which consists of two identical monomers, each of which comprises, from the N-terminus to the C-terminus, an IgG1 Fc LALA (SEQ ID NO: 35), a connecting peptide (SEQ ID NO: 52), and an IL-2 mutant (SEQ ID NOs: 5-34).

[0429] The monovalent form of the IL-2 mutant and Fc fusion protein comprises a single IL-2 mutant molecule: it comprises two different monomers, one of which is fused to the N-terminus or C-terminus of one Fc subunit, and the other is another Fc subunit. The two asymmetric Fc subunits are facilitated to pair with each other by knob and hole mutations. In a preferred form, it comprises two different monomers, one of which is fused to the C-terminus of one Fc subunit via a connecting peptide. The monomer comprises, from the N-terminus to the C-terminus, the IgG1 Fc LALA knob (SEQ ID NO:41), a connecting peptide (SEQ ID NO:52), and the IL-2 mutant (SEQ ID NOs:5-34); and the other monomer is the IgG1 Fc LALA hole (SEQ ID NO:42). An exemplary structure of this fusion protein is shown in Figure 2.

[0430] The specific construction process of the IL-2 mutant and Fc fusion protein (using the bivalent fusion protein Fc-IL-2 of the IL-2 mutant and Fc as an example) is as follows: Recognition sites for the restriction endonucleases HindIII and XhoI were introduced by PCR. The nucleic acid sequence encoding the IgG1 Fc LALA was ligated to the nucleic acid sequence encoding the IL-2 mutant via an oligonucleotide sequence encoding GGGGS. The construct was then inserted into the pcDNA3.1 eukaryotic expression vector, transfected into 293F cells, and cultured at 37°C, 5% CO2, and 120 rpm for 5 days. The culture medium was then collected and the Fc-IL-2 mutant fusion protein was purified by protein A affinity chromatography followed by gel filtration using a Superdex 200 column. The specific steps are as described in Example 1.

[0431] Activity testing of different forms of IL-2 mutants and Fc fusion proteins:

[0432] Apply HEK-BLUE TM Cell line experiments were conducted to test whether different fusion forms would affect the activity of IL-2 mutants. TM The specific experimental steps are as described in Example 2.

[0433] The bivalent forms of (81-85)-mut8 and (81-85)-mut10 in the first group of IL-2 mutants and S75R83-mut16 in the second group of IL-2 mutants and Fc fusion proteins: Fc-(81-85)-mut8, Fc-(81-85)-mut10 and Fc-S75R83-mut16 were tested as exemplary fusion proteins, which contained two identical monomers, in each monomer the IL-2 mutant was fused to the C-terminus of Fc LALA via the linker peptide GGGGS.

[0434] At the same time, the monovalent forms of the above-mentioned mutants and Fc fusion proteins: Fc knob-(81-85)-mut8 / Fc-hole, Fc knob-(81-85)-mut10 / Fc-hole, and Fc knob-S75R83-mut16 / Fc-hole were tested as exemplary fusion proteins, which contained two different monomers, one of which contained an IL-2 mutant molecule fused to the C-terminus of the Fc LALA knob via the connecting peptide GGGGS, and the other monomer was the Fc LALA hole.

[0435] 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 to IL-2Rβγ or IL-2Rαβγ, and the fusion protein of IL-2 mutant and Fc still has a preference for binding to different receptors.

[0436] Table 13 (Note: The values ​​marked with * are due to the low activation ability of these mutants on IL-2Rβγ cells, and accurate EC50 values ​​cannot be obtained through fitting within the concentration range used in the experiment)

[0437] Table 14

[0438] Example 4: IL-2 mutant BIACORE affinity determination

[0439] Biacore assays were used to determine the binding affinity of exemplary Fc-IL-2 mutant bivalent fusion proteins, Fc-(81-85)-mut8 or Fc-S75R83-mut16, to human IL-2Rβγ and human IL-2Rαβγ, respectively. IL-2Rβγ employed the IL-2Rβγ-Fc heterodimer prepared in Example 1, while IL-2Rαβγ employed a heterodimeric fusion protein composed of an IL-2Rαγ-Fc knob monomer and an IL-2Rβ-Fc hole monomer (IL-2Rαβγ-Fc). The Fc-wild-type IL-2 bivalent fusion protein served as a control in this experiment. The specific steps are as follows: IL-2Rβγ-Fc or IL-2Rαβγ-Fc was captured using a Protein A sensor chip on a Biacore instrument (Biacore T200, GE). IL-2Rβγ-Fc or IL-2Rαβγ-Fc was diluted to 1 μg / mL and maintained at a flow rate of 10 μL / min for 30 seconds. A concentration gradient of wild-type IL-2 or its mutant bivalent fusion protein was then passed over the chip surface at a flow rate of 30 μL / min. Binding lasted 120 seconds, and dissociation lasted 360 seconds. The reaction signals were monitored in real time using the Biacore instrument to generate binding and dissociation curves. Kon, Koff, and Kd values ​​are shown in Table 15.

[0440] Table 15

[0441] Example 5: In vitro biological activity assay of IL-2 mutants

[0442] When IL-2 binds to its cell surface receptor, it activates the JAK-STAT signaling pathway, phosphorylating STAT5. STAT5 phosphorylation is a necessary step in the IL-2 signal transduction pathway. Once phosphorylated, STAT5 enters the cell nucleus and participates in downstream signal transduction processes. Therefore, the phosphorylated STAT5 signal in cells can reflect IL-2 activation.

[0443] CD8 + T lymphocytes express IL-2Rβγ but not IL-2Rα. Treg cells express both IL-2Rβγ and IL-2Rα. CD8 + The level of STAT5 phosphorylation (pSTAT5) in T lymphocytes and Treg cells indirectly reflects the activation of CD8 by IL-2 mutants. + Activity of T lymphocytes and Treg cells (for example, see Peterson LB, et al. J Autoimmun. 2018 Dec; 95: 1-14).

[0444] The specific operation steps are as follows: PBMC cells (Shanghai Miaoshun Biotechnology Co., Ltd., cat# PB050C) were revived one day in advance, PBMCs were collected overnight, washed with PBS, and collected by centrifugation. After mixing with 2% complete medium, the cells were counted and 60 μL of cell suspension was added to each well, about 5 to 6 × 10 5 Cells were seeded into cell culture plates and placed in a cell culture incubator for later use. The test samples included an exemplary first group of bivalent fusion proteins 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, as well as an exemplary second group of bivalent fusion proteins of IL-2 mutants and Fc (Fc-S75R83-mut7 and Fc-S75R83-mut16). After these samples were serially diluted, they were added to cells for stimulation for 30 min. Cell fixative was added, and after washing the cells, anti-CD4 antibody (BD cat#562424), anti-CD25 antibody (Biolegend cat#356128), and anti-CD8 antibody (Biolegend cat#300932) were added. The cells were incubated in the dark at 4°C for 30 min. After washing the cells, 150 μL / well of pre-cooled Phosflow TM Incubate on ice for 30 minutes in Perm Buffer III (BD cat#558050). Wash the cells with FACS buffer and add 100 μL / well of PE Mouse Anti-Stat5 (pY694) (BD cat#612567) and anti-Foxp3 antibody (Biolegend cat#320112) diluted in FACS buffer. Incubate at 4°C in the dark for 30 minutes. Wash the cells with FACS buffer, centrifuge, and discard the supernatant. Resuspend the cells in 100 μL / well of FACS buffer and analyze on an analyzer. CD8 + The surface marker of T lymphocytes is CD8. In this experiment, CD4 - CD8 + CD8 + T lymphocytes. The surface markers of Treg cells usually include CD4, CD25 and Foxp3. In this experiment, CD4 + CD25 + Foxp3 +The cells that are affected by agonist inhibition are 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 value into a logarithm, i.e., X = log(X), and then fit the curve according to log(agonist) vs. response-Variable slope (four parameters) to calculate the Treg cells and CD8 + EC50 values ​​for T lymphocytes.

[0445] Figures 3A, 3C, 3E, and 3G show the results of an exemplary first group of IL-2 mutants and Fc bivalent fusion proteins inducing STAT5 phosphorylation in Treg cells; Figures 3B, 3D, 3F, and 3H show the results of an exemplary first group of IL-2 mutants and Fc bivalent fusion proteins inducing STAT5 phosphorylation in CD8 + Results of the induction of STAT5 phosphorylation in T lymphocytes.

[0446] FIG3I shows the results of an exemplary second group of IL-2 mutants and Fc bivalent fusion proteins inducing STAT5 phosphorylation in Treg cells; FIG3J shows the results of an exemplary second group of IL-2 mutants and Fc bivalent fusion proteins inducing STAT5 phosphorylation in CD8 + Results of inducing STAT5 phosphorylation in T cells.

[0447] From the above results, it can be seen that both IL-2 mutant and Fc bivalent fusion protein can induce STAT5 phosphorylation in Treg cells, but in CD8 + In T lymphocytes, the IL-2 mutant and Fc bivalent fusion protein did not significantly induce STAT5 phosphorylation. The above results further demonstrate that the IL-2 mutant of the present invention can selectively activate Treg cells without activating CD8 + T lymphocytes have good Treg cell targeting and are safer in human treatment than wild-type IL-2.

[0448] Example 6: Activity determination of IL-2 mutants in mice

[0449] To demonstrate the selectivity of IL-2 mutants for Treg cells in rodents, the expression of Treg cells and CD8 T cells in the blood of mice was detected after a single administration of Fc-IL-2 mutant bivalent fusion protein. + Proliferation of T lymphocytes.

[0450] The specific procedures are as follows: C57LB / 6 mice were purchased from Vital River and housed in a specific pathogen-free environment. All animal experiments were performed in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC).

[0451] Experimental animal treatment: 21 SPF female C57BL / 6 mice were divided into 7 groups, with 3 mice in each group. Each group of mice was subcutaneously injected with the following bivalent fusion proteins of exemplary IL-2 mutants and Fc: 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) was used as the control sample in this experiment.

[0452] First, a single dose of 1 mg / kg was administered to each mouse on day 0. Before administration on day 0 and 3 days after administration, 100 μL of venous blood was collected and the animal number was marked on an EDTA anticoagulant blood collection tube.

[0453] The assay was performed using the Trye-Nulear Transcription Factor Buffer Set (Biolegend cat#424401). Briefly, 80 μL of blood sample was added to 1 mL of 1× Lyse / Fix buffer (BD cat#558049), mixed, and placed in a 37°C incubator for 30 minutes. The sample was then centrifuged at 400 g for 8 minutes, and the supernatant was discarded. For flow cytometry, cells were first fixed, and 1000 μL / tube of 1× Transcription Factor Fix working solution was added. The cells were incubated at room temperature in the dark for 40 minutes. The cell membrane was then perforated, and the cells were centrifuged at 400 g for 8 minutes, and the supernatant was discarded. 2 mL / tube of 1× Perm buffer was added, the cells were resuspended, and the cells were centrifuged at 400 g for 8 minutes, and the supernatant was discarded. The perforation step was repeated once. The cells were transferred to a V-shaped 96-well plate. Resuspend the cells in 1× Perm buffer and add anti-CD4 (Biolegend cat#317429), anti-CD25 (Biolegend cat#356128), anti-Foxp3 (Biolegend cat#320112), and anti-CD8 (Biolegend cat#344742) antibodies, respectively. Incubate at room temperature for 1 hour, centrifuge at 600g for 8 minutes, and discard the supernatant. Wash twice with 200 μL / well of 1% BSA, centrifuge, discard the supernatant, and resuspend in 100 μL of FACS buffer before analysis.

[0454] The flow cytometry software CytExpert was used for statistics. The lymphocyte population (P1) was gated at 30,000, and the percentage of Treg cells in CD4 + The percentage of cell populations, and CD8 + The percentage of T lymphocytes in total lymphocytes. CD8 + The surface marker of T lymphocytes is CD8. + CD8 + T lymphocytes. The surface markers of Treg cells are CD4, CD25 and Foxp3. + CD25 + Foxp3 + The cells are defined as Treg cells.

[0455] As shown in Figure 4A, the first group of IL-2 mutants and Fc bivalent fusion proteins: Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12 were able to stimulate the proliferation of Treg cells in mice at a dose of 1 mg / kg. Furthermore, compared with the control Fc-AMG592 (SEQ ID NO: 106), the activity of Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12 in stimulating Treg cell proliferation was comparable to or greater than that of Fc-AMG592. Furthermore, as shown in Figure 4B, Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12 did not stimulate the proliferation of CD8+ T lymphocytes. This demonstrates their specificity in promoting Treg cell expansion in vivo.

[0456] The results are shown in Figure 4C. The second group of IL-2 mutants and Fc bivalent fusion proteins Fc-S75R83-mut5, Fc-S75R83-mut9 and Fc-S75R83-mut16 were able to stimulate the proliferation of Treg cells in mice at a dose of 1 mg / kg. Compared with the control Fc-AMG592, the activity of Fc-S75R83-mut5 and Fc-S75R83-mut9 in stimulating Treg cell proliferation was comparable to that of Fc-AMG592, while the activity of Fc-S75R83-mut16 in stimulating Treg cell proliferation was higher. In addition, as shown in Figure 4D, Fc-S75R83-mut5, Fc-S75R83-mut9 and Fc-S75R83-mut16 did not stimulate CD8 Treg cell proliferation while stimulating Treg cell proliferation. + Proliferation of T lymphocytes.

[0457] The above results indicate that the IL-2 mutant molecules of the present invention can specifically activate Treg cells in rodents without affecting CD8 + T cells have an impact.

[0458] Example 7: Activity determination of IL-2 mutants in cynomolgus monkeys

[0459] To demonstrate the selectivity of IL-2 mutants for Treg cells in primates, the expression of Treg cells and CD8 T cells in the blood was detected after a single administration of Fc-IL-2 mutant bivalent fusion protein in cynomolgus monkeys. + Proliferation of T lymphocytes.

[0460] The specific procedures are as follows: Six standard-grade cynomolgus macaques, weighing 2.5–3 kg, were purchased from Beijing Zhongke Lingrui Biotechnology Co., Ltd. and housed in a specific pathogen-free environment. All animal experiments were conducted in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC).

[0461] First, a single dose was administered on day 0. Each cynomolgus monkey was subcutaneously injected with 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 of 0.3 mg / kg and a high-dose group of 1.5 mg / kg. Among them, 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 low-dose and high-dose groups. 1 mL of blood was collected intravenously on day 0 before and 7 days after dosing.

[0462] Trye-Nulear Transcription Factor Buffer Set (Biolegend cat#424401) was used for detection. The specific operation method was as described in Example 6.

[0463] The results are shown in Figures 5A-5B. The first group of IL-2 mutants and Fc bivalent fusion proteins Fc-(81-85)-mut8 and Fc-(81-85)-mut10 were able to stimulate the proliferation of Treg cells in cynomolgus monkeys at a low dose of 0.3 mg / kg (Figure 5A). Compared with the control drug Fc-AMG592, Fc-(81-85)-mut8 and Fc-(81-85)-mut10 had higher activity in stimulating Treg cell proliferation. At the same time, it did not stimulate CD8 + T lymphocytes proliferated, but their cell numbers did not change significantly ( Figure 5B ).

[0464] The results are shown in Figures 5C-5D. The second group of IL-2 mutants and Fc bivalent fusion protein Fc-S75R83-mut5 can stimulate the proliferation of Treg cells in cynomolgus monkeys at a low dose of 0.3 mg / kg (Figure 5C). At the same time, it does not stimulate CD8 + T lymphocytes proliferated, but the number of cells did not change significantly ( Figure 5D ).

[0465] Fc-S75R83-mut16 could stimulate the proliferation of Treg cells in cynomolgus monkeys at both the low-dose group (0.3 mg / kg) and the high-dose group (1.5 mg / kg). The effect of promoting Treg cell proliferation increased with the increase of the administration dose (Figure 5C). At the same time, Fc-S75R83-mut16 did not stimulate CD8 Treg cells in cynomolgus monkeys at both the low-dose and high-dose groups. + T lymphocytes proliferated, but the number of cells did not change significantly ( Figure 5D ).

[0466] Moreover, compared with the control Fc-AMG592, at the same dose, Fc-S75R83-mut5 and Fc-S75R83-mut16 had higher activity in stimulating Treg cell proliferation ( Figure 5C ).

[0467] The above results indicate that the IL-2 mutant of the present invention can specifically activate Treg cells in primates without affecting CD8 + The effect was comparable to or even better than that of Fc-AMG592, a control.

[0468] Example 8: PK experiment of IL-2 mutants in mice

[0469] C57LB / 6 mice were purchased from Vital River and housed in a specific pathogen-free environment. All animal experiments were performed in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC).

[0470] The in vivo PK study in mice was performed as follows: 24 C57LB / 6 mice were randomly divided into four groups of six mice each, half male and half female. Each group received a single tail vein injection of 1 mg / kg of the bivalent fusion proteins of the exemplary IL-2 mutants and Fc (Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-S75R83-mut5, or Fc-S75R83-mut16. Anticoagulated whole blood was collected at 0 hour before dosing and 15 minutes, 5 hours, 24 hours, 72 hours, 168 hours, 240 hours, 336 hours, 432 hours, and 600 hours after dosing, and plasma was separated. Plasma drug concentrations in each sample were determined by ELISA. Pharmacokinetic parameters were calculated using the non-compartmental model (NCA) software WinNonlin 6.0.

[0471] Following intravenous injection of 1 mg / kg of the exemplary IL-2 mutant-Fc bivalent fusion proteins, Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-S75R83-mut5, and Fc-S75R83-mut16, into mice, plasma concentrations gradually decreased over time. The results of the PK experiments are shown in Figure 6 , demonstrating that the IL-2 mutant-Fc bivalent fusion proteins of the present invention exhibited favorable pharmacokinetic properties in mice (PK curves for Fc-(81-85)-mut8 and Fc-S75R83-mut5 are not shown).

Claims

1. An IL-2 mutant comprising a deletion or mutation of amino acid residues RPRDL at positions 81 to 85 relative to the amino acid sequence of wild-type human IL-2.

2. The IL-2 mutant according to claim 1, wherein the amino acid sequence of the human wild-type IL-2 is shown in SEQ ID NO:

1.

3. The IL-2 mutant according to claim 1 or 2, comprising a complete deletion of amino acid residues RPRDL from positions 81 to 85 relative to the amino acid sequence of wild-type human IL-2.

4. The IL-2 mutant according to claim 1 or 2, which comprises a mutation of amino acid residues RPRDL at positions 81 to 85 relative to the amino acid sequence of human wild-type IL-2 to RPL.

5. The IL-2 mutant according to claim 1 or 2, which comprises a mutation of amino acid residues RPRDL at positions 81 to 85 relative to the amino acid sequence of human wild-type IL-2 to RHL.

6. The IL-2 mutant according to claim 1 or 2, which comprises a mutation of amino acid residues RPRDL at positions 81 to 85 relative to the amino acid sequence of human wild-type IL-2 to SPL.

7. The IL-2 mutant according to claim 3, further comprising mutations at E67 and N71 relative to the human wild-type IL-2 amino acid sequence.

8. The IL-2 mutant according to claim 7, wherein the mutations of E67 and N71 are E67K and N71S, respectively.

9. The IL-2 mutant according to claim 4, further comprising a mutation of E67 relative to the human wild-type IL-2 amino acid sequence.

10. The IL-2 mutant according to claim 9, wherein the mutation of E67 is E67A.

11. The IL-2 mutant according to claim 4, further comprising mutations in N71 and S75 relative to the human wild-type IL-2 amino acid sequence.

12. The IL-2 mutant according to claim 11, wherein the mutations in N71 and S75 are N71W and S75I, respectively.

13. The IL-2 mutant according to claim 5, further comprising a mutation in E67 and / or V91 relative to the human wild-type IL-2 amino acid sequence. The IL-2 mutant according to claim 13 , wherein the E67 mutation is E67T. The IL-2 mutant according to claim 13 , wherein the mutation of V91 is V91K.

16. The IL-2 mutant according to claim 13, wherein the mutations of E67 and V91 are E67T and V91K, respectively.

17. The IL-2 mutant according to claim 5, further comprising mutations in L70 and S75 relative to the human wild-type IL-2 amino acid sequence.

18. The IL-2 mutant according to claim 17, wherein the mutations of L70 and S75 are L70I and S75F, respectively.

19. The IL-2 mutant according to claim 5, further comprising a mutation at N71 relative to the human wild-type IL-2 amino acid sequence.

20. The IL-2 mutant according to claim 19, wherein the mutation of N71 is N71I.

21. The IL-2 mutant according to claim 6, further comprising mutations at E67 and N71 relative to the human wild-type IL-2 amino acid sequence.

22. The IL-2 mutant according to claim 21, wherein the mutations of E67 and N71 are E67M and N71S, respectively.

23. The IL-2 mutant according to any one of claims 1 to 22, further comprising a mutation of C125 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutation of C125 is C125S or C125A.

24. An IL-2 mutant, characterized in that The mutant comprises an amino acid sequence shown in any one of SEQ ID NOs: 5-18 or a variant thereof, wherein the variant has at least about 90% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 5-18.

25. An IL-2 mutant comprising mutations of amino acid residue S at position 75 (S75) and amino acid residue R at position 83 (R83) relative to the amino acid sequence of human wild-type IL-2.

26. The IL-2 mutant according to claim 25, wherein the amino acid sequence of human wild-type IL-2 is shown in SEQ ID NO:

1.

27. The IL-2 mutant according to claim 25 or 26, wherein S75 is mutated to an uncharged amino acid residue.

28. The IL-2 mutant according to any one of claims 25 to 27, wherein the mutation of S75 is S75P, S75F, S75Y, S75G, S75A, S75V, S75T, S75I or S75L, and the mutation of R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K or R83E. The IL-2 mutant according to claim 28 , wherein the mutation of S75 is S75P, and the mutation of R83 is R83Y or R83A.

30. The IL-2 mutant according to claim 29, further comprising mutations in E67 and D84 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutations in E67 and D84 are E67A and D84N, respectively; more preferably, the mutant comprises mutations S75P, R83Y, E67A and D84N.

31. The IL-2 mutant according to claim 29, further comprising mutations in H16, L70 and N71 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutations in H16, L70 and N71 are H16D, L70I and N71G, respectively; more preferably, the mutant comprises mutations S75P, R83A, H16D, L70I and N71G.

32. The IL-2 mutant according to claim 28, wherein the mutation of S75 is S75F, and the mutation of R83 is R83P or R83L.

33. The IL-2 mutant according to claim 32, further comprising a mutation in H16 and / or N71 relative to the human wild-type IL-2 amino acid sequence.

34. The IL-2 mutant according to claim 33, wherein the mutation of H16 is H16Q; preferably, the mutant comprises mutations S75F, R83P and H16Q.

35. The IL-2 mutant according to claim 33, wherein the mutations in H16 and N71 are H16N and N71S, respectively; preferably, the mutant comprises mutations S75F, R83P, H16N and N71S.

36. The IL-2 mutant according to claim 32, further comprising mutations in E67 and L70 relative to the human wild-type IL-2 amino acid sequence; preferably, wherein the mutations in E67 and L70 are E67R and L70V, respectively; more preferably, the mutant comprises mutations S75F, R83L, E67R and L70V. The IL-2 mutant according to claim 28 , wherein the mutation of S75 is S75I, and the mutation of R83 is R83L or R83K.

38. The IL-2 mutant according to claim 37, further comprising mutations of E67 and L70 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutations of E67 and L70 are E67G and L70V, or E67S and L70V, respectively; more preferably, the mutant comprises mutations S75I, R83L, E67G and L70V, or comprises mutations S75I, R83K, E67S and L70V. The IL-2 mutant according to claim 28 , wherein the mutation of S75 is S75T, and the mutation of R83 is R83A or R83F.

40. The IL-2 mutant according to claim 39, further comprising mutations in H16 and E67 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutations in H16 and E67 are H16N and E67K, respectively; more preferably, the mutant comprises mutations S75T, R83A, H16N and E67K. The IL-2 mutant according to claim 28 , wherein the mutation of S75 is S75V, and the mutation of R83 is R83N or R83F.

42. The IL-2 mutant according to claim 41, further comprising mutations in E15 and H16 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutations in E15 and H16 are E15K and H16Q, respectively; more preferably, the mutant comprises mutations S75V, R83F, E15K and H16Q.

43. The IL-2 mutant according to claim 41, further comprising mutations in L70 and V91 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutations in L70 and V91 are L70F and V91K, respectively; more preferably, the mutant comprises mutations S75V, R83N, L70F and V91K.

44. The IL-2 mutant according to claim 25 or 26, comprising the following mutation: S75G, R83L, L17I, V91E, and I92L; or S75Y, R83N, L17I, and V91K; or S75A, R83K, and L17I; or S75L, R83E, L63V and E67F.

45. The IL-2 mutant according to any one of claims 25 to 44, further comprising a mutation of C125 relative to the human wild-type IL-2 amino acid sequence; preferably, the mutation of C125 is C125S or C125A.

46. ​​An IL-2 mutant, characterized in that The mutant comprises an amino acid sequence shown in any one of SEQ ID NOs: 19-34 or a variant thereof, wherein the variant has at least about 90% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 19-34.

47. The IL-2 mutant according to any one of claims 1 to 46, which has reduced binding affinity to IL-2Rβγ compared to human wild-type IL-2, while retaining binding to IL-2Rα.

48. A fusion protein comprising the IL-2 mutant of any one of claims 1-47.

49. The fusion protein according to claim 48, comprising Fc; preferably, wherein the Fc comprises a mutation that can alter effector function, and / or a mutation that can prolong half-life, and / or a mutation that can promote dimer formation of heterologous polypeptides; more preferably, the Fc is derived from human IgG1 or human IgG4.

50. The fusion protein of claim 49, wherein the Fc comprises: (i) L234A and L235A mutations; and / or (ii) N297G mutation; and / or (iii) N297A mutation; and / or (iv) L234A, L235A and P331S mutations; and / or (v) L234A, L235E, G237A, A330S and P331S mutations, wherein the numbering is according to the EU numbering system.

51. The fusion protein of claim 49, wherein the Fc comprises S228P, F234A, and L235A mutations, wherein the numbering is the EU numbering system.

52. The fusion protein of any one of claims 49-51, wherein the Fc further comprises: (i) S354C and T366W mutations; and / or (ii) Y349C, T366S, L368A, and Y407V mutations, wherein the numbering is the EU numbering system.

53. The fusion protein according to any one of claims 49-52, wherein the IL-2 mutant is linked to Fc; preferably, the IL-2 mutant is linked to Fc via a connecting peptide; preferably, the connecting peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 43-74; more preferably, the connecting peptide comprises the amino acid sequence GGGGS (SEQ ID NO: 52).

54. The fusion protein according to any one of claims 49 to 53, wherein the IL-2 mutant is located at the N-terminus and / or C-terminus of Fc.

55. A fusion protein comprising an IL-2 mutant, characterized in that The fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 76-105 or a variant thereof, wherein the variant has at least about 80% sequence homology with the amino acid sequence shown in any one of SEQ ID NOs: 76-105.

56. A nucleic acid encoding the IL-2 mutant of any one of claims 1-47 or the fusion protein of any one of claims 48-55.

57. A vector comprising the nucleic acid of claim 56.

58. A host cell comprising the vector of claim 57.

59. A method for preparing an IL-2 mutant or a fusion protein comprising an IL-2 mutant, comprising: a) culturing the host cell according to claim 58 under conditions that effectively express the IL-2 mutant or a fusion protein comprising the IL-2 mutant; and b) obtaining the expressed IL-2 mutant or the fusion protein comprising the IL-2 mutant from the host cell.

60. A pharmaceutical composition comprising the IL-2 mutant of any one of claims 1-47, the fusion protein of any one of claims 48-55, the nucleic acid of claim 56, the vector of claim 57, the host cell of claim 58, or the IL-2 mutant or fusion protein comprising the IL-2 mutant prepared by the method of claim 59, and a pharmaceutically acceptable carrier and / or excipient.

61. A method for treating and / or preventing a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of the IL-2 mutant of any one of claims 1-47, or the fusion protein of any one of claims 48-55, or the nucleic acid of claim 56, or the vector of claim 57, or the host cell of claim 58, or the IL-2 mutant or fusion protein comprising the IL-2 mutant prepared by the method of claim 59, or the pharmaceutical composition of claim 60.

62. The method of claim 61, wherein the disease or condition comprises an inflammatory disease or an autoimmune disease.

63. The method of claim 61 or 62, wherein the disease or condition comprises lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, atopic disease, 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.

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