WO2023241653A1 - 白介素-2(il-2)突变体及其用途 - Google Patents

白介素-2(il-2)突变体及其用途 Download PDF

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WO2023241653A1
WO2023241653A1 PCT/CN2023/100442 CN2023100442W WO2023241653A1 WO 2023241653 A1 WO2023241653 A1 WO 2023241653A1 CN 2023100442 W CN2023100442 W CN 2023100442W WO 2023241653 A1 WO2023241653 A1 WO 2023241653A1
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mutant
amino acid
mutations
acid sequence
type
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PCT/CN2023/100442
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French (fr)
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李平
李楠
晏丽
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舒泰神（北京）生物制药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • 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, and Pharmaceutical compositions and therapeutic uses thereof, and methods for preparing IL-2 mutants and fusion proteins thereof are further provided.
  • IL-2 interleukin-2
  • Interleukin-2 also known as T cell growth factor (TCGF)
  • TCGF T cell growth factor
  • A, B, C, D ⁇ -helices
  • IL-2 achieves its function by binding to IL-2 receptors on the surface of target cells.
  • IL-2 has three receptor subunits: ⁇ chain (IL-2R ⁇ or CD25), ⁇ chain (IL-2R ⁇ or CD122) and universal cytokine receptor ⁇ chain ( ⁇ c or IL-2R ⁇ or CD132)
  • ⁇ chain IL-2R ⁇ or CD25
  • IL-2R ⁇ or CD122 ⁇ chain
  • ⁇ c or IL-2R ⁇ or CD132 universal cytokine receptor ⁇ chain
  • IL-2 receptors can be divided into the following types:
  • the first type is a low-affinity receptor, consisting of a separate IL-2R ⁇ subunit (Kd value Approximately 10 nM), since IL-2R ⁇ does not have an intracellular signaling domain, IL-2 cannot transmit signals and regulate cells by binding to this type of receptor (HM Wang, KA Smith, J. Exp. Med. 166, 1055 (1987)) .
  • the second type is a medium-affinity receptor, composed of IL-2R ⁇ and ⁇ c subunits (Kd value is about 1nM). This type of receptor is mainly expressed on natural killer cells, macrophages and resting T cells.
  • IL-2R ⁇ alone has a very low affinity for IL-2 (Kd value approximately 100 nM), ⁇ c alone has almost no detectable binding to IL-2 (M. Rickert, MJ Boulanger, N. Goriatcheva, KC Garcia, J .Mol.Biol.339,1115(2004)), but by IL-2R ⁇ and ⁇ c The complex formed (IL-2R ⁇ ) is necessary for effective cell signaling by IL-2 (Y.
  • the third type is a high-affinity receptor, consisting of three subunits: IL-2R ⁇ , IL-2R ⁇ and ⁇ c (Kd value is about 10pM). It is usually found on activated lymphocytes and CD4 + CD25 + Foxp3 + 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).
  • IL-2 can enhance the function of effector T cells and natural killer cells, it can be used as an immune activator to treat lymphocyte proliferation-related 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 realizing the function, development and induction of suppressive characteristics of Treg cells.
  • Treg cells are typically defined by their expression of CD4, CD25, and the transcription factor forkhead P3 (Foxp3), which suppress autoreactive lymphocytes and regulate innate and adaptive immune responses, leading to immunosuppression.
  • Treg cells are one of the key effector cells of immunosuppression and play a crucial role in inducing and maintaining peripheral self-tolerance to antigens (including those expressed by tumors), and are important in the occurrence and development of autoimmune diseases. significance.
  • Molek 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).
  • IL-2 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 of IL-2 therapy and/or improve its Efficacy and improving the safety window of IL-2 in treating specific diseases are of great significance to improving the safety and effectiveness of IL-2 in treating diseases. There is still a need in this field to develop new IL-2 molecules with improved properties.
  • One aspect of 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 human wild-type IL-2 amino acid sequence.
  • the amino acid sequence of human wild-type IL-2 is shown in SEQ ID NO: 1.
  • the IL-2 mutant described in the present application contains all deletions of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutants described in the present application further comprise mutations of E67 and N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and N71 are E67K and N71S, respectively.
  • this application Please describe the IL-2 mutant, which contains all deletions of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence, and E67K and E67K relative to the human wild-type IL-2 amino acid sequence. N71S mutation.
  • the IL-2 mutant described in the present application includes amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RPL. In some embodiments, the IL-2 mutants described in the present application further comprise a mutation of E67 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of E67 is E67A. In some embodiments, the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RPL, and relative to human wild-type IL -E67A mutation of the 2 amino acid sequence.
  • the IL-2 mutants described in the present application 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 amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence.
  • the mutations of N71 and S75 are N71W and S75I, respectively.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RPL, and relative to human wild-type IL -2 N71W and S75I mutations of the amino acid sequence.
  • the IL-2 mutant described in the present application includes amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RHL.
  • the IL-2 mutants described in the present application further comprise mutations of E67 and/or V91 relative to the human wild-type IL-2 amino acid sequence.
  • the mutation of E67 is E67T.
  • the mutation of V91 is V91K.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and relative to human wild-type IL -2 E67T and/or V91K mutations of the amino acid sequence.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and further comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence.
  • the mutations of L70 and S75 are L70I and S75F, respectively.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and relative to human wild-type IL -2 L70I and S75F mutations of the amino acid sequence.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and further comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence.
  • the mutation of N71 is N71I.
  • the IL-2 mutant described in the present application includes amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to SPL. In some embodiments, the IL-2 mutants described in the present application further comprise mutations of E67 and N71 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutations of E67 and N71 are E67M and N71S, respectively. In some embodiments, the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to SPL, and relative to human wild-type IL -2 E67M and N71S mutations in the amino acid sequence.
  • the IL-2 mutants described in the present application in addition to the above mutations, further comprise a C125 mutation relative to the amino acid sequence of human wild-type IL-2.
  • the C125 mutation is C125S or C125A.
  • the IL-2 mutants described in the present application comprise the amino acid sequence shown in any one of SEQ ID NOs: 5-18 or a variant thereof, which variant is the same as that in SEQ ID NOs: 5-18 Any of the amino acid sequences shown has at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology.
  • Another aspect of the present application relates to another IL-2 mutant comprising amino acid residue S at position 75 (S75) and amino acid residue R at position 83 (R83) relative to the human wild-type IL-2 amino acid sequence. mutation.
  • the amino acid sequence of human wild-type IL-2 is shown in SEQ ID NO: 1.
  • the IL-2 mutants described herein are wherein S75 is mutated to an uncharged amino acid residue.
  • the mutation of S75 is S75P, S75F, S75Y, S75G, S75A, S75V, S75T, S75I or S75L.
  • the mutation of R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K, or R83E.
  • the IL-2 mutant described in the present application has a mutation of S75 to S75P and a mutation of R83 to R83Y.
  • the IL-2 mutants described in the present application further comprise mutations of E67 and D84 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and D84 are E67A and D84N, respectively.
  • the IL-2 mutants described herein comprise S75P, R83Y, E67A and D84N mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application has a mutation of S75 to S75P and a mutation of R83 to R83A.
  • the IL-2 mutants described in the present application further comprise mutations of H16, L70 and N71 relative to the amino acid sequence of human wild-type IL-2.
  • H16, L70 The mutations of N71 and N71 are H16D, L70I and N71G respectively.
  • 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.
  • the IL-2 mutant described in the present application has a mutation of S75 to S75F and a mutation of R83 to R83P.
  • the IL-2 mutants described in the present application further comprise mutations of H16 and/or N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutation of H16 is H16Q or H16N.
  • the mutation of N71 is N71S.
  • the IL-2 mutants described herein comprise S75F, R83P and H16Q mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutants described herein comprise S75F, R83P, H16N and N71S mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application has a mutation of S75 to S75F and a mutation of R83 to R83L.
  • the IL-2 mutants described in the present application further comprise mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and L70 are E67R and L70V, respectively.
  • the IL-2 mutants described herein comprise S75F, R83L, E67R and L70V mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application wherein the mutation of S75 is S75I, and the mutation of R83 is R83L.
  • the IL-2 mutants described in the present application further comprise mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and L70 are E67G and L70V, respectively.
  • the IL-2 mutants described herein comprise S75I, R83L, E67G and L70V mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application wherein the mutation of S75 is S75I, and the mutation of R83 is R83K.
  • the IL-2 mutants described in the present application further comprise mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and L70 are E67S and L70V, respectively.
  • the IL-2 mutants described herein comprise S75I, R83K, E67S and L70V mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application wherein the mutation of S75 is S75T, and the mutation of R83 is R83A or R83F.
  • the IL-2 mutants described in the present application further comprise mutations of H16 and E67 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of H16 and E67 are H16N and E67K respectively.
  • the IL-2 mutants described herein comprise S75T, R83A, H16N and E67K mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application has a mutation of S75 to S75V and a mutation of R83 to R83F.
  • the IL-2 mutants described in the present application further comprise mutations of E15 and H16 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E15 and H16 are E15K and H16Q, respectively.
  • the IL-2 mutants described herein comprise S75V, R83F, E15K and H16Q mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application has a mutation of S75 to S75V and a mutation of R83 to R83N.
  • the IL-2 mutants described in the present application further comprise mutations of L70 and V91 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L70 and V91 are L70F and V91K respectively.
  • the IL-2 mutants described herein comprise S75V, R83N, L70F and V91K mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application has a mutation of S75 to S75G and a mutation of R83 to R83L.
  • the IL-2 mutants described in the present application further comprise mutations of L17, V91 and I92 relative to the human wild-type IL-2 amino acid sequence.
  • the mutations of L17, V91, and I92 are L17I, V91E, and I92L, respectively.
  • 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.
  • the IL-2 mutant described in the present application wherein the mutation of S75 is S75Y, and the mutation of R83 is R83N.
  • the IL-2 mutants described in the present application further comprise mutations of L17 and V91 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L17 and V91 are L17I and V91K, respectively.
  • the IL-2 mutants described herein comprise S75Y, R83N, L17I and V91K mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application wherein the mutation of S75 is S75A, and the mutation of R83 is R83K.
  • the IL-2 mutants described in the present application further comprise a mutation of L17 relative to the amino acid sequence of human wild-type IL-2.
  • the mutation of L17 is L17I.
  • the IL-2 mutants described herein comprise S75A, R83K and L17I mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application has a mutation of S75 to S75L and a mutation of R83 to R83E.
  • the IL-2 mutants described in the present application further comprise mutations of L63 and E67 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L63 and E67 are L63V and E67F, respectively.
  • the IL-2 mutants described herein comprise S75L, R83E, L63V and E67F mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutants described in the present application in addition to the above-mentioned mutations, further comprise mutations relative to the C125 amino acid sequence of human wild-type IL-2.
  • the C125 mutation is C125S or C125A.
  • the IL-2 mutants described in the present application comprise the amino acid sequence shown in any one of SEQ ID NOs: 19-34 or a variant thereof, which variant is the same as that in SEQ ID NOs: 19-34 Any of the amino acid sequences shown has at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology.
  • the IL-2 mutants described herein have reduced binding affinity to IL-2R ⁇ compared to wild-type IL-2 and are able to maintain binding to IL-2R ⁇ .
  • the fusion protein includes Fc.
  • the Fc includes mutations that alter effector function, and/or mutations that extend half-life, and/or mutations that promote dimer formation of heterologous polypeptides.
  • the Fc is of human origin.
  • the Fc is derived from human IgG, including IgG1, IgG2, IgG3 or IgG4, preferably IgG1 or IgG4.
  • the Fc is derived from a 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 the P331S mutation, where the numbering is the EU numbering system.
  • the Fc is derived from human IgG4 comprising mutations S228P, F234A and L235A, wherein the numbering is EU numbering system.
  • the Fc further comprises S354C and T366W mutations; and/or Y349C, T366S, L368A, and Y407V mutations, wherein the numbering is EU numbering system.
  • the IL-2 mutant and Fc are linked through a linking peptide.
  • the connecting peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 43-74.
  • the linking peptide comprises the amino acid sequence GGGGS (SEQ ID NO:52).
  • the fusion protein of IL-2 mutant and Fc described in the present application wherein the IL-2 mutant is located at the N-terminal and/or C-terminal of Fc.
  • the fusion protein described in the present application includes the amino acid sequence shown in any one of SEQ ID NOs: 76-105 or a variant thereof, and the variant is identical to any one of SEQ ID NOs: 76-105.
  • the present application relates to isolated nucleic acids encoding any IL-2 mutants or fusion proteins comprising IL-2 mutants described herein, 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 articles of manufacture comprising any IL-2 mutant or fusion protein comprising an IL-2 mutant described herein.
  • vectors comprising the nucleic acids
  • host cells comprising the nucleic acids or vectors
  • compositions e.g., pharmaceutical compositions
  • kits, and articles of manufacture comprising any IL-2 mutant or fusion protein comprising an IL-2 mutant described herein.
  • IL-2 mutant or fusion protein thereof described herein for the treatment of inflammatory or autoimmune diseases (e.g., lupus, Graft versus host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopy, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolysis anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, and Behcet's disease).
  • inflammatory or autoimmune diseases e.g., lupus, Graft versus host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopy, asthma, inflammatory bowel disease,
  • Figure 1A shows a schematic diagram of an exemplary structure of a bivalent fusion protein of IL-2 mutant and Fc, which contains two identical monomers, and the IL-2 mutant molecule in each monomer is connected to the N-terminus of Fc.
  • Figure 1B shows a schematic diagram of an exemplary structure of a bivalent fusion protein of IL-2 mutant and Fc, which contains two identical monomers, and the IL-2 mutant molecule in each monomer is connected to the C-terminus of Fc. .
  • Figure 2 shows a schematic diagram of an exemplary structure of a monovalent fusion protein of IL-2 mutant and Fc, which contains two different monomers, one of which contains an IL-2 mutant molecule linked to the Fc knob. At the C-terminal, the other monomer is the Fc hole molecule.
  • Figures 3A, 3C, 3E, and 3G show the bivalent fusion proteins of the first group of IL-2 mutants and Fc respectively: Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc- Results of (81-85)-mut2, Fc-(81-85)-mut3, Fc-(81-85)-mut9 and Fc-(81-85)-mut14 inducing STAT5 phosphorylation in Treg cells;
  • Figure 3B , 3D, 3F, and 3H show the bivalent fusion proteins of the first group of IL-2 mutants and Fc respectively: Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-(81 Results of -85)-mut2, Fc-(81-85)-mut3, Fc-(81-85)-mut9 and Fc-(81-85)-mut14 inducing STAT5 phosphorylation in CD8 + T lymphocytes.
  • Figure 3I shows the results of the second group of bivalent fusion proteins of IL-2 mutants and Fc: Fc-S75R83-mut7 and Fc-S75R83-mut16 inducing STAT5 phosphorylation in Treg cells;
  • Figure 3J shows the second Group IL-2 mutations Results of bivalent fusion proteins of body and Fc: Fc-S75R83-mut7 and Fc-S75R83-mut16 inducing STAT5 phosphorylation in CD8 + T lymphocytes.
  • Figure 4A shows the first group of bivalent fusion proteins of IL-2 mutants and Fc: Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)- mut12 stimulates the proliferation of Treg cells in mice at a dose of 1 mg/kg.
  • Figure 4B shows the first group of bivalent fusion proteins of IL-2 mutants and Fc: Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)- Effect of mut12 on CD8 + T lymphocyte proliferation in mice at a dose of 1 mg/kg.
  • Figure 4C shows the bivalent fusion proteins Fc-S75R83-mut5, Fc-S75R83-mut9 and Fc-S75R83-mut16 of the second group of IL-2 mutants and Fc in mice at a dose of 1 mg/kg. Stimulate the proliferation of Treg cells.
  • Figure 4D shows the bivalent fusion proteins Fc-S75R83-mut5, Fc-S75R83-mut9 and Fc-S75R83-mut16 of the second group of IL-2 mutants and Fc in mice at a dose of 1 mg/kg. Effect on CD8 + T lymphocyte proliferation.
  • Figure 5A shows the bivalent fusion proteins Fc-(81-85)-mut8 and Fc-(81-85)-mut10 of the first group of IL-2 mutants and Fc when the dosage is 0.3 mg/kg. Stimulation of Treg cell proliferation in cynomolgus monkeys.
  • Figure 5B shows the bivalent fusion proteins Fc-(81-85)-mut8 and Fc-(81-85)-mut10 of the first group of IL-2 mutants and Fc when the dosage is 0.3 mg/kg. Effects on CD8 + T lymphocyte proliferation in cynomolgus monkeys.
  • Figure 5C shows the bivalent fusion protein Fc-S75R83-mut5 of the second group of IL-2 mutants and Fc at a dosage of 0.3 mg/kg and Fc-S75R83-mut16 at a dosage of 0.3 mg/kg respectively. And at 1.5 mg/kg, it stimulates the proliferation of Treg cells in cynomolgus monkeys.
  • Figure 5D shows the bivalent fusion protein Fc-S75R83-mut5 of the second group of IL-2 mutants and Fc at a dosage of 0.3 mg/kg and Fc-S75R83-mut16 at a dosage of 0.3 mg/kg respectively. and the effect on CD8 + T lymphocyte proliferation in cynomolgus monkeys at 1.5 mg/kg.
  • Figure 6 shows the PK of exemplary IL-2 mutants and Fc bivalent fusion proteins Fc-(81-85)-mut10 and Fc-S75R83-mut16 in mice at a dose of 1 mg/kg. curve.
  • the present application relates to IL-2 mutants, fusion proteins containing IL-2 mutants, and the like.
  • the IL-2 mutant described in this application can selectively activate Treg cells and stimulate Treg cell proliferation. have a higher safety window for certain diseases (e.g., inflammatory or autoimmune diseases) (e.g., in HEK-Blue TM IL-2 cell line screening experiments, compared with wild-type IL-2, by HEK -The EC50 value of the Blue TM CD122/CD132 cell line to detect the binding of IL-2 mutants to IL-2R ⁇ and the EC50 value of the HEK-Blue TM IL-2 cell line to detect the binding of IL-2 mutants to IL-2R ⁇ higher ratio).
  • diseases e.g., inflammatory or autoimmune diseases
  • HEK-Blue TM IL-2 cell line screening experiments compared with wild-type IL-2, by HEK -The EC50 value of the Blue TM CD122/CD132 cell line to detect the binding of
  • the fusion protein of IL-2 mutant and Fc described in this application reduces or minimizes antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (ADCC) by modifying the Fc fragment.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCC complement-dependent cytotoxicity
  • the present application also relates to nucleic acids encoding the IL-2 mutants or fusion proteins comprising the IL-2 mutants, vectors comprising the nucleic acids, host cells comprising the nucleic acids or vectors, and the above-mentioned IL-2 mutants or Methods for producing fusion proteins, pharmaceutical compositions and manufactured articles containing the same, and use of the IL-2 mutants or fusion proteins or pharmaceutical compositions containing IL-2 mutants to treat diseases (e.g., lupus, graft versus host) disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopy, asthma or inflammatory bowel disease).
  • diseases e.g., lupus, graft versus host
  • wild-type IL-2 or "WT IL-2” refers to naturally occurring IL-2, which may be derived from natural IL-2 from any vertebrate source, such as mammals, including but not Restricted to domestic animals (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 (eg, containing a signal peptide) and processed mature IL-2 forms (eg, not containing a signal peptide).
  • the term also includes naturally occurring IL-2 allelic variants and splice variants, isoforms, homologues, and species homologues.
  • the term also includes naturally occurring IL-2 variants and the like.
  • the naturally occurring IL-2 variant has at least 95% (eg, 95%, 96%, 97%, 98% or 99%) sequence homology to native IL-2.
  • an exemplary human wild-type IL-2 amino acid sequence is set forth in SEQ ID NO: 1.
  • unprocessed human wild-type IL-2 additionally includes an N-terminal 20 amino acid signal peptide, an exemplary amino acid sequence of which is shown in SEQ ID NO: 4.
  • wild-type IL-2 may further comprise one or more amino acid mutations that do not change the binding effect to the IL-2 receptor, for example, introduced at amino acid residue 125 of wild-type human IL-2.
  • Serine mutation C125S
  • Serine mutation can prevent mismatching or aggregation caused by cysteine. Its exemplary amino acid sequence is shown in SEQ ID NO:2; introducing alanine mutation (C125A) at position 125 can promote efficient folding and improve Expression, its exemplary amino acid sequence is shown in SEQ ID NO:3.
  • “Mutation” of amino acids as described in this application includes substitutions, deletions and insertions. Any combination of substitutions, deletions and insertions can be made to obtain a construct with desired properties (eg, reduced binding affinity to IL-2R ⁇ ).
  • deletions or insertions of amino acids include deletions or insertions in the polypeptide sequence.
  • amino acid substitutions may be conservative amino acid substitutions.
  • the amino acid substitution may be a non-conservative amino acid substitution, that is, one amino acid is replaced with another amino acid having different structural and/or chemical properties.
  • Amino acid substitutions also include the use of non-naturally occurring amino acids or naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5- hydroxylysine) is replaced.
  • amino acid mutations include a combination of amino acid substitutions and deletions, for example, within a certain region of the polypeptide sequence (for example, amino acid residues 81-85 RPRDL), including both amino acid substitutions and deletions.
  • 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 other methods.
  • IL-2 mutants as described in this application include one or more substitutions, deletions, insertions, or any form thereof in the wild-type IL-2 amino acid sequence (e.g., SEQ ID NO. 1). combination.
  • an "IL-2 mutant” described herein is capable of reducing its binding affinity to IL-2R ⁇ and maintaining binding to IL-2R ⁇ .
  • the positions of amino acid mutations were determined based on the amino acid positions in wild-type IL-2.
  • the amino acid sequence of wild-type IL-2 is as shown in SEQ ID NO: 1, and can be obtained by SEQ ID NO: 1 is compared to the amino acid sequence (for example, using BLAST) to identify the position corresponding to the amino acid mutation in the IL-2 mutant.
  • S75 the amino acid residue represented at position 75 relative to the wild-type IL-2 amino acid sequence is serine (S). "Mutation of S75" represents a mutation in the serine residue (S) at position 75. In some embodiments, when IL-2 mutants are described, this is done as follows.
  • amino acid substitution is expressed as "original amino acid residue/substituted amino acid position/substituted amino acid residue"
  • S75G represents the amino acid sequence relative to wild-type IL-2 (for example, SEQ ID NO. 1)
  • the serine residue (S) at position 75 is replaced by a glycine residue (G).
  • G glycine residue
  • S75V+R83N represents the amino acid sequence relative to the wild-type IL-2 (for example, SEQ ID NO. .1)
  • the serine residue (S) at position 75 is replaced by a valine residue (V)
  • the arginine residue (R) at position 83 is replaced by an asparagine residue (N).
  • IL-2 mutants “selectively activate Treg cells” means that they can more effectively activate Treg cells in the presence of other T cell subsets (such as CD4 + T cells, CD8 + T cells) or NK cells. Specifically target Treg cells and activate Treg cells.
  • the ability of IL-2 mutants to "activate Treg cells” can be determined by methods known in the art or by methods disclosed in the examples of this application, including but not limited to: IL-2 mutants induce IL-2 in Treg cells Receptor signal transduction or induction of Treg cell proliferation.
  • IL-2 receptor signaling can be defined by STAT5 phosphorylation levels.
  • STAT5 phosphorylation is an essential 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 activated Treg cells.
  • Induced proliferation can be defined by changes in the number of Treg cells following IL-2 stimulation, for example by measuring an increase in the number of Treg cells in a mixed cell population by flow cytometry or by measuring proliferation-associated cell cyclins (e.g. Ki-67) in Treg cells. expression increases.
  • autoimmune disease refers to a non-malignant disease or condition that originates from and targets an individual's own tissues, in which the immune system attacks its own proteins, cells, and tissues.
  • 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, and rheumatoid arthritis.
  • atopy asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, malnutrition Epidermolysis bullosa and Behcet's disease.
  • treatment is an approach to obtain beneficial or desired results, including clinical results.
  • the beneficial or desired results include, but are 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 progression of the disease) ), prevent or delay the spread of the disease, prevent or delay the recurrence of the disease, delay or slow down the progression of the disease, improve the disease state, alleviate the disease (partially or completely), reduce the dose of one or more other drugs required to treat the disease, delay the disease development, improve quality of life and/or extend survival.
  • treatment also includes reducing the pathological consequences of the disease.
  • the methods of the present application contemplate 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 symptoms caused by the disease, improving the quality of life of patients with the disease, reducing the dosage of other medications needed to treat the disease, and/ Or prolong the survival of the individual, the patient is considered to be successfully "treated”.
  • prevention and similar words such as “prevented”, “preventing” and the like refer to methods of preventing, inhibiting or reducing the likelihood of occurrence or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition. As used herein, "prevention” and similar words also include reducing the intensity, impact, symptoms and/or burden of a disease or condition before its occurrence or recurrence, or reducing the likelihood of the occurrence or recurrence of a disease or condition.
  • delaying the progression of a disease means delaying, hindering, slowing down, slowing down, stabilizing and/or postponing the progression of a disease.
  • the length of delay may vary depending on the disease history and/or the individual receiving treatment.
  • a method that "slows" the progression of a disease is one that reduces the probability of development of the disease within a given time frame and/or reduces the extent of the disease within a given time frame compared to not using the method. In some embodiments, this comparison can be based on animal experiments, with observations and/or statistics performed in individual animals. In other embodiments, such comparisons may be based on clinical studies using statistically significant numbers of individuals.
  • an effective amount refers to a dosage of a drug or pharmaceutical composition sufficient to treat a particular disorder, condition, or disease, such as ameliorating, alleviating, attenuating, and/or delaying one or more symptoms.
  • an effective amount is an amount sufficient to delay the progression of disease.
  • an effective amount is an amount sufficient to prevent or delay the onset or recurrence of disease.
  • the effective amount can be administered in one or more doses.
  • an effective amount of a drug or pharmaceutical composition is an amount capable of increasing the number of Treg cells in diseased tissue of a patient.
  • an "individual” or “subject” refers to a mammal including, but not limited to, a human, bovine, equine, cat, dog, rodent, or primate. In some embodiments, the individual is a human.
  • constant region refers to the portion of an immunoglobulin molecule (Ig) as opposed to the "variable region” of the immunoglobulin molecule that contains the antigen-binding site.
  • the constant region has a more conserved amino acid sequence and contains The heavy chain constant region ( CH ), which contains the CH1 , CH2 and CH3 domains, and the light chain constant region ( CL ).
  • CH The heavy chain constant region
  • CL the light chain constant region
  • immunoglobulins can be divided into different classes or subtypes.
  • immunoglobulins There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with the heavy chains being alpha, delta, epsilon, gamma, and mu. Gamma and alpha are further divided into subclasses based on relatively small differences in CH sequence and function. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.
  • IgG immunoglobulins defined by the chemical and antigenic properties of the constant regions.
  • immunoglobulins There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and many of these 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 ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are described in detail in Abbas et al., Cellular and Molecular Immunology, 4th Edition (W.B. Saunders, Co., 2000).
  • Fc Fc domain
  • Fc fragment Fc region
  • Crystallizable fragment are used to define the C-terminal region of an immunoglobulin heavy chain, including native Fc and mutant Fc.
  • Body Fc Exemplary Fcs include the CH2 and CH3 domains of immunoglobulins.
  • the human IgG heavy chain Fc is generally defined as starting at the amino acid residue at position Cys226 or starting at Pro230 and extending to its carboxyl terminus.
  • the C-terminal lysine of the Fc may be removed, for example, during production or purification of the protein, or by recombinant engineering of the nucleic acid encoding the protein.
  • Suitable native sequence Fc regions for use in constructs described herein are derived from, but are not limited to, human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region in an Fc-containing molecule (eg, an antibody or an Fc-containing fusion protein).
  • the preferred FcR is the native human FcR sequence.
  • an exemplary FcR is one that binds an IgG antibody (a gamma receptor) and includes the receptor subclasses Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII, as well as allelic variants and alternatively spliced forms of these receptors.
  • Fc ⁇ RII receptors include Fc ⁇ RIIA (“activating receptor”) and Fc ⁇ RIIB (“inhibitory receptor”), which have similar amino acid sequences and differ mainly in the cytoplasmic domain.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain.
  • the inhibitory receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain (see M. Annu. Rev. Immunol. 15:203-234 (1997)).
  • ITIM immunoreceptor tyrosine inhibitory motif
  • 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 (see, e.g., 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.
  • the half-life of a human FcRn high-affinity binding polypeptide bound 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 mice administered with a polypeptide having a variant Fc region.
  • WO 2004/42072 (Presta) details antibody variants that enhance or weaken binding to FcRs.
  • FcRn see Shield et al., J. Biol. Chem. 9(2):6591-6604 (2001).
  • Fc effector functions refer to those biological activities caused by the Fc region (either a native sequence Fc region or an Fc region containing amino acid sequence mutations) in an Fc-containing molecule (e.g., an antibody or an Fc-containing fusion protein), according to which Fc Varies depending on the source of immunoglobulin subtype.
  • effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor) and B cell activation.
  • Reduced or minimized effector function means an effector function that is reduced by at least 50% (or 60%, 65%, 70%, 75%) compared to wild-type Fc or an unmodified Fc-containing molecule (e.g., antibody) , 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%). Determination of effect 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 cytotoxicity will all be affected.
  • effector function is eliminated by a mutation in the constant region that eliminates glycosylation, e.g., a "no-effector function mutation.”
  • the non-effector functional mutant is N297A or the DANA mutation (D265A+N297A) in the CH2 region, see Shields et al., J. Biol. Chem. 276(9):6591-6604 ( 2001).
  • other mutations leading to reduced or eliminated effector function include: K322A and L234A/L235A (LALA).
  • effector functions can be reduced or eliminated through changes in production technology, such as expression in host cells that do not undergo glycosylation (e.g., E. coli) or host cells that result in altered glycosylation patterns. Pattern changes are ineffective or less effective in promoting effector function (eg, Shinkaw et al., J. Biol. Chem. 278(5):3466-3473 (2003)).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells e.g., natural killer cells Binding to Fc receptors (FcRs) on (NK), neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells carrying antigen (or carrying ligand receptors) and subsequently use the cells Toxin kills target cell.
  • Antibodies or other Fc-containing molecules "arm" the cytotoxic cells and are necessary to kill target cells by this mechanism.
  • the main cells that mediate ADCC include NK cells and monocytes.
  • NK cells only express Fc ⁇ RIII, while monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • Fc expression in hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • an in vitro ADCC assay can be performed, as detailed in US Pat. No. 5,500,362 or 5,821,337. Effector cells suitable for such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK).
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • the ADCC activity of the target molecule can also be assessed in vivo, for example, in animal models as disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).
  • “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 the binding of the first component of the complement system (Clq) to an Fc-containing molecule (of an appropriate subclass) to its cognate receptor via an Fc-fused ligand combine.
  • the CDC assay can be performed as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).
  • U.S. Pat. No. 6,194,551B1 and WO99/51642 detail antibody variants in which the amino acid sequence of the Fc region is altered to increase or decrease C1q binding ability. The contents of these patent publications are incorporated herein by reference. See Idusogie et al. J. Immunol. 164:4178-4184 (2000).
  • Tregs are a subset of CD4 + T cells with significant immunosuppressive effects that can avoid suppressing the immune response of other cells.
  • Tregs are characterized by expressing the ⁇ subunit of IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3), and play an important role in maintaining the body's immune balance 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.
  • effector cells refers to a population of lymphocytes that mediate the cytotoxic effects induced by IL-2. Effector cells mainly include effector T cells such as CD8 + cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages.
  • effector T cells such as CD8 + cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages.
  • the terms “specific binding,”"specificrecognition,” or “specific for” refer to a measurable and reproducible interaction, such as binding between a ligand and a receptor, when present including
  • the presence of ligands can be determined in the case of heterogeneous molecular populations, including biomolecules.
  • a ligand that specifically binds to a receptor will bind the target receptor with greater affinity, affinity, easier, and/or longer duration when compared to binding to other receptors.
  • the ligand binds to the irrelevant receptor to a degree that is less than 10% of the ligand binding to the target receptor, as measured, for example, by radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the ligand that specifically binds the target receptor has an equilibrium dissociation constant (Kd) ⁇ 10 -5 M, ⁇ 10 -6 M, ⁇ 10 -7 M, ⁇ 10 -8 M, ⁇ 10 - 9M , ⁇ 10 -10 M, ⁇ 10 -11 M or ⁇ 10 -12 M.
  • Kd equilibrium dissociation constant
  • a ligand can specifically bind to a receptor that is conserved across species.
  • specific binding may include, but does not require, exclusive binding.
  • the binding specificity of a ligand can be determined experimentally using methods known in the art. Examples include, but are not limited to, Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM -tests and peptide scans.
  • Binding affinity generally refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (eg, a ligand) and its binding partner (eg, a receptor). Unless otherwise stated, "binding affinity” as used herein refers to intrinsic binding affinity, which may 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, measurable by a kinetic selection device, and expressed in units of s -1 .
  • the term "Kon” refers to the binding rate constant of a ligand binding to a receptor to form a ligand/receptor complex, expressed in units of M -1 s -1 .
  • the term equilibrium dissociation constant "Kd” refers to the dissociation constant for a specific ligand-receptor interaction when, in a receptor solution, the ligand occupies half of all receptor binding sites and The ligand concentration required to reach equilibrium is equal to Koff/Kon.
  • the dissociation constant (Kd) can be used as an indicator to reflect the affinity between the ligand and the receptor.
  • the Kd value obtained using the described method is expressed in units of M (mol/L). The prerequisite for measuring Kd is that all binding molecules are in solution.
  • the corresponding dissociation rate constant is expressed as an EC50 value.
  • the EC50 value is a good approximation of Kd.
  • the affinity constant Ka is the reciprocal of the dissociation constant Kd, expressed in units of M -1 .
  • the dissociation constant (Kd) can be used as an indicator to reflect the affinity between the ligand and the receptor.
  • the half-inhibitory concentration is a measure of the effectiveness of a substance (eg, ligand) in inhibiting a specific biological or biochemical function. It indicates how much of a specific drug or other substance (inhibitor, eg, ligand) is required to inhibit a given biological process by half. IC50 values are usually expressed as molar concentrations. The IC50 is comparable to the "EC50" of an agonist drug or other substance (eg, a ligand). EC50 also represents the plasma concentration required to obtain 50% of the maximum effect in the body. As used herein, "IC50" is used to represent the effective concentration of ligand required to neutralize 50% of the biological activity of the receptor in vitro.
  • the IC50 or EC50 can be determined by biometric measurements, such as inhibition of ligand binding by FACS analysis (competitive binding assay), cell-based cytokine release assays, or amplified luminescence homogeneous enzyme-linked immunoassay (AlphaLISA).
  • FACS analysis competitive binding assay
  • cell-based cytokine release assays cell-based cytokine release assays
  • AlphaLISA amplified luminescence homogeneous enzyme-linked immunoassay
  • fusion refers to the direct connection of various components by peptide bonds or the connection of various components through one or more connecting peptides (also known as “linkers”).
  • the sequence of a “linking peptide (or linker)" may be a single amino acid or polypeptide sequence.
  • the connecting peptide (or linker) comprises or consists of a glycine-serine linker.
  • glycine-serine linker refers to a linker composed of glycine and serine Peptide composed of residues.
  • Exemplary glycine-serine linkers include amino acid sequences of the general formula (Gly4Ser)n, where n is a positive integer (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • GGGGS amino acid sequences of the general formula (Gly4Ser)n, where n is a positive integer (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • a preferred glycine-serine linker is GGGGS.
  • a "covalent bond” refers to a stable bond formed between two atoms through the sharing of 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 bound by one or more disulfide bonds. One or more disulfide bonds between two fragments may be formed by linking thiol groups in the two fragments.
  • one or more disulfide bonds may be formed between one or more cysteines of two Fc fragments. Oxidation of two thiol groups forms a disulfide bond.
  • the covalent linkage is directly linked by a covalent bond. In some embodiments, the covalent linkage is directly by peptide bonds or disulfide bonds.
  • derivative refers to a molecule that has a certain protein amino acid sequence or an analog thereof, but also has one or more amino acid groups, an alpha carbon atom, an amino terminus, or a carboxyl terminus. Extra touch-ups.
  • modifications include, but are not limited to, chemical modifications, amino acid side group modifications, amino terminal modifications, and carboxyl terminal modifications. Chemical modifications include, but are not limited to, adding chemical groups, generating new chemical bonds, and removing chemical groups.
  • Modifications of amino acid side groups include, but are not limited to, epsilon aminoacylation of lysine, N-alkylation of arginine, histidine, or lysine, and carboxyalkylation of glutamic acid or aspartic acid. , deamination of glutamine or asparagine. Modifications to the amino terminus include, but are not limited to, deamination, N-lower alkyl, N-dilower alkyl and N-acyl modifications. Modifications to the carboxyl terminus include, but are not limited to, amide, lower alkyl acyl, dialkyl amide, and lower alkyl ester modifications.
  • the lower alkyl group is a C1-C4 alkyl group.
  • one or more side or terminal groups may be protected by one skilled in the art of chemistry using known protecting groups.
  • the alpha carbon of an amino acid can be monomethylated or dimethylated.
  • percent amino acid sequence identity (%) or "homology" of a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to a specific polypeptide or polypeptide sequence, when the sequences are aligned and gaps are introduced. (if necessary) to maximize percent sequence identity and not consider any conservative substitutions as part of the sequence identity.
  • various alignment methods within the technical scope of the art can be used, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software.
  • One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared.
  • C-terminus of a polypeptide refers to the last amino acid residue of the polypeptide, the amine group of which forms a peptide bond with the carboxyl group of its adjacent amino acid residue.
  • N-terminus of a polypeptide refers to the first amino acid residue of the polypeptide, the carboxyl group of which forms a peptide bond with the amine group of its adjacent amino acid residue.
  • an "isolated" polypeptide refers to a polypeptide that has been identified, separated and/or recovered from components of the environment in which it was produced (eg, natural or recombinant).
  • the isolated polypeptide is free from all other components of its production environment. Contaminating components of the manufacturing environment, such as those produced by recombinantly transfected cells, often interfere with peptide research, diagnosis, or treatment, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified to: (1) the polypeptide content is greater than 95% by weight, as determined by the Lowry method, and in some embodiments, the polypeptide content is greater than 99% by weight; (2) By using a spin cup sequencer to a degree sufficient to obtain at least 15 N-terminal residues or internal amino acid sequences; or (3) by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining achieve homogeneity.
  • Isolated polypeptides include polypeptides in situ within recombinant cells because at least one element of the polypeptide's natural environment is not present. Typically, however, an isolated peptide undergoes at least one purification step.
  • amino acid is used herein in its broadest definition to include 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, those skilled in the art will find that amino acids described herein include, for example, natural L-amino acids that form proteins; D-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; do not form Natural amino acids of proteins, such as norleucine, ⁇ -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 through metabolic pathways into a peptide, polypeptide or protein of the cell.
  • An "isolated" nucleic acid molecule encoding a protein or polypeptide is one identified from a nucleic acid molecule containing at least one impurity and isolated nucleic acid molecules, said impurity nucleic acid molecules often associated with the environment in which it was produced.
  • the isolated nucleic acid is free from all components of its production environment.
  • An isolated nucleic acid molecule encoding a polypeptide as described herein exists in a form or morphology other than that in which it is found in nature.
  • an isolated nucleic acid molecule is different from the nucleic acid naturally occurring in a cell encoding a polypeptide described herein.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.
  • control sequences refers to DNA sequences necessary for expression in a particular host organism of an operably linked coding sequence.
  • suitable control sequences for prokaryotes include a promoter, optional operator sequences, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
  • a nucleic acid is "operably linked" to another nucleic acid sequence when the nucleic acid establishes a functional relationship with the other nucleic acid sequence.
  • the DNA encoding the presequence or secretion leader sequence participates in the secretion process of a polypeptide expressed as a precursor protein, it is operably connected to the DNA encoding the polypeptide molecule; if the 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.
  • operably linked means that the linked DNA sequences are contiguous and, for a secretion leader, are not only contiguous but also in the reading phase. However, enhancers need not be contiguous. Ligation is accomplished by ligation at appropriate restriction sites. If no such sites exist, synthetic oligonucleotide aptamers or linkers are used following conventional practice.
  • vector refers to a nucleic acid molecule 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.”
  • transfection refers 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.
  • the cells include primary test cells and their progeny.
  • host cell refers to cells into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny resulting therefrom, regardless of passage number.
  • the progeny may not be nucleic acid identical to the parent cell, for example, may contain mutations. This article includes screening or selecting mutant progeny having the same function or biological activity in the original transformed cells.
  • pharmaceutical formulation or “pharmaceutical composition” refers to a formulation that is in a form that is effective for the biological activity of the active ingredient and does not contain additional ingredients that would be unacceptable toxicities to the subject administered the formulation .
  • This preparation is sterile.
  • a "sterile" preparation is sterile or does not contain any viable microorganisms and their spores.
  • Embodiments described in this application should be understood to include embodiments “consisting of” and/or “consisting essentially of.”
  • references to "about” in this application are to a numerical value or parameter, including (and describing) variations on the numerical value or parameter itself. For example, descriptions referring to “about X” include descriptions of "X”.
  • references to "not” a value or parameter generally mean and describe "except for” a value or parameter.
  • the method cannot be used to treat type X cancer, meaning that the method is generally used to treat other types of disease besides type X cancer.
  • the IL-2 mutants provided herein have reduced affinity to IL-2R ⁇ and/or IL-2R ⁇ compared to human wild-type IL-2 (e.g., SEQ ID NO: 1). In some embodiments, the IL-2 mutant has a reduced affinity for IL-2R ⁇ and/or IL-2R ⁇ by at least 5% (e.g., at least 10%, 20%, 30%) compared to human wild-type IL-2. %, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%).
  • the IL-2 mutants provided herein have increased affinity to IL-2R ⁇ compared to human wild-type IL-2 (e.g., SEQ ID NO: 1). In some embodiments, the IL-2 mutant has an increased affinity for IL-2R ⁇ that is at least 1-fold (e.g., at least 1-fold, 2-fold, 3-fold, 5-fold, 10-fold) compared to human wild-type IL-2. times, or 20 times).
  • the affinity of IL-2 mutants provided by the application to IL-2R ⁇ remains unchanged or is not significantly weakened or has no affinity with IL-2R ⁇ . Increased affinity for -2R ⁇ .
  • the IL-2 mutants provided herein have reduced affinity to IL-2R ⁇ and/or IL-2R ⁇ compared to human wild-type IL-2 (e.g., SEQ ID NO: 1), while simultaneously binding to IL-2R ⁇ and/or IL-2R ⁇ .
  • the affinity for -2R ⁇ remained unchanged or there was no significant weakening or increase in affinity to IL-2R ⁇ .
  • the affinity between the IL-2 mutant and the receptor can be determined by conventional methods known in the art, such as binding ELISA experiments, HEK-Blue TM IL-2 cell line detection experiments, or surface plasmon resonance (SPR).
  • the IL-2 mutants provided by the application have a higher safety profile than human wild-type IL-2 (e.g., SEQ ID NO: 1) in the treatment of certain specific diseases.
  • human wild-type IL-2 e.g., SEQ ID NO: 1
  • IL-2 mutants were detected by the HEK-Blue TM CD122/CD132 cell line compared to human wild-type IL-2.
  • the EC50 value of binding to IL-2R ⁇ (hereinafter referred to as the second receptor) is consistent with the detection of IL-2 mutants and IL-2R ⁇ (below) through the HEK-Blue TM IL-2 cell line.
  • the ratio between the EC50 values of binding of three receptors is higher, that is, it has a higher therapeutic safety window and therefore has higher safety.
  • the EC50 value of IL-2 or IL-2 mutants binding to IL-2R ⁇ is determined by HEK-Blue TM CD122/CD132 cell line assay, by HEK-Blue TM IL-2 cell line assay. To determine the EC50 value of IL-2 or IL-2 mutants binding to IL-2R ⁇ .
  • the EC50 di-receptor /EC50 tri-receptor ratio of the IL-2 mutant is at least 2 times the EC50 di-receptor /EC50 tri-receptor ratio of human wild-type IL-2, for example: 2 times, 5 times, 10 times, 50 times, 100 times, 200 times, 500 times, 700 times, 1000 times, 2000 times, 10000 times, 20000 times, 30000 times or higher.
  • the IL-2 mutants provided herein can selectively activate Treg cells and reduce the activation of other T cells, such as reducing Activation of CD8 + T cells; reduced activation of NK cells.
  • the IL-2 mutants provided herein can selectively activate Treg cells.
  • the activation ability of IL-2 mutants on Treg cells is reflected by detecting the activation of STAT5 phosphorylation signals in Treg cells by IL-2 or IL-2 mutants. Detected by flow cytometry, GraphPad Prism software analyzes and calculates the EC50 value of IL-2 or IL-2 mutants in activating STAT5 phosphorylation in Treg cells. The EC50 value indirectly reflects the effect of IL-2 or IL-2 mutants on Treg cells. The activation ability of Treg cells shows that IL-2 mutants can effectively activate Treg cells.
  • the IL-2 mutants provided herein can reduce the activation of CD8 + T cells.
  • the activation of STAT5 phosphorylation signals by IL-2 or IL-2 mutants in CD8 + T lymphocytes is measured to reflect the activation of CD8 + T lymphocytes by IL-2 mutants.
  • ability Detected by flow cytometry, GraphPad Prism software analyzed and calculated the EC50 value of IL-2 mutants activating STAT5 phosphorylation in CD8 + T lymphocytes.
  • the EC50 value indirectly reflects the effect of IL-2 or IL-2 mutants on CD8 + T lymphocyte activation ability.
  • the ability of the IL-2 mutant to activate CD8 + T lymphocytes is reduced by at least 10% compared to human wild-type IL-2. For example: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
  • the IL-2 mutants provided herein can effectively expand Treg cells in vivo or in vitro.
  • the IL-2 mutant is capable of increasing the proportion of Treg cells in the CD4 + T cell population in the subject (e.g., increasing the proportion of CD4 + CD25 + Foxp3 + cells in the CD4 + T cell population ).
  • the IL-2 mutant can increase the ratio of Treg cells to non-Treg cells in the subject (eg, the ratio of CD4 + CD25 + Foxp3 + cells to CD8 + cells is increased).
  • Tregs in the peripheral blood of the test animal cynomolgus monkey
  • the number of cells increased without significant effects on the number of CD8 + T lymphocytes.
  • the proportion of Treg cells in CD4 + T lymphocytes in the subject animal is increased by at least 1-fold. For example: 1x, 2x, 3x, 5x, 7x, 10x, 15x, 20x or higher.
  • the IL-2 mutants described herein have at least one amino acid mutation, such as an amino acid substitution, deletion, insertion, or Contains any combination of the above mutant forms.
  • the IL-2 mutants described in the present application comprise a deletion 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) or mutation.
  • the IL-2 mutants described in the present application further comprise one or more mutations of E67, L70, N71, S75 and V91.
  • the mutation of E67 is E67K, E67A, E67M, or E67T.
  • the mutation of L70 is L70I.
  • the mutation of N71 is N71I, N71S, or N71W.
  • the mutation of S75 is S75I or S75F.
  • the mutation of V91 is V91K.
  • the IL-2 mutant described in the present application has 1, 2, 3, 4 or all 5 amino acids missing from amino acid residues RPRDL at positions 81-85.
  • the IL-2 mutant described in the present application contains all deletions of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application includes 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 includes E67 and N71 one or more mutations in .
  • the mutations of E67 and N71 are E67K and N71S, respectively.
  • the IL-2 mutants described in the present application comprise all deletions of amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence, and relative to the human wild-type IL -2 E67K and N71S mutations in the amino acid sequence.
  • amino acid residues in the RPRDL relative to amino acid residues 81-85 of the human wild-type IL-2 amino acid sequence in the IL-2 mutant described herein are substituted.
  • the IL-2 mutant described in the present application includes amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RPL.
  • the IL-2 mutants described in the present application 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 a mutation of E67 .
  • the mutation of E67 is E67A.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RPL, and relative to the human wild-type IL-2 amino acid sequence E67A mutation of IL-2 amino acid sequence.
  • the IL-2 mutants described in the present application 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 amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence.
  • the mutations of N71 and S75 are N71W and S75I, respectively.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RPL, and relative to human wild-type IL -2 N71W and S75I mutations of the amino acid sequence.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RHL.
  • the IL-2 mutants described in the present application 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 E67 and/or or mutations in V91.
  • the mutation of E67 is E67T.
  • the mutation of V91 is V91K.
  • 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 relative to the human wild-type IL-2 amino acid sequence E67T mutation of IL-2 amino acid sequence.
  • 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 relative to the human wild-type IL-2 amino acid sequence V91K mutation of IL-2 amino acid sequence.
  • 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 relative to the human wild-type IL-2 amino acid sequence E67T and V91K mutations in the IL-2 amino acid sequence.
  • the IL-2 mutants described in the present application 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 amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence. Mutation of L70 and S75 of wild-type IL-2 amino acid sequence. In some embodiments, the L70 and S75 mutations are respectively For L70I and S75F.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and relative to human wild-type IL -2 L70I and S75F mutations of the amino acid sequence.
  • the IL-2 mutants described in the present application 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 amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence. Mutation of N71 of wild-type IL-2 amino acid sequence. In some embodiments, the mutation of N71 is N71I. In some embodiments, the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and relative to human wild-type IL N71I mutation of -2 amino acid sequence.
  • the IL-2 mutants described in the present application comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to SPL.
  • the IL-2 mutants described in the present application which comprise amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence, are mutated to SPL, further comprise One or more mutations in E67 and N71 of the wild-type IL-2 amino acid sequence.
  • the mutations of E67 and N71 are E67M and N71S, respectively.
  • 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 SPL, and relative to the human wild-type IL-2 amino acid sequence E67M and N71S mutations in the IL-2 amino acid sequence.
  • the IL-2 mutant described in the present application includes amino acid residue S (S75) at position 75 relative to the human wild-type IL-2 amino acid sequence (e.g., SEQ ID NO: 1). Mutation of amino acid residue R (R83) at position 83.
  • the mutation of S75 is S75P, S75F, S75Y, S75G, S75A, S75V, S75T, S75I, or S75L.
  • the mutation of R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K, or R83E.
  • the IL-2 mutants described in the application further comprise one or more mutations in E15, H16, L17, L63, E67, L70, N71, D84, V91 and I92.
  • the mutation of E15 is E15K.
  • the mutation of H16 is H16D, H16Q, or H16N.
  • the mutation of L17 is L17I.
  • the mutation of L63 is L63V.
  • the mutation of E67 is E67A, E67R, E67G, E67S, E67K, or E67F.
  • the mutation of L70 is L70I, L70V, or L70F.
  • 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.
  • 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 of 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.
  • the IL-2 mutants described herein comprise S75P and R83A 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 H16, L70 and N71 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of H16, L70 and N71 are H16D, L70I and N71G respectively. In some embodiments, the IL-2 mutants described herein comprise S75P, R83A, H16D, L70I and N71G mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutants described herein comprise S75F and R83P 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 H16 and 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 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.
  • the IL-2 mutants described herein comprise S75F 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 human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of 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.
  • the IL-2 mutants described herein, relative to human wild-type IL-2 comprise S75I and R83L mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutants described herein further comprise one of E67 and L70 relative to the human wild-type IL-2 amino acid sequence or Multiple mutations.
  • the mutations of E67 and L70 are E67G and L70V, respectively.
  • the IL-2 mutants described herein comprise S75I, R83L, E67G and L70V mutations relative to the human wild-type IL-2 amino acid sequence.
  • 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 of 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.
  • the IL-2 mutants described herein comprise S75T and R83F mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutants described herein comprise S75T and R83A 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 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 mutants described herein comprise S75T, R83A, H16N and E67K mutations relative to the human wild-type IL-2 amino acid sequence.
  • 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 of 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.
  • 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 of 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.
  • 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 of 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.
  • 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 of 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.
  • 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 in the present application further comprise 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 mutants described herein comprise S75A, R83K and L17I mutations relative to the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutants described herein comprise S75L and R83E 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 L63 and E67 relative to the human wild-type IL-2 amino acid sequence. In some embodiments, the mutations of L63 and E67 are L63V and E67F, respectively. In some embodiments, the IL-2 mutants described herein comprise S75L, R83E, L63V and E67F mutations relative to the human wild-type IL-2 amino acid sequence.
  • 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 meet the requirements of IL-2 of the present invention.
  • the -2 mutant has reduced selective preference for the receptor IL-2R ⁇ , and/or has one or more beneficial properties.
  • the IL-2 mutants described in the present application also include mutants that do not change the affinity to the IL-2 receptor relative to the amino acid sequence of human wild-type IL-2 (e.g., SEQ ID NO: 1). mutations at other locations.
  • 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 details U.S. Patent No. 4,518,584).
  • SEQ ID NO: 1 human wild-type IL-2 amino acid sequence
  • C125S, C125A , C125T or C125V see details U.S. Patent No. 4,518,584
  • 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.
  • the IL-2 mutants described in the present application comprise the amino acid sequence shown in any one of SEQ ID NOs: 5-18 or a variant thereof, which variant is the same as that in SEQ ID NOs: 5-18 Any of the amino acid sequences shown has at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology.
  • the IL-2 mutants described in the present application comprise the amino acid sequence shown in any one of SEQ ID NOs: 19-34 or a variant thereof, which variant is the same as that in SEQ ID NOs: 19-34 Any of the amino acid sequences shown has at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology.
  • the one or more mutations at the other positions or regions comprise one or more conservative amino acid substitutions.
  • a “conservative substitution” refers to a substitution with another amino acid that has the same net charge and is approximately the same size and shape as the amino acid being substituted.
  • Amino acids with aliphatic or substituted aliphatic amino acid side chains are approximately the same size when the total number of carbon atoms and heteroatoms in the side chain differs by no more than 4. Amino acids have roughly the same shape when the number of branches on their side chains differs by no more than 1.
  • Amino acids having a phenyl or substituted phenyl group on the side chain can be considered to be approximately the same size and shape.
  • conservative substitutions preferably employ natural amino acids. Exemplary conservative substitutions are shown in Table 1.
  • Amino acids can be classified according to common side chain properties: (1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn , Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain direction: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions require the replacement of a member of one of these classes with a member of another class. Amino acid substitutions can be introduced into protein constructs and the products screened for the desired activity as described above.
  • the insertion of unnatural amino acids, including synthetic unnatural amino acids or one or more D-amino acids, into the IL-2 mutant or IL-2 mutant fusion protein of the present application can have multiple kind of benefit.
  • peptides containing D-amino acids show higher stability in vitro and in vivo. Therefore, the construction of polypeptides by adding D-amino acids is particularly useful when better intracellular stability is required.
  • D-peptides and their analogs are resistant to endogenous peptidases and proteases, thereby increasing the bioavailability of the molecule and extending its lifespan in the body when needed.
  • D-peptide and its analogs cannot be efficiently processed for limited presentation to helper T cells via class II major histocompatibility complex (MHC) and therefore are less likely to induce humoral immune responses in subjects.
  • MHC major histocompatibility complex
  • the IL-2 mutants described herein are modified IL-2 mutants, such as pegylated IL-2 mutants, or covalently modified IL-2 mutants, or glycosyl Chemically modified IL-2 mutants.
  • the IL-2 mutants described herein are full-length sequences.
  • the N-terminus of the IL-2 mutant contains a signal peptide either from a different molecule or from wild-type IL-2.
  • the IL-2 mutants described in the present application comprise the amino acid sequence shown in any one of SEQ ID NOs: 5-34 or a variant thereof, which variant is the same as that in SEQ ID NOs: 5-34.
  • Any of the amino acid sequences shown has at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology.
  • amino acid sequences of exemplary wild-type IL-2 and its C125 mutant are shown in Table 2
  • amino acid sequences of the exemplary first group of IL-2 mutants are shown in Table 3
  • the exemplary second group of IL-2 The amino acid sequence of the 2 mutant is shown in Table 4.
  • the application also provides fusion proteins comprising IL-2 mutants.
  • IL-2 mutants as described herein can be fused to other proteins to increase circulating half-life by increasing the size of the molecule and/or reducing renal clearance.
  • the IL-2 mutants described herein are fused to Fc.
  • the IL-2 mutants described herein are fused to human serum albumin (HSA).
  • HSA human serum albumin
  • the IL-2 mutants described herein are fused to short peptides (eg, XTEN).
  • Fc fusion proteins can be produced by recombinant DNA technology in which the translational reading frame of the Fc domain of a mammalian immunoglobulin (eg, IgG) is fused to another protein to produce a new single recombinant polypeptide.
  • Fc fusion proteins can generally be produced as disulfide-linked dimers held together by disulfide bonds located in the hinge region of the Fc domain.
  • IL-2 mutants can be fused to the N-terminus and/or C-terminus of Fc.
  • the IL-2 mutant is fused to the C-terminus of Fc.
  • the IL-2 mutant and Fc are directly fused via a peptide bond.
  • a linker peptide is included between the IL-2 mutant and Fc.
  • the Fc described herein is from any one of IgA, IgD, IgE, IgG, and IgM, and their subclasses. Among all immunoglobulins, IgG has the highest content in serum and the longest half-life. Unlike other immunoglobulins, IgG can be efficiently recovered after binding to Fc receptors (FcRs).
  • the Fc is from IgG (eg, IgGl, IgG2, IgG3 or IgG4). In some embodiments, the Fc is from human IgG. In some embodiments, the Fc includes CH2 and CH3 domains. In some embodiments, Fc further comprises all or part of the hinge region.
  • the Fc is from human IgGl or human IgG4.
  • two subunits of Fc dimerize through one or more (eg, 1, 2, 3, 4, or more) disulfide bonds.
  • each subunit of the Fc contains full-length Fc sequence.
  • each subunit of Fc includes an N-terminal truncated Fc sequence, such as a truncated Fc containing less N-terminal cysteine to reduce disulfide bond errors during dimerization. match.
  • the Fc is truncated at the N-terminus, e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or even more amino acids of the intact immunoglobulin Fc are deleted .
  • the Fc contains one or more mutations, such as insertions, deletions, and/or substitutions.
  • the Fc described herein has reduced or eliminated Fc effector function. In some embodiments, the Fc has reduced Fc-mediated effector functions, such as reduced ADCC, CDC, and/or ADCP effector functions.
  • Fc-containing fusion proteins can activate complement and interact with Fc receptors (FcRs).
  • FcRs Fc receptors
  • this intrinsic immunoglobulin property has been considered disadvantageous, as the fusion protein may target cells expressing Fc receptors rather than the preferred cells expressing IL-2 receptors, which may May cause undesirable cytotoxicity.
  • further consideration is given to the long half-life of Fc fusion proteins, which makes it difficult to apply for treatment or has limited therapeutic application due to systemic toxicity.
  • the Fc is engineered (e.g., contains one or more amino acid mutations) to alter its binding to the FcR, in particular to alter binding to the Fc ⁇ receptor (responsible for ADCC) and/or to alter effector function,
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • amino acid mutations do not reduce binding to the FcRn receptor (related to half-life).
  • the Fc (e.g., the Fc of human IgG1) is mutated to remove one or more effector functions, such as ADCC, ADCP, and/or CDC, hereafter referred to as a "no effect” or “nearly effector” Fc .
  • the Fc is a null-effect human IgG1 Fc comprising mutations L234A and L235A.
  • the human IgG1 Fc comprises one or more of the following mutations (eg, in each Fc subunit): L234A, L235E, G237A, A330S, and P331S.
  • the combination of K322A, L234A and L235A in the IgG1 Fc is sufficient to completely eliminate Fc ⁇ R and Clq binding (Hezareh et al., J Virol 75, 12161-12168, 2001). MedImmune found that Fc containing a set of triple mutations L234F/L235E/P331S had very similar effects (Oganesyan et al., Acta Crystallographica 64, 700–704, 2008).
  • the Fc comprises a glycosylation modification on N297 of the IgG1 Fc region, which is known to be required for optimal FcR interactions.
  • the modification of Fc can be any suitable engineered IgG Fc mentioned by Wang et al. (“IgG Fc engineering to modulate antibody effector functions,” Protein Cell. 2018 Jan; 9(1):63–73), the contents of which are all approved Incorporated herein by reference.
  • fusion proteins of IL-2 mutants and Fc have no ADCC and/or CDC, or have detectable ADCC and/or CDC, as described herein. In some embodiments, as described herein, fusion proteins of IL-2 mutants with Fc produce ADCC and /or the CDC is reduced by at least 5% (such as at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%).
  • the degree of glycosylation of the construct is increased or decreased by altering the fusion protein of the Fc or IL-2 mutant with Fc.
  • Glycosylation sites can be added or deleted in the Fc by changing the amino acid sequence to create or remove one or more glycosylation sites.
  • Natural Fc-containing proteins produced by mammalian cells typically contain a branched chain of biantennary oligosaccharides linked to Asn297 of the Fc CH2 domain, usually via an N-bond. 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 GlcNAc on the "stem" of the biantennary oligosaccharide structure.
  • the oligosaccharides in the Fc can be modified to produce certain improved properties.
  • fusion proteins of Fc or IL-2 mutants and Fc as described herein have a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region.
  • the fucose content may range from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%.
  • the fucose content was measured by MALDI-TOF mass spectrometry. The average fucose content within the sugar chain attached to Asn297 relative to that attached to Asn297 (e.g., complex, hybrid and high mannose structures) Determined by the sum of all sugar structures.
  • Asn297 refers to the asparagine residue located at position 297 of the Fc region (EU numbering system for Fc region residues); however, due to minor sequence changes in the Fc region, Asn297 can also be located approximately ⁇ 3 amino acids upstream or downstream of position 297, That is between 294 and 300 bits. Such fucosylation variants may have enhanced ADCC function. See US Patent Publication Nos. US 2003/0157108 (Presta, L.), US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to "afucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/ US 2004/0109865 6; 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 generating afucosylated Fc-containing proteins include Lec13 CHO cells lacking protein fucosylation function (Ripka et al., Arch.
  • the present application contemplates an Fc that possesses some, but not all, Fc effector functions, making it an ideal candidate for an application in which a fusion protein of an IL-2 mutant with an Fc Half-life in the body Periods are important, but some effector functions (such as CDC and ADCC) are non-essential or harmful.
  • Cytotoxicity assays can be performed in vitro or in vivo to determine reduction/depletion of CDC and/or ADCC activity.
  • an Fc receptor (FcR) binding assay can be performed to ensure that the Fc or fusion protein containing this Fc and IL-2 mutant lacks Fc ⁇ R binding (and therefore may lack ADCC activity), but retains FcRn binding ability.
  • the ADCC activity of the target molecule can also be assessed in vivo, for example, in an animal model as 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 fusion proteins of IL-2 mutants and Fc are unable to bind to C1q and therefore lack CDC activity. See C1q and C3c combined enzyme-linked immunosorbent assay in WO 2006/029879 and WO 2005/100402.
  • CDC assays can be performed to assess complement activity (see Gazzano-Santoro et al., J. Immunol.
  • FcRn binding and in vivo clearance/half-life assays can be performed using methods known in the art (see Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).
  • Fcs with reduced effector function include those with substitutions of one or more residues at positions 238, 265, 269, 270, 297, 327 and 329 of the Fc region (US Patent No. 6,737,056).
  • Such Fc mutants include substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" substitutions of residues 265 and 297 with alanine Fc mutants (US Patent No. 7,332,581).
  • Certain antibody variants that enhance or reduce binding to FcRs are detailed (see US Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 ( 2001).
  • the Fc region is engineered to alter (i.e., increase or decrease) Clq binding and/or CDC, such as US Patent No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164 :4178-4184(2000).
  • the Fc contains one or more amino acid substitutions that increase half-life and/or enhance binding to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • 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 detailed in US2005/0014934A1 (Hinton et al.).
  • Those antibodies containing an Fc region with one or more substitutions thus increase the binding of the Fc region to FcRn.
  • Such Fc variants include those with one or more Fc region residue substitutions, for example, Fc region residue 434 substitution (US Patent No. 7,371,826).
  • the substitution residue occurs at an accessible site on the Fc or fusion protein of the IL-2 mutant and Fc.
  • the reactive thiol group is thus positioned at an accessible site of Fc or fusion proteins of IL-2 mutants with Fc and can be used to conjugate the molecule to other moieties, e.g. Drug moiety or linker-drug moiety to create conjugates of IL-2 mutants and Fc fusion proteins.
  • 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 produced as described in U.S. Patent No. 7,521,541.
  • the Fc is from an IgG1 Fc. In some embodiments, the Fc is from human IgG1 Fc. In some embodiments, the Fc is from human wild-type IgG1 Fc. In some embodiments, the Fc does not comprise the hinge region of the IgG1 Fc. In some embodiments, the Fc contains one or more null mutations and/or deglycosylation mutations.
  • the Fc comprises one or more mutations relative to amino acid residue L 234 (L234) and amino acid residue L 235 (L235) of the human wild-type IgG1 Fc amino acid sequence. In some embodiments, the Fc comprises mutations at 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.
  • Fc comprises (or consists essentially of, or consists of) the amino acid sequence SEQ ID NO:35 or a variant thereof; the variant is at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • the Fc comprises a mutation relative to amino acid residue N (N297) at position 297 of the human wild-type IgG1 Fc amino acid sequence.
  • the Fc package Contains mutation N297G or N297A.
  • the Fc comprises (or consists essentially of, or consists of) the amino acid sequence SEQ ID NO: 36, or a variant thereof; said variant is identical to SEQ ID NO: 36 or a variant thereof; NO:36 has at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology.
  • the Fc comprises (or consists essentially of, or consists of) the amino acid sequence SEQ ID NO:37 or a variant thereof; the variant is at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • the Fc comprises amino acid residue L at position 234 (L234), amino acid residue L at position 235 (L235), and amino acid residue P (P331) at position 331 relative to the human wild-type IgG1 Fc amino acid sequence. one or more mutations. 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 includes mutations L234A, L235A, and P331S relative to the human wild-type IgG1 Fc amino acid sequence.
  • Fc comprises (or consists essentially of, or consists of) the amino acid sequence SEQ ID NO:38 or a variant thereof; the variant is at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • the Fc comprises amino acid residue L at position 234 (L234), amino acid residue at position 235 (L235), amino acid residue G at position 237 (G237), relative to the human wild-type IgG1 Fc amino acid sequence.
  • the Fc comprises mutations of L234, L235, G237, A330, and P331 relative to the human wild-type IgG1 Fc amino acid sequence.
  • the Fc includes mutations L234A, L235E, G237A, A330S, and P331S relative to the human wild-type IgG1 Fc amino acid sequence.
  • Fc comprises (or consists essentially of, or consists of) the amino acid sequence SEQ ID NO:39 or a variant thereof; the variant is identical to SEQ ID NO:39 Have at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology.
  • the Fc is from an IgG4 Fc. In some embodiments, the Fc is from human IgG4 Fc. In some embodiments, the Fc is from human wild-type IgG4 Fc. In some embodiments, the Fc does not comprise the hinge region of IgG4. In some embodiments, the Fc contains one or more null mutations and/or deglycosylation mutations.
  • the Fc comprises amino acid residue S at position 228 (S228), amino acid residue F at position 234 (F234), and amino acid residue L (L235) at position 235 relative to the human wild-type IgG4 Fc amino acid sequence. one or more mutations.
  • the Fc comprises S228, F234, and L235 mutations relative to the human wild-type IgG4 Fc amino acid sequence.
  • the Fc Contains mutations S228P, F234A and L235A.
  • the Fc portion comprises (or consists essentially of, or consists of) the amino acid sequence SEQ ID NO: 40, or a variant thereof; the variant has at least the same amino acid sequence as SEQ ID NO: 40 About 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology.
  • Additional mutations that can be made in Fc include those that promote heterodimer formation between Fc polypeptides.
  • the heterodimer assembly problem is solved through KIH (Knob-into-Hole) technology, which introduces an asymmetric mutation structure in the CH3 domain ("knob” mutation refers to the C In the H3 domain, a larger amino acid residue is used to replace a smaller residue, while "hole” mutations refer to using a smaller amino acid residue to replace a larger residue).
  • the modified Fc is more prone to heterodimerization than homodimerization due to steric hindrance (Ridgway J B, et al.
  • the amino acid residues (V) form a "holes” structure while the two cysteine residues (C) mutations that form a stabilizing disulfide bridge are introduced (S354C on the "knob” side, and Y349C on the "hole” side). side), enhance the stability of KIH, wherein the numbering is in accordance with the EU index such as 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).
  • the fusion protein of IL-2 mutant and Fc of the present application is in a bivalent form, and the fusion protein contains two IL-2 mutant molecules.
  • a homodimer form composed of two of the above-mentioned fused polypeptide chain monomers is shown in Figures 1A and 1B.
  • the fusion protein of IL-2 mutant and Fc of the present application is in a monovalent form, and the fusion protein only contains one IL-2 mutant molecule.
  • an exemplary structure is shown in Figure 2. in others In the embodiment, the fusion protein of IL-2 mutant and Fc of the present application is in a monovalent form and consists of one fusion polypeptide chain monomer mentioned above.
  • the IL-2 mutant and Fc can be connected through a linker (eg, connecting peptide, non-linking peptide).
  • the joint is a flexible joint.
  • the joint is a stabilizing joint.
  • ideal linkers do not affect or do not significantly affect the correct folding and conformation of the fusion proteins of IL-2 mutants and Fc described herein.
  • the linker confers 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 fusion protein of the IL-2 mutant and Fc.
  • the linker is a stable linker (eg, cannot be cleaved by proteases, particularly MMP).
  • 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 in length amino acids.
  • the linking peptide is no longer than necessary to prevent undesired domain interactions and/or to optimize biological function and/or stability.
  • the linking peptide is up to 30 amino acids in length, such as up to 20 amino acids, or up to 15 amino acids in length. In some embodiments, the linking peptide is 5 to 30 amino acids in length, or 5 to 18 amino acids in length.
  • the linking peptide may have a naturally occurring sequence or a non-naturally occurring sequence.
  • sequences from the hinge region of an antibody heavy chain can be used as a linker. See, for example, WO1996/34103.
  • the linking peptide is a human IgGl, IgG2, IgG3 or IgG4 hinge region.
  • the linking peptide is a mutated human IgGl, IgG2, IgG3 or IgG4 hinge region.
  • the joint is a flexible joint.
  • Glycine and glycine-serine polymers are relatively unstructured and therefore can act as a neutral chain between the 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.
  • the designed fusion protein of IL-2 mutant and Fc can include all or part of the flexible linker, such that the linker can include a flexible linker portion and one or more structures that provide less flexibility. section to provide the structure and function of an ideal IL-2 mutant fusion protein with Fc.
  • the connecting peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 43-74 or a variant thereof, which variant has the same amino acid sequence as any one of SEQ ID NOs: 43-74. There is at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology.
  • linker include the effect on the physical or pharmacokinetic properties of the resulting fusion protein of IL-2 mutant and Fc, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (or more). or less stability and planned degradation), rigidity, flexibility, immunogenicity, binding of IL-2 mutants to IL-2 receptors, binding ability of colloids or liposomes, etc.
  • the fusion protein of IL-2 mutant and Fc described in the present application includes a bivalent form and a monovalent form.
  • the bivalent form, which contains two IL-2 mutant molecules can be composed of two identical or different monomers, for example, the monomers are IL-2 mutant and Fc fusion polypeptide chain.
  • the monovalent form, which contains one IL-2 mutant molecule can be composed of two different monomers, e.g., one monomer is an IL-2 mutant with an Fc subunit. Fusion polypeptide chain, the other monomer is the Fc subunit.
  • the monovalent form, which includes an IL-2 mutant molecule consists of only one monomer, for example, the monomer is a fusion polypeptide chain of the IL-2 mutant and an Fc subunit.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, in the form of a bivalent or monovalent fusion protein), contains Deletion or mutation of amino acid residues RPRDL at positions 81-85 of the IL-2 amino acid sequence.
  • the amino acid sequence of human wild-type IL-2 is shown in SEQ ID NO: 1.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, in the form of a bivalent or monovalent fusion protein), contains All amino acid residues RPRDL at positions 81-85 of the IL-2 amino acid sequence are deleted.
  • the IL-2 mutant further comprises mutations of E67 and N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and N71 are E67K and N71S respectively.
  • the IL-2 mutant includes all deletions of amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2, and relative to human wild-type IL-2 Amino acid sequence of E67K and N71S mutations.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, in the form of a bivalent or monovalent fusion protein), contains Amino acid residues RPRDL at positions 81-85 of the IL-2 amino acid sequence are mutated to RPL.
  • the IL-2 mutant further comprises a mutation of E67 relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the mutation of E67 is E67A.
  • the IL-2 mutant comprises amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RPL, and relative to human wild-type IL-2 Amino acid sequence of the E67A mutation.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, in the form of a bivalent or monovalent fusion protein), contains Amino acid residues RPRDL at positions 81-85 of the IL-2 amino acid sequence are mutated to RPL, and further include mutations at N71 and S75 relative to the human wild-type IL-2 amino acid sequence.
  • the mutations of N71 and S75 are N71W and S75I, respectively.
  • the IL-2 mutant comprises amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RPL, and relative to human wild-type IL-2 N71W and S75I mutations of the amino acid sequence.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, in the form of a bivalent or monovalent fusion protein), contains Amino acid residues RPRDL at positions 81-85 of the IL-2 amino acid sequence are mutated to RHL.
  • the IL-2 mutant further comprises mutations of E67 and/or V91 relative to the amino acid sequence of human wild-type IL-2.
  • the mutation of E67 is E67T.
  • the mutation of V91 is V91K.
  • the IL-2 mutant includes amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and relative to human wild-type IL-2 E67T and/or V91K mutations in the amino acid sequence.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, in the form of a bivalent or monovalent fusion protein), contains Amino acid residues RPRDL at positions 81-85 of the IL-2 amino acid sequence are mutated to RHL, and further comprise mutations of L70 and S75 relative to the human wild-type IL-2 amino acid sequence.
  • the mutations of L70 and S75 are L70I and S75F, respectively.
  • the IL-2 mutant includes an amino acid sequence relative to human wild-type IL-2 Amino acid residues 81-85 were mutated from RPRDL to RHL, as well as L70I and S75F mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, in the form of a bivalent or monovalent fusion protein), contains Amino acid residues RPRDL at positions 81-85 of the IL-2 amino acid sequence are mutated to RHL, and further comprise a mutation relative to N71 of the human wild-type IL-2 amino acid sequence.
  • the mutation of N71 is N71I.
  • the IL-2 mutant includes amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and relative to human wild-type IL-2 N71I mutation of the amino acid sequence.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, in the form of a bivalent or monovalent fusion protein), contains Amino acid residues RPRDL at positions 81-85 of the IL-2 amino acid sequence are mutated to SPL.
  • the IL-2 mutant further comprises mutations of E67 and N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and N71 are E67M and N71S, respectively.
  • the IL-2 mutant includes amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to SPL, and relative to human wild-type IL-2 Amino acid sequence of E67M and N71S mutations.
  • the IL-2 mutant contains Mutation of amino acid residue S at position 75 (S75) and amino acid residue R (R83) at position 83 of the IL-2 amino acid sequence.
  • the amino acid sequence of human wild-type IL-2 is shown in SEQ ID NO: 1.
  • the IL-2 mutant in which S75 is mutated to not contain Charged amino acid residues.
  • the mutation of S75 is S75P, S75F, S75Y, S75G, S75A, S75V, S75T, S75I or S75L.
  • the mutation of R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K, or R83E.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75P, The mutation of R83 is R83Y.
  • the IL-2 mutant further comprises mutations of E67 and D84 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and D84 are E67A and D84N, respectively.
  • the IL-2 mutants comprise S75P, R83Y, E67A and D84N mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75P,
  • the mutation of R83 is R83A.
  • the IL-2 mutant further comprises mutations of H16, L70 and N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of H16, L70 and N71 are H16D, L70I and N71G respectively.
  • the IL-2 mutants comprise S75P, R83A, H16D, L70I and N71G mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75F,
  • the mutation of R83 is R83P.
  • the IL-2 mutant further comprises mutations of H16 and/or N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutation of H16 is H16Q or H16N.
  • the mutation of N71 is N71S.
  • the IL-2 mutants comprise S75F, R83P and H16Q mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutants comprise S75F, R83P, H16N and N71S mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75F, The mutation of R83 is R83L.
  • the IL-2 mutant further comprises mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and L70 are E67R and L70V, respectively.
  • the IL-2 mutants comprise S75F, R83L, E67R and L70V mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75I,
  • the mutation of R83 is R83L.
  • the IL-2 mutant further comprises mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and L70 are E67G and L70V, respectively.
  • the IL-2 mutants comprise S75I, R83L, E67G and L70V mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75I, The mutation of R83 is R83K.
  • the IL-2 mutant further comprises mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and L70 are respectively E67S and L70V.
  • the IL-2 mutants comprise S75I, R83K, E67S and L70V mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75T,
  • the mutation of R83 is R83A or R83F.
  • the IL-2 mutant further comprises mutations of H16 and E67 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of H16 and E67 are H16N and E67K respectively.
  • the IL-2 mutants comprise S75T, R83A, H16N and E67K mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75V, The mutation of R83 is R83F.
  • the IL-2 mutant further comprises mutations of E15 and H16 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E15 and H16 are E15K and H16Q, respectively.
  • the IL-2 mutants comprise S75V, R83F, E15K and H16Q mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75V, The mutation of R83 is R83N.
  • the IL-2 mutant further comprises mutations of L70 and V91 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L70 and V91 are L70F and V91K respectively.
  • the IL-2 mutants comprise S75V, R83N, L70F and V91K mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75G,
  • the mutation of R83 is R83L.
  • the IL-2 mutant further comprises mutations of L17, V91 and I92 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L17, V91, and I92 are L17I, V91E, and I92L, respectively.
  • the IL-2 mutants comprise S75G, R83L, L17I, V91E and I92L mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75Y, The mutation of R83 is R83N.
  • the IL-2 mutant further comprises mutations of L17 and V91 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L17 and V91 are L17I and V91K.
  • the IL-2 mutants comprise S75Y, R83N, L17I and V91K mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75A,
  • the mutation of R83 is R83K.
  • the IL-2 mutant further comprises a mutation of L17 relative to the amino acid sequence of human wild-type IL-2.
  • the mutation of L17 is L17I.
  • the IL-2 mutants comprise S75A, R83K and L17I mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), the IL-2 mutant, wherein the mutation of S75 is S75L,
  • the mutation of R83 is R83E.
  • the IL-2 mutant further comprises mutations of L63 and E67 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L63 and E67 are L63V and E67F, respectively.
  • the IL-2 mutants comprise S75L, R83E, L63V and E67F mutations relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutant in addition to the above mutations, further Contains a mutation at C125 relative to the human wild-type IL-2 amino acid sequence.
  • the C125 mutation is C125S or C125A.
  • the IL-2 mutant in the fusion protein of IL-2 mutant and Fc described in the present application (for example, bivalent or monovalent fusion protein form), includes SEQ ID NOs: 5-34
  • the amino acid sequence shown in any one of SEQ ID NOs: 5-34 or a variant thereof, the variant has at least about 90% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • 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 exemplary amino acid sequences of the second group of bivalent fusion proteins of IL-2 mutants and Fc are shown in Table 7. 8 shown.
  • each monomer from the N terminus to the C terminus, or from the C terminus to the N terminus includes: (i) IL-2 mutants and (ii) Fc.
  • each monomer from the N terminus to the C terminus, or from the C terminus to the N terminus includes: (i) IL-2 mutants, (ii) linker peptide and (iii) Fc.
  • the Fc in the bivalent fusion protein form of the IL-2 mutant and Fc described herein, is an IgG1 Fc, and the Fc includes L234A and L235A (LALA) mutations. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO:35.
  • the Fc in the bivalent fusion protein form of the IL-2 mutant and Fc described herein, is an IgG1 Fc, and the Fc includes the N297G mutation. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO: 36.
  • the Fc in the bivalent fusion protein form of the IL-2 mutant and Fc described herein, is an IgG1 Fc, and the Fc includes the N297A mutation. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO: 37.
  • the Fc in the bivalent fusion protein form of the IL-2 mutant and Fc described herein, is an IgG1 Fc, and the Fc includes L234A, L235A and P331S mutations. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO: 38.
  • the Fc in the bivalent fusion protein form of the IL-2 mutant and Fc described herein, is an IgG1 Fc, and the Fc includes L234A, L235E, G237A, A330S and P331S mutations. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO:39.
  • the Fc in the bivalent fusion protein form of the IL-2 mutant and Fc described herein, is an IgG4 Fc, and the Fc includes S228P, F234A and L235A mutations. In some embodiments, the Fc comprises the amino acid sequence SEQ ID NO: 40.
  • each monomer from the N terminus to the C terminus, or from the C terminus to the N terminus includes: (i ) IL-2 mutant, which includes the amino acid sequence shown in any one of SEQ ID NOs: 5-34 or a variant thereof, which variant has the same amino acid sequence as any one of SEQ ID NOs: 5-34 At least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology and (ii) an Fc, comprising as The amino acid sequence shown in any one of SEQ ID NOs: 35-40 or a variant thereof, which variant has at least about 90% (such as at least 90%) with the amino acid sequence shown in any one of SEQ ID NOs: 35-40 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology.
  • each monomer from the N terminus to the C terminus, or from the C terminus to the N terminus includes: (i ) IL-2 mutant, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 5-34 or a variant thereof, which variant has the same amino acid sequence as any one of SEQ ID NOs: 5-34 At least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology, (ii) a connecting peptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 43-74 or a variant thereof, which variant is identical to SEQ ID NOs : The amino acid sequence shown in any one of 43-74 has at least about 90% (such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) Sequence
  • the bivalent fusion protein form of IL-2 mutant and Fc described in the present application includes the amino acid sequence shown in any one of SEQ ID NOs: 76-105 or a variant thereof, which variant is identical to The amino acid sequence shown in any one of SEQ ID NOs: 76-105 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.
  • the Fc in one of the monovalent fusion protein forms of the IL-2 mutant and Fc described herein, is an IgG1 Fc.
  • the Fc comprises a KIH mutation (referred to as Fc knob and Fc hole, respectively).
  • the Fc comprises a LALA mutation and a KIH mutation (referred to as Fc LALA knob and Fc LALA hole, respectively).
  • 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.
  • the IgG1 Fc LALA knob comprises the amino acid sequence SEQ ID NO: 41 and the Fc LALA hole comprises the amino acid sequence SEQ ID NO: 42.
  • one of the monomers from the N terminus to the C terminus or from the C terminus to the N terminus includes: (i )IL-2 mutants and (ii) Fc knob, and a monomer containing: Fc hole.
  • one of the monomers includes from the N terminus to the C terminus, or from the C terminus to the N terminus: (i) IL-2 mutants comprising the amino acid sequence shown in any one of SEQ ID NOs: 5-34 or a variant thereof, which variant is identical to the amino acid sequence shown in any one of SEQ ID NOs: 5-34
  • the sequence has at least about 90% (eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology
  • the Fc LALA knob which comprises the amino acid sequence SEQ ID NO: 41 or a variant thereof that is at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%) identical to SEQ ID NO: 41 , 96%, 97%, 98% or 99%) sequence homology
  • another monomer is the Fc LALA
  • one of the monomers from the N terminus to the C terminus or from the C terminus to the N terminus includes: (i )IL-2 mutant and (ii) Fc hole; and another monomer containing: Fc knob.
  • one of the monomers includes from the N terminus to the C terminus or from the C terminus to the N terminus: ( i) IL-2 mutant, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 5-34 or a variant thereof, which variant is the same as the amino acid sequence shown in any one of SEQ ID NOs: 5-34 Having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology and (ii) an Fc LALA hole, It comprises the amino acid sequence SEQ ID NO: 42 or a variant thereof that is at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology; and the other monomer is an Fc LALA knob comprising the amino acid sequence
  • one of the monomers from the N terminus to the C terminus or from the C terminus to the N terminus includes: (i ) IL-2 mutant, (ii) linker peptide and (iii) Fc knob; and another monomer containing: Fc hole.
  • one of the monomers includes from the N terminus to the C terminus or from the C terminus to the N terminus: ( i) IL-2 mutant, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 5-34 or a variant thereof, which variant is the same as the amino acid sequence shown in any one of SEQ ID NOs: 5-34 Having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology (ii) a linking peptide comprising The amino acid sequence shown in any one of SEQ ID NOs: 43-74 or a variant thereof, which variant is at least about 90% (for example, at least 90%) identical to the amino acid sequence shown in any one of SEQ ID NOs: 43-74 , 91%, 92%, 93%, 94%, 95%, 96%, 97%,
  • one of the monomers from the N terminus to the C terminus or from the C terminus to the N terminus includes: (i ) IL-2 mutant, (ii) linker peptide and (iii) Fc hole, and another monomer containing: Fc knob.
  • one of the monomers includes from the N terminus to the C terminus or from the C terminus to the N terminus: ( i) IL-2 mutant, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 5-34 or a variant thereof, which variant is the same as the amino acid sequence shown in any one of SEQ ID NOs: 5-34 Having at least about 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence homology (ii) a linking peptide comprising The amino acid sequence shown in any one of SEQ ID NOs: 43-74 or a variant thereof, which has at least about 90% (e.g., at least 90%) similarity with the amino acid sequence shown in any one of SEQ ID NOs: 43-74 , 91%, 92%, 93%, 94%, 95%, 96%, 9
  • the fusion protein of an IL-2 mutant and Fc described herein has a half-life of at least 10 hours (e.g., at least for 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).
  • a molecule e.g., an IL-2 mutant or a fusion protein containing an IL-2 mutant
  • its binding partner e.g., an IL-2 receptor such as IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ , or The binding affinity of IL-2R ⁇
  • IL-2 receptor such as IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ , or The binding affinity of IL-2R ⁇
  • IL-2 receptor e.g., an IL-2 receptor such as IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ , or The binding affinity of IL-2R ⁇
  • ligand binding assay e.g., Western blot, enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence, bead-based Multiplex immunoassay (MIA), RIA, surface plasmon resonance (SPR), ECL, IRMA
  • Mutant fusion proteins or their receptors e.g., IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ , IL-2R ⁇ , or IL-2R ⁇
  • BiacoreX is an over-the-counter measurement kit or similar kit that can be operated according to the user manual and experimental instructions provided with the kit.
  • protein microarrays are used for large-scale analysis of the interactions, functions, and activities of IL-2 mutants described herein or fusion proteins containing IL-2 mutants and their receptors.
  • Protein microarrays have a support surface that binds a series of capture proteins (eg, IL-2 receptors or subunits thereof). Fluorescently labeled probe molecules (eg, IL-2 mutants described herein or fusion proteins containing IL-2 mutants) are then added to the array and interact with the bound capture proteins, releasing a fluorescent signal and Read via laser scanner.
  • Binding affinity can also be measured using Biacore.
  • EDC/NHS chemistry was used to couple IL-2R ⁇ or IL-2R ⁇ to the CM-5 sensor chip surface as a ligand.
  • a series of dilutions of the IL-2 mutants or fusion proteins of IL-2 mutants and Fc described herein are then combined as analytes with ligands coupled to the chip, and IL-2 and IL can be monitored in real time.
  • Affinity (Kd) can be determined by kinetic analysis using BIA evaluation software.
  • Biacore experiments can be applied to determine the Kd value of the IL-2 mutant or the fusion protein of IL-2 mutant and Fc provided in this application and IL-2R ⁇ to determine the binding affinity.
  • the fusion protein of IL-2 mutant and Fc of the present application has lower binding affinity to IL-2R ⁇ .
  • the affinity of the fusion protein of IL-2 mutant and Fc to IL-2R ⁇ and/or IL-2R ⁇ is reduced by at least 5% relative to the fusion protein of wild-type IL-2 and Fc (e.g., At least any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%).
  • the fusion protein of IL-2 mutant and Fc described in the present application has a higher affinity with IL-2R ⁇ than the fusion protein of wild-type IL-2 and Fc (e.g., human wild-type IL-2). Affinity increases or remains unchanged or does not decrease significantly.
  • the binding ability of the fusion protein of IL-2 mutant and Fc of the present application to IL-2R ⁇ is increased, remains unchanged, or is not significantly weakened.
  • 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 medium affinity receptor (IL-2R ⁇ ) is greater than that of wild-type IL-2 or wild-type The Kd value of the binding between IL-2 and Fc fusion protein and its same receptor. In some embodiments, the Kd value of the binding between the IL-2 mutant or the fusion protein of IL-2 mutant and Fc and its high affinity receptor (IL-2R ⁇ ) is close to (for example, equal to, Less than or slightly greater than) the binding Kd value between wild type and the same receptor (IL-2R ⁇ ).
  • Pharmacokinetics refers to the absorption, distribution, metabolism, and excretion of a drug (eg, an IL-2 mutant or a fusion protein comprising an IL-2 mutant described herein) after administration to a subject.
  • Pharmacokinetic parameters that can be used to determine clinical utility include, but are not limited to, serum/plasma concentration, serum/plasma concentration over time, maximum serum/plasma concentration ( Cmax ), time to maximum concentration ( Tmax ), half-life (T 1/2 ), the area under the concentration-time curve within the dosing interval (AUC ⁇ ), etc.
  • PK profiles for drugs 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.
  • the PK profile of an IL-2 mutant or a fusion protein comprising an IL-2 mutant as described herein is measured in an individual's blood, plasma or serum sample.
  • the PK profile of an IL-2 mutant or a fusion protein comprising an IL-2 mutant as described herein is measured in an individual using mass spectrometry techniques (eg, LC-MS/MS or ELISA).
  • PK analysis can be performed on a PK curve by any method known in the art, e.g., noncompartmental analysis, using PKSolver V2 software (Zhang Y. et al., "PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel,” Comput Methods Programs Biomed. 2010;99(3):306-1). See Example 8 for an example method.
  • C represents the concentration of drug (e.g., IL-2 mutant or fusion protein containing IL-2 mutant) in the subject's plasma, serum, or any suitable body fluid or tissue, usually expressed as mass per unit volume, For example nanograms/ml.
  • drug concentrations in serum or plasma are referred to herein as “serum concentrations” or "plasma concentrations.”
  • the serum/plasma concentration at any time after administration eg, intravenous, intraperitoneal, or subcutaneous injection of an IL-2 mutant or a fusion protein polypeptide comprising an IL-2 mutant
  • C time or C t The maximum serum/plasma drug concentration during the dosing period 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.
  • bioavailability refers to the extent or rate at which a drug (eg, an IL-2 mutant or a fusion protein containing an IL-2 mutant) passes through the systemic circulation and thereby enters the site of action.
  • a drug eg, an IL-2 mutant or a fusion protein containing an IL-2 mutant
  • 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 over the dosing interval (AUC ⁇ ), “total exposure” or “"Total drug exposure over a period of time” (AUC 0- ⁇ ), area under the concentration-time curve at time t after administration (AUC 0-t ), etc.
  • Time to peak serum/plasma concentration is the time to reach peak serum/plasma concentration (C max ) after administration of (eg, an IL-2 mutant or a fusion protein containing an IL-2 mutant).
  • Half-life is the decrease in the concentration of a drug (e.g., IL-2 mutant or fusion protein containing an IL-2 mutant) measured in plasma or serum (or other biological matrix) to its value at a specific time The time required to reach half the concentration or volume.
  • a drug e.g., IL-2 mutant or fusion protein containing an IL-2 mutant
  • plasma or serum or other biological matrix
  • the elimination half-life is determined by the terminal or elimination (primary) phase of the plasma/serum concentration-time curve. See Michael Schrag and Kelly Regal, “Chapter 3 - Pharmacokinetics and Toxokinetics” of "Comprehensive Toxicology Guide to Preclinical Drug Development", 2013.
  • a fusion protein comprising an IL-2 mutant (e.g., a bivalent fusion protein form of an IL-2 mutant and Fc) described herein has a half-life of at least 10 hours (e.g., intravenously, subcutaneously, or Intramuscular injection, such as injection into 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.
  • the IL-2 mutants or fusion proteins comprising IL-2 mutants referred to herein can be further modified to include additional non-protein moieties known and readily available in the art.
  • additional 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 copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1 , 3-dioxolane, poly-1,3,6-trioxopentane, ethylene/maleic anhydride copolymer, polyamic acid (homopolymer or random copolymer), dextran or poly( n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylene polyol (such as glycerol), polyvinyl alcohol and mixtures thereof.
  • PEG polyethylene glycol
  • ethylene glycol/propylene glycol copolymer carboxymethylcellulose
  • dextran polyvinyl alcohol
  • polyvinylpyrrolidone poly-1 , 3-dioxolane, poly
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymers can be of any molecular weight and can be branched or unbranched.
  • the number of polymers attached to an IL-2 mutant or a fusion protein containing an IL-2 mutant may vary, and if multiple polymers are attached, they may be the same or different molecules.
  • the amount and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties of the IL-2 mutant to be improved or the fusion protein containing the IL-2 mutant; Function, whether IL-2 mutants or derivatives of fusion proteins containing IL-2 mutants will be useful in treatments under specific conditions, etc.
  • the IL-2 mutants or fusion proteins comprising IL-2 mutants described herein further comprise a tag selected from a chromophore, a fluorophore (e.g., coumarin, xanthene, cyanine, pyrene, boron polybenzazepines, oxazines and their derivatives), fluorescent proteins (such as GFP, phycobiliprotein and their derivatives), phosphorescent dyes (such as dioxetane, oxa Anthracene or carbocyanine dyes, lanthanide chelates), tandem dyes (e.g., cyanine-phycobiliprotein derivatives and xanthene-phycobiliprotein derivatives), particles (e.g., gold clusters, colloidal gold, microspheres, quantum dots), haptens, enzymes (eg, peroxidase, phosphatase, glycosidase, luciferase) and radioactive isotope
  • IL-2 mutants or fusion proteins containing IL-2 mutants can be further modified to include one or more other biologically active proteins, polypeptides, or fragments thereof.
  • biologically active or “biologically active” are used interchangeably and refer to exhibiting biological activity in the body to perform a specific function. For example, it may mean binding to a specific biomolecule, such as a protein, DNA, etc., and then promoting or inhibiting the activity of that biomolecule.
  • bioactive proteins or fragments thereof include proteins and polypeptides that are administered to patients as active pharmaceuticals to prevent or treat disease or symptoms, as well as proteins and polypeptides that are used for diagnostic purposes, such as for use in diagnostic tests or in vitro assays.
  • the biologically active protein or fragment thereof has immunostimulatory/immunomodulatory, membrane transport, or enzymatic activity.
  • the biologically active protein, polypeptide, or fragment thereof is an enzyme, hormone, growth factor, cytokine, or mixture thereof.
  • a biologically active protein, polypeptide, or fragment specifically recognizes a peptide of interest (eg, an antigen or other protein).
  • the biologically active protein or fragment thereof that may be included in an IL-2 mutant or a fusion protein comprising an IL-2 mutant described herein is an antigen-binding protein (eg, an antibody).
  • the biologically active protein or fragment thereof that may be included in an IL-2 mutant or a fusion protein comprising an IL-2 mutant as described herein is an antibody mimetic that is pronounced of Antibodies are small engineered proteins containing antigen-binding domains, (GGeering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015). These molecules are derived from existing human scaffold proteins and consist of a single polypeptide.
  • antibody mimetics that may be included in IL-2 mutants or fusion proteins containing IL-2 mutants as described herein may be, but are not limited to, designed ankyrin repeat proteins (DARPin; containing 3-5 fully synthetic Ankyrin repeat sequence, flanked by N-terminal and C-terminal cap domains), an affinity multimer (avimer; a high-affinity protein containing multiple A domains, each domain has an affinity for the target lower), or an anticoagulin (based on a lipid scaffold with four accessible loops, each of which can be randomized in sequence).
  • DARPin designed ankyrin repeat proteins
  • avimer avimer
  • an anticoagulin based on a lipid scaffold with four accessible loops, each of which can be randomized in sequence
  • the biologically active protein or fragment thereof that may be included in an IL-2 mutant or a fusion protein comprising an IL-2 mutant as described herein is an armadillo repeat protein (e.g., ⁇ -catenin, Alpha-importin, plakoglobin, adenomatous polyposis coli (APC)), contains armadillo repeating units (characteristic, the length of the repeated amino acid sequence is approximately 40 residues).
  • Each armadillo repeat unit consists of a pair of alpha helices forming a hairpin structure. Multiple repeated copies form the alpha solenoid structure.
  • Armadillo repeat proteins are able to bind different types of peptides, relying on a constant binding pattern along the peptide backbone without requiring specific conserved side chains or interactions with the free N- or C-terminus of the peptide.
  • the present application also relates to isolated nucleic acids encoding any IL-2 mutant or fusion protein comprising an IL-2 mutant described herein, including fusions encoding any IL-2 mutant or comprising an IL-2 mutant described herein.
  • Protein nucleic acid vectors are also contemplated.
  • isolated host cells eg, CHO cells, HEK293 cells, Hela cells or COS cells
  • the isolated nucleic acid further includes a nucleic acid sequence encoding an N-terminal signal peptide of an IL-2 mutant or a fusion protein comprising an IL-2 mutant.
  • a 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).
  • eukaryotic cells such as mammalian cells (e.g., CHO cells, HEK 293 cells, HeLa cells, COS cells).
  • the vector is a viral vector.
  • the vector is a non-viral vector, such as pTT5.
  • viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, herpes simplex virus vectors, and derivatives thereof.
  • Viral vector technology is well known in the art and is described in detail in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology handbooks. narrate.
  • 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 under in vitro or ex vivo conditions.
  • Many retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • self-inactivating lentiviral vectors are used.
  • 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 are ideal for achieving long-term Suitable tools for transduction because they allow long-term, stable integration of transgenes and propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.
  • 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 shaped macromolecules.
  • PLGA poly(lactic-co-glycolic acid)
  • PLA polylactic acid
  • PEI poly(ethyleneimine)
  • dendrimers shaped macromolecules dendrimers shaped macromolecules.
  • the carrier is a cationic lipid-based non-viral vector, such as cationic liposomes, lipid nanoemulsions, and solid lipid nanoparticles (SLN).
  • 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 a nucleic acid encoding an IL-2 mutant or a fusion protein containing an IL-2 mutant into a host cell. See Yin H. et al.., Nature Rev.
  • any one or more nucleic acids or vectors encoding an IL-2 mutant described herein or a fusion protein comprising an IL-2 mutant are introduced into a host cell (e.g., CHO, HEK 293, HeLa or COS), including but not limited to electroporation, sonoporation, photoporation, magnetofection, and hydroporation.
  • a host cell e.g., CHO, HEK 293, HeLa or COS
  • the vector contains a selectable marker gene or reporter gene for selecting from a population of host cells transfected with the vector (e.g., lentiviral vector, pTT5 vector) expressing an IL-2 mutant described herein or cells containing fusion proteins of IL-2 mutants.
  • the selectable marker and the reporter gene may be surrounded by appropriate regulatory sequences to enable their expression in the host cell.
  • a vector may contain transcriptional and translational terminators, an initiation sequence, and a promoter for regulating expression of the nucleic acid sequence.
  • any molecular cloning method known in the art may be used, including, for example, the use of restriction endonuclease sites and one or more selectable markers to clone the nucleic acid into the vector.
  • the nucleic acid is operably linked to a promoter.
  • promoters have been developed for gene expression in prokaryotic or eukaryotic cells (eg, mammalian cells), and any promoter known in the art may be used in this application. Promoters can be broadly classified as constitutive promoters or regulated promoters, such as inducible promoters.
  • a nucleic acid encoding an IL-2 mutant or a fusion protein comprising an IL-2 mutant described herein is operably linked to a constitutive promoter.
  • Constitutive promoters allow constitutive expression of heterologous genes (also called transgenes) in host cells.
  • promoters considered herein include, but are not limited to, the CMV promoter (CMV), human elongation factor-1 ⁇ (hEF1 ⁇ ), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), simian virus 40 early promoter (SV40), chicken ⁇ -actin promoter and CMV early enhancer (CAGG) coupling, Roche sarcoma virus (RSV) promoter, polyoma virus enhancer/herpes simplex thymidine kinase (MC1) promoter, beta actin (beta-ACT) promoter, "bone marrow Proliferative sarcoma virus enhancer, negative control region deletion, d1587rev primer binding site substitution (MND)” promoter.
  • CMV CMV promoter
  • hEF1 ⁇ human elongation factor-1 ⁇
  • UbiC ubiquitin C promoter
  • PGK phosphoglycerol kinase promoter
  • a nucleic acid encoding an IL-2 mutant or a fusion comprising an IL-2 mutant described herein is operably linked to a CMV promoter.
  • a nucleic acid encoding an IL-2 mutant or a fusion protein comprising an IL-2 mutant described herein is operably linked to an inducible promoter.
  • Inducible promoters belong to the category of regulated promoters.
  • An inducible promoter 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 inducing agent (i.e., an inducing agent), or a combination thereof. In some embodiments, the inducing conditions do not induce expression of endogenous genes in the host cell.
  • the induction conditions are selected from: inducer, radiation (eg, ionizing radiation, light), temperature (eg, heat), redox state, and activation state of the host cell.
  • the inducible promoter can be the NFAT promoter, promoter or NF ⁇ B promoter.
  • a method for preparing an IL-2 mutant or a fusion protein comprising an IL-2 mutant comprising: (a) in a state where the encoded IL-2 mutant or a fusion protein comprising IL-2 can be effectively expressed; Host cells (e.g., CHO cells, HEK 293 cells, Hela cells or COS cells); and (b) obtaining an expressed IL-2 mutant or a fusion protein comprising an IL-2 mutant from the host cell.
  • Host cells e.g., CHO cells, HEK 293 cells, Hela cells or COS cells
  • 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.
  • IL-2 mutants or fusion proteins comprising IL-2 mutants described herein can be prepared using any method known in the art or described herein.
  • 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, IL-2 mutants or fusion proteins comprising IL-2 mutants described herein can be expressed in prokaryotic cells.
  • Polynucleic acid 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 may be used in this application. Selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cells being transformed by the vector.
  • Each vector contains various components depending on the function of the vector (amplification or expression of a heterologous polynucleotide, or both) and the compatibility between the vector and the particular host cell in which it is used.
  • Vector components typically include, but are not limited to: replication origin sites, selectable marker genes, promoters, ribosome binding sites (RBS), signal sequences, heterologous nucleic acid inserts, and transcription termination sequences.
  • plasmid vectors contain replicon and control sequences from species that are compatible with the host cells with which they are used. Vectors typically carry a replication site and marker sequences that provide phenotypic selection in transformed cells.
  • E. coli is commonly 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 to identify transformed cells.
  • pBR322 its derivatives, or other bacterial plasmids or phages may also contain or be modified to contain promoters that can be used by microorganisms to express endogenous proteins. Carter et al., U.S. Pat. No. 5,648,237 details examples of pBR322 derivatives used to express specific antibodies.
  • phage vectors containing replicons and control sequences that are compatible with the host microorganism can be used as transformation vectors with these host cells.
  • phages such as GEM TM -11 can be used to prepare recombinant vectors that can be used to transform susceptible host cells, such as E. coli LE392.
  • the promoter is an untranslated regulatory sequence located upstream (5′) of the cistron that regulates downstream gene expression.
  • Prokaryotic promoters are generally divided into two categories, inducible and constitutive.
  • An inducible promoter is a promoter that can initiate and increase the level of cistronic transcription in response to changes in culture conditions (for example, the presence or absence of nutrients or changes in temperature).
  • promoters recognized by potential host cells are known.
  • the promoter is removed from the source DNA by restriction enzymes and the isolated promoter sequence is inserted into the vector of the present application, and the selected promoter is operably linked to the cistron DNA encoding the polypeptide.
  • Both native promoter sequences and many heterologous promoters can be used to direct amplification and/or expression of target genes.
  • heterologous promoters are utilized because heterologous promoters generally allow for greater transcription and higher yields of expressed target genes compared to native target polypeptide promoters.
  • Promoters suitable for prokaryotic hosts include the PhoA promoter, -galactosidase and lactose promoter systems, tryptophan (trp) promoter systems and hybrid promoters such as tac or trc promoters.
  • trp tryptophan
  • hybrid promoters such as tac or trc promoters.
  • Other promoters functional in bacteria such as other known bacterial or phage promoters
  • Their nucleic acid sequences have been published, allowing the skilled person to connect them to the cistron encoding the light and heavy chains of interest using linkers or adapters to provide any required restriction sites (Siebenlist et al.,( 1980)Cell 20:269).
  • each cistron within the recombinant vector contains a secretion signal sequence component that directly directs the transmembrane transfer of the expressed polypeptide.
  • the signal sequence can be a component of the vector or a part of the target polypeptide DNA inserted into the vector.
  • the signal sequence selected for this application should be one that is recognized and processed by the host cell (ie, cleaved by a signal peptidase).
  • the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp, or thermostable enterotoxin II (STII) Leader, LamP, PhoE, PelB, OmpA and MBP.
  • a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp, or thermostable enterotoxin II (STII) Leader, LamP, PhoE, PelB, OmpA and MBP.
  • production of the IL-2 mutants of the present application or fusion proteins containing IL-2 mutants can occur in the cytoplasm of the host cell, thus eliminating the need for a secretion signal sequence to be present within each cistron.
  • the polypeptide components are expressed, folded, and assembled to form a protein construct within the cytoplasm.
  • Certain host strains eg, E. coli trxB-strain
  • Prokaryotic host cells suitable for expression of the proteins of the present application include archaea and eubacteria, such as Gram-negative or Gram-positive bacteria.
  • useful bacteria include Escherichia coli (e.g., E. coli), Bacillus (e.g., Bacillus subtilis), Enterobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Oscillator or Paracoccus.
  • Gram-negative cells are used.
  • E. coli cells serve as hosts for this application. Examples of E.
  • coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and its derivatives, including those having Genotype W3110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompT A(nmpc fepE)degP41 kanR strain 33D3 (US Pat. No. 5,639,635).
  • 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.
  • host cells should secrete minimal amounts of proteolytic enzymes and require the appropriate addition of additional protease inhibitors to the cell culture.
  • Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA can be replicated as an extrachromosomal element or through chromosomal integration. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment with calcium chloride is typically used for bacterial cells that contain a large cell wall barrier. Another transformation method uses polyethylene glycol/dimethyl sulfoxide. Another technique is electroporation.
  • Prokaryotic cells used to produce the protein constructs of the present application are grown in media known in the art and suitable for culturing the host cell of choice. Suitable media include luria broth (LB) and necessary nutritional supplements.
  • the culture medium further contains a selection agent selected based on the structure of the expression vector to selectively allow growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium for the growth of cells expressing an ampicillin resistance gene.
  • any necessary supplements may also be added at appropriate concentrations, alone or as a mixture with other supplements or media (such as complex nitrogen sources).
  • the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycine, dithioerythritol and dithiothreitol.
  • Prokaryotic host cells are cultured at the appropriate temperature. For example, for the growth of E. 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 of the culture medium can be anywhere between pH 5 and 9, depending primarily on the host organism. For E. coli, the pH value is preferably 6.8 to 7.4, and more preferably 7.0.
  • an inducible promoter is used in the expression vector of the present application, protein expression is induced under conditions suitable for promoter activation.
  • the PhoA promoter is used to control the transcription of a polypeptide. Therefore, transformed host cells are cultured in phosphate-limiting medium for induction.
  • the phosphate-limited medium is CRAP medium (see Simmons et al., J. Immunol. Methods (2002), 263:133-147).
  • CRAP medium see Simmons et al., J. Immunol. Methods (2002), 263:133-147.
  • a variety of other inducers known in the art may be used and are known in the art.
  • the protein constructs expressed herein are secreted into and recovered from the periplasm of the host cell. Protein recovery typically involves the destruction of microorganisms, often through osmotic shock, sonication, or lysis. Once cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. For example, the protein can be further purified by affinity resin chromatography. Alternatively, the protein can be transported to the culture medium and isolated therein. The cells can be removed from the culture medium and the culture supernatant filtered and concentrated to further purify the resulting protein. The expressed polypeptides can be further separated and identified by common methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot testing.
  • PAGE polyacrylamide gel electrophoresis
  • protein can be produced on a large scale through a fermentation process.
  • Various large-scale fed-batch fermentation procedures are available for the production of recombinant proteins.
  • the capacity of large-scale fermentation is at least 1000 liters, preferably 1000 to 100000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, specifically glucose (preferred carbon/energy source).
  • Small-scale fermentation usually refers to fermentation in fermenters with a capacity volume of no more than 100 liters, ranging from 1 liter to 100 liters.
  • protein expression is usually induced after the cells have grown to the desired density under appropriate conditions, for example, when the OD550 is about 180-220, when the cells are in the early stationary phase.
  • inducers known in the art and described above may be used. Cells can be grown for a short period of time before induction. Cells are typically induced for approximately 12-50 hours, although induction times that may be longer or shorter may be used.
  • host prokaryotic cells can be cotransformed using additional vectors that overexpress chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, or DsbG) or FkpA (peptides with chaperone activity).
  • chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, or DsbG) or FkpA (peptides with chaperone activity).
  • prolyl cis-trans isomerase Chaperones have been shown to help promote the correct folding and solubilization of heterologous proteins produced in bacterial host cells.
  • certain host strains lacking proteolytic enzymes can be used in this application.
  • the host cell strain can be modified to genetically mutate genes encoding known bacterial proteases, such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof.
  • E. coli protease deletion strains can be used, as described in Joly et al., (1998), supra; Georgiou et al., US Pat. No. 5,264,365; Georgiou et al., No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72 (1996).
  • E. coli strains lacking proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins can be used in expression systems encoding IL-2 mutants or fusion proteins containing IL-2 mutants as described herein. as host cells.
  • 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 employed.
  • the following procedures are examples of suitable purification procedures: Fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reversed phase liquid chromatography HPLC, silica or cation exchange resin (e.g. DEAE) chromatography, chromatographic focusing, SDS-PAGE , ammonium sulfate precipitation and gel filtration, for example, Sephadex G-75.
  • Protein A immobilized on a solid phase is used for immunoaffinity purification of protein constructs comprising an Fc region described herein.
  • Protein A is a 42 kDa surface protein from Staphylococcus aureus that has high binding affinity to Fc-containing structures, such as, for example, the IL-2 mutant-Fc-containing fusion proteins described herein. Lindmark et al., (1983) J. Immunol. Meth. 62:1-1.
  • the solid phase for immobilizing protein A preferably contains a column with a glass or silica surface, and is more preferably a controlled-pore glass column or a silicic acid column.
  • columns are coated with reagents, such as glycerol, to prevent nonspecific adhesion of contaminants.
  • reagents such as glycerol
  • the solid phase is then washed to remove contaminants that are non-specifically bound to the solid phase.
  • the target protein construct is recovered from the solid phase by elution.
  • vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence.
  • Vectors for eukaryotic hosts may also be an insert encoding a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected is preferably one that is recognized and processed by the host cell (ie, cleaved by a signal peptidase). In mammalian cell expression, it is useful to obtain mammalian signal sequences as well as viral secretion leaders, for example, the herpes simplex gD signal.
  • the DNA of this precursor region is linked in reading frame to the DNA encoding the protein construct of the present application.
  • mammalian expression vectors do not require an origin of replication element (the SV40 origin is usually used simply because it contains an early promoter).
  • Selection and cloning vectors can contain selectable genes, also known as selectable markers.
  • selectable genes encode proteins that (a) provide resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic proteins, or (c) provide Proteins of key nutrients that complex media cannot provide, such as the gene encoding Bacillus D-alanine racemase.
  • drugs to prevent the growth of host cells. Those cells successfully transformed with the heterologous gene produce a drug-resistant protein and thus survive the selection regimen. Examples of this advantageous selection use the drugs neomycin, mycophenolic acid, and hygromycin.
  • selectable markers suitable for use in mammalian cells are those capable of identifying cells capable of carrying nucleic acids encoding protein constructs described herein, such as DHFR, thymidine kinase, metallothionein- I and -II are preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.
  • DHFR human hamster ovary
  • a host cell (especially a wild-type host 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 growth of the cells in culture medium containing a selectable marker, such as an aminoglycoside antibiotic, for example, kanamycin, neomycin or G418. See U.S. Pat. No. 4,965,199.
  • APH aminoglycoside 3'-phosphotransferase
  • 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. There is an AATAAA sequence at the 3' end of most eukaryotes, which may be a signal for adding a polyA tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.
  • a promoter for example, by a promoter obtained from the genome of a virus, such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian Sarcoma viruses, cytomegalovirus, retroviruses, hepatitis B virus and most preferably simian virus 40 (SV40), promoters from heterologous mammalian sources, e.g., actin promoters or immunoglobulin promoters, from heat shock promoters, provided such promoters are compatible with the host cell system.
  • a promoter obtained from the genome of a virus, such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian Sarcoma viruses, cytomegalovirus, retroviruses, hepatitis B virus and most preferably simian virus 40 (SV40), promote
  • the SV40 viral early and late promoters are readily available as SV40 restriction fragments, which also contain the SV40 viral origin of replication.
  • the human cytomegalovirus immediate early promoter is readily available as a HindIII E restriction fragment.
  • U.S. Pat. No. 4,419,446 discloses a system for expressing DNA in a mammalian host using bovine papilloma virus as a vector. Improvements to this system are detailed in U.S. 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.
  • the transcription of DNA encoding the protein construct of the present application by higher eukaryotes is usually increased by inserting an enhancer sequence into the vector.
  • enhancer sequences have been found in mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Often, however, enhancers from eukaryotic viruses are used. Examples include the SV40 enhancer distal to the origin of replication (100-270 bp), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer distal to the origin of replication, and the adenovirus enhancer. See Yaniv, Nature 297:17-18 (1982), on enhancer 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.
  • Expression vectors used in eukaryotic host cells also contain sequences necessary for transcription termination and stabilization of the mRNA. These sequences are usually obtained from the 5' untranslated region of eukaryotic or viral DNA or cDNA, and occasionally the 3' end. These regions comprise nucleic acid fragments that are transcribed as polyadenylate fragments in the untranslated portion of the mRNA encoding the polypeptide.
  • One suitable transcription termination element is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vectors disclosed therein.
  • Suitable host cells for cloning or expressing DNA in vectors described herein include higher eukaryotic cells, including vertebrate host cells, as described herein.
  • the culture and propagation of vertebrate cells has become a routine procedure sequence.
  • 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 293 cell subtype Cloning, for growth in suspension culture, Graham et al., J. Gen Virol.
  • monkey kidney cells (CV1ATCC 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 liver cells (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TR1 cells (Mather et al ., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells and human hepatoma cell line (Hep G2).
  • the host cells are transformed with the above-mentioned expression vectors or cloning vectors to produce protein constructs and cultured in appropriately modified conventional nutrient media to induce promoters, select transformants or amplify genes encoding the desired sequences.
  • Host cells used to produce protein constructs of the present application can be cultured in a variety of media.
  • Commercial 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.
  • Ham et al. Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), US Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655 or 5,122,469, WO 90 /03430, WO 87/00195 or US Pat. Re.
  • any culture medium can be used as the culture medium for host cells. Any of these media can be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers as needed (such as HEPE), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug gentamicinTM ), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or etc. Efficient energy. 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 the ordinarily skilled artisan.
  • compositions comprising any IL-2 mutant described herein or a fusion protein comprising an IL-2 mutant, or encoding any IL-2 mutant described herein or comprising an IL-2 mutant fusion protein
  • These compositions may optionally further comprise pharmaceutically acceptable carriers and/or excipients.
  • the excipients include excipients and/or stabilizers.
  • compositions can be prepared by combining an IL-2 mutant or a fusion protein comprising an IL-2 mutant with the desired purity described herein and an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) are mixed and prepared into a lyophilized preparation or aqueous solution.
  • the IL-2 mutants or fusion proteins comprising IL-2 mutants or compositions comprising the same (eg, pharmaceutical compositions) described herein can be used in a variety of applications, such as diagnosis, molecular detection, and therapy.
  • the present application relates to a method of treating a disease in an individual (e.g., a human), comprising administering to the individual an effective dose of any IL-2 mutant, fusion protein comprising an IL-2 mutant, as described herein , nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells containing the nucleic acids, or pharmaceutical compositions containing the same.
  • the fusion protein comprising an IL-2 mutant is a fusion protein of an IL-2 mutant and Fc.
  • the disease is associated with modulation of Treg cells in the subject.
  • the disease includes an inflammatory disease or an autoimmune disease.
  • the IL-2 mutant or a fusion protein comprising an IL-2 mutant or a pharmaceutical composition comprising an IL-2 mutant or a fusion protein thereof is administered by intravenous injection, intramuscular injection, or subcutaneous injection.
  • the disease includes lupus, graft versus host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopy, 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 Behcet's disease.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL- 2 mutants, fusion proteins containing IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells containing the nucleic acids, or pharmaceutical compositions containing the same.
  • an inflammatory or autoimmune disease e.g., lupus
  • the IL-2 mutant comprises a deletion or mutation of amino acid residues RPRDL at positions 81-85 of the human wild-type IL-2 amino acid sequence.
  • the amino acid sequence of human wild-type IL-2 is shown in SEQ ID NO: 1.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein Body, fusion protein containing IL-2 mutant, nucleic acid encoding IL-2 mutant or its fusion protein, vector containing the nucleic acid and Host cells, or pharmaceutical compositions containing the same.
  • the IL-2 mutant includes all deletions of amino acid residues RPRDL at positions 81 to 85 of the human wild-type IL-2 amino acid sequence.
  • the IL-2 mutant further comprises mutations of E67 and N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and N71 are E67K and N71S, respectively.
  • the IL-2 mutant includes all deletions of amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2, and relative to human wild-type IL-2 Amino acid sequence of E67K and N71S mutations.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the IL-2 mutant comprises amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RPL.
  • the IL-2 mutant further comprises a mutation of E67 relative to the amino acid sequence of human wild-type IL-2.
  • the mutation of E67 is E67A.
  • the IL-2 mutant comprises amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RPL, and relative to human wild-type IL-2 Amino acid sequence of the E67A mutation.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the IL-2 mutant comprises amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RPL, and further comprises N71 and S75 relative to the human wild-type IL-2 amino acid sequence. mutation.
  • the mutations of N71 and S75 are N71W and S75I, respectively.
  • the IL-2 mutant comprises amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RPL, and relative to human wild-type IL-2 N71W and S75I mutations of the amino acid sequence.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the IL-2 mutant comprises amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RHL.
  • the IL-2 mutant further comprises mutations of E67 and/or V91 relative to the amino acid sequence of human wild-type IL-2.
  • the mutation of E67 is E67T.
  • the mutation of V91 is V91K.
  • the IL-2 mutant includes amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and relative to human wild-type IL-2 E67T and/or V91K mutations in the amino acid sequence.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the IL-2 mutant comprises amino acid residues RPRDL at positions 81-85 relative to the human wild-type IL-2 amino acid sequence mutated to RHL, and further comprises L70 and S75 relative to the human wild-type IL-2 amino acid sequence. mutation.
  • the mutations of L70 and S75 are L70I and S75F, respectively.
  • the IL-2 mutant includes amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and relative to human wild-type IL-2 L70I and S75F mutations of the amino acid sequence.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the IL-2 mutant comprises a mutation of amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 to RHL, and further comprises a mutation of N71 relative to the amino acid sequence of human wild-type IL-2 .
  • the mutation of N71 is N71I.
  • the IL-2 mutant includes amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to RHL, and relative to human wild-type IL-2 N71I mutation of the amino acid sequence.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the IL-2 mutant comprises amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to SPL.
  • the IL-2 mutant further comprises mutations of E67 and N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and N71 are E67M and N71S, respectively.
  • the IL-2 mutant includes amino acid residues RPRDL at positions 81-85 relative to the amino acid sequence of human wild-type IL-2 mutated to SPL, and relative to human wild-type IL-2 Amino acid sequence of E67M and N71S mutations.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the IL-2 mutant comprises mutations of amino acid residue S at position 75 (S75) and amino acid residue R (R83) at position 83 relative to the amino acid sequence of human wild-type IL-2.
  • the amino acid sequence of human wild-type IL-2 is shown in SEQ ID NO: 1.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the IL-2 mutant comprises mutations of amino acid residue S at position 75 (S75) and amino acid residue R (R83) at position 83 relative to the amino acid sequence of human wild-type IL-2.
  • S75 in the IL-2 mutant is mutated to an uncharged amino acid residue.
  • the mutation of S75 is S75P, S75F, S75Y, S75G, S75A, S75V, S75T, S75I or S75L.
  • the mutation of R83 is R83N, R83F, R83Y, R83P, R83L, R83A, R83K, or R83E.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75P
  • the mutation of R83 is R83Y.
  • the IL-2 mutant further comprises mutations of E67 and D84 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and D84 are E67A and D84N, respectively.
  • the IL-2 mutants comprise S75P, R83Y, E67A and D84N mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75P
  • the mutation of R83 is R83A.
  • the IL-2 mutant further comprises mutations of H16, L70 and N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of N71 and N71 are H16D, L70I and N71G respectively.
  • the IL-2 mutants comprise S75P, R83A, H16D, L70I and N71G mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75F
  • the mutation of R83 is R83P.
  • the IL-2 mutant further comprises mutations of H16 and/or N71 relative to the amino acid sequence of human wild-type IL-2.
  • the mutation of H16 is H16Q or H16N. In some embodiments, the mutation of N71 is N71S. In some embodiments, the IL-2 mutants comprise S75F, R83P and H16Q mutations relative to the amino acid sequence of human wild-type IL-2. In some embodiments, the IL-2 mutants comprise S75F, R83P, H16N and N71S mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75F
  • the mutation of R83 is R83L.
  • the IL-2 mutant further comprises mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and L70 are E67R and L70V, respectively.
  • the IL-2 mutants comprise S75F, R83L, E67R and L70V mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75I
  • the mutation of R83 is R83L.
  • the IL-2 mutant further comprises mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and L70 are E67G and L70V, respectively.
  • the IL-2 mutants comprise S75I, R83L, E67G and L70V mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein body, include Fusion proteins of IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells containing the nucleic acids, or pharmaceutical compositions containing the same.
  • an inflammatory or autoimmune disease e.g., lupus
  • the mutation of S75 is S75I
  • the mutation of R83 is R83K.
  • the IL-2 mutant further comprises mutations of E67 and L70 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E67 and L70 are E67S and L70V, respectively.
  • the IL-2 mutants comprise S75I, R83K, E67S and L70V mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75T
  • the mutation of R83 is R83A or R83F.
  • the IL-2 mutant further comprises mutations of H16 and E67 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of H16 and E67 are H16N and E67K respectively.
  • the IL-2 mutants comprise S75T, R83A, H16N and E67K mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75V
  • the mutation of R83 is R83F.
  • the IL-2 mutant further comprises mutations of E15 and H16 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of E15 and H16 are E15K and H16Q, respectively.
  • the IL-2 mutant or fusion protein thereof contains S75V, R83F, E15K and H16Q mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75V
  • the mutation of R83 is R83N.
  • the IL-2 mutant further comprises mutations of L70 and V91 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L70 and V91 are L70F and V91K respectively.
  • the IL-2 mutants comprise S75V, R83N, L70F and V91K mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75G
  • the mutation of R83 is R83L.
  • the IL-2 mutant further comprises mutations of L17, V91 and I92 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L17, V91, and I92 are L17I, V91E, and I92L, respectively.
  • the IL-2 mutants comprise S75G, R83L, L17I, V91E and I92L mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75Y
  • the mutation of R83 is R83N.
  • the IL-2 mutants described in the present application further comprise mutations of L17 and V91 relative to the amino acid sequence of human wild-type IL-2.
  • the mutations of L17 and V91 are L17I and V91K, respectively.
  • the IL-2 mutants comprise S75Y, R83N, L17I and V91K mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75A
  • the mutation of R83 is R83K.
  • the IL-2 mutant further comprises a mutation of L17 relative to the amino acid sequence of human wild-type IL-2.
  • the mutation of L17 is L17I.
  • the IL-2 mutants comprise S75A, R83K and L17I mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • the mutation of S75 is S75L
  • the mutation of R83 is R83E.
  • the IL-2 mutants described in the present application further comprise mutations of L63 and E67 relative to the amino acid sequence of human wild-type IL-2.
  • the IL-2 mutants comprise S75L, R83E, L63V and E67F mutations relative to the amino acid sequence of human wild-type IL-2.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein body, or a fusion protein containing IL-2 mutants.
  • the IL-2 mutant or its fusion protein in addition to the above-mentioned mutations, further includes a mutation relative to the amino acid sequence C125 of human wild-type IL-2.
  • the C125 mutation is C125S or C125A.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein body, or a fusion protein containing IL-2 mutants.
  • the IL-2 mutant includes the amino acid sequence shown in any one of SEQ ID NOs: 5-34 or a variant thereof, which has at least the same amino acid sequence with the amino acid sequence shown in any one of SEQ ID NOs: 5-34.
  • About 90% eg, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology.
  • the fusion protein comprising an IL-2 mutant is a fusion protein of an IL-2 mutant and Fc.
  • the fusion protein of IL-2 mutant and Fc is in a bivalent form.
  • the bivalent fusion protein of IL-2 mutant and Fc comprises the amino acid sequence shown in any one of SEQ ID NOs: 76-105 or a variant thereof, and the variant is the same as SEQ ID NOs:
  • the amino acid sequence shown in any one of 76-105 has at least about 80% (for example, at least 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98% or 99%) sequence homology.
  • the fusion protein of IL-2 mutant and Fc is in a monovalent form.
  • the disease or disorder is selected from the group consisting of lupus, graft versus host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopy Diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophic bullous Epidermolysis and Behcet's disease.
  • the present application relates to a method of treating an individual suffering from an inflammatory or autoimmune disease (e.g., lupus), comprising administering to the individual an effective amount of an IL-2 mutation described herein
  • One or more additional active ingredients required for the particular indication being treated preferably with active ingredients that do not interfere with the therapeutic effect of each other.
  • the immunosuppressive agent is selected from: glucocorticoid; azathioprine; cyclosporine A; methotrexate; anti-CD3 antibody; or anti-TNF- ⁇ antibody, etc.
  • IL-2 mutants may be by any known and convenient means, including by injection or infusion. Routes of administration are in accordance with known and accepted methods, such as by single or multiple bolus injections or by prolonged infusion in an appropriate manner.
  • the dosage and desired drug concentration of the pharmaceutical compositions of the present application may vary depending on the specific use. Determining the appropriate dosage or route of administration is well within the skill of the ordinarily skilled person. Animal experiments provide reliable guidance for determining effective doses for human treatment. It can be based on the principles in 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 Make interspecies analogies of effective doses.
  • IL-2 mutants When using IL-2 mutants, fusion proteins containing IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells containing such nucleic acids, or pharmaceutical compositions containing the same, they are administered in vivo
  • the therapeutically effective amount may depend, for example, on the context and goals of treatment.
  • dosage levels for treatment will depend on the molecule being delivered, the indication for treatment with the IL-2 mutant, the route of administration, and the patient's body size (body weight, body surface or organ size ) and/or condition (age and general health).
  • kits comprising any of the pharmaceutical compositions described herein are contemplated, and instructions are preferably provided for use thereof, such as for treating a disease described herein (eg, an autoimmune disease).
  • a disease described herein eg, an autoimmune disease
  • the kit of the present application includes one or more nucleic acids containing IL-2 mutants described herein, fusion proteins containing IL-2 mutants, encoding IL-2 mutants or fusion proteins thereof, vectors containing this nucleic acid, and Host cells, or containers of pharmaceutical compositions containing them, are used, for example, to treat disease.
  • Instructions for treating diseases (such as autoimmune diseases).
  • the kit may further include instructions for selecting individuals (eg, humans) suitable for treatment based on identifying whether the individual has the disease and the stage of the disease.
  • individuals eg, humans
  • Containers may be unit doses, bulk packages (eg, multi-dose packages), or subunit doses.
  • kits of this application are usually written instructions on the label or package insert (e.g., paper included with the kit), but machine-readable instructions (e.g., instructions stored on a magnetic or optical disk) are also acceptable. of.
  • the kit of this application is packaged appropriately. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed Mylar or plastic bags), and the like. Also considered are packaging for use with specific devices, such as infusion devices such as micropumps.
  • the kit may have a sterile access port (eg, the container may be an intravenous solution bag or a vial with a stopper that can be pierced 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 a fusion protein thereof, a vector comprising such nucleic acid and host cells, or pharmaceutical compositions containing the same.
  • the container may further comprise a second pharmaceutically active agent.
  • Kits may optionally provide additional components such as buffers and interpretive information.
  • a kit consists of a container and a label or package insert on or associated with the container.
  • the kit includes: a) a nucleic acid comprising any of the IL-2 mutants described herein, a fusion protein comprising an IL-2 mutant, or a nucleic acid encoding an IL-2 mutant or a fusion protein thereof; , vectors and host cells comprising this nucleic acid, or pharmaceutical compositions comprising the same, and b) at least one effective amount of other agents capable of enhancing IL-2 mutants, fusion proteins comprising IL-2 mutants, encoding Effects (such as therapeutic effects) of nucleic acids of IL-2 mutants or fusion proteins thereof, vectors and host cells containing the nucleic acids, or pharmaceutical compositions containing the same.
  • the kit includes: a) any IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a nucleic acid comprising vectors and host cells of such nucleic acid, or pharmaceutical compositions comprising the same, and b) administering to an individual a protein comprising an IL-2 mutant, a fusion protein comprising an IL-2 mutant, or encoding an IL-2 mutant or a fusion protein thereof Instructions for use of nucleic acids, vectors and host cells containing the nucleic acids, or pharmaceutical compositions containing the same for the treatment of autoimmune diseases (eg, lupus).
  • autoimmune diseases eg, lupus
  • the kit includes: a) any IL-2 mutant described herein, a fusion protein comprising an IL-2 mutant, a nucleic acid encoding an IL-2 mutant or a fusion protein thereof, a nucleic acid comprising Vectors and host cells of this nucleic acid, or pharmaceutical compositions containing the same, and b) at least one effective amount of other agents capable of enhancing IL-2 mutants, fusion proteins containing IL-2 mutants, encoding IL-2 2.
  • the IL-2 mutants, fusion proteins containing IL-2 mutants, nucleic acids encoding IL-2 mutants or fusion proteins thereof, vectors and host cells containing the nucleic acids, or pharmaceutical compositions and other substances containing the same Can exist in separate containers or in the same container.
  • the kit may comprise one specific composition or two or more compositions, one of which includes an IL-2 mutant, a fusion protein comprising an IL-2 mutant, a protein encoding an IL-2 mutant Or a nucleic acid of a fusion protein thereof, a vector and a host cell containing the nucleic acid, or a pharmaceutical composition containing the same, another composition including another pharmaceutical agent.
  • the kit includes a nucleic acid (or set of nucleic acids) encoding an IL-2 mutant or a fusion protein thereof (eg, a fusion protein of an IL-2 mutant and Fc).
  • the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an IL-2 mutant or a fusion protein thereof (eg, a fusion protein of an IL-2 mutant and Fc), and b) a A host cell that expresses a nucleic acid (or a set of nucleic acids).
  • the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an IL-2 mutant or a fusion protein thereof (eg, a fusion protein of an IL-2 mutant and Fc), and b) using Instructions for: i) expressing an IL-2 mutant or a fusion protein thereof in a host cell, ii) preparing a composition comprising an IL-2 mutant or a fusion protein thereof, and iii) administering to an individual a composition comprising the IL-2 mutant or a fusion protein thereof or fusion proteins thereof to treat autoimmune diseases (e.g., lupus).
  • autoimmune diseases e.g., lupus
  • the kit includes: a) a nucleic acid (or a set of nucleic acids) encoding an IL-2 mutant or a fusion protein thereof (eg, a fusion protein of an IL-2 mutant and Fc), b) an expression a host cell for a nucleic acid (or set of nucleic acids), and c) instructions for use, suitable for: i) expressing an IL-2 mutant or a fusion protein thereof in a host cell, ii) preparing an IL-2 mutant or a fusion protein thereof; a composition, and iii) administering to an individual a composition comprising an IL-2 mutant or a fusion protein thereof to treat an autoimmune disease (eg, lupus).
  • an autoimmune disease eg, lupus
  • the present application also relates to articles, including vials (such as sealed vials), bottles, cans, flexible packaging, etc.
  • the article contains a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, and the like.
  • Containers can be made from a variety of materials, such as glass or plastic.
  • the container contains a composition that is effective in treating a disease or disorder described herein (e.g., an autoimmune disease) and may have a Bacterial access port (eg, the container may 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 in an individual.
  • the label or package insert further contains instructions for administering the composition to an individual. Labels may indicate instructions for reconstruction and/or use.
  • the container holding the pharmaceutical composition can be a multi-use vial, allowing repeated administration of the reconstituted formulation (eg, 2-6 administrations).
  • Package insert means the instructions usually included in the commercial packaging of a therapeutic product that contain information on the indications, usage, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic product.
  • the diseases indicated by the instructions as treatable include lupus, graft versus host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, atopy Diseases, asthma, inflammatory bowel disease, autoimmune hepatitis, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, alopecia areata, psoriasis, vitiligo, dystrophic bullous Epidermolysis and Behcet's disease.
  • the article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution.
  • a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate buffered saline such as phosphate buffered saline
  • Ringer's solution phosphate buffered saline
  • dextrose solution a pharmaceutically acceptable buffer
  • other required materials may further be included, including other buffers, diluents, filters, needles and syringes.
  • kits or article of manufacture includes multiple unit doses of a pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and compounding pharmacies.
  • Example 1 Design and expression of human wild-type IL-2 and IL-2 mutants
  • the nucleic acid sequence encoding human wild-type IL-2 (the amino acid sequence is shown in SEQ ID NO: 1) was synthesized by Nanjing Genscript Biotechnology Co., Ltd., and at the 5' end and 3' end of the synthesized wild-type IL-2 nucleic acid sequence Restriction sites were introduced into the ' ends respectively. After the synthesized nucleic acid fragment is digested, it is connected to the vector fragment that has been digested by the same double enzyme to obtain the wild-type IL-2 expression vector.
  • the C125S mutation was introduced at position 125 of wild-type IL-2 (the amino acid sequence is shown in SEQ ID NO: 2, this mutation can prevent cysteine mismatching or aggregation), that is Obtain the expression vector of the IL-2 C125S mutant; introduce the C125A mutation at position 125 of wild-type IL-2 (the amino acid sequence is shown in SEQ ID NO: 3, this mutation can promote efficient folding and improve expression), to obtain IL -2 Expression vector for C125A mutant.
  • the above two IL-2 mutants did not change their binding effect on the IL-2 receptor.
  • IL-2R ⁇ The extracellular region coding sequence of IL-2R ⁇ (Uniprot ID: P01589) was fully gene synthesized by Nanjing GenScript Biotechnology Co., Ltd. after codon optimization. The synthesized gene sequence was constructed into an expression vector carrying the His tag gene and expressed in 293F cells. According to the operating instructions, a (Ni) nickel column was used to purify IL-2R ⁇ . Briefly, immobilized metal affinity chromatography (IMAC) was performed using Ni-NTA from QIAGEN. First, use buffer A1 (50mM Na 3 PO 4 , 0.15M NaCl, pH 7.2) to balance the nickel column, with a flow rate of 150cm/h.
  • buffer A1 50mM Na 3 PO 4 , 0.15M NaCl, pH 7.2
  • the pH of the culture supernatant was adjusted to 7.2, and the sample was loaded at room temperature with a flow rate of 150cm/h. Subsequently, the column was equilibrated again with 6 column volumes of A1 buffer at a flow rate of 150 cm/h. Finally, 10 times the column volume of 50mM PB solution (containing 0.15M NaCl and 0.2M imidazole, pH 7.2) was used for elution, and the eluate was collected.
  • 50mM PB solution containing 0.15M NaCl and 0.2M imidazole, pH 7.2
  • IL-2R ⁇ -Fc heterodimer constructed by KIH (Knob-into-Hole) technology.
  • IL-2R ⁇ -Fc heterodimer constructed by KIH (Knob-into-Hole) technology.
  • IL The extracellular region coding sequences of -2R ⁇ (Uniprot ID: P14784) and IL-2R ⁇ (Uniprot ID: P31785) were cloned and fused to Fc hole molecule (SEQ ID NO: 109) and Fc knob molecule (SEQ ID NO: 108) respectively. ), used to prepare IL-2R ⁇ -Fc heterodimer.
  • IL-2R ⁇ -Fc hole and IL-2R ⁇ -Fc knob were constructed into pcDNA3.1 eukaryotic expression vector respectively, and then co-transfected into 293F cells, and the cell culture medium was collected.
  • IL-2R ⁇ -Fc heterodimer was purified by protein A affinity chromatography and gel filtration Superdex 200 column chromatography. Briefly, a Protein A affinity chromatography column was used to purify the culture medium of 293F cells. First, the Protein A column was equilibrated with 50mM PBS buffer containing 0.15M NaCl (pH7.2). The flow rate was 150cm/h and the volume was the column volume. 6 times.
  • the supernatant of the culture medium (pH value 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. Then, after purification by gel filtration Superdex 200 column chromatography, IL-2R ⁇ -Fc heterodimer was obtained.
  • Biotinylation labeling of IL-2R ⁇ and IL-2R ⁇ According to the instructions of the Biotin-Protein Ligase/BirA Enzyme (GeneCopoeia cat#BI001) kit, use biotinylation ligase to biotinylate IL-2R ⁇ and IL-2R ⁇ . mark. Briefly, the corresponding Biotin Ligase BufferA, Buffer B and BirA ligase were added to the IL-2R ⁇ and IL-2R ⁇ protein solutions respectively and incubated at 30°C for 2 hours. The biotinylation efficiency was detected by ELISA method to confirm successful biotin labeling.
  • FACS screening The designed IL-2 mutant library was constructed into a yeast display system, and flow cytometric fluorescence sorting technology (FACS) was used to screen the library for reduced affinity to IL-2R ⁇ , but basically maintained affinity to IL-2R ⁇ . mutants that are or are not significantly attenuated.
  • FACS screening method is well known to those skilled in the art.
  • biotinylated IL-2R ⁇ Biotin-IL-2R ⁇
  • Biotin-IL-2R ⁇ is used to conduct 4 rounds of FACS screening to enrich yeast-positive groups with weakened affinity to IL-2R ⁇ .
  • yeast cells induced in SGCAA medium were precipitated, 1 mL of PBSM was added, centrifuged at 14000 g for 30 sec, the supernatant was discarded, and the cell pellet was washed.
  • PBSM buffer containing Biotin-IL-2R ⁇ to resuspend the yeast cells and incubate at room temperature for 1 hour. After washing, cells were stained with Streptavidin-PE (BD Biosciences, 554061) and anti-V5 tag antibody [iFluor 647] (GenScript, A01805-100) for cell sorting.
  • the enriched positive yeast group with weakened binding to Biotin-IL-2R ⁇ was used to continue to use the FACS method to screen for those whose affinity to biotinylated-labeled IL-2R ⁇ (Biotin-IL-2R ⁇ ) remained basically unchanged or not significantly weakened. Monoclonal. Finally, the single clones obtained by screening were sequenced, constructed into an expression vector carrying the His tag gene, samples were prepared, and corresponding biological evaluations were performed.
  • the first group of IL-2 mutants has mutations, including but not limited to deletions and/or substitutions, at amino acid residues 81-85 relative to the amino acid sequence of human wild-type IL-2.
  • Detailed information for 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 respectively RPL, RHL or SPL.
  • “All (81-85) RPRDL is missing” means that all amino acid residues RPRDL at positions 81-85 of IL-2 are missing.
  • "+" represents simultaneous mutations.
  • the second group of IL-2 mutants has amino acid mutations at both S75 and R83 relative to the amino acid sequence of human wild-type IL-2.
  • the specific information of each mutant is shown in Table 10. “+” represents simultaneous mutation.
  • Expression and purification of IL-2 mutants Obtain the gene coding sequence of the above IL-2 mutants through PCR, and after enzyme digestion, connect it to an expression vector carrying a His tag gene that has been digested by the same double enzymes to obtain IL-2 mutants. 2 Recombinant expression vectors of each mutant. The above recombinant expression vector was transfected into 293F cells and incubated at 37°C, 5% CO 2 , After culturing for 5 days at 120 rpm, collect the cell culture supernatant and follow the (Ni) nickel column purification instructions to complete the purification of the IL-2 mutant. The specific steps are as described above.
  • Whether the two groups of IL-2 mutants finally obtained can selectively activate Treg cells were further tested through the following experiments.
  • 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 IL- 2
  • the EC50 value of the mutant binding to IL-2R ⁇ or IL-2R ⁇ , the greater the difference between the two for example, while the IL-2 mutant retains binding to IL-2R ⁇ , its binding to IL-2R ⁇
  • the larger the EC50 value the better the selectivity of the IL-2 mutant for Treg cells; for another example, it can also be judged by the change in the number of Treg cells after stimulation by the IL-2 mutant, such as through flow cytometry Techniques are used to measure changes in the number of Treg cells in mixed cell populations.
  • Example 2 Determination of binding affinity of IL-2 mutants to IL-2R ⁇ and IL-2R ⁇
  • HEK-Blue TM IL-2 cell line (InvivoGen, cat#hkb-il2) is a HEK293 cell line stably transfected with human IL-2R ⁇ (CD25), IL-2R ⁇ (CD122) and IL-2R ⁇ (CD132) genes.
  • Another cell line, HEK-Blue TM CD122/CD132 (InvivoGen, cat#hkb-il2bg) only expresses IL-2R ⁇ (CD122) and IL-2R ⁇ (CD132), but not IL-2R ⁇ (CD25).
  • 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.
  • QUANTI can be used -Blue TM solution (InvivoGen, cat#rep-qbs) detects the expression level of SEAP induced by STAT5.
  • the HEK-Blue TM IL-2 cell line is used to detect the combination of IL-2 or its mutants and IL-2R ⁇ .
  • HEK-Blue TM CD122/CD132 cell line is used to detect the binding of IL-2 or its mutants and IL-2R ⁇ .
  • IL-2 or its mutants 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-2R ⁇ or the medium-affinity receptor IL-2R ⁇ , activating the intracellular Janus family protease.
  • the amino acid kinase signaling cascade (JAK1 and JAK3) initiates the phosphorylation of signal transducer and activator of transcription 5 (STAT5), thereby inducing the expression of SEAP.
  • the HEK-Blue TM CD122/CD132 cell line was used to determine the EC50 value of the IL-2 mutant binding to IL-2R ⁇ , and the HEK-Blue TM IL-2 cell line was used to determine the EC50 value of the IL-2 mutant binding to IL-2R ⁇ . value, will The ratio between the two, namely the EC50 of HEK-IL-2R ⁇ /EC50 of HEK-IL-2R ⁇ , is defined as the therapeutic safety window for IL-2 to select and activate Treg cells.
  • the safety window value can reflect the selective targeting of Treg cells by IL-2 mutants. The higher the value, the stronger the selective targeting of Treg cells by IL-2 mutants.
  • the IL-2 mutant to be detected was first diluted to 800 ⁇ g/mL with detection culture medium, and then 4-fold gradient diluted into a 96-well U-shaped plate (Coring), with a total of 12 concentrations for later use. After adjusting the digested cell (HEK-Blue TM IL-2 cell line or HEK-Blue TM CD122/CD132 cell line) suspension to 2.8 ⁇ 10 5 cells/mL, inoculate 96 cells at 180 ⁇ L cell suspension/per well. - Well flat-bottomed cell culture plate, add 20 ⁇ L of diluted IL-2 or IL-2 mutant to each well, mix gently with the cell suspension, and then place it in a 37°C, 5% CO2 incubator for 24 hours.
  • the detection results of the first and second groups of IL-2 mutants are shown in Table 11 and Table 12.
  • the results showed that compared with wild-type IL-2, both groups of IL-2 mutants were able to bind to IL-2R ⁇ , and their EC50 value for binding to IL-2R ⁇ was increased, their affinity was reduced, and their therapeutic safety window was significantly improved. This indicates that these mutants have excellent targeting properties for Treg cell selection.
  • Example 3 Expression and purification of IL-2 mutant and Fc fusion protein
  • IgG1 Fc LALA refers to IgG1 Fc with L234A and L235A mutations, and the numbering is numbered according to the EU Kabat numbering system (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al. , U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991)).
  • the bivalent form of the fusion protein of IL-2 mutant and Fc contains two IL-2 mutant molecules: it contains two identical monomers, in each monomer the IL-2 mutant is connected to the N of Fc terminal or C terminal.
  • An exemplary structure of one such form is shown in Figure 1A (IL-2-Fc), which consists of two identical monomers, each containing from N-terminus to C-terminus: IL-2 mutant ( SEQ ID NOs:5-34), linker peptide (SEQ ID NO:52) and IgG1 Fc LALA (SEQ ID NO:35).
  • Fc-IL-2 An exemplary structure of another such form is shown in Figure 1B (Fc-IL-2), which consists of two identical monomers, each containing from N-terminus to C-terminus: IgG1 Fc LALA (SEQ ID NO:35), linker peptide (SEQ ID NO:52) and IL-2 mutants (SEQ ID NOs:5-34).
  • the monovalent form of IL-2 mutant and Fc fusion protein contains one IL-2 mutant molecule: it contains two different monomers.
  • the IL-2 mutant is fused to one of the Fc subunits.
  • the other monomer is another Fc subunit, and the two asymmetric Fc subunits promote mutual pairing through knob and hole mutations.
  • it contains two different monomers.
  • the IL-2 mutant is fused to the C-terminus of one of the Fc subunits through a connecting peptide, and the monomer is from the N-terminus to the C-terminus.
  • the end contains IgG1 Fc LALA knob (SEQ ID NO:41), connecting peptide (SEQ ID NO:52) and IL-2 mutant (SEQ ID NOs:5-34); and the other monomer is IgG1 Fc LALA hole ( SEQ ID NO:42).
  • IgG1 Fc LALA knob SEQ ID NO:41
  • connecting peptide SEQ ID NO:52
  • IL-2 mutant SEQ ID NOs:5-34
  • the other monomer is IgG1 Fc LALA hole ( SEQ ID NO:42).
  • the specific construction process of IL-2 mutant and Fc fusion protein (taking the bivalent fusion protein form Fc-IL-2 of IL-2 mutant and Fc as an example): Recognition of restriction endonucleases HindIII and XhoI by introduction of PCR site, the nucleic acid sequence encoding IgG1 Fc LALA was connected to the nucleic acid sequence encoding the IL-2 mutant through the oligonucleotide sequence encoding GGGGS, and constructed into the pcDNA3.1 eukaryotic expression vector, and transfected into 293F cells, 37 °C, 5% CO 2 , After culturing at 120 rpm for 5 days, the culture medium was collected, and the Fc-IL-2 mutant fusion protein was purified by protein A affinity chromatography and gel filtration Superdex 200 column chromatography. The specific operating steps are as described in Example 1.
  • HEK-BLUE TM cell line experiments were used to test and verify whether different fusion forms would affect the activity of IL-2 mutants.
  • the specific experimental procedures of HEK-BLUE TM are as described in Example 2.
  • Fc knob-(81-85)-mut8/Fc-hole Fc knob-(81-85)-mut10/Fc-hole
  • Fc knob-S75R83-mut16 /Fc-hole was tested as an exemplary fusion protein, which contains two different monomers, one of which contains an IL-2 mutant molecule fused to the C terminus of Fc LALA knob through the connecting peptide GGGGS, and the other monomer.
  • the body is Fc LALA hole.
  • Biacore experiments were used to determine the affinity of exemplary Fc-IL-2 mutant bivalent fusion proteins Fc-(81-85)-mut8 or Fc-S75R83-mut16 to human IL-2R ⁇ and human IL-2R ⁇ , respectively.
  • IL-2R ⁇ uses the IL-2R ⁇ -Fc heterodimer prepared in Example 1, and IL-2R ⁇ uses IL-2R ⁇ -Fc knob monomer and IL-2R ⁇ -Fc hole monomer.
  • Heterodimeric fusion protein IL-2R ⁇ -Fc.
  • Fc-wild-type IL-2 bivalent fusion protein was used as a control in this experiment.
  • the specific operation steps are as follows: Use the Protein A sensor chip of Biacore instrument (Biacore T200, GE) to capture IL-2R ⁇ -Fc or IL-2R ⁇ -Fc, in which IL-2R ⁇ -Fc or IL-2R ⁇ -Fc is diluted to 1 ⁇ g. /mL, at a flow rate of 10 ⁇ L/min for 30 seconds. Then, a series of concentration gradients of wild-type IL-2 or its mutant bivalent fusion protein flow through the chip surface at a flow rate of 30 ⁇ L/min. The binding lasts for 120 seconds and the dissociation lasts for 360 seconds.
  • the Biacore instrument is used to detect the reaction signal in real time. Obtain association and dissociation curves.
  • the Kon, Koff and Kd values are shown in Table 15.
  • Example 5 In vitro biological activity assay of IL-2 mutants
  • IL-2 When IL-2 binds to its cell surface receptor, it activates the JAK-STAT signaling pathway and phosphorylates STAT5. Phosphorylation of STAT5 is an essential step in the IL-2 signal transduction pathway. After phosphorylation, STAT5 enters the nucleus and participates in the downstream signal transduction process. Therefore, the phosphorylated STAT5 signal in cells can reflect the activation of IL-2.
  • CD8 + T lymphocytes express IL-2R ⁇ but not IL-2R ⁇ on their surface.
  • Treg cells express both IL-2R ⁇ and IL-2R ⁇ on their surface.
  • the levels of STAT5 phosphorylation (pSTAT5) in CD8 + T lymphocytes and Treg cells were measured by flow cytometry, respectively, to indirectly reflect the activity of IL-2 mutants in activating CD8 + T lymphocytes and Treg cells (for example, see Literature Peterson LB, et al. J Autoimmun. 2018 Dec; 95:1-14).
  • PBMC cells (Shanghai Mieshun Biotechnology Co., Ltd., cat#PB050C) were resuscitated one day in advance, PBMC were collected overnight, cells were washed with PBS, and collected by centrifugation. Mix with 2% complete culture medium and count. Add 60 ⁇ L of cell suspension to each well. About 5 to 6 ⁇ 10 5 cells are seeded on the cell culture plate and placed in a cell culture incubator for later use.
  • the samples to be tested include the exemplary first group of bivalent fusion proteins of IL-2 mutants and Fc, Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)- mut2, Fc-(81-85)-mut3, Fc-(81-85)-mut9 and Fc-(81-85)-mut14 and exemplary second group of bivalent fusion proteins of IL-2 mutants and Fc Fc-S75R83-mut7 and Fc-S75R83-mut16. After gradient dilution of these samples, add them to the cells and stimulate for 30 minutes. Add cell fixative.
  • CD8 + T lymphocytes The surface marker of CD8 + T lymphocytes is CD8.
  • CD4 - CD8 + cells were defined as CD8 + T lymphocytes.
  • Surface markers of Treg cells usually include CD4, CD25 and Foxp3.
  • cells with CD4 + CD25 + Foxp3 + were defined as Treg cells.
  • Data processing Use GraphPad Prism 8.0.1 for statistics on the pSTAT5 fluorescence value (MFI) of the above two cell populations after treatment with IL-2 mutants at different concentrations, and calculate the EC50 value.
  • the fitting method is to first convert the concentration value into a logarithm, that is, EC50 value.
  • Figures 3A, 3C, 3E, and 3G show the results of the first group of exemplary bivalent fusion proteins of IL-2 mutants and Fc inducing STAT5 phosphorylation in Treg cells;
  • Figures 3B, 3D, 3F, and 3H show This is an exemplary first set of results of bivalent fusion proteins of IL-2 mutants and Fc inducing STAT5 phosphorylation in CD8 + T lymphocytes.
  • Figure 3I shows the results of the bivalent fusion protein of the second group of IL-2 mutants and Fc inducing STAT5 phosphorylation in Treg cells;
  • Figure 3J shows the exemplary second group of IL-2 mutants. Results of bivalent fusion protein with Fc inducing STAT5 phosphorylation in CD8 + T cells.
  • IL-2 mutant and Fc bivalent fusion protein can induce STAT5 phosphorylation in Treg cells, but in CD8 + T lymphocytes, IL-2 mutant and Fc bivalent fusion protein do not. Significantly induces STAT5 phosphorylation.
  • the above results further illustrate that the IL-2 mutant of the present invention can selectively activate Treg cells without activating CD8 + T lymphocytes, and has good Treg cell targeting. Compared with wild-type IL-2, it is more effective in human treatment There is greater safety in the process.
  • Treg cells and CD8 + T cells in the blood were detected after a single administration of Fc-IL-2 mutant bivalent fusion protein in mice. Lymphocyte proliferation.
  • mice were purchased from Viton Lever and raised in a special pathogen-free environment. All animal experiments were performed in accordance with the protocols and guidelines of the Institutional Animal Care and Use Committee (IACUC).
  • IACUC Institutional Animal Care and Use Committee
  • mice 21 C57BL/6 mice, SPF grade, female, divided into 7 groups, 3 mice in each group, mice in each group were subcutaneously injected with an exemplary bivalent fusion protein of IL-2 mutant 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) is the control sample in this experiment.
  • a single dose was administered on day 0, with a dose of 1 mg/kg per mouse.
  • the flow cytometry software CytExpert was used for statistics.
  • the lymphocyte population (P1) gate was counted at 30,000.
  • the percentage of Treg cells in the CD4 + cell population and the percentage of CD8 + T lymphocytes in the total lymphocytes were calculated.
  • the surface marker of CD8 + T lymphocytes is CD8, and CD8 + cells are defined as CD8 + T lymphocytes.
  • the surface markers of Treg cells are CD4, CD25 and Foxp3, and CD4 + CD25 + Foxp3 + cells are defined as Treg cells.
  • the results are shown in Figure 4A.
  • the first group of bivalent fusion proteins of IL-2 mutants and Fc: Fc-(81-85)-mut8, Fc-(81-85)-mut10 and Fc-(81-85) -mut12 can stimulate the proliferation of Treg cells in mice at a dose of 1 mg/kg, and Fc-(81-85)-mut8, Fc-(81-85)-mut10 and Fc-(81- 85)-mut12 compared with the control Fc-AMG592 (SEQ ID NO: 106), its activity in stimulating Treg cell proliferation is equivalent to or higher than that of Fc-AMG592.
  • Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-(81-85)-mut12 did not stimulate the proliferation of CD8 + T lymphocytes. This demonstrates its specificity in promoting Treg cell expansion in vivo.
  • Fc-S75R83-mut5, Fc-S75R83-mut9 and Fc-S75R83-mut16 stimulated the proliferation of Treg cells but did not stimulate the proliferation of CD8 + T lymphocytes.
  • Example 7 Activity assay of IL-2 mutants in cynomolgus monkeys
  • Treg cells and CD8 in the blood were detected after a single administration of Fc-IL-2 mutant bivalent fusion protein in cynomolgus monkeys. + The proliferation of T lymphocytes.
  • Each cynomolgus monkey was injected subcutaneously 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 is divided into low-dose groups of 0.3mg/kg. and 1.5 mg/kg in the high-dose group.
  • 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 the low-dose group at the same time.
  • group and high-dose group experiments 1 mL of intravenous blood was taken 0 days before administration and 7 days after administration.
  • Fc-S75R83-mut16 whether in the low-dose group 0.3 mg/kg or the high-dose group 1.5 mg/kg, can stimulate the proliferation of Treg cells in cynomolgus monkeys, and as the dosage increases, it promotes the proliferation of Treg cells The effect of proliferation was enhanced ( Figure 5C). At the same time, whether at low or high doses, Fc-S75R83-mut16 did not stimulate CD8 + T lymphocyte proliferation in cynomolgus monkeys, and there was no significant change in the number of cells (Figure 5D ).
  • Fc-S75R83-mut5 and Fc-S75R83-mut16 had higher activity in stimulating Treg cell proliferation (Figure 5C).
  • IL-2 mutant of the present invention can specifically activate Treg cells in primates without affecting CD8 + T cells. Its effect is equivalent to that of Fc-AMG592 as a control, or even better.
  • Example 8 PK experiment of IL-2 mutants in mice
  • C57LB/6 mice were purchased from Viton Lever and maintained in a specific pathogen-free environment. All animal experiments were performed in accordance with Institutional Animal Care and Use Committee (IACUC) protocols and guidelines.
  • IACUC Institutional Animal Care and Use Committee
  • mice were randomly divided into 4 groups, with 6 mice in each group, half male and half female.
  • Each group of mice was injected with a single tail vein injection of exemplary bivalent fusion proteins of IL-2 mutants and Fc, Fc-(81-85)-mut8, Fc-(81-85)-mut10, and Fc-S75R83-mut5. or Fc-S75R83-mut16, the injection dose is 1mg/kg. respectively at 0 hours before administration, 15 minutes, 5 hours, 24 hours, 72 hours and 168 hours after administration.
  • Anticoagulated whole blood was collected at hour, 240 hours, 336 hours, 432 hours, and 600 hours, and plasma was separated.
  • the plasma drug concentration in each sample was determined by ELISA method.
  • WinNonlin 6.0 non-compartmental model (NCA) was used to calculate the pharmacokinetic parameters of each animal.
  • Exemplary bivalent fusion proteins of IL-2 mutants and Fc Fc-(81-85)-mut8, Fc-(81-85)-mut10, Fc-S75R83-mut5 and Fc-S75R83-mut16 at 1 mg/kg After intravenous injection into mice, the plasma concentration gradually decreased over time.
  • the results of the PK experiment are shown in Figure 6, which shows that the bivalent fusion protein of IL-2 mutant and Fc of the present invention has good pharmacokinetic properties in mice (wherein Fc-(81-85)-mut8 and Fc-S75R83-mut5 PK curves not shown).

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Abstract

涉及IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物，同时还包括IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物的治疗用途。同时还涉及IL-2突变体及其融合蛋白的制备方法等。

Description

白介素-2(IL-2)突变体及其用途
以下提交的序列表的内容通过整体引用并入本文中，文件名称：IL-2-1 20230516.xml，记录日期：2023.05.16，大小：145KB。
技术领域
本申请涉及白介素-2(IL-2)突变体，还包含所述IL-2突变体的融合蛋白、编码IL-2突变体或融合蛋白的核酸分子、包含核酸分子的载体和宿主细胞，以及包含其的药物组合物和治疗用途，还进一步提供了IL-2突变体及其融合蛋白的制备方法。
发明背景
白介素-2(IL-2)也称作T细胞生长因子(TCGF)，是由4个α螺旋(A,B,C,D)组成的束状I型细胞生长因子，其主要由抗原激活T细胞(antigen-activated T cells)产生，并促进成熟T细胞和B细胞的增殖、分化和存活，以及在先天免疫防御中自然杀伤细胞(NK cell)的溶细胞活性(K.A.Smith,Science 240,1169(1988)；B.H.Nelson,D.M.Willerford,Adv.Immunol.70,1(1998))。
IL-2是通过与靶细胞表面的IL-2受体相结合来实现其功能的。IL-2有三个受体亚基：α链(IL-2Rα或CD25)，β链(IL-2Rβ或CD122)和通用细胞因子受体γ链(γc或IL-2Rγ或CD132)(W.J.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))。
根据结合亚基及与IL-2的亲和力的不同，可将IL-2受体划分为以下几种类型：第一种类型为低亲和力受体，由单独的IL-2Rα亚基组成(Kd值约为10nM)，由于IL-2Rα没有胞内信号传导区域，所以IL-2与此类受体结合不能传递信号及调控细胞(H.M.Wang,K.A.Smith,J.Exp.Med.166,1055(1987))。第二种类型为中等亲和力受体，由IL-2Rβ和γc亚基组成(Kd值约为1nM)，该类受体主要在自然杀伤细胞，巨噬细胞和处于静息状态的T细胞上表达，在较高的IL-2浓度下可以激活信号通路(B.H.Nelson,D.M.Willerford,Adv.Immunol.70,1(1998))。尽管单独的IL-2Rβ与IL-2的亲和力非常低(Kd值约为100nM)，而单独的γc几乎检测不到与IL-2的结合(M.Rickert,M.J.Boulanger,N.Goriatcheva,K.C.Garcia,J.Mol.Biol.339,1115(2004))，但由IL-2Rβ和γc 组成的复合物(IL-2Rβγ)对IL-2有效的细胞信号传导是十分必要的(Y.Nakamura,et al.,Nature 369,330(1994)；B.H.Nelson,et al.,Nature 369,333(1994))。第三种类型为高亲和力受体，由IL-2Rα、IL-2Rβ和γc三个亚基组成(Kd值约为10pM)，通常在激活的淋巴细胞和CD4+CD25+Foxp3+调节性T细胞(Treg)上表达(T.Takeshita et al.,Science 257,379(1992)；Boyman O,et al.,Nat Rev Immunol.2012 Feb 17；12(3):180-90)。
由于IL-2能够增强效应T细胞和自然杀伤细胞的功能，其可作为免疫激活剂来治疗淋巴细胞增生相关疾病。近年的研究发现，IL-2对于Treg细胞的分化、功能和存活也是一个关键的细胞因子，对实现Treg细胞的功能、发育以及其抑制性特征的诱导具有重要作用。Treg细胞通常由表达CD4、CD25和转录因子叉头P3(Foxp3)来进行定义，其能够抑制自身反应性淋巴细胞，并调控先天性和适应性免疫反应，从而导致免疫抑制。Treg细胞作为免疫抑制的关键效应细胞之一，并且在诱导和维持对抗原(包括肿瘤表达的那些抗原)的外周自我耐受性中发挥至关重要的作用，对自身免疫疾病的发生发展具有重要意义。(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)。
基于IL-2广泛的生物学效应，通过改变IL-2对不同受体的选择性或偏好性，使IL-2选择性地激活靶细胞，以降低IL-2治疗的毒性和/或提高其功效，提高IL-2治疗特定疾病的安全窗，对提高IL-2治疗疾病的安全性和有效性具有重要意义，本领域仍然存在着开发具有改善性质的新IL-2分子的需求。
本文提及的所有出版物、专利、专利申请和已公开的专利申请所披露的内容，全部以引用方式并入本文。
发明概述
本申请一方面涉及一种IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的缺失或突变。在一些实施例中，所述人野生型IL-2的氨基酸序列如SEQ ID NO:1所示。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的全部缺失。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和N71的突变。在一些实施例中，所述E67和N71的突变分别为E67K和N71S。在一些实施例中，本申 请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的全部缺失，以及相对于人野生型IL-2氨基酸序列的E67K和N71S突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67的突变。在一些实施例中，所述E67的突变为E67A。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，以及相对于人野生型IL-2氨基酸序列的E67A突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，且进一步包含相对于人野生型IL-2氨基酸序列的N71和S75的突变。在一些实施例中，所述N71和S75的突变分别为N71W和S75I。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，以及相对于人野生型IL-2氨基酸序列的N71W和S75I突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和/或V91的突变。在一些实施例中，所述E67的突变为E67T。在一些实施例中，所述V91的突变为V91K。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的E67T和/或V91K突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，且进一步包含相对于人野生型IL-2氨基酸序列的L70和S75的突变。在一些实施例中，所述L70和S75的突变分别为L70I和S75F。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的L70I和S75F突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，且进一步包含相对于人野生型IL-2氨基酸序列的N71的突变。在一些实施例中，所述N71的突变为N71I。在一些实施例 中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的N71I突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为SPL。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和N71的突变。在一些实施例中，所述E67和N71的突变分别为E67M和N71S。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为SPL，以及相对于人野生型IL-2氨基酸序列的E67M和N71S突变。
在一些实施例中，本申请所述的IL-2突变体，除上述突变外，还进一步包含相对于人野生型IL-2氨基酸序列的C125的突变。在一些实施例中，所述C125的突变为C125S或C125A。
在一些实施例中，本申请所述的IL-2突变体包含SEQ ID NOs:5-18中任一所示的氨基酸序列或者其变体，所述变体与SEQ ID NOs:5-18中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
本申请另一方面涉及另一种IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第75位氨基酸残基S(S75)和第83位氨基酸残基R(R83)的突变。在一些实施例中，所述人野生型IL-2的氨基酸序列如SEQ ID NO:1所示。
在一些实施例中，本申请所述的IL-2突变体，其中S75被突变为不带电荷的氨基酸残基。在一些实施例中，其中S75的突变为S75P、S75F、S75Y、S75G、S75A、S75V、S75T、S75I或S75L。在一些实施例中，其中R83的突变为R83N、R83F、R83Y、R83P、R83L、R83A、R83K或R83E。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75P，R83的突变为R83Y。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和D84的突变。在一些实施例中，其中E67和D84的突变分别为E67A和D84N。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P、R83Y、E67A和D84N突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75P，R83的突变为R83A。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16、L70和N71的突变。在一些实施例中，其中H16、L70 和N71的突变分别为H16D、L70I和N71G。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P、R83A、H16D、L70I和N71G突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75F，R83的突变为R83P。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和/或N71的突变。在一些实施例中，其中H16的突变为H16Q或H16N。在一些实施例中，其中N71的突变为N71S。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83P和H16Q突变。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83P、H16N和N71S突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75F，R83的突变为R83L。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变。在一些实施例中，其中E67和L70的突变分别为E67R和L70V。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83L、E67R和L70V突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75I，R83的突变为R83L。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变。在一些实施例中，其中E67和L70的突变分别为E67G和L70V。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75I、R83L、E67G和L70V突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75I，R83的突变为R83K。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变。在一些实施例中，其中E67和L70的突变分别为E67S和L70V。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75I、R83K、E67S和L70V突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75T，R83的突变为R83A或R83F。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和E67的突变。在一些实施例中，其中H16和E67的突变分别为H16N和E67K。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75T、R83A、H16N和E67K突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75V，R83的突变为R83F。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E15和H16的突变。在一些实施例中，其中E15和H16的突变分别为E15K和H16Q。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75V、R83F、E15K和H16Q突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75V，R83的突变为R83N。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L70和V91的突变。在一些实施例中，其中L70和V91的突变分别为L70F和V91K。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75V、R83N、L70F和V91K突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75G，R83的突变为R83L。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17、V91和I92的突变。在一些实施例中，L17、V91和I92的突变分别为L17I、V91E和I92L。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75G、R83L、L17I、V91E和I92L突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75Y，R83的突变为R83N。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17和V91的突变。在一些实施例中，L17和V91的突变分别为L17I和V91K。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75Y、R83N、L17I和V91K突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75A，R83的突变为R83K。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17的突变。在一些实施例中，L17的突变为L17I。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75A、R83K和L17I突变。
在一些实施例中，本申请所述的IL-2突变体，其中S75的突变为S75L，R83的突变为R83E。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L63和E67的突变。在一些实施例中，L63和E67的突变分别为L63V和E67F。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75L、R83E、L63V和E67F突变。
在一些实施例中，本申请所述的IL-2突变体，除上述突变外，还进一步包含相对于人野生型IL-2氨基酸序列C125的突变。在一些实施例中，所述C125的突变为C125S或C125A。
在一些实施例中，本申请所述的IL-2突变体包含SEQ ID NOs:19-34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:19-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，与野生型IL-2相比，本申请所述的IL-2突变体与IL-2Rβγ的结合亲和力降低，并且能够保持与IL-2Rα的结合。
本申请另一方面涉及包含IL-2突变体的融合蛋白。在一些实施例中，所述融合蛋白包含Fc。在一些实施例中，所述Fc包含能够改变效应功能的突变，和/或能够延长半衰期的突变，和/或能够促进异源多肽形成二聚体的突变。在一些实施例中，所述Fc来源于人。在另一些实施例中，所述Fc来源于人的IgG，包括IgG1，IgG2，IgG3或IgG4，优选IgG1或IgG4。
在一些实施例中，所述Fc来源于人IgG1，其包含突变L234A和L235A；和/或N297G；和/或N297A；和/或L234A、L235A和P331S；和/或L234A、L235E、G237A、A330S和P331S突变，其中所述编号为EU编号系统。
在一些实施例中，所述Fc来源于人IgG4，其包含突变S228P、F234A和L235A突变，其中所述编号为EU编号系统。
在一些实施例中，所述Fc进一步包含S354C和T366W突变；和/或Y349C、T366S、L368A、和Y407V突变，其中所述编号为EU编号系统。
在一些实施例中，如本申请所述的融合蛋白，其中IL-2突变体与Fc连接。在一些实施例中，其中IL-2突变体与Fc通过连接肽连接。在一些实施例中，所述连接肽包含SEQ ID NOs:43-74中任一所示的氨基酸序列。在一些实施例中，所述连接肽包含氨基酸序列GGGGS(SEQ ID NO:52)。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白，其中IL-2突变体位于Fc的N端和/或C端。
在一些实施例中，本申请所述的融合蛋白，其包含SEQ ID NOs:76-105中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:76-105中任一所示的氨基酸序列 具有至少约80％(例如至少80％、85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
同时，本申请还涉及编码本文所述任何IL-2突变体或包含IL-2突变体的融合蛋白的分离的核酸、包含所述核酸的载体、包含所述核酸或载体的宿主细胞(例如，CHO细胞、HEK 293细胞、Hela细胞或COS细胞)、组合物(例如，药物组合物)、试剂盒以及包含本文所述任何IL-2突变体或包含IL-2突变体的融合蛋白的制品。还涉及使用本文所述任何IL-2突变体或其融合蛋白或包含其的药物组合物用于在有需要的个体中(例如，人类)治疗炎性疾病或自身免疫性疾病(例如，狼疮、移植物抗宿主病、C型肝炎诱发的血管炎、Ⅰ型糖尿病、Ⅱ型糖尿病、多发性硬化、类风湿性关节炎、特应性疾病、哮喘、炎症性肠病、自身免疫性肝炎、溶血性贫血、风湿热、甲状腺炎、克罗恩病、重症肌无力、肾小球肾炎、斑秃、牛皮癣、白癜风、营养不良性大疱性表皮松解症和白塞病)的方法。
附图说明
图1A所示为IL-2突变体与Fc的二价融合蛋白示例性结构的示意图，其包含两个相同的单体，每个单体中的IL-2突变体分子连接至Fc的N端。图1B所示为IL-2突变体与Fc的二价融合蛋白示例性结构的示意图，其包含两个相同的单体，每个单体中的IL-2突变体分子连接至Fc的C端。
图2所示为IL-2突变体与Fc的单价融合蛋白示例性结构的示意图，其包含两个不同的单体，其中一个单体包含一个IL-2突变体分子，其连接至Fc knob的C端，另一个单体为Fc hole分子。
图3A、3C、3E、3G所示分别为第一组IL-2突变体与Fc的二价融合蛋白：Fc-(81-85)-mut8、Fc-(81-85)-mut10、Fc-(81-85)-mut2、Fc-(81-85)-mut3、Fc-(81-85)-mut9和Fc-(81-85)-mut14在Treg细胞中诱导STAT5磷酸化的结果；图3B、3D、3F、3H所示分别为第一组IL-2突变体与Fc的二价融合蛋白：Fc-(81-85)-mut8、Fc-(81-85)-mut10、Fc-(81-85)-mut2、Fc-(81-85)-mut3、Fc-(81-85)-mut9和Fc-(81-85)-mut14在CD8+T淋巴细胞中诱导STAT5磷酸化的结果。
图3I所示为第二组IL-2突变体与Fc的二价融合蛋白：Fc-S75R83-mut7和Fc-S75R83-mut16在Treg细胞中诱导STAT5磷酸化的结果；图3J所示为第二组IL-2突变 体与Fc的二价融合蛋白：Fc-S75R83-mut7和Fc-S75R83-mut16在CD8+T淋巴细胞中诱导STAT5磷酸化的结果。
图4A所示为第一组IL-2突变体与Fc的二价融合蛋白：Fc-(81-85)-mut8，Fc-(81-85)-mut10，和Fc-(81-85)-mut12在给药剂量为1mg/kg时，在小鼠体内刺激Treg细胞的增殖。
图4B所示为第一组IL-2突变体与Fc的二价融合蛋白：Fc-(81-85)-mut8，Fc-(81-85)-mut10，和Fc-(81-85)-mut12在给药剂量为1mg/kg时，在小鼠体内对CD8+T淋巴细胞增殖的影响。
图4C所示为第二组IL-2突变体与Fc的二价融合蛋白Fc-S75R83-mut5、Fc-S75R83-mut9和Fc-S75R83-mut16在给药剂量为1mg/kg时在小鼠体内刺激Treg细胞的增殖。
图4D所示为第二组IL-2突变体与Fc的二价融合蛋白Fc-S75R83-mut5、Fc-S75R83-mut9和Fc-S75R83-mut16在给药剂量为1mg/kg时在小鼠体内对CD8+T淋巴细胞增殖的影响。
图5A所示为第一组IL-2突变体与Fc的二价融合蛋白Fc-(81-85)-mut8和Fc-(81-85)-mut10在给药剂量为0.3mg/kg时，在食蟹猴体内刺激Treg细胞的增殖。
图5B所示为第一组IL-2突变体与Fc的二价融合蛋白Fc-(81-85)-mut8和Fc-(81-85)-mut10在给药剂量为0.3mg/kg时，在食蟹猴体内对CD8+T淋巴细胞增殖的影响。
图5C所示为第二组IL-2突变体与Fc的二价融合蛋白Fc-S75R83-mut5在给药剂量为0.3mg/kg以及Fc-S75R83-mut16分别在给药剂量为0.3mg/kg以及1.5mg/kg时，在食蟹猴体内刺激Treg细胞的增殖。
图5D所示为第二组IL-2突变体与Fc的二价融合蛋白Fc-S75R83-mut5在给药剂量为0.3mg/kg以及Fc-S75R83-mut16分别在给药剂量为0.3mg/kg以及1.5mg/kg时，在食蟹猴体内对CD8+T淋巴细胞增殖的影响。
图6所示为示例性的IL-2突变体与Fc的二价融合蛋白Fc-(81-85)-mut10和Fc-S75R83-mut16在给药剂量为1mg/kg时，小鼠体内的PK曲线。
本申请的详细描述
本申请涉及IL-2突变体，以及包含IL-2突变体的融合蛋白等。与野生型IL-2相比，本申请所述的IL-2突变体能够选择性活化Treg细胞，刺激Treg细胞增殖，在治疗某 些特定疾病(例如，炎性疾病或自身免疫性疾病)时具有更高的安全窗(例如，在HEK-BlueTM IL-2细胞系筛选实验中，与野生型IL-2相比，通过HEK-BlueTM CD122/CD132细胞系检测IL-2突变体与IL-2Rβγ结合的EC50值与通过HEK-BlueTM IL-2细胞系检测IL-2突变体与IL-2Rαβγ结合的EC50值之间的比值更高)。进一步地，本申请所述的IL-2突变体与Fc的融合蛋白，通过改造Fc片段，降低或最小化了抗体依赖性细胞介导的细胞毒性(ADCC)和/或补体依赖性细胞毒性(CDC)，从而避免在治疗期间产生不期望的免疫反应，并且能够增加IL-2突变体的体内循环半衰期，从而减少了给药频率和总给药次数，为患者提供了方便并降低了治疗成本。
本申请还涉及编码所述IL-2突变体或包含IL-2突变体的融合蛋白的核酸、包含所述核酸的载体、包含所述核酸或载体的宿主细胞、以及上述IL-2突变体或融合蛋白的生产方法、药物组合物和包含其的生产制品，以及使用所述IL-2突变体或包含IL-2突变体的融合蛋白或药物组合物治疗疾病(例如，狼疮、移植物抗宿主病、C型肝炎诱发的血管炎、Ⅰ型糖尿病、Ⅱ型糖尿病、多发性硬化、类风湿性关节炎、特应性疾病、哮喘或炎症性肠病)的方法。
I.定义
除非上下文另有明确说明，本申请的实施将采用本领域技术范围内的病毒学、免疫学、微生物学、分子生物学和重组DNA技术的常规方法，许多所述方法将在下文详述以供说明。所述技术在文献中有充分解释。参见Current Protocols in Molecular Biology or Current Protocols in Immunology,John Wiley&Sons,New York,N.Y.(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)和其它相似参考资料。
如本申请所述，术语“野生型IL-2”或“WT IL-2”是指天然存在的IL-2，其可来自任何脊椎动物来源的天然IL-2，例如哺乳动物，包括但不限于家畜(例如，牛、绵羊、山羊、猫、狗、驴和马)、灵长类动物(例如，人类和非人类灵长类动物，如猴子或 黑猩猩)、兔子和啮齿动物(例如，小鼠、大鼠、沙鼠和仓鼠)。该术语包括未经加工的IL-2形式(例如，包含信号肽)和经过加工的成熟的IL-2形式(例如，不包含信号肽)。该术语也包括天然存在的IL-2等位基因变体和剪接变体、同种型、同源物、和物种同源物。该术语还包括天然存在的IL-2变体等。例如，所述天然存在的IL-2变体与天然IL-2相比具有至少95％(例如，95％，96％，97％，98％或99％)的序列同源性。在一些实施例中，示例性的人野生型IL-2氨基酸序列如SEQ ID NO:1所示。在一些实施例中，未经加工的人野生型IL-2额外包含N端20个氨基酸的信号肽，其示例性氨基酸序列如SEQ ID NO:4所示。
在一些实施例中，野生型IL-2还可以进一步包含一个或多个不改变与IL-2受体结合作用的氨基酸突变，例如，在野生型人IL-2的125位氨基酸残基处引入丝氨酸突变(C125S)能够防止半胱氨酸导致的错配或聚集，其示例性的氨基酸序列如SEQ ID NO:2所示；在125位引入丙氨酸突变(C125A)能够促进有效折叠，改善表达，其示例性的氨基酸序列如SEQ ID NO:3所示。
如本申请所述的氨基酸的“突变”，包括取代、缺失和插入。可以进行取代、缺失和插入的任意组合来获得具有期望性质(例如，降低与IL-2Rβγ的结合亲和力)的构建体。在一些实施例中，氨基酸的缺失或插入包括在多肽序列中的缺失或插入。在一些实施例中，氨基酸的取代可以为保守性的氨基酸取代。在另一些实施例中，氨基酸的取代可以为非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。氨基酸的取代还包括应用非天然存在的氨基酸或由20种标准氨基酸的天然存在的氨基酸衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟基赖氨酸)进行替换。在一些实施例中，氨基酸的突变包括氨基酸的取代和缺失的组合，例如，在多肽序列的某一区域内(例如第81-85位氨基酸残基RPRDL)，同时包括氨基酸的取代和缺失。可以利用本领域中公知的遗传或化学方法生成氨基酸突变，包括定点突变、PCR、基因合成、化学修饰等方法。
如本申请所述的“IL-2突变体”，其包括在野生型IL-2氨基酸序列(例如，SEQ ID NO.1)中包含一个或多个取代、缺失、插入或者包含其任何形式的组合。在一些实施例中，本申请所述的“IL-2突变体”能够降低其与IL-2Rβγ的结合亲和力，并且保持与IL-2Rα的结合。
在IL-2突变体中，氨基酸突变的位置是基于野生型IL-2中的氨基酸位置来进行确定的。在一些实施例中，野生型IL-2的氨基酸序列如SEQ ID NO:1所示，可以通过与 SEQ ID NO:1进行氨基酸序列比对(例如，应用BLAST)，来鉴定IL-2突变体中氨基酸发生突变所对应的位置。在一些实施例中，当描述例如S75时，代表相对于野生型IL-2氨基酸序列的第75位的氨基酸残基为丝氨酸(S)。“S75的突变”代表第75位的丝氨酸残基(S)发生突变。在一些实施例中，当描述IL-2突变体时，按照如下方式描述。例如“氨基酸取代”表示为“原始氨基酸残基/发生取代的氨基酸位置/取代后的氨基酸残基”，例如“S75G”代表相对于野生型IL-2氨基酸序列(例如，SEQ ID NO.1)的第75位丝氨酸残基(S)被甘氨酸残基(G)所取代。本申请中关于突变位点组合方案中所述的“+”表示在多个特定的位置同时发生突变，例如，“S75V+R83N”代表相对于野生型IL-2氨基酸序列(例如，SEQ ID NO.1)的第75位丝氨酸残基(S)被缬氨酸残基(V)所取代，以及第83位精氨酸残基(R)被天冬酰胺残基(N)所取代。
如本申请所述，IL-2突变体“选择性活化Treg细胞”是指在同时存在其它T细胞亚群(如CD4+T细胞，CD8+T细胞)或NK细胞的情况下，其能够更特异性地靶向Treg细胞，活化Treg细胞。IL-2突变体“活化Treg细胞”的能力可以通过本领域公知的方法或通过本申请实施例中公开的方法来进行测定，包括但不限于：IL-2突变体诱导Treg细胞中IL-2受体信号转导或诱导Treg细胞增殖。在一些实施例中，IL-2受体信号转导可以通过STAT5磷酸化水平进行定义。STAT5磷酸化是IL-2信号转导途径中的必要步骤，因此Treg细胞中STAT5的磷酸化水平(pSTAT5)被认为是IL-2活化Treg细胞的反映。诱导增殖可以通过IL-2刺激后Treg细胞的数量变化进行定义，例如通过流式细胞术测量混合细胞群中Treg细胞数量的增加或通过测量Treg细胞中增殖相关细胞周期蛋白(例如Ki-67)的表达增加。
如本申请所述，“自身免疫性疾病”指源自和针对个体自身组织的非恶性疾病或病症，免疫系统攻击其自身蛋白、细胞和组织。自身免疫性疾病的列表和综述可以参考The Autoimmune Diseases(Noel Rose,Ian Mackay,2014,Academic Press)。在一些实施例中，自身免疫性疾病的例子包括，但不限于狼疮、移植物抗宿主病、C型肝炎诱发的血管炎、Ⅰ型糖尿病、Ⅱ型糖尿病、多发性硬化、类风湿性关节炎、特应性疾病、哮喘、炎症性肠病、自身免疫性肝炎、溶血性贫血、风湿热、甲状腺炎、克罗恩病、重症肌无力、肾小球肾炎、斑秃、牛皮癣、白癜风、营养不良性大疱性表皮松解症和白塞病。
如本申请所述，“治疗(treatment)”或“治疗(treating)”是一种为获得有益或期望的结果的方法，包括临床结果。鉴于本申请的目的，所述有益或期望的结果包括但不限于下列一种或多种：缓解由疾病引起的一种或多种症状、减少疾病范围、稳定疾病(例如，预防或延缓疾病恶化)、预防或延缓疾病传播、预防或延缓疾病复发、延缓或减缓疾病发展、改善疾病状态、缓解疾病(部分或全部)、减少治疗疾病所需的一种或多种其它药物的剂量、延缓疾病发展、提高生存质量和/或延长生存期。同时，“治疗”还包括减少疾病的病理结果。本申请的方法考虑了这些治疗的任何一个或多个方面。例如，如果一个或多个与该疾病相关的症状得到缓解或消除，包括但不限于减少该疾病引起的症状，提高该疾病患者的生活质量，减少治疗疾病所需的其它药物的剂量，和/或延长个体的生存期，则认为该患者被成功“治疗”。
术语“预防(prevent)”和类似的词语，如“预防(prevented)”、“预防(preventing)”等表示预防、抑制或降低疾病或病症的发生或复发可能性的方法。它还指延缓疾病或病症的发生或复发。如本申请所述，“预防”和类似词语还包括在疾病或病症发生或复发之前降低疾病或病症的强度、影响、症状和/或负担，或降低疾病或病症发生或复发的可能性。
如本申请所述，“延缓”疾病的发展表示推迟、阻碍、减缓、减慢、稳定和/或推迟疾病的发展。根据疾病史和/或接受治疗的个体不同，延缓的时间可能不同。一种“延缓”疾病发展的方法是指与不使用该方法相比，在给定时间范围内降低疾病发展概率和/或在给定时间范围内降低疾病程度的方法。在一些实施例中，这种比较可以基于动物试验，在动物个体中进行观测和/或统计。在另一些实施例中，这种比较可以基于临床研究，使用具有统计学意义的个体数量。
如本申请所述，术语“有效量”是指足以治疗特定紊乱、病症或疾病的药物剂量或药物组合物的剂量，如改善、减轻、减弱和/或延缓一个或多个症状。在一些实施例中，有效量是足以延缓疾病发展的量。在一些实施例中，有效量是足以预防或延缓疾病发生或复发的量。有效量可在一次给药或多次给药。在一些实施例中，药物或药物组合物的有效量是指能够增加患者患病组织内Treg细胞数量的量。
如本申请所述，“个体”或“主体”是指哺乳动物，包括但不限于人类、牛、马、猫、狗、啮齿动物或灵长类动物。在一些实施例中，所述个体是指人类。
术语“恒定区”是指免疫球蛋白分子(Ig)的一部分，相对于免疫球蛋白分子中包含抗原结合位点的“可变区”的另一部分。恒定区具有更保守的氨基酸序列，包含 重链恒定区(CH)，其包含CH1、CH2和CH3结构域和轻链恒定区(CL)。根据免疫球蛋白重链恒定区(CH)的氨基酸序列，免疫球蛋白可分为不同类别或亚型。有五类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，重链分别为α、δ、ε、γ和μ。根据CH序列和功能的相对较小差异，将γ和α进一步划分为亚类，例如，人类表达以下亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgA2。
如本申请所述，术语IgG“亚型”或“亚类”是指通过恒定区的化学和抗原特性定义的免疫球蛋白的任何亚类。免疫球蛋白主要分为五大类：IgA、IgD、IgE、IgG和IgM，并且其中多个可进一步分为亚类(亚型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。与不同免疫球蛋白类别相对应的重链分别应用希腊字母α、δ、ε、γ和μ来命名。不同类别免疫球蛋白的亚单位结构和三维构型已众所周知，并在Abbas等人的《细胞和分子免疫学》第四版(W.B.Saunders，Co.，2000)中进行了详述。
如本申请所述，术语“Fc”、“Fc结构域”、“Fc片段”、“Fc区”或“可结晶片段”用于定义免疫球蛋白重链的C端区域，包括天然Fc和变体Fc。示例性的Fc包括免疫球蛋白的CH2和CH3结构域。尽管免疫球蛋白重链Fc的边界可能不同，但人类IgG重链Fc通常定义为从Cys226位置的氨基酸残基或从Pro230开始，延伸至其羧基末端。Fc的C端赖氨酸(根据EU编号系统为447残基)可能被移除，例如，在蛋白质的生产或纯化过程中，或通过重组工程编码蛋白质的核酸而移除。用于本文所述构建体的合适的天然序列Fc区来源于，包括但不限于，人类IgG1、IgG2(IgG2A、IgG2B)、IgG3和IgG4。
“Fc受体”或“FcR”描述了与包含Fc的分子(例如，抗体或包含Fc的融合蛋白)中的Fc区结合的受体。首选的FcR是人类FcR天然序列。此外，示例性的FcR是一种结合IgG抗体的FcR(一种γ受体)，包括FcγRI、FcγRII和FcγRIII等受体亚类，以及这些受体的等位基因变体和可变剪接形式。FcγRII受体包括FcγRIIA(“激活受体”)和FcγRIIB(“抑制受体”)，它们具有相似的氨基酸序列，主要在胞质结构域有所不同。激活受体FcγRIIA在其胞质结构域中包含免疫受体酪氨酸活化基序(ITAM)。抑制受体FcγRIIB在其胞质结构域中包含免疫受体酪氨酸抑制基序(ITIM)(见M.Annu.Rev.Immunol.15:203-234(1997))。在Ravetch and Kinet,Annu.Rev.Immunol 9:457-92(1991)；Capel et al.,Immunomethods 4:25-34(1994)和de Haas et al.,J.Lab.Clin.Med.126:330-41(1995)中对FcRs进行了综述。本文中术语“FcR”涵盖其它FcRs，包括那些在未来将被鉴定的FcRs。
术语“Fc受体”或“FcR”也包括新生儿受体FcRn，所述受体负责将母体IgG转运给胎儿。Guyer et al.,J.Immunol.117:587(1976)和Kim et al.,J.Immunol.24:249(1994)。测定与FcRn结合的方法是本领域已知的(例如，参见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.))。可以测定人类FcRn高亲和力结合多肽在体内和血清中与FcRn结合的半衰期，例如，在表达人类FcRn的转基因小鼠或转染的人类细胞系中，或在施用具有变体Fc区的多肽的灵长类动物中。WO 2004/42072(Presta)详述了增强或减弱与FcRs结合的抗体变体。关于FcRn，可参见Shield et al.,J.Biol.Chem.9(2):6591-6604(2001)。
“Fc效应功能”是指那些由包含Fc的分子(例如，抗体或包含Fc的融合蛋白)中的Fc区(天然序列Fc区或包含氨基酸序列突变的Fc区)引起的生物活性，根据Fc所来源的免疫球蛋白亚型的不同而不同。效应功能的示例包括：C1q结合和补体依赖的细胞毒性作用(CDC)；Fc受体结合；抗体依赖的细胞介导的细胞毒性作用(ADCC)；吞噬作用；细胞表面受体的下调(例如，B细胞受体)和B细胞活化。“减少或最小化”效应功能表示与野生型Fc或未经修饰的包含Fc的分子(例如，抗体)相比，其效应功能减少至少50％(或者60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％或99％)。测定效应功能可由本领域普通技术人员轻而易举地测定和测量。在优选实施例中，补体结合、补体依赖性细胞毒性和抗体依赖性细胞毒性的效应功能都将受到影响。在一些实施例中，通过恒定区中的突变消除糖基化来消除效应功能，例如，“无效应功能突变”。在一些实施例中，无效应功能突变体是在CH2区的N297A或DANA突变(D265A+N297A)，可参见Shields et al.,J.Biol.Chem.276(9):6591-6604(2001)。另外，导致效应功能降低或消除的其它突变包括：K322A和L234A/L235A(LALA)。另外，可以通过生产技术的改变来减少或消除效应功能，如在不进行糖基化的宿主细胞中(例如，大肠杆菌)或导致糖基化模式改变的宿主细胞中表达，所述糖基化模式改变在促进效应功能方面无效或效果较小(例如，Shinkaw et al.,J.Biol.Chem.278(5):3466-3473(2003))。
“抗体依赖的细胞介导的细胞毒性作用”或“ADCC”是指一种细胞毒性形式，其中分泌型Ig(或配体-Fc结构)与存在于某些细胞毒性细胞(例如，自然杀伤细胞(NK)、中性粒细胞和巨噬细胞)上的Fc受体(FcRs)结合，使这些细胞毒性效应细胞能够特异性结合携带抗原(或携带配体受体)的靶细胞，随后用细胞毒素杀死靶 细胞。抗体(或其他包含Fc的分子)“武装”细胞毒性细胞，是通过这种机制杀死靶细胞所必需的。介导ADCC的主要细胞包括NK细胞和单核细胞，其中，NK细胞只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。在Ravetch和Kinet,Annu.Rev.Immunol 9:457-92(1991)第464页的Table 2中总结了造血细胞中Fc的表达。为评估目标分子的ADCC活性，可以进行体外ADCC试验，如U.S.Pat.No.5,500,362或5,821,337中进行了详述。适用于此类试验的效应细胞包括外周血单个核细胞(PBMC)和自然杀伤细胞(NK)。或者，或额外地，也可以在体内评估目标分子的ADCC活性，例如，在动物模型中如在Clynes et al.,PNAS(USA)95:652-656(1998)中所公开的。
“补体依赖的细胞毒性作用”或“CDC”是指在补体存在的情况下裂解靶细胞。经典的补体途径的激活是由补体系统第一组分(C1q)与(合适亚类的)包含Fc的分子结合起始的，所述包含Fc的分子通过Fc融合的配体与其同源受体结合。为了评估补体激活，可以进行CDC试验，如Gazzano-Santoro et al.,J.Immunol.Methods 202:163(1996)中所述。U.S.Pat.No.6,194,551B1和WO99/51642中详述了Fc区氨基酸序列的改变从而增加或减少C1q结合能力的抗体变体。这些专利出版物的内容通过引用并入本文。参见Idusogie et al.J.Immunol.164:4178-4184(2000)。
如本申请所述，“调节性T细胞”或“Treg”是一类具有显著免疫抑制作用的CD4+T细胞亚群，能免抑制其他细胞的免疫应答。Treg的特征在于表达IL-2受体的α亚基(CD25)和转录因子叉头框P3(FOXP3)，并在维持机体免疫平衡和预防自身免疫疾病、移植排斥方面发挥着重要作用。Treg需要IL-2来实现其功能和发育以及其抑制性特征的诱导。
如本申请所述，“效应细胞”是指介导由IL-2诱导的细胞毒性效果的淋巴细胞群体。效应细胞主要包括效应T细胞如CD8+细胞毒性T细胞、NK细胞、淋巴因子激活的杀伤(LAK)细胞和巨噬细胞等。
如本申请所述，术语“特异性结合”、“特异性识别”或“特异性用于”是指可测量和可再现的相互作用，如配体和受体之间的结合，当存在包括生物分子在内的异质分子群的情况下可确定配体的存在。例如，与受体特异性结合的配体，当与结合其它受体相比时，在结合目标受体时具有更大的亲和性、亲和力、更容易和/或持续时间更长。在一些实施例中，通过例如放射免疫分析(RIA)法进行测定，配体与无关受体的结合程度小于配体与目标受体结合程度的10％。在一些实施例中，特异性结合靶受体的配体的平衡解离常数(Kd)≤10-5M、≤10-6M、≤10-7M、≤10-8M、≤10-9M、≤10-10 M、≤10-11M或≤10-12M。在一些实施例中，配体可特异性结合在不同物种中保守的受体。在一些实施例中，特异性结合可以包括但不要求排他性结合。可利用本领域已知的方法通过实验确定配体的结合特异性。如包括但不限于Western blots、ELISA-、RIA-、ECL-、IRMA-、EIA-、BIACORETM-测试和肽扫描。
“亲和力”或“结合亲和力”通常指分子(例如，配体)的单个结合位点和其结合搭档(例如，受体)之间的非共价相互作用总和的强度。除非另有说明，如本申请所述的“结合亲和力”指内在结合亲和力，该内在亲和力可以反映结合对成员之间的1:1相互作用。结合亲和力可以采用Kd、Koff、Kon或Ka表示。如本申请所述，术语“Koff”是指配体从配体/受体复合物中解离的速率常数，可通过动力学选择装置测定，以s-1为单位来表示。如本申请所述，术语“Kon”是指配体与受体结合形成配体/受体复合物的结合速率常数，以M-1s-1为单位来表示。如本申请所述，术语平衡解离常数“Kd”是指特定配体-受体相互作用时的解离常数，是指在受体溶液中，配体占据所有受体结合位点的一半并且达到平衡时所需的配体浓度，等于Koff/Kon。解离常数(Kd)可以作为反映配体与受体亲和力的指标。使用所述方法得到的Kd值，以M(mol/L)为单位来表示。测定Kd的前提是所有的结合分子均在溶液中。当受体在细胞膜上的情况下，相应的解离速率常数用EC50值表示。EC50值是Kd的一个很好的近似值。亲和常数Ka，是解离常数Kd的倒数，以M-1为单位来表示。解离常数(Kd)可以作为反映配体与受体亲和力的指标。
半抑制浓度(IC50)是对物质(例如，配体)在抑制特定生物或生化功能中有效性的度量。它表示需要多少特定药物或其它物质(抑制剂，例如，配体)能将给定的生物过程抑制一半。IC50值通常表示为摩尔浓度。IC50与激动剂药物或其它物质(例如，配体)的“EC50”相当。EC50也代表在体内获得最大效应的50％所需的血浆浓度。如本申请所述，“IC50”用于表示在体外中和50％的受体生物活性所需配体的有效浓度。可通过生物测量测定IC50或EC50，如通过FACS分析(竞争结合试验)抑制配体结合、基于细胞的细胞因子释放试验或放大发光的均相酶联免疫试验(AlphaLISA)。
如本申请所述“融合”是指由肽键直接连接或通过一个或多个连接肽(或称为“接头”)将各个组分进行连接。“连接肽(或接头)”的序列可以是单个氨基酸或多肽序列。在一些实施例中，所述连接肽(或接头)包含甘氨酸-丝氨酸接头或由甘氨酸-丝氨酸接头组成。如本申请所述，术语“甘氨酸-丝氨酸接头”指由甘氨酸和丝氨酸 残基组成的肽。示例性的甘氨酸-丝氨酸接头包括通式为(Gly4Ser)n的氨基酸序列，其中n是正整数(例如，1、2、3、4、5、6、7、8、9或10)。一个优选的甘氨酸-丝氨酸接头为GGGGS。
如本申请所述，“共价键”是指两个原子之间通过共用一个或多个电子形成的稳定键。共价键的示例包括但不限于肽键和二硫键。如本申请所述，“肽键”是指氨基酸的羧基与相邻氨基酸的胺基之间形成的共价键。如本申请所述，“二硫键”是指两个硫原子之间形成的共价键，如两个Fc片段通过一个或多个二硫键结合。两个片段之间的一个或多个二硫键可能通过连接两个片段中的硫醇基形成。在一些实施例中，两个Fc片段的一个或多个半胱氨酸之间可能形成一个或多个二硫键。氧化两个硫醇基可形成二硫键。在一些实施例中，共价连接是由共价键直接连接而成的。在一些实施例中，共价连接直接由肽键或二硫键连接。
如本申请所述，术语“衍生物”是指一种具有某种蛋白质氨基酸序列或其类似物的分子，但在一个或多个氨基酸基团、α碳原子、氨基末端或羧基末端上还具有额外的修饰。如本申请所述，修饰包括但不限于化学修饰、氨基酸侧基团修饰、氨基末端修饰、羧基末端修饰。其中化学修饰包括但不限于，添加化学基团、生成新化学键和移除化学基团。氨基酸侧基团的修饰包括但不限于赖氨酸的ε氨基酰基化，精氨酸、组氨酸或赖氨酸的N-烷基化，谷氨酸或天冬氨酸的羧基烷基化，谷氨酰胺或天冬酰胺的脱氨。氨基末端的修饰包括但不限于脱氨、N-低烷基，N-二低烷基和N-酰基修饰。羧基末端的修饰包括但不限于酰胺、低烷基酰基、二烷基酰胺和低烷基酯修饰。在一些实施例中，低烷基基团为C1-C4烷基基团。此外，一个或多个侧基或末端基团可以由化学领域的技术人员利用已知的保护基团来保护。氨基酸的α碳可以是单甲基化或二甲基化。
肽或多肽序列的“氨基酸序列同一性百分比(％)”或“同源性”被定义为候选序列中与特定多肽或多肽序列中相同氨基酸残基所占的百分比，在序列比对并引入间隙(如果必要)后以实现序列同一性百分比的最大化，并且不考虑任何保守替换作为序列同一性的一部分。为了确定氨基酸序列同一性百分比，可以通过本领域技术范围内的多种比对方式，例如，使用如BLAST、BLAST-2、ALIGN、Megalign(DNASTAR)、或MUSCLE软件等可公开获得的计算机软件。本领域技术人员可以确定用于测量比对的合适参数，包括在所比较的序列的全长上实现最大比对所需的任何算法。
如本申请所述，多肽的“C端”是指该多肽的最后一个氨基酸残基，该残基的胺基以与其相邻氨基酸残基的羧基形成肽键。如本申请所述，多肽的“N端”是指该多肽的第一个氨基酸残基，该残基的羧基以与其相邻氨基酸残基的胺基形成肽键。
“分离的”多肽是指一种已从其生产环境(例如，天然或重组)的组分中鉴定、分离和/或回收的多肽。优选地，所分离的多肽与其生产环境中的所有其它成分没有关联。生产环境的污染成分，如重组转染细胞产生的污染成分，通常会干扰多肽的研究、诊断或治疗，并且可能包括酶、激素和其它蛋白溶质或非蛋白溶质。在一些实施例中，多肽将被纯化至：(1)按重量计算，多肽含量大于95％，如通过Lowry法确定，在一些实施例中，按重量计算，多肽含量大于99％；(2)通过使用旋杯测序仪达到足以获得至少15个N端残基或内部氨基酸序列的程度；或(3)通过SDS-PAGE在非还原性或还原性条件下使用考马斯蓝或优选为银染色达到同质性。分离的多肽包括在重组细胞内的原位多肽，因为多肽天然环境中的至少一个要素是不存在的。然而，通常情况下，一个分离的多肽至少要经过一个纯化步骤。
“氨基酸”在本文中以其最广泛的定义使用，即包括天然存在的氨基酸，也包括非天然存在的氨基酸，包括氨基酸的类似物和衍生物。后者包括含有氨基酸部分的分子。根据这一宽泛的定义，本领域的技术人员会发现，本文所述氨基酸包括，例如，形成蛋白质的天然L-氨基酸；D-氨基酸；化学修饰的氨基酸，如氨基酸类似物和衍生物；不形成蛋白质的天然氨基酸，如正亮氨酸、β-丙氨酸、鸟氨酸、GABA等；以及本领域已知的具有氨基酸特征的化学合成的化合物。如本申请所述，术语“形成蛋白质”是指可以通过代谢途径合成细胞的肽、多肽或蛋白质的氨基酸。
一种编码蛋白质或多肽(例如，本文所述的IL-2突变体或包含IL-2突变体的融合蛋白)的“分离的”核酸分子为一种从至少含一种杂质的核酸分子中鉴定和分离出来的核酸分子，所述杂质核酸分子通常与生产它的环境有关。优选地，所分离的核酸与其生产环境中的所有成分没有关联。如本文所述的编码多肽的分离的核酸分子是以一种并非它在自然界中发现时的形式或形态存在。因此，分离的核酸分子不同于自然存在于细胞中编码本文所述多肽的核酸。一种分离的核酸包括含有该核酸分子的细胞中所含的核酸分子，但该核酸分子存在于染色体外或与其自然染色体位置不同的染色体位置上。
术语“控制序列”是指在特定宿主生物体中表达可操作连接编码序列所必需的DNA序列。例如，适合原核生物的控制序列包括启动子、任选的操作序列和核糖体结合位点。已知真核细胞利用启动子、多聚腺苷酸化信号和增强子。
当核酸与另一个核酸序列建立功能性关系时，所述核酸与另一核酸序列之间即为“可操作地连接”。例如，编码前序列或分泌前导序列的DNA参与表达形式为前体蛋白的多肽的分泌过程，则其与编码该多肽分子的DNA可操作性的连接；如果启动子或增强子影响编码序列的转录，则所述启动子或增强子可操作连接到编码序列；或如果核糖体结合位点处在便于翻译的位置，则所述核糖体结合位点可操作连接到编码序列。一般来说，“可操作连接”意味着连接的DNA序列是连续的，并且，对于分泌先导物，其不仅是连续的并且处于读取阶段(reading phase)。然而，增强子不必是连续的。连接是通过在适宜的限制性位点进行连接来完成的。如果不存在此类位点，则按照常规做法使用合成的寡核苷酸适配体或接头。
如本申请所述，术语“载体”是指一种核酸分子，其能够扩增与其连接的另一核酸分子。该术语包括作为自我复制核酸结构的载体以及被引入已知宿主细胞基因组的载体。某些载体能够指导与之连接的核酸表达。在本文中称此类载体为“表达载体”。
如本申请所述，术语“转染”、“转化”或“转导”是指将外源核酸转移或引入宿主细胞的过程。“转染的”、“转化的”或“转导的”细胞是用外源核酸转染、转化或转导的细胞。所述细胞包括原代受试细胞及其子代。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，指引入外源核酸的细胞，包括此类细胞的子代。宿主细胞包括“转化体”和“转化细胞”，其中包括原代转化细胞和由此产生的子代，而不考虑传代次数。子代在核酸上可能与母细胞不完全相同，例如，可能含有突变。本文包括在原始转化细胞中筛选或选择与其具有相同功能或生物活性的突变子代。
术语“药物制剂”或“药物组合物”是指一种制剂，所述制剂为使活性成分的生物活性有效的形式，并且不包含对施用所述制剂的受试者具有不可接受毒性的额外成分。这种制剂是无菌的。“无菌”制剂是无菌的或不含任何活的微生物及其孢子。
本申请中所述的实施例应理解为包括“由…组成”和/或“基本上由…组成”的实施例。
本申请中提及“约”为一个数值或参数，包含(和描述)针对该数值或参数本身的变体。例如，涉及“约X”的描述包括“X”的描述。
如本申请所述，提及“不是”一个数值或参数，通常表示并描述“除了”某一数值或参数之外。例如，该方法不能用于治疗X型癌症，意味着该方法通常用于治疗除X型癌症之外的其它类型的疾病。
如本申请所述，术语“约X-Y”与“约X到约Y”意思相同。
除非上下文另有明确说明，本文和所述权利要求中所采用的单数形式“一”，“一个”和“该”包括复数对象。
II.IL-2突变体
本申请的IL-2突变体对IL-2受体选择和对淋巴细胞激活/增殖的偏好性及生物学性质：
在一些实施例中，与人野生型IL-2(例如，SEQ ID NO:1)相比，本申请提供的IL-2突变体与IL-2Rβ和/或IL-2Rβγ的亲和力降低。在一些实施例中，与人野生型IL-2相比，所述IL-2突变体与IL-2Rβ和/或IL-2Rβγ的亲和力减少了至少5％(例如至少10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或100％)。
在一些实施例中，与人野生型IL-2(例如SEQ ID NO:1)相比，本申请提供的IL-2突变体，与IL-2Rα的亲和力增加。在一些实施例中，与人野生型IL-2相比，所述IL-2突变体与IL-2Rα的亲和力增加了至少1倍(例如至少1倍、2倍、3倍、5倍、10倍、或20倍)。
在一些实施例中，与人野生型IL-2(例如SEQ ID NO:1)相比，本申请提供的IL-2突变体，与IL-2Rα的亲和力保持不变或者没有明显减弱或与IL-2Rα的亲和力增加。
在一些实施例中，与人野生型IL-2(例如SEQ ID NO:1)相比，本申请提供的IL-2突变体与IL-2Rβ和/或IL-2Rβγ的亲和力降低，同时与IL-2Rα的亲和力保持不变或者没有明显减弱或与IL-2Rα的亲和力增加。IL-2突变体与受体之间的亲和力可以通过本领域已知的常规方法测定，例如结合ELISA实验、HEK-BlueTM IL-2细胞系检测实验或表面等离子体共振法(SPR)等。
在一些实施例中，本申请提供的IL-2突变体，与人野生型IL-2(例如SEQ ID NO:1)相比，在治疗某些特定疾病中，具有更高的安全性。
例如，在HEK-BlueTM IL-2细胞系(InvivoGen,cat#hkb-il2)实验中，与人野生型IL-2相比，通过HEK-BlueTM CD122/CD132细胞系检测IL-2突变体与IL-2Rβγ(下称二受体)结合的EC50值与通过HEK-BlueTM IL-2细胞系检测IL-2突变体与IL-2Rαβγ(下 称三受体)结合的EC50值之间的比值更高，即具有更高的治疗安全窗，因此具有更高的安全性。在一些实施例中，通过HEK-BlueTM CD122/CD132细胞系检测实验来确定IL-2或IL-2突变体与IL-2Rβγ结合的EC50值，通过HEK-BlueTM IL-2细胞系检测实验来确定IL-2或IL-2突变体与IL-2Rαβγ结合的EC50值。在一些实施例中，IL-2突变体的EC50二受体/EC50三受体比值是人野生型IL-2的EC50二受体/EC50三受体比值的至少2倍，例如：2倍、5倍、10倍、50倍、100倍、200倍、500倍、700倍、1000倍、2000倍、10000倍、20000倍、30000倍或更高。
在一些实施例中，与人野生型IL-2(例如SEQ ID NO:1)相比，本申请提供的IL-2突变体能够选择性活化Treg细胞，降低对其它T细胞的活化，例如降低对CD8+T细胞的活化；降低对NK细胞的活化。
在一些实施例中，与人野生型IL-2(例如SEQ ID NO:1)相比，本申请提供的IL-2突变体能够选择性活化Treg细胞。例如，在STAT5磷酸化实验中，通过检测IL-2或IL-2突变体在Treg细胞中对STAT5磷酸化信号的激活，来反映IL-2突变体对Treg细胞的活化能力。通过流式细胞仪进行检测，GraphPad Prism软件分析并计算IL-2或IL-2突变体在Treg细胞中激活STAT5磷酸化的EC50值，该EC50值间接反映IL-2或IL-2突变体对Treg细胞的活化能力，表明IL-2突变体能够有效地活化Treg细胞。
在一些实施例中，与人野生型IL-2(例如SEQ ID NO:1)相比，本申请提供的IL-2突变体，能够降低对CD8+T细胞的活化。例如，在STAT5磷酸化实验中，通过检测IL-2或IL-2突变体在CD8+T淋巴细胞中对STAT5磷酸化信号的激活，来反映IL-2突变体对CD8+T淋巴细胞的活化能力。通过流式细胞仪进行检测，GraphPad Prism软件分析并计算IL-2突变体在CD8+T淋巴细胞中激活STAT5磷酸化的EC50值，该EC50值间接反映IL-2或IL-2突变体对CD8+T淋巴细胞的活化能力。在一些实施例中，与人野生型IL-2相比，IL-2突变体对CD8+T淋巴细胞的活化能力下降至少10％。例如：10％、20％、30％、40％、50％、60％、70％、80％、90％或100％。
在一些实施例中，本申请提供的IL-2突变体能够在体内或体外能够有效扩增Treg细胞。在一些实施例中，所述IL-2突变体能够增加受试者体内CD4+T细胞群中Treg细胞的占比(例如，CD4+T细胞群体内CD4+CD25+Foxp3+细胞的占比增加)。
在一些实施例中，所述IL-2突变体，能够增加受试者体内Treg细胞与非Treg细胞的比率(例如，CD4+CD25+Foxp3+细胞与CD8+细胞的比率增加)。例如，在本申请实施例中，通过IL-2突变体刺激后，在一定时间段内，受试动物食蟹猴外周血中Treg 细胞数量增加，而对CD8+T淋巴细胞的细胞数量无显著影响。在一些实施例中，通过IL-2突变体刺激，受试动物体内Treg细胞在CD4+T淋巴细胞中的占比增加了至少1倍。例如：1倍、2倍、3倍、5倍、7倍、10倍、15倍、20倍或更高。
本申请的IL-2突变体：
在一些实施例中，与野生型IL-2(例如人野生型IL-2)相比，本申请所述的IL-2突变体具有至少一个氨基酸突变，例如氨基酸的取代、缺失、插入，或者包含上述突变形式的任意组合。
第一组IL-2突变体：
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列(例如SEQ ID NO:1)第81-85位的氨基酸残基RPRDL的缺失或突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含E67、L70、N71、S75和V91中的一个或多个突变。在一些实施例中，E67的突变为E67K、E67A、E67M或E67T。在一些实施例中，L70的突变为L70I。在一些实施例中，N71的突变为N71I、N71S或N71W。在一些实施例中，S75的突变为S75I或S75F。在一些实施例中，V91的突变为V91K。
在一些实施例中，本申请所述的IL-2突变体，第81-85位的氨基酸残基RPRDL缺失1、2、3、4或全部5个氨基酸。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的全部缺失。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL的全部缺失，还进一步包含E67和N71中的一个或多个突变。在一些实施例中，所述E67和N71的突变分别为E67K和N71S。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL全部缺失、以及相对于人野生型IL-2氨基酸序列的E67K和N71S突变。
在一些实施例中，本申请所述的IL-2突变体中相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL中的部分或全部氨基酸残基被取代。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RPL。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RPL，且进一步包含E67的突变。在一些实施例中，所述E67的突变为E67A。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RPL，以及相对于人野生型IL-2氨基酸序列的E67A突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RPL，且进一步包含相对于人野生型IL-2氨基酸序列的N71和S75的突变。在一些实施例中，所述N71和S75的突变分别为N71W和S75I。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，以及相对于人野生型IL-2氨基酸序列的N71W和S75I突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RHL。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RHL，且进一步包含E67和/或V91的突变。在一些实施例中，所述E67的突变为E67T。在一些实施例中，所述V91的突变为V91K。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的E67T突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的V91K突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的E67T和V91K突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RHL，且进一步包含相对于人野生型IL-2氨基酸序列的L70和S75的突变。在一些实施例中，所述L70和S75的突变分别 为L70I和S75F。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的L70I和S75F突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为RHL，且进一步包含相对于人野生型IL-2氨基酸序列的N71的突变。在一些实施例中，所述N71的突变为N71I。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的N71I突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为SPL。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为SPL，还进一步包含相对于人野生型IL-2氨基酸序列的E67和N71中的一个或多个突变。在一些实施例中，所述E67和N71的突变分别为E67M和N71S。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第81-85位的氨基酸残基RPRDL突变为SPL，以及相对于人野生型IL-2氨基酸序列的E67M和N71S突变。
第二组IL-2突变体：
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列(例如SEQ ID NO:1)的第75位氨基酸残基S(S75)和第83位氨基酸残基R(R83)的突变。在一些实施例中，S75的突变为S75P、S75F、S75Y、S75G、S75A、S75V、S75T、S75I或S75L。在一些实施例中，R83的突变为R83N、R83F、R83Y、R83P、R83L、R83A、R83K或R83E。在一些实施例中，本申请所述的IL-2突变体，其进一步包含E15、H16、L17、L63、E67、L70、N71、D84、V91和I92中的一个或多个突变。在一些实施例中，E15的突变为E15K。在一些实施例中，H16的突变为H16D、H16Q或H16N。在一些实施例中，L17的突变为L17I。在一些实施例中，L63的突变为L63V。在一些实施例中，E67的突变为E67A、E67R、E67G、E67S、E67K或E67F。在一些实施例中，L70的突变为L70I、L70V或L70F。在一些实施例中， N71的突变为N71G或N71S。在一些实施例中，D84的突变为D84N。在一些实施例中，V91的突变为V91E或V91K。在一些实施例中，I92的突变为I92L。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P和R83Y突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和D84中的一个或多个突变。在一些实施例中，其中E67和D84的突变分别为E67A和D84N。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P、R83Y、E67A和D84N突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P和R83A突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16、L70和N71中的一个或多个突变。在一些实施例中，其中H16、L70和N71的突变分别为H16D、L70I和N71G。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P、R83A、H16D、L70I和N71G突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F和R83P突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和N71中的一个或多个突变。在一些实施例中，其中H16的突变为H16Q或H16N。在一些实施例中，其中N71的突变为N71S。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83P和H16Q突变。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83P、H16N和N71S突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F和R83L突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70中的一个或多个突变。在一些实施例中，其中E67和L70的突变分别为E67R和L70V。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83L、E67R和L70V突变。
在一些实施例中，本申请所述的IL-2突变体，相对于人野生型IL-2，其包含相对于人野生型IL-2氨基酸序列的S75I和R83L突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70中的一个或 多个突变。在一些实施例中，其中E67和L70的突变分别为E67G和L70V。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75I、R83L、E67G和L70V突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75I和R83K突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70中的一个或多个突变。在一些实施例中，其中E67和L70的突变分别为E67S和L70V。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75I、R83K、E67S和L70V突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75T和R83F突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75T和R83A突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和E67中的一个或多个突变。在一些实施例中，其中H16和E67的突变分别为H16N和E67K。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75T、R83A、H16N和E67K突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75V和R83F突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E15和H16中的一个或多个突变。在一些实施例中，其中E15和H16的突变分别为E15K和H16Q。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75V、R83F、E15K和H16Q突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75V和R83N突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L70和V91中的一个或多个突变。在一些实施例中，其中L70和V91的突变分别为L70F和V91K。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75V、R83N、L70F和V91K突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75G和R83L突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17、V91和I92中的一个或多个突变。在一些实施例中，L17、V91和I92的突变分别为L17I、V91E和I92L。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75G、R83L、L17I、V91E和I92L突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75Y和R83N突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17和V91中的一个或多个突变。在一些实施例中，L17和V91的突变分别为L17I和V91K。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75Y、R83N、L17I和V91K突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75A和R83K突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17的突变。在一些实施例中，L17的突变为L17I。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75A、R83K和L17I突变。
在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75L和R83E突变。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L63和E67中的一个或多个突变。在一些实施例中，L63和E67的突变分别为L63V和E67F。在一些实施例中，本申请所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75L、R83E、L63V和E67F突变。
其它位点的突变：
在一些实施例中，本申请的IL-2突变体还可以进一步在相对于人野生型IL-2氨基酸序列的其它位置或区域上具有一个或多个突变，只要其满足本发明所述的IL-2突变体对受体IL-2Rβγ的选择偏好性降低，和/或一个或多个有益的性质即可。在一些实施例中，本申请所述的IL-2突变体，还包括在相对于人野生型IL-2氨基酸序列(例如SEQ ID NO:1)上的不改变与IL-2受体亲和力的其它位置的突变。在一些实施例中，本申请所述的IL-2突变体，在相对于人野生型IL-2氨基酸序列(例如，SEQ ID NO：1)的第125位上存在氨基酸突变，例如C125S、C125A、C125T或C125V(具体参见 美国专利NO.4,518,584)。本领域技术人员知晓如何在本申请所述的突变之外，增加其它的额外突变的方法。
在一些实施例中，本申请所述的IL-2突变体进一步包含相对于人野生型IL-2氨基酸序列的C125S突变。在一些实施例中，本申请所述的IL-2突变体进一步包含相对于人野生型IL-2氨基酸序列的C125A突变。
在一些实施例中，本申请所述的IL-2突变体包含SEQ ID NOs:5-18中任一所示的氨基酸序列或者其变体，所述变体与SEQ ID NOs:5-18中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，本申请所述的IL-2突变体包含SEQ ID NOs:19-34中任一所示的氨基酸序列或者其变体，所述变体与SEQ ID NOs:19-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，所述其他位置或区域上的一个或多个突变包含一个或多个保守的氨基酸取代。“保守取代”是指应用另一种氨基酸进行取代，所述另一氨基酸与被取代氨基酸带有相同的净电荷和大致相同的大小和形状。当侧链上的碳原子和杂原子总数相差不超过4个时，带有脂肪族或取代脂肪族氨基酸侧链的氨基酸大小大致相同。当其侧链上的分支数相差不超过1个时，氨基酸的形状大致相同。在侧链上具有苯基或取代苯基的氨基酸，可以认为其大小和形状大致相同。除非另有说明，保守取代优选应用天然氨基酸。示例性的保守性取代如表1所示。
在表1“取代示例”标题下提供了更多实质性取代，如下文关于氨基酸侧链类别部分进一步详述。氨基酸可以根据常见的侧链性质分类：(1)疏水性：正亮氨酸(Norleucine)、Met、Ala、Val、Leu、Ile；(2)中性亲水性：Cys、Ser、Thr、Asn、Gln；(3)酸性：Asp、Glu；(4)碱性：His、Lys、Arg；(5)影响链方向的残基：Gly、Pro；(6)芳香族：Trp、Tyr、Phe。非保守型取代需要将这些类别中的一个成员替换为另一类别的成员。可以将氨基酸取代引入到蛋白质构建体中，并筛选符合上文所述所需活性的产品。
表1氨基酸取代

在一些实施例中，将包括合成的非天然氨基酸或一种或多种D-氨基酸在内的非天然氨基酸插入到本申请的IL-2突变体或IL-2突变体融合蛋白中可具有多种益处。与含有L-氨基酸的多肽相比，含有D-氨基酸的多肽等在体外和体内表现出更高的稳定性。因此，当需要更好的细胞内稳定性时，如通过加入D-氨基酸进行多肽的构建是特别有用的。特别是D-肽和其类似物对内源性肽酶和蛋白酶具有抗性，从而在需要时提高分子的生物有效性并延长其在体内的寿命。此外，D-肽和其类似物不能被有效加工以通过II型主要组织相容性复合物(MHC)有限地呈递给辅助T细胞，因此不易在受试者中诱导体液免疫反应。
在一些实施例中，本申请所述的IL-2突变体为修饰的IL-2突变体，如聚乙二醇化IL-2突变体，或共价修饰的IL-2突变体，或糖基化修饰的IL-2突变体。
在一些实施例中，本申请所述的IL-2突变体为全长序列。在另一些实施例中，IL-2突变体的N端包含信号肽，所述信号肽或来自不同分子，或来自野生型IL-2。
在一些实施例中，本申请所述的IL-2突变体包含SEQ ID NOs:5-34中任一所示的氨基酸序列或者其变体，所述变体与SEQ ID NOs:5-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
示例性的野生型IL-2及其C125突变体的氨基酸序列如表2所示，示例性的第一组IL-2突变体的氨基酸序列如表3所示，示例性的第二组IL-2突变体的氨基酸序列如表4所示。
表2示例性的野生型IL-2及C125的突变体
表3示例性的第一组IL-2突变体

表4示例性的第二组IL-2突变体序列

表5示例性的Fc突变体序列


表6示例性的连接肽序列

表7示例性的第一组IL-2突变体与Fc的二价融合蛋白序列


表8示例性的第二组IL-2突变体与Fc的二价融合蛋白序列


III.包含IL-2突变体的融合蛋白
本申请还提供包含IL-2突变体的融合蛋白。如本申请所述的IL-2突变体可以与其它蛋白进行融合，通过增加分子的大小和/或降低肾清除率来达到增加循环半衰期的目的。在一些优选实施例中，本申请所述的IL-2突变体与Fc进行融合。在另一些优选实施例中，本申请所述的IL-2突变体与人血清白蛋白(HSA)进行融合。在另一些优选实施例中，本申请所述的IL-2突变体与一些短肽(例如，XTEN)进行融合。
IL-2突变体与Fc的融合蛋白：
Fc融合蛋白可通过重组DNA技术产生，其中哺乳动物免疫球蛋白(例如，IgG)的Fc结构域的翻译阅读框被融合到另一蛋白以产生新的单个重组多肽。Fc融合蛋白通常可以作为二硫键连接的二聚体产生，其由位于Fc结构域中铰链区的二硫键连接在一起。
可以将IL-2突变体融合于Fc的N端和/或C端。优选地，将IL-2突变体融合于Fc的C端。在一些实施例中，IL-2突变体与Fc之间通过肽键直接融合。在一些实施例中，IL-2突变体与Fc之间包含连接肽。
在一些实施例中，本申请所述的Fc来自IgA、IgD、IgE、IgG和IgM及其亚类中的任何一种。在所有免疫球蛋白中，IgG在血清中的含量最高，半衰期最长。与其它免疫球蛋白不同，在与Fc受体(FcRs)结合后，IgG可有效回收。在一些优选实施例中，Fc来自IgG(例如，IgG1、IgG2、IgG3或IgG4)。在一些实施例中，Fc来自人IgG。在一些实施例中，Fc包含CH2和CH3结构域。在一些实施例中，Fc进一步包含全部或部分铰链区。在一些实施例中，Fc来自人IgG1或人IgG4。在一些实施例中，Fc的两个亚基通过一个或多个(例如，1、2、3、4或更多)二硫键二聚化。在一些实施例中，Fc的每个亚基都包含全长Fc序列。在一些实施例中，Fc的每个亚基都包含N端截短的Fc序列，如截短的Fc含有较少的N端半胱氨酸，以减少二聚化过程中二硫键的错配。在一些实施例中，Fc在N端被截短，例如，缺失完整免疫球蛋白Fc的前1、2、3、4、5、6、7、8、9、10个或甚至更多个氨基酸。在一些实施例中，Fc包含一个或多个突变，如插入、缺失和/或取代。
在一些实施例中，本申请所述的Fc具有减弱的或消除的Fc效应功能。在一些实施例中，Fc具有减少的由Fc介导的效应功能，例如减少的ADCC、CDC和/或ADCP效应功能。
包含Fc的融合蛋白可以激活补体并与Fc受体(FcRs)相互作用。在某些情况下，这种固有的免疫球蛋白特性已经被认为是不利的，因为这种融合蛋白可能靶向表达Fc受体的细胞，而不是优选的表达IL-2受体的细胞，这可能造成不期望的细胞毒性。而且进一步考虑到Fc融合蛋白的半衰期长，由于全身毒性，使其难以应用于治疗，或治疗应用受限。因此，在一些实施例中，Fc被改造(例如，包含一个或多个氨基酸突变)以改变其与FcR的结合，特别是改变与Fcγ受体(负责ADCC)的结合和/或改变效应功能，如改变抗体依赖性细胞介导的细胞毒性(ADCC)、抗体依赖型细胞吞噬作用(ADCP)和/或补体依赖性细胞毒性(CDC)。优选地，此类氨基酸突变不会减少与FcRn受体的结合(与半衰期有关)。
在一些实施例中，Fc(例如，人IgG1的Fc)发生突变，以去除一个或多个效应功能，如ADCC、ADCP和/或CDC，以下称为“无效应”或“几乎无效应”Fc。例如，在一些实施例中，Fc为一种无效应人IgG1 Fc，所述人IgG1 Fc包含突变L234A和L235A。在一些实施例中，所述人IgG1 Fc包含一个或多个以下突变(如在每一个Fc亚基中)：L234A、L235E、G237A、A330S和P331S。如本领域技术人员所已知的，IgG1 Fc中K322A、L234A和L235A的组合足以完全消除FcγR和C1q的结合(Hezareh et al.,J Virol 75,12161–12168,2001)。MedImmune发现包含一组三突变L234F/L235E/P331S的Fc具有非常相似的效应(Oganesyan et al.,Acta Crystallographica 64,700–704,2008)。在一些实施例中，Fc包含IgG1 Fc区N297上的糖基化修饰，已知这是产生最优FcR相互作用所必需的。Fc的修饰可以是Wang等人提到的任何合适的工程化IgG Fc(“IgG Fc engineering to modulate antibody effector functions,”Protein Cell.2018 Jan；9(1):63–73)，其内容全部通过引用并入本文。
在一些实施例中，如本申请所述，IL-2突变体与Fc的融合蛋白不具有ADCC和/或CDC，或不具有可检测的ADCC和/或CDC。在一些实施例中，如本申请所述，与包含相同IL-2突变体部分但与野生型或未修饰Fc融合的融合蛋白相比，IL-2突变体与Fc的融合蛋白产生的ADCC和/或CDC减少了至少5％(如至少为10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或100％)。
糖基化变体
在一些实施例中，通过改变Fc或IL-2突变体与Fc的融合蛋白来增加或减少构建体的糖基化程度。可以通过改变氨基酸序列在Fc中加入或删除糖基化位点，以创建或移除一个或多个糖基化位点。
哺乳动物细胞产生的天然含Fc蛋白通常包含一个分支链的双触角寡糖，所述双触角寡糖通常通过N-键连接到Fc CH2结构域的Asn297上。参见Wright et al.,TIBTECH 15:26-32(1997)。寡糖可以包括各种碳水化合物，例如，甘露糖、N-乙酰葡萄糖胺(GlcNAc)、半乳糖和唾液酸，以及附着在双触角寡糖结构的“茎”上GlcNAc的岩藻糖。在一些实施例中，可对Fc中的寡糖进行修饰以产生某些改良的特性。
在一些实施例中，如本申请所述的Fc或IL-2突变体与Fc的融合蛋白具有碳水化合物结构，该结构缺乏附着(直接或间接)在Fc区的岩藻糖。例如，在这种Fc或IL-2突变体与Fc的融合蛋白中，岩藻糖的含量可能从1％至80％、从1％至65％、从5％至65％或从20％至40％。如WO 2008/077546所述，岩藻糖的含量通过MALDI-TOF质谱测量连接到Asn297上的糖链内岩藻糖平均含量相对于附着在Asn297(例如，复合、杂交和高甘露糖结构)上的所有糖结构的总和来确定的。Asn297是指位于Fc区297位的天冬酰胺残基(Fc区残基EU编号系统)；然而，由于Fc区的微小序列变化，Asn297也可位于297位的上游或下游约±3个氨基酸，即在294和300位之间。这类岩藻糖基化变体可能具有增强的ADCC功能。参见US Patent Publication Nos.US 2003/0157108(Presta,L.)，US 2004/0093621(Kyowa Hakko Kogyo Co.,Ltd)。与“去岩藻糖基化”或“岩藻糖缺乏”的抗体变体相关的出版物示例包括：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)。能够产生去岩藻糖基化含Fc蛋白的细胞系的示例包括缺乏蛋白岩藻糖基化功能的Lec13 CHO细胞(Ripka et al.,Arch.Biochem.Biophys.249:533-545(1986)；US Pat Appl No US 2003/0157108 A1,Presta,L；和WO 2004/056312 A1,Adams et al.,尤其是实施例11)，和基因敲除细胞系，如α-1,6-岩藻糖基转移酶基因、FUT8基因敲除的CHO细胞(参见Yamane-Ohnuki et al.,Biotech.Bioeng.87:614(2004)；Kanda,Y.et al.,Biotechnol.Bioeng.,94(4):680-688(2006)；和WO2003/085107)。
效应功能变体
在一些实施例中，本申请考虑了一种Fc，其具有一些但非全部Fc效应功能，使得其成为某种应用的理想候选，在该应用中，IL-2突变体与Fc的融合蛋白在体内的半衰 期很重要，但某些效应功能(如CDC和ADCC)是非必需的或有害的。可以在体外或体内进行细胞毒性试验，以确定CDC和/或ADCC活性的减少/耗尽。例如，可以进行Fc受体(FcR)结合试验，以确保Fc或包含此Fc和IL-2突变体的融合蛋白缺乏FcγR结合(因此可能缺乏ADCC活性)，但保留FcRn结合能力。介导ADCC的主要细胞，自然杀伤细胞(NK)，仅表达FcγRIII，然而单核细胞表达FcγRI、FcγRII和FcγRIII。Ravetch和Kinet,Annu.Rev.Immunol.9:457-492(1991)第464页的表2中汇总了FcR在造血细胞上的表达。U.S.Patent No.5,500,362(参见Hellstrom,I.et al.,Proc.Nat’l Acad.Sci.USA 83:7059-7063(1986))和Hellstrom,I.et al.,Proc.Nat’l Acad.Sci.USA 82:1499-1502(1985)；5,821,337(见于Bruggemann,M.et al.,J.Exp.Med.166:1351-1361(1987))中详述了用于评估目标分子ADCC活性的体外试验的非限制性示例。或者，可采用非放射性检测方法(参见用于流式细胞术的ACTITM非放射性毒性试验(CellTechnology,Inc.Mountain View,CA)和CytoTox非放射性毒性试验(Promega,Madison,WI))。适用于这种检测的效应细胞包括外周血单核细胞(PBMC)和NK细胞。此外，也可以在体内评估目标分子的ADCC活性，例如，在如Clynes et al.,Proc.Nat’l Acad.Sci.USA 95:652-656(1998)所公开的动物模型中。也可进行C1q结合试验以确定IL-2突变体与Fc的融合蛋白不能与C1q结合并因此缺失CDC活性。参见WO 2006/029879和WO 2005/100402中C1q和C3c结合酶联免疫吸附试验。可以进行CDC试验以评估补体活性(参见Gazzano-Santoro et al.,J.Immunol.Methods 202:163(1996)；Cragg,M.S.et al.,Blood 101:1045-1052(2003)和Cragg,M.S.和M.J.Glennie,Blood 103:2738-2743(2004))。可以利用本领域已知的方法进行FcRn结合和体内清除/半衰期测定(参见Petkova,S.B.et al..,Int’l.Immunol.18(12):1759-1769(2006))。
效应功能降低的Fc包含那些在Fc区第238、265、269、270、297、327和329位中一个或多个残基的取代(U.S.专利No.6,737,056)。这类Fc突变体包括在氨基酸位点265、269、270、297和327中的两个或更多位点上的取代，包括所谓的将265和297残基替换成丙氨酸的“DANA”Fc突变体(US专利No.7,332,581)。详述了增强或减少与FcRs的结合的某些抗体变体(参见U.S.专利No.6,737,056；WO 2004/056312,和Shields et al.,J.Biol.Chem.9(2):6591-6604(2001)。在一些实施例中，改造Fc区以改变(即，增加或减少)C1q结合和/或CDC，例如US专利No.6,194,551、WO 99/51642和Idusogie et al.,J.Immunol.164:4178-4184(2000)中所述的。
在一些实施例中，Fc包含一个或多个氨基酸替换，这增加了半衰期和/或增强了与新生儿Fc受体(FcRn)的结合。半衰期增加和与新生儿FcRn结合增强了的抗体负责将母体IgGs转运给胎儿(Guyer et al.,J.Immunol.117:587(1976)和Kim et al.,J.Immunol.24:249(1994))，并在US2005/0014934A1(Hinton等)中进行了详述。那些包含带有一个或多个替换的Fc区的抗体因此增加了Fc区与FcRn的结合。这类Fc变体包括那些带有一个或多个Fc区残基替换的变体，例如，Fc区434残基替换(US专利No.7,371,826)。
参见Duncan和Winter,Nature 322:738-40(1988)；U.S.专利No.5,648,260；U.S.专利No.5,624,821；和WO 94/29351关于Fc变体的其它示例。
半胱氨酸工程变体
在一些实施例中，可能需要创建半胱氨酸工程化的Fc或包含此Fc与IL-2突变体的融合蛋白，其中Fc的一个或多个残基被半胱氨酸残基取代。在一些实施例中，取代残基出现在Fc或IL-2突变体与Fc的融合蛋白上易接近的位点。通过将这些残基替换成半胱氨酸，活性硫醇基因此被定位在Fc或IL-2突变体与Fc的融合蛋白的易接近位点，并可用于将分子与其它部分共轭，如药物部分或接头-药物部分，以创建IL-2突变体与Fc的融合蛋白的共轭物。在一些实施例中，下列残基中的任一个和多个可能被半胱氨酸取代：重链A118(EU编号系统)和重链Fc结构域S400(EU编号系统)。半胱氨酸工程分子可以按照U.S.Patent No.7,521,541中所述的那样产生。
在一些实施例中，Fc来自IgG1 Fc。在一些实施例中，Fc来自人IgG1 Fc。在一些实施例中，Fc来自人野生型IgG1 Fc。在一些实施例中，Fc不包含IgG1 Fc的铰链区。在一些实施例中，Fc包含一个或多个无效应突变和/或去糖基化突变。
在一些实施例中，Fc包含相对于人野生型IgG1 Fc氨基酸序列第234位氨基酸残基L(L234)和第235位氨基酸残基L(L235)中的一个或多个突变。在一些实施例中，Fc包含相对于人野生型IgG1 Fc氨基酸序列的L234和L235的突变。在一些实施例中，相对于人野生型IgG1 Fc氨基酸序列，Fc包含突变L234A和L235A。在一些实施例中，Fc包含(或基本上由...组成、或由...组成)氨基酸序列SEQ ID NO:35或其变体；所述变体与SEQ ID NO:35具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，Fc包含相对于人野生型IgG1 Fc氨基酸序列第297位氨基酸残基N(N297)的突变。在一些实施例中，相对于人野生型IgG1 Fc氨基酸序列，Fc包 含突变N297G或N297A。在一些实施例中，Fc包含(或基本上由......组成、或由......组成)氨基酸序列SEQ ID NO:36或其变体；所述变体与SEQ ID NO:36具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。在一些实施例中，Fc包含(或基本上由...组成、或由...组成)氨基酸序列SEQ ID NO:37或其变体；所述变体与SEQ ID NO:37具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，Fc包含相对于人野生型IgG1 Fc氨基酸序列第234位氨基酸残基L(L234)、第235位氨基酸残基L(L235)和第331位氨基酸残基P(P331)中的一个或多个突变。在一些实施例中，Fc包含相对于人野生型IgG1 Fc氨基酸序列的L234、L235和P331的突变。在一些实施例中，相对于人野生型IgG1 Fc氨基酸序列，Fc包含突变L234A、L235A和P331S。在一些实施例中，Fc包含(或基本上由...组成、或由...组成)氨基酸序列SEQ ID NO:38或其变体；所述变体与SEQ ID NO:38具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，Fc包含相对于人野生型IgG1 Fc氨基酸序列第234位氨基酸残基L(L234)、第235位氨基酸残基L(L235)、第237位氨基酸残基G(G237)、第330位氨基酸残基A(A330)和第331位氨基酸残基P(P331)中的一个或多个突变。在一些实施例中，Fc包含相对于人野生型IgG1 Fc氨基酸序列的L234、L235、G237、A330和P331的突变。在一些实施例中，相对于人野生型IgG1 Fc氨基酸序列，Fc包含突变L234A、L235E、G237A、A330S和P331S。在一些实施例中，Fc包含(或基本上由...组成、或由......组成)氨基酸序列SEQ ID NO:39或其变体；所述变体与SEQ ID NO:39具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，Fc来自IgG4 Fc。在一些实施例中，Fc来自人IgG4 Fc。在一些实施例中，Fc来自人野生型IgG4 Fc。在一些实施例中，Fc不包含IgG4的铰链区。在一些实施例中，Fc包含一个或多个无效应突变和/或去糖基化突变。
在一些实施例中，Fc包含相对于人野生型IgG4 Fc氨基酸序列第228位氨基酸残基S(S228)、第234位氨基酸残基F(F234)和第235位氨基酸残基L(L235)中的一个或多个突变。在一些实施例中，Fc包含相对于人野生型IgG4 Fc氨基酸序列的S228、F234和L235突变。在一些实施例中，相对于人野生型IgG4 Fc氨基酸序列，Fc 包含突变S228P、F234A和L235A。在一些实施例中，Fc部分包含(或基本上由...组成、或由...组成)氨基酸序列SEQ ID NO:40或其变体；所述变体与SEQ ID NO:40具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
KIH(Knob-into-Hole)技术：
可以对Fc产生的另外的突变包含促进Fc多肽之间异源二聚体形成的那些突变。在一些实施例中，通过KIH(Knob-into-Hole)技术来解决异源二聚体的组装问题，KIH技术即在CH3结构域引入不对称的突变结构(“knob”突变指在CH3结构域中，用一个大的氨基酸残基替换一个较小的残基，而“hole”突变指使用小的氨基酸残基来替换较大的残基)。经过改造的Fc由于空间位阻的影响更倾向于发生异二聚化而不是同二聚化(Ridgway J B，et al.“Knobs-into-holes”engineering of antibody CH3 domains for heavy chain heterodimerization[J].Protein Eng.1996，9(7):617-621)。即将一个CH3结构域366位苏氨酸残基(T)替换为色氨酸残基(W)形成“杵”(knobs)结构，并将配对的另一个CH3结构域366位苏氨酸残基(T)替换为丝氨酸残基(S)，368位亮氨酸残基(L)替换为丙氨酸残基(A)，407位酪氨酸残基(Y)替换为缬氨酸残基(V)形成“臼”(holes)结构同时引入形成稳定化二硫桥的两个半胱氨酸残基(C)突变(S354C在“knob”侧，并且Y349C在“hole”侧)，增强KIH的稳定性，其中所述编号依照如Kabat的EU索引(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)。
示例性的Fc突变体序列如表5所示。
在一些实施例中，本申请的IL-2突变体与Fc的融合蛋白是二价形式的，在该融合蛋白中，包含2个IL-2突变体分子。例如，由2个上述的融合多肽链单体构成的同源二聚体形式。在一些实施例中，其示例性结构如图1A和图1B所示。
在一些实施例中，本申请的IL-2突变体与Fc的融合蛋白是单价形式的，在该融合蛋白中，仅包含1个IL-2突变体分子。例如，由1个上述的融合多肽链单体和1个Fc亚基构成的异源二聚体形式。在一些实施例中，其示例性结构如图2所示。在另一些 实施例中，本申请的IL-2突变体与Fc的融合蛋白是单价形式的，由1个上述的融合多肽链单体构成。
接头
在一些实施例中，IL-2突变体和Fc之间可以通过接头(例如，连接肽、非连接肽)连接。在一些实施例中，该接头为一种柔性接头。在一些实施例中，该接头为一种稳定接头。一般来说，理想的接头不会影响或不会显著影响本文所述的IL-2突变体与Fc的融合蛋白的正确折叠和构象。优选地，该接头赋予了IL-2突变体与Fc的融合蛋白的灵活性，保留或提高了IL-2突变体的生物功能，和/或不影响或不显著影响IL-2突变体与Fc的融合蛋白在体内的半衰期和/或稳定性。在一些实施例中，接头为稳定接头(例如，不能被蛋白酶，特别是MMP切割)。
在一些实施例中，该接头为连接肽。连接肽可以是任意长度。在一些实施例中，连接肽长度为1到10个氨基酸，3到18个氨基酸，1到20个氨基酸，10到20个氨基酸，21到30个氨基酸，1到30个氨基酸，10到30个氨基酸，1到50个氨基酸，5到40个氨基酸，12到18个氨基酸，4到25个氨基酸。在一些实施例中，该连接肽长度为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个氨基酸。在一些实施例中，该连接肽长度为21、22、23、24、25、26、27、28、29或30个氨基酸。在一些实施例中，该连接肽长度为31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50个氨基酸。在一些实施例中，连接肽长度不超过防止不期望的结构域相互作用和/或优化生物功能和/或稳定性所必需的长度。在一些实施例中，连接肽的长度最多为30个氨基酸，例如最多20个氨基酸，或最多15个氨基酸。在一些实施例中，连接肽长度为5到30个氨基酸，或5到18个氨基酸。
连接肽可以具有天然存在的序列，也可以具有非天然存在的序列。例如，可以使用来自抗体重链铰链区的序列作为接头。例如，参见WO1996/34103。在一些实施例中，连接肽为人IgG1、IgG2、IgG3或IgG4铰链区。在一些实施例中，连接肽为突变的人IgG1、IgG2、IgG3或IgG4铰链区。在一些实施例中，接头是柔性接头。典型的柔性接头包括但不限于甘氨酸聚合物(G)n(SEQ ID NO:43)、甘氨酸-丝氨酸聚合物(包括例如，(GS)n(SEQ ID NO:44)、(GGS)n(SEQ ID NO:45)、(GGGS)n(SEQ ID NO:46)、(GGGGS)n(SEQ ID NO:47)或(GGS)m(GGGS)n(SEQ ID NO:48)，其中m和n为至少是1的整数)、甘氨酸-丙氨酸聚合物、丙氨酸-丝氨酸聚合物和其它本领域 已知的柔性接头。甘氨酸和甘氨酸-丝氨酸聚合物为相对非结构化的，并且因此可以作为组分之间的一种中性链。甘氨酸比丙氨酸拥有更多的phi-psi空间，且相比具有更长侧链的残基，受到的限制更少(见于Scheraga,Rev.Computational Chem.11 173-142(1992))。柔性接头的示例包括但不限于表6中所示的氨基酸序列。一般来说，本领域技术人员会意识到所设计的IL-2突变体与Fc的融合蛋白可以包括全部或部分柔性接头，使得接头可以包括一个柔性接头部分以及一个或多个提供较少柔性结构的部分，以提供一个理想的IL-2突变体与Fc的融合蛋白的结构和功能。在一些实施例中，连接肽包含SEQ ID NOs:43-74中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:43-74中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
示例性的连接肽序列如表6所示。
其它关于接头的考虑因素包括对产生的IL-2突变体与Fc的融合蛋白的物理或药代动力学性质的影响，例如溶解性、亲脂性、亲水性、疏水性、稳定性(或多或少的稳定性以及计划内的降解)、刚性、柔韧性、免疫原性、IL-2突变体与IL-2受体的结合、胶体或脂质体的结合能力等。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白包括二价形式和单价形式。在另一些实施例中，所述二价形式，其包含两个IL-2突变体分子，可以由两个相同的或不同的单体组成，例如，所述单体是IL-2突变体与Fc的融合多肽链。在另一些实施例中，所述单价形式，其包含一个IL-2突变体分子，可以由两个不同的单体组成，例如，其中一个单体为IL-2突变体与一个Fc亚基的融合多肽链，另一个单体为Fc亚基。在另一些实施例中，所述单价形式，其包含一个IL-2突变体分子，仅由一个单体组成，例如，该单体为IL-2突变体与一个Fc亚基的融合多肽链。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的缺失或突变。在一些实施例中，所述人野生型IL-2的氨基酸序列如SEQ ID NO:1所示。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的全部缺失。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和N71的突变。在一些实施例中， 所述E67和N71的突变分别为E67K和N71S。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的全部缺失，以及相对于人野生型IL-2氨基酸序列的E67K和N71S突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67的突变。在一些实施例中，所述E67的突变为E67A。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，以及相对于人野生型IL-2氨基酸序列的E67A突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，且进一步包含相对于人野生型IL-2氨基酸序列的N71和S75的突变。在一些实施例中，所述N71和S75的突变分别为N71W和S75I。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，以及相对于人野生型IL-2氨基酸序列的N71W和S75I突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和/或V91的突变。在一些实施例中，所述E67的突变为E67T。在一些实施例中，所述V91的突变为V91K。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的E67T和/或V91K突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，且进一步包含相对于人野生型IL-2氨基酸序列的L70和S75的突变。在一些实施例中，所述L70和S75的突变分别为L70I和S75F。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列 第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的L70I和S75F突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，且进一步包含相对于人野生型IL-2氨基酸序列的N71的突变。在一些实施例中，所述N71的突变为N71I。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的N71I突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为SPL。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和N71的突变。在一些实施例中，所述E67和N71的突变分别为E67M和N71S。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为SPL，以及相对于人野生型IL-2氨基酸序列的E67M和N71S突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第75位氨基酸残基S(S75)和第83位氨基酸残基R(R83)的突变。在一些实施例中，所述人野生型IL-2的氨基酸序列如SEQ ID NO:1所示。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75被突变为不带电荷的氨基酸残基。在一些实施例中，其中S75的突变为S75P、S75F、S75Y、S75G、S75A、S75V、S75T、S75I或S75L。在一些实施例中，其中R83的突变为R83N、R83F、R83Y、R83P、R83L、R83A、R83K或R83E。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75P，R83的突变为R83Y。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和D84的突变。在一些实施例中，其中E67和D84的突变分别为E67A和D84N。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P、R83Y、E67A和D84N突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75P，R83的突变为R83A。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16、L70和N71的突变。在一些实施例中，其中H16、L70和N71的突变分别为H16D、L70I和N71G。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P、R83A、H16D、L70I和N71G突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75F，R83的突变为R83P。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和/或N71的突变。在一些实施例中，其中H16的突变为H16Q或H16N。在一些实施例中，其中N71的突变为N71S。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83P和H16Q突变。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83P、H16N和N71S突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75F，R83的突变为R83L。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变。在一些实施例中，其中E67和L70的突变分别为E67R和L70V。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83L、E67R和L70V突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75I，R83的突变为R83L。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变。在一些实施例中，其中E67和L70的突变分别为E67G和L70V。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75I、R83L、E67G和L70V突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75I，R83的突变为R83K。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变。在一些实施例中，其中E67和L70的突变分别为E67S 和L70V。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75I、R83K、E67S和L70V突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75T，R83的突变为R83A或R83F。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和E67的突变。在一些实施例中，其中H16和E67的突变分别为H16N和E67K。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75T、R83A、H16N和E67K突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75V，R83的突变为R83F。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E15和H16的突变。在一些实施例中，其中E15和H16的突变分别为E15K和H16Q。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75V、R83F、E15K和H16Q突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75V，R83的突变为R83N。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L70和V91的突变。在一些实施例中，其中L70和V91的突变分别为L70F和V91K。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75V、R83N、L70F和V91K突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75G，R83的突变为R83L。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17、V91和I92的突变。在一些实施例中，L17、V91和I92的突变分别为L17I、V91E和I92L。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75G、R83L、L17I、V91E和I92L突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75Y，R83的突变为R83N。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17和V91的突变。在一些实施例中，L17和V91的突变分别为L17I和 V91K。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75Y、R83N、L17I和V91K突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75A，R83的突变为R83K。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17的突变。在一些实施例中，L17的突变为L17I。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75A、R83K和L17I突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，其中S75的突变为S75L，R83的突变为R83E。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L63和E67的突变。在一些实施例中，L63和E67的突变分别为L63V和E67F。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75L、R83E、L63V和E67F突变。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体，除上述突变外，还进一步包含相对于人野生型IL-2氨基酸序列的C125的突变。在一些实施例中，所述C125的突变为C125S或C125A。
在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白中(例如，二价或单价融合蛋白形式)，所述的IL-2突变体包含SEQ ID NOs:5-34中任一所示的氨基酸序列或者其变体，所述变体与SEQ ID NOs:5-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
示例性的第一组IL-2突变体与Fc的二价融合蛋白的氨基酸序列如表7所示，示例性的第二组IL-2突变体与Fc的二价融合蛋白的氨基酸序列如表8所示。
在一些实施例中，本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，每个单体从N端到C端，或者从C端到N端均包含：(i)IL-2突变体和(ii)Fc。
在一些实施例中，本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，每个单体从N端到C端，或者从C端到N端均包含：(i)IL-2突变体、(ii)连接肽和(iii)Fc。
在一些实施例中，本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，所述Fc为IgG1 Fc，并且所述Fc包含L234A和L235A(LALA)突变。在一些实施例中，所述Fc包含氨基酸序列SEQ ID NO:35。
在一些实施中，本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，所述Fc为IgG1 Fc，并且所述Fc包含N297G突变。在一些实施例中，所述Fc包含氨基酸序列SEQ ID NO:36。
在一些实施中，本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，所述Fc为IgG1 Fc，并且所述Fc包含N297A突变。在一些实施例中，所述Fc包含氨基酸序列SEQ ID NO:37。
在一些实施中，本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，所述Fc为IgG1 Fc，并且所述Fc包含L234A、L235A和P331S突变。在一些实施例中，所述Fc包含氨基酸序列SEQ ID NO:38。
在一些实施中，本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，所述Fc为IgG1 Fc，并且所述Fc包含L234A、L235E、G237A、A330S和P331S突变。在一些实施例中，所述Fc包含氨基酸序列SEQ ID NO:39。
在一些实施中，本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，所述Fc为IgG4 Fc，并且所述Fc包含S228P、F234A和L235A突变。在一些实施例中，所述Fc包含氨基酸序列SEQ ID NO:40。
在一些实施例中，在本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，每个单体从N端到C端，或者从C端到N端均包含：(i)IL-2突变体，其包含SEQ ID NOs:5-34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:5-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性和(ii)Fc，其包含如SEQ ID NOs:35-40中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:35-40中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，在本申请所述的IL-2突变体与Fc的二价融合蛋白形式中，每个单体从N端到C端，或者从C端到N端均包含：(i)IL-2突变体，其包含SEQ ID NOs:5-34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:5-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、 97％、98％或99％)序列同源性，(ii)连接肽，其包含SEQ ID NOs:43-74中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:43-74中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性和(iii)Fc，其包含如SEQ ID NOs:35-40中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:35-40中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，本申请所述的IL-2突变体与Fc的二价融合蛋白形式包含SEQ ID NOs:76-105中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:76-105中任一所示的氨基酸序列具有至少约80％(例如至少80％、85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，在本申请所述的IL-2突变体与Fc的其中一种单价融合蛋白形式中，其中Fc为IgG1 Fc。在一些实施例中，所述Fc包含KIH突变(分别称为Fc knob和Fc hole)。在一些优选的实施例中，所述Fc包含LALA突变和KIH突变(分别称为Fc LALA knob和Fc LALA hole)。在一些更优选的实施例中，Fc LALA knob可包含突变L234A、L235A、T366W和S354C，Fc LALA hole可包含突变L234A、L235A、T366S、L368A、Y407V和Y349C。
在一些实施例中，IgG1 Fc LALA knob包含氨基酸序列SEQ ID NO:41，Fc LALA hole包含氨基酸序列SEQ ID NO:42。
在一些实施例中，在本申请所述的IL-2突变体与Fc的其中一种单价融合蛋白形式中，其中一个单体从N端到C端或从C端到N端包含：(i)IL-2突变体和(ii)Fc knob，以及一个单体包含：Fc hole。
在一些实施例中，在本申请所述的IL-2突变体与Fc的其中一种单价融合蛋白形式中，其中一个单体从N端到C端包含，或者从C端到N端包含：(i)IL-2突变体，其包含SEQ ID NOs:5-34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:5-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性和(ii)Fc LALA knob，其包含氨基酸序列SEQ ID NO:41或其变体，所述变体与SEQ ID NO:41具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性；以及另一个单体为Fc LALA hole，其包含氨基酸序列SEQ ID NO:42或其变体，所述变体与SEQ ID  NO:42具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，在本申请所述的IL-2突变体与Fc的其中一种单价融合蛋白形式中，其中一个单体从N端到C端或从C端到N端包含：(i)IL-2突变体和(ii)Fc hole；以及另一个单体包含：Fc knob。
在一些实施例中，在本申请所述的IL-2突变体与Fc的其中一种单价融合蛋白形式中，其中一个单体从N端到C端包含或者从C端到N端包含：(i)IL-2突变体，其包含SEQ ID NOs:5-34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:5-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性和(ii)Fc LALA hole，其包含氨基酸序列SEQ ID NO:42或其变体，所述变体与SEQ ID NO:42具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性；以及另一个单体为Fc LALA knob，其包含氨基酸序列SEQ ID NO:41或其变体，所述变体与SEQ ID NO:41具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，在本申请所述的IL-2突变体与Fc的其中一种单价融合蛋白形式中，其中一个单体从N端到C端或从C端到N端包含：(i)IL-2突变体、(ii)连接肽和(iii)Fc knob；以及另一个单体包含：Fc hole。
在一些实施例中，在本申请所述的IL-2突变体与Fc的其中一种单价融合蛋白形式中，其中一个单体从N端到C端包含或者从C端到N端包含：(i)IL-2突变体，其包含SEQ ID NOs:5-34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:5-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性(ii)连接肽，其包含SEQ ID NOs:43-74中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:43-74中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性和(iii)Fc LALA knob，其包含氨基酸序列SEQ ID NO:41或其变体，所述变体与SEQ ID NO:41具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性；以及另一个单体为Fc LALA hole，其包含氨基酸序列SEQ ID NO:42或其变体，所述变体与SEQ ID NO:42 具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，在本申请所述的IL-2突变体与Fc的其中一种单价融合蛋白形式中，其中一个单体从N端到C端或从C端到N端包含：(i)IL-2突变体、(ii)连接肽和(iii)Fc hole，以及另一个单体包含：Fc knob。
在一些实施例中，在本申请所述的IL-2突变体与Fc的其中一种单价融合蛋白形式中，其中一个单体从N端到C端包含或者从C端到N端包含：(i)IL-2突变体，其包含SEQ ID NOs:5-34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:5-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性(ii)连接肽，其包含SEQ ID NOs:43-74中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:43-74中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性和(iii)Fc LALA hole，其包含氨基酸序列SEQ ID NO:42或其变体，所述变体与SEQ ID NO:42具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性；以及另一个单体为Fc LALA knob，其包含氨基酸序列SEQ ID NO:41或其变体，所述变体与SEQ ID NO:41具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。
在一些实施例中，当通过静脉注射、肌肉注射或皮下注射给个体(例如，人类)时，本申请所述的IL-2突变体与Fc的融合蛋白的半衰期至少为10小时(例如，至少为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或100小时或更长)。
结合亲和力
分子(例如，IL-2突变体或包含IL-2突变体的融合蛋白)与其结合对象(例如，IL-2受体，如IL-2Rα、IL-2Rβ、IL-2Rγ、IL-2Rβγ、或IL-2Rαβγ)的结合亲和力可以通过任何本领域已知的合适的配体结合试验来确定，例如，Western blot、酶联免疫吸附试验(ELISA)、Meso Scale Discovery(MSD)电化学发光、基于珠子的多重免疫分析(MIA)、RIA、表面等离子共振(SPR)、ECL、IRMA、EIA、Biacore试验、Octet分析、肽扫描等。例如，通过使用各种标记试剂标记的IL-2突变体或包含IL-2 突变体的融合蛋白或其受体(例如，IL-2Rα、IL-2Rβ、IL-2Rγ、IL-2Rβγ、或IL-2Rαβγ)，或其亚单位来进行简单分析，同样可以使用BiacoreX(Amersham Biosciences)，这是一种非处方的测量试剂盒或类似试剂盒，可根据试剂盒随附的用户手册和实验操作方法来操作。
在一些实施例中，蛋白质微阵列用于大规模分析本申请所述的IL-2突变体或包含IL-2突变体的融合蛋白与其受体的相互作用、功能和活性。蛋白质微阵列具有与一系列捕获蛋白(例如，IL-2受体或其亚单位)结合的支撑表面。随后将荧光标记的探针分子(例如，本申请所述的IL-2突变体或包含IL-2突变体的融合蛋白)添加到阵列中，并且与结合的捕获蛋白相互作用，释放荧光信号并通过激光扫描仪读取。
结合亲和力也可以使用Biacore进行测量。例如，使用EDC/NHS化学法将IL-2Rβγ或IL-2Rαβγ偶联到CM-5传感器芯片表面作为配体。然后将本文所述的IL-2突变体或IL-2突变体与Fc的融合蛋白的一系列稀释液作为分析物与偶联到芯片上的配体结合，并且可以实时监测IL-2与IL-2Rβγ或IL-2Rαβγ的结合和解离。亲和力(Kd)可通过使用BIA评估软件进行动力学分析来确定。
在一些实施例中，可以应用Biacore实验来测定本申请提供的IL-2突变体或IL-2突变体与Fc的融合蛋白与IL-2Rβγ的Kd值来确定结合亲和力，在一些实施例中，相对于野生型IL-2与Fc的融合蛋白，本申请的IL-2突变体与Fc的融合蛋白与IL-2Rβγ具有更低的结合亲和力。在一些实施例中，相对于野生型IL-2与Fc的融合蛋白，所述IL-2突变体与Fc的融合蛋白与IL-2Rβ和/或IL-2Rβγ的亲和力减少了至少5％(例如至少10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或100％中的任何一个)。在一些实施例中，本申请所述的IL-2突变体与Fc的融合蛋白，相对于野生型IL-2与Fc的融合蛋白(例如人野生型IL-2)，其与IL-2Rα的亲和力增加或者保持不变或者没有明显减弱。在一些实施例中，相对于野生型IL-2与Fc的融合蛋白，本申请的IL-2突变体与Fc的融合蛋白与IL-2Rαβγ的结合力增加、保持不变或者没有明显减弱。
在一些实施例中，本文所述IL-2突变体或IL-2突变体与Fc的融合蛋白与中等亲和力受体(IL-2Rβγ)之间结合的Kd值大于野生型IL-2或野生型IL-2与Fc的融合蛋白与其相同受体之间结合的Kd值。在一些实施例中，本申请所述的IL-2突变体或IL-2突变体与Fc的融合蛋白与其高亲和力受体(IL-2Rαβγ)之间结合的Kd值接近于(例如，等于、小于或略大于)野生型与相同受体(IL-2Rαβγ)之间结合Kd值。
药代动力学(PK)
药代动力学(PK)是指药物(例如，本文所述的IL-2突变体或包含IL-2突变体的融合蛋白)在向受试者施用后的吸收、分布、代谢和排泄。可用于确定临床效用的药代动力学参数包括但不限于血清/血浆浓度、随时间变化的血清/血浆浓度、最大血清/血浆浓度(Cmax)、达到最大浓度的时间(Tmax)、半衰期(T1/2)、给药间隔内浓度-时间曲线下的面积(AUCτ)等。
用于获得药物(例如，本文所述的IL-2突变体或包含IL-2突变体的融合蛋白)的PK曲线的技术是本领域已知的。参见Heller et al.,Annu Rev Anal Chem,11,2018；和Ghandforoush Sattari et al.,J Amino Acids,Article ID 346237,Volume 2010。在一些实施例中，在个体的血液、血浆或血清样本中测量如本文所述的IL-2突变体或包含IL-2突变体的融合蛋白的PK曲线。在一些实施例中，使用质谱技术(例如，LC-MS/MS或ELISA)测量个体中如本文所述的IL-2突变体或包含IL-2突变体的融合蛋白的PK曲线。可通过本领域已知的任何方法在PK曲线上进行PK分析，例如，非室间分析，使用PKSolver V2软件(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)。示例方法参见实施例8。
“C”表示受试者血浆、血清或任何合适的体液或组织中的药物(例如，IL-2突变体或包含IL-2突变体的融合蛋白)浓度，通常表示为每单位体积的质量，例如纳克/毫升。为方便起见，血清或血浆中的药物浓度在本文中称为“血清浓度”或“血浆浓度”。给药后任何时间(例如，IL-2突变体或包含IL-2突变体的融合蛋白多肽通过静脉注射、腹腔注射或皮下注射)的血清/血浆浓度称为Ctime或Ct。给药期间的最大血清/血浆药物浓度被称为Cmax；Cmin是指给药间隔结束时的最小血清/血浆药物浓度；Cave指给药间隔期间的平均浓度。
术语“生物利用度”是指药物(例如，IL-2突变体或包含IL-2突变体的融合蛋白)通过体循环，从而进入作用部位的程度或速率。
“AUC”是血清/血浆浓度-时间曲线下的面积，被认为是对生物利用度最可靠的测量方式，如给药间隔内浓度-时间曲线下的面积(AUCτ)，“总暴露”或“一段时间内的总药物暴露”(AUC0-∞)，给药后t时间的浓度-时间曲线下的面积(AUC0-t)等。
血清/血浆浓度峰值时间(Tmax)是给药(例如，IL-2突变体或包含IL-2突变体的融合蛋白)后达到血清/血浆浓度(Cmax)峰值的时间。
半衰期(T1/2)是指在血浆或血清(或其它生物基质)中测得的药物浓度(例如，IL-2突变体或包含IL-2突变体的融合蛋白)降至其在特定时间点的浓度或量的一半所需的时间。例如，静脉给药后，由于药物的分布和消除，血浆或血清中的药物浓度下降。在静脉给药后血浆或血清药物浓度随时间变化的曲线中，第一阶段或快速下降阶段被认为主要是由于分布导致，而后期的下降通常较慢，主要是由于消除导致，尽管这两个过程在这两个阶段都会发生。分布被认为是在足够的时间后完成的。一般来讲，消除半衰期由血浆/血清浓度-时间曲线的终末期或消除(主要)阶段决定。参见Michael Schrag和Kelly Regal，“临床前药物开发毒理学综合指南”的“第3章-药代动力学和毒代动力学”，2013。
在一些实施例中，本文所述的包含IL-2突变体的融合蛋白(例如IL-2突变体与Fc的二价融合蛋白形式)具有至少10小时的半衰期(例如，静脉注射、皮下注射或肌肉注射，如给人类注射)，如至少为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或100小时，或更长。
IL-2突变体或包含IL-2突变体的融合蛋白的衍生物
在一些实施例中，本文所涉及的IL-2突变体或包含IL-2突变体的融合蛋白可被进一步修饰，以包含本领域已知且容易获得的额外的非蛋白部分。示例性的非蛋白部分包括但不限于水溶性聚合物。水溶性聚合物的非限制性示例包括但不限于聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二氧环戊烷、聚-1,3,6-三氧戊烷、乙烯/马来酸酐共聚物，聚酰胺酸(均聚物或无规共聚物)、葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、环氧丙烷/环氧乙烷共聚物、聚氧乙烯基多元醇(例如甘油)、聚乙烯醇及其混合物。由于聚乙二醇丙醛在水中的稳定性，其在制造中可能具有优势。聚合物可以是任何分子量的，并且可以是支链或非支链的。连接到IL-2突变体或包含IL-2突变体的融合蛋白上的聚合物的数量可能不同，如果连接了多个聚合物，则它们可以是相同或不同的分子。一般来说，用于衍生化的聚合物的数量和/或类型可基于以下考虑来确定，包括但不限于待改进的IL-2突变体或包含IL-2突变体的融合蛋白的特定性质或功能，IL-2突变体或包含IL-2突变体的融合蛋白的衍生物是否会在特定条件下用于治疗等。
在一些实施例中，本文所述的IL-2突变体或包含IL-2突变体的融合蛋白进一步包含标签，所述标签选自发色团、荧光团(例如，香豆素、氧杂蒽、菁、芘、硼聚苯并氮杂吲哚、恶嗪及其衍生物)、荧光蛋白(例如GFP、藻胆蛋白及其衍生物)，磷光染料(例如，二氧杂环丁烷、氧杂蒽或碳菁染料、镧系螯合物)、串联染料(例如，菁-藻胆蛋白衍生物和氧杂蒽-藻胆蛋白衍生物)、粒子(例如，金簇、胶体金、微球、量子点)、半抗原、酶(例如，过氧化物酶、磷酸酶、糖苷酶、荧光素酶)和放射性同位素(例如，125I、3H、14C、32P)。
在一些实施例中，IL-2突变体或包含IL-2突变体的融合蛋白可被进一步修饰以包含一种或多种其他的生物活性蛋白质、多肽或其片段。如本申请所述，“生物活性”或“生物学上的活性”可互换使用，是指在体内显示生物活性以执行特定功能。例如，它可能意味着与特定生物分子结合，如蛋白质、DNA等，然后促进或抑制该生物分子的活性。在一些实施例中，生物活性蛋白质或其片段包括为预防或治疗疾病或症状作为活性药物施用给患者的蛋白质和多肽，以及用于诊断目的的蛋白质和多肽，例如用于诊断试验或体外检测的酶，以及给患者施用以预防疾病的蛋白质和多肽，例如疫苗。在一些实施例中，生物活性蛋白质或其片段具有免疫刺激/免疫调节、膜转运或酶活性。在一些实施例中，生物活性蛋白、多肽或其片段为酶、激素、生长因子、细胞因子或其混合物。在一些实施例中，生物活性蛋白质、多肽或片段可特异性识别目标肽(例如抗原或其它蛋白质)。
在一些实施例中，可包含在本文所述的IL-2突变体或包含IL-2突变体的融合蛋白中的生物活性蛋白质或其片段是抗原结合蛋白质(例如，抗体)。在一些实施例中，可包含在如本文所述的IL-2突变体或包含IL-2突变体的融合蛋白中的生物活性蛋白质或其片段是抗体模拟物，其是一种使人联想到抗体的包含抗原结合域的小型工程蛋白质，(GGeering和Fussenegger,Trends Biotechnol.,33(2):65-79,2015)。这些分子来自现有的人类支架蛋白，由单一多肽组成。可包含在如本文所述的IL-2突变体或包含IL-2突变体的融合蛋白中的抗体模拟物示例可以是但不限于设计的锚蛋白重复蛋白(DARPin；包含3-5个完全合成的锚蛋白重复序列，两侧为N端和C端帽结构域)，一种亲和力多聚体(avimer；一种高亲和力蛋白质，包含多个A结构域，每个结构域对靶点的亲和力较低)，或一种抗凝素(基于脂质支架，具有四个可接近的环，每个环的序列可以随机)。在一些实施例中，可包含在如本文所述的IL-2突变体或包含IL-2突变体的融合蛋白中的生物活性蛋白质或其片段是犰狳重复蛋白(例如，β-连环蛋白、 α-导入蛋白、斑珠蛋白、大肠腺瘤性息肉病(APC))，包含犰狳重复单元(特性，重复氨基酸序列的长度约为40个残基)。每个犰狳重复单元由一对形成发夹结构的α螺旋组成。多个重复拷贝形成了所述的α螺线管结构。犰狳重复蛋白能够结合不同类型的肽，依赖于肽主链的恒定结合方式，而不需要特定的保守侧链或与肽的游离N-或C-末端相互作用。通过残基识别肽残基的可能性，再加上重复蛋白的内在模块性，使得犰狳重复蛋白有望成为肽结合通用支架的候选。
IV.编码IL-2突变体或包含IL-2突变体的融合蛋白的核酸
本申请还涉及编码本文所述任何IL-2突变体或包含IL-2突变体的融合蛋白的分离的核酸，包含编码本文所述的任何IL-2突变体或包含IL-2突变体的融合蛋白的核酸的载体。还涉及包含上述核酸或上述载体的分离的宿主细胞(例如，CHO细胞、HEK293细胞、Hela细胞或COS细胞)。在一些实施例中，分离的核酸进一步包括编码IL-2突变体或包含IL-2突变体的融合蛋白的N端信号肽的核酸序列。
在一些实施例中，包含编码本文所述任何IL-2突变体或包含IL-2突变体的融合蛋白的核酸的载体适于在真核细胞中复制和整合，如哺乳动物细胞(例如，CHO细胞、HEK 293细胞、Hela细胞、COS细胞)。在一些实施例中，载体是一种病毒载体。在一些实施例中，载体是非病毒载体，如pTT5。
已经开发出许多基于病毒的系统用于将基因转移到哺乳动物细胞中。病毒载体的示例包括但不限于腺病毒载体、腺相关病毒载体、慢病毒载体、逆转录病毒载体、单纯疱疹病毒载体及其衍生物。病毒载体技术是本领域公知的，例如，在Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)中，及其它病毒学和分子生物学手册中均对其进行了详述。逆转录病毒为基因传递系统提供了一个方便的平台。可使用本领域已知的技术将异源核酸插入载体并包装在逆转录病毒微粒中。然后，可分离重组病毒并在体外或离体条件下将其输送至工程哺乳动物细胞。许多逆转录病毒系统是本领域已知的。在一些实施例中，使用腺病毒载体。许多腺病毒载体是本领域已知的。在一些实施例中，使用慢病毒载体。在一些实施例中，使用自失活慢病毒载体。例如，携带构建体蛋白编码序列的自失活慢病毒载体可以用本领域已知的实验方法进行包装。所得的慢病毒载体可用于使用本领域已知的方法转导至哺乳动物细胞。来自逆转录病毒(如慢病毒)的载体是实现长期基 因转导的合适工具，因为它们允许转基因长期、稳定地整合并在子代细胞中的繁殖。慢病毒载体也具有低免疫原性，并且可以转导非增殖细胞。
在一些实施例中，载体是非病毒载体。在一些实施例中，载体是pTT5载体。在一些实施例中，载体是转座子，例如睡美人(SB)转座子系统或PiggyBac转座子系统。在一些实施例中，载体是基于聚合物的非病毒载体，包括，例如，聚(乳酸-羟基乙酸共聚物)(PLGA)和聚乳酸(PLA)、聚(乙烯亚胺)(PEI)和树枝状大分子。在一些实施例中，载体是基于阳离子脂质的非病毒载体，如阳离子脂质体、脂质纳米乳和固体脂质纳米粒(SLN)。在一些实施例中，载体是基于肽的非病毒基因载体，例如聚-L-赖氨酸。适用于基因组编辑的任何已知非病毒载体都可用于将编码IL-2突变体或包含IL-2突变体的融合蛋白的核酸引入宿主细胞。参见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和Zhao S.et al.,“PiggyBac transposon vectors:the tools of the human gene editing.”Transl.Lung Cancer Res.(2016)5(1):120-125，通过引用并入本文。在一些实施例中，通过物理方法将编码本文所述IL-2突变体或包含IL-2突变体的融合蛋白的任何一个或多个核酸或载体引入宿主细胞(例如，CHO、HEK 293、Hela或COS)，包括但不限于电穿孔、声穿孔、光穿孔、磁转染、水穿孔。
在一些实施例中，载体包含可选择的标记基因或报告基因，用于从载体(例如，慢病毒载体、pTT5载体)转染的宿主细胞群中选择出表达本文所述的IL-2突变体或包含IL-2突变体的融合蛋白的细胞。可选择的标记和报告基因都可能被适当的调控序列包围，以使其能够在宿主细胞中表达。例如，载体可包含转录和翻译终止子、起始序列和用于调节核酸序列表达的启动子。
可使用本领域已知的任何分子克隆方法，包括，例如，使用限制性内切酶位点和一个或多个可选择的标记将核酸克隆到载体中。在一些实施例中，核酸可操作地连接到启动子上。已经探索出多种用于原核细胞或真核细胞(例如，哺乳动物细胞)中进行基因表达的启动子，并且本领域已知的任何启动子都可用于本申请。启动子大致可分为组成型启动子或调控型启动子，如诱导型启动子。
在一些实施例中，编码本文所述的IL-2突变体或包含IL-2突变体的融合蛋白的核酸可操作地连接到组成型启动子上。组成型启动子允许异源基因(也称为转基因)在宿主细胞中组成型表达。本文所考虑的启动子示例包括但不限于CMV启动子 (CMV)、人类延伸因子-1α(hEF1α)、泛素C启动子(UbiC)、磷酸甘油激酶启动子(PGK)、猿猴病毒40早期启动子(SV40)、鸡β-肌动蛋白启动子与CMV早期增强子(CAGG)偶联，罗氏肉瘤病毒(RSV)启动子、多瘤病毒增强子/单纯疱疹胸苷激酶(MC1)启动子、β肌动蛋白(β-ACT)启动子、“骨髓增生性肉瘤病毒增强子、阴性对照区缺失、d1587rev引物结合位点取代(MND)”启动子。大量研究中已广泛比较了这些组成型启动子在驱动转基因表达方面的效率。在一些实施例中，编码本文所述的IL-2突变体或包含IL-2突变体的融合的核酸可操作地连接到CMV启动子上。
在一些实施例中，编码本文所述的IL-2突变体或包含IL-2突变体的融合蛋白的核酸可操作地连接到诱导型启动子。诱导型启动子属于调控型启动子的范畴。诱导型启动子可被一个或多个条件诱导，如物理条件、宿主细胞的微环境或宿主细胞的生理状态、诱导剂(即诱导药剂)或其组合物。在一些实施例中，诱导条件不诱导宿主细胞中内源性基因的表达。在一些实施例中，诱导条件选自：诱导剂、辐射(如电离辐射、光)、温度(如热)、氧化还原状态和宿主细胞的激活状态。在一些实施例中，诱导型启动子可以是NFAT启动子、启动子或NFκB启动子。
V.制备方法
本申请还涉及制备本文所述任何IL-2突变体或包含IL-2突变体的融合蛋白的方法。因此，在一些实施例中，涉及一种制备IL-2突变体或包含IL-2突变体的融合蛋白的方法，包括：(a)在可有效表达所编码的IL-2突变体或包含IL-2突变体的融合蛋白的条件下，培养包含编码本文所述任何IL-2突变体或包含IL-2突变体的融合蛋白的核酸或载体的宿主细胞(例如，CHO细胞、HEK 293细胞、Hela细胞或COS细胞)；和(b)从所述宿主细胞获得表达的IL-2突变体或包含IL-2突变体的融合蛋白。在一些实施例中，步骤(a)的方法进一步包含产生宿主细胞，所述宿主细胞包含编码本文所述的IL-2突变体或包含IL-2突变体的融合蛋白的核酸或载体。本文所述的IL-2突变体或包含IL-2突变体的融合蛋白可使用本领域已知或本文所述的任何方法制备。
在一些实施例中，本文所述的IL-2突变体或包含IL-2突变体的融合蛋白可以用真核细胞表达，如哺乳动物细胞。在一些实施例中，本文所述的IL-2突变体或包含IL-2突变体的融合蛋白可以用原核细胞表达。
1.原核细胞的重组产物
a)载体构建
可以使用标准重组技术获得编码本申请所述的蛋白质构建体的多核酸序列。可以使用核苷酸合成仪或PCR技术合成多核苷酸。一旦获得编码多肽的序列，将其插入能够在原核宿主中复制和表达异源多核苷酸的重组载体中。本领域中已知且可使用的许多载体都可用于本申请。选择合适的载体主要取决于要插入载体的核酸大小以及载体所转化的特定宿主细胞。每个载体包含各种成分，这取决于该载体的功能(异源多核苷酸的扩增或表达，或两者兼有)及该载体与其所在的特定宿主细胞之间的兼容性。载体组件通常包括但不限于：复制起始位点、选择标记基因、启动子、核糖体结合位点(RBS)、信号序列、异源核酸插入和转录终止序列。
一般来说，质粒载体含有来自于与宿主细胞相容物种的复制子和控制序列，其与这些宿主细胞一起使用。载体通常携带一个复制位点，以及能够在转化细胞中提供表型选择的标记序列。例如，大肠杆菌通常使用pBR322转化，pBR322是一种来源于大肠杆菌的质粒。pBR322包含编码氨苄青霉素(Amp)和四环素(Tet)抗性的基因，并因此提供了识别转化细胞的简单方法。pBR322、其衍生物或其它细菌质粒或噬菌体也可含有或经修饰后含有可被微生物用于表达内源性蛋白质的启动子。Carter et al.,U.S.Pat.No.5,648,237详述了用于表达特定抗体的pBR322衍生物的示例。
此外，包含与宿主微生物相容的复制子和控制序列的噬菌体载体可作为转化载体与这些宿主细胞一起使用。例如，噬菌体如GEMTM-11可用于制备重组载体，所述重组载体可用于转化易感宿主细胞，如大肠杆菌LE392。
启动子是一段位于顺反子上游(5′)的非翻译调控序列，可调节下游基因表达。原核启动子通常分为两类，诱导型和组成型。诱导型启动子是一种可以响应培养条件改变(例如，营养素的存在或缺乏或温度的变化)，从而启动并提高顺反子转录水平的启动子。
可被潜在宿主细胞识别的许多启动子都是公知的。通过限制性内切酶将启动子从源DNA中移出并将分离的启动子序列插入本申请的载体中，所选启动子可操作地连接到编码多肽的顺反子DNA上。天然启动子序列和许多异源启动子都可用于指导靶基因的扩增和/或表达。在一些实施例中，利用异源启动子，因为与天然靶多肽启动子相比，异源启动子通常允许更大的转录，并且表达靶基因的产量更高。
适用于原核宿主的启动子包括PhoA启动子、-半乳糖苷酶和乳糖启动子系统、色氨酸(trp)启动子系统和杂交启动子，如tac或trc启动子。然而，在细菌中具有功能的其它启动子(如其它已知的细菌或噬菌体启动子)也适用。它们的核酸序列已经公开，从而使技术人员能够使用接头或适配子来提供任何所需的限制位点将它们连接到编码目标轻链和重链的顺反子上(Siebenlist et al.,(1980)Cell 20:269)。
在一些实施例中，重组载体内的每个顺反子都包含一个分泌信号序列组分，该组分直接引导所表达的多肽跨膜转移。一般来说，信号序列可以是载体的组成部分，也可以是插入载体的靶多肽DNA的一部分。为本申请而选择的信号序列应为能被宿主细胞识别和加工(即，被信号肽酶切割)的序列。对于不能识别和加工异源多肽固有信号序列的原核宿主细胞，信号序列被原核信号序列所取代，该原核信号序列选自，例如，碱性磷酸酶、青霉素酶、Ipp或热稳定性肠毒素II(STII)先导物、LamP、PhoE、，PelB、OmpA和MBP。
在一些实施例中，生产本申请的IL-2突变体或包含IL-2突变体的融合蛋白可发生在宿主细胞的细胞质中，因此不需要在每个顺反子内都存在分泌信号序列。在一些实施例中，多肽组分被表达、折叠和组装以在细胞质内形成蛋白质构建体。某些宿主菌株(例如，大肠杆菌trxB-菌株)提供有利于二硫键形成的细胞质条件，从而允许表达的蛋白质亚基适当折叠和组装。参见Proba和Pullthun,Gene,159:203(1995)。
b)原核宿主细胞
适于表达本申请的蛋白质的原核宿主细胞包括古细菌和真细菌，如革兰氏阴性菌或革兰氏阳性菌。可用细菌的示例包括大肠杆菌(例如，大肠杆菌)、杆菌(例如，枯草杆菌)、肠杆菌、假单胞菌(例如，铜绿假单胞菌)、鼠伤寒沙门氏菌、粘质沙雷氏菌、克雷伯菌、变形杆菌、志贺菌、根瘤菌、透明颤菌或副球菌。在一些实施例中，使用革兰氏阴性细胞。在一些实施例中，大肠杆菌细胞作为本申请的宿主。大肠杆菌菌株示例包括菌株W3110(Bachmann,Cellular and Molecular Biology,vol.2(Washington,D.C.:American Society for Microbiology,1987),pp.1190-1219；ATCC Deposit No.27,325)及其衍生物，包括具有基因型W3110 AfhuA(AtonA)ptr3 lac Iq lacL8 AompT A(nmpc fepE)degP41 kanR的菌株33D3(U.S.Pat.No.5,639,635)。其它菌株及其衍生物，如E.coli 294(ATCC 31446)、E.coli B、E.coli 1776(ATCC 31537)和E.coli RV308(ATCC 31608)也同样适用。这些例子是说明性的，而不是限制性。构建任何上述提及的已知基因型的细菌衍生物的方法在本领域已知，并在例 如Bass et al.,Proteins,8:309-314(1990)中进行了详述。考虑到复制子在细菌细胞中的可复制性，通常需要选择合适的细菌。例如，当使用公知的质粒如pBR322、pBR325、pACYC177或pKN410来提供复制子时，大肠杆菌、沙雷氏菌或沙门氏菌适于用作宿主。
通常，宿主细胞应分泌最少量的蛋白水解酶，并需要在细胞培养物中适当加入额外的蛋白酶抑制剂。
c)蛋白生产
用上述表达载体转化宿主细胞，并在经适当改良的传统营养培养基中培养，以诱导启动子、选择转化子或扩增编码所需序列的基因。转化是指将DNA导入原核宿主，使DNA可以作为染色体外的元件或通过染色体整合进行复制。根据所用的宿主细胞，使用适合此类细胞的标准技术进行转化。采用氯化钙的钙处理通常用于含有大量细胞壁屏障的细菌细胞。另一种转化方法采用聚乙二醇/二甲基亚砜。另一种技术是电穿孔。
用于生产本申请的蛋白质构建体的原核细胞在本领域已知的且适于培养所选宿主细胞的培养基中生长。合适的培养基包括luria broth(LB)和必要的营养补充剂。在一些实施例中，培养基还包含基于表达载体的结构所选择的选择剂，以选择性地允许包含表达载体的原核细胞生长。例如，将氨苄青霉素添加到培养基中，用于表达氨苄青霉素抗性基因的细胞生长。
除碳源、氮源和无机磷酸盐源外，任何必要的补充剂也可单独或作为与其它补充剂或介质(如复合氮源)的混合物以适当的浓度加入。可选地，培养基可包含一种或多种选自谷胱甘肽、半胱氨酸、胱胺、硫代甘氨酸、二硫代赤藓糖醇和二硫苏糖醇的还原剂。原核宿主细胞在合适的温度下培养。例如，对于大肠杆菌的生长，优选的温度范围为20℃至39℃，更优选的为25℃至37℃，甚至更优选的为30℃。培养基的pH值可以是5到9之间的任何pH值，主要取决于宿主生物。对于大肠杆菌来说，pH值优选为6.8至7.4，且更优选为7.0。
如果在本申请的表达载体中使用诱导型启动子，则在适合启动子激活的条件下诱导蛋白质表达。在本申请的一方面，PhoA启动子用于控制多肽的转录。因此，转化的宿主细胞在磷酸盐限制培养基中培养以进行诱导。优选地，磷酸盐限制培养基为C.R.A.P培养基(参见Simmons et al.,J.Immunol.Methods(2002),263:133-147)。根据所采用的载体结构，可使用本领域已知的多种其它诱导剂，这在本领域是已知的。
本申请所表达的蛋白质构建体分泌到宿主细胞的周质中并从中回收。蛋白质回收通常涉及破坏微生物，通常通过渗透压休克、超声处理或裂解等方式。一旦细胞被破坏，可通过离心或过滤去除细胞碎片或整个细胞。例如，可通过亲和树脂色谱法进一步纯化蛋白质。或者，蛋白质可以被运输到培养基中并在其中被分离出来。可从培养基中移除细胞，过滤和浓缩培养基上清液以进一步纯化所产生的蛋白质。所表达的多肽可通过聚丙烯酰胺凝胶电泳(PAGE)和Western blot试验等常用方法进一步分离和鉴定。
或者，通过发酵过程大规模生产蛋白质。各种大规模补料分批发酵程序可用于生产重组蛋白。大规模发酵的容量至少为1000升，最好为1000至100000升。这些发酵罐使用搅拌器叶轮来分配氧气和营养物质，特别是葡萄糖(首选碳/能源)。小规模发酵通常指发酵罐中的发酵，其容量体积不超过100升，范围从1升到100升。
在发酵过程中，通常是细胞在合适的条件下生长至所需密度后开始诱导蛋白质表达，例如，在OD550约为180-220时，此时细胞处于早期静止期。根据所采用的载体结构，可使用本领域已知和上文所述的多种诱导剂。细胞在诱导前可以生长较短的时间。细胞通常诱导约12-50小时，尽管可以使用可能更长或更短的诱导时间。
为了提高本申请的IL-2突变体或包含IL-2突变体的融合蛋白的产量和质量，可以改良各种发酵条件。例如，为了提高分泌多肽的正确组装和折叠，可以使用过表达伴侣蛋白的附加载体来共转化宿主原核细胞，如Dsb蛋白(DsbA、DsbB、DsbC、DsbD或DsbG)或FkpA(具有伴侣活性的肽脯氨酰顺反异构酶)。已证明伴侣蛋白有助于促进细菌宿主细胞中所产生的异源蛋白正确折叠和溶解。Chen et al.,(1999)J Bio Chem 274:19601-19605；Georgiou et al.,U.S.Pat.No.6,083,715；Georgiou et al.,U.S.Pat.No.6,027,888；Bothmann和Pluckthun(2000)J.Biol.Chem.275:17100-17105；Ramm和Pluckthun(2000)J.Biol.Chem.275:17106-17113；Arie et al.,(2001)Mol.Microbiol.39:199-210。
为了最大限度地减少表达的异源蛋白质(尤其是蛋白水解敏感的蛋白质)的水解，某些缺乏蛋白水解酶的宿主菌株可用于本申请。例如，宿主细胞菌株可经修饰使得编码已知细菌蛋白酶的基因发生基因突变，如蛋白酶III、OmpT、DegP、Tsp、蛋白酶I、蛋白酶Mi、蛋白酶V、蛋白酶VI及其组合物。可以使用一些大肠杆菌蛋白酶缺失菌株，在Joly et al.,(1998),supra；Georgiou et al.,U.S.Pat.No.5,264,365；Georgiou et al., U.S.Pat.No.5,508,192；Hara et al.,Microbial Drug Resistance,2:63-72(1996)中进行了详述。
缺乏蛋白水解酶且用过表达一种或多种伴侣蛋白的质粒转化的大肠杆菌菌株，可在编码本申请所述的IL-2突变体或包含IL-2突变体的融合蛋白的表达系统中作为宿主细胞。
d)蛋白纯化
进一步纯化本文所生产的蛋白质构建体，以获得用于进一步分析和使用的基本上均匀的制剂。可采用本领域已知的标准蛋白质纯化方法。以下程序为适用的纯化程序示例：免疫亲和柱或离子交换柱上的分馏、乙醇沉淀、反相液相色谱HPLC、二氧化硅或阳离子交换树脂(如DEAE)色谱、色谱聚焦、SDS-PAGE、硫酸铵沉淀和凝胶过滤，例如，葡聚糖凝胶G-75。
在一些实施例中，固定在固相上的蛋白质A被用于蛋白质构建体的免疫亲和纯化，所述蛋白质构建体包含本申请所述的Fc区域。蛋白A为来自金黄色葡萄球菌的42kDa表面蛋白，其与含Fc的结构具有很高的结合亲和力，例如，本文所述的包含IL-2突变体-Fc融合蛋白。Lindmark et al.,(1983)J.Immunol.Meth.62:1-1。固定蛋白质A的固相优选包含玻璃或二氧化硅表面的柱子，更优选为可控孔径玻璃柱或硅酸柱。在某些应用中，色谱柱涂有试剂，如甘油，以防止污染物的非特异性粘附。然后清洗固相以去除非特异性结合到固相的污染物。最后，通过洗脱从固相中回收目标蛋白构建体。
2.真核细胞的重组产物
对于真核表达，载体组分通常包括但不限于下列一个或多个：信号序列、复制起始点、一个或多个标记基因、增强子元件、启动子和转录终止序列。
a)信号序列元件
用于真核宿主的载体还可以是一个插入物，该插入物编码信号序列或在成熟蛋白或多肽的N端具有特定裂解位点的其它多肽。选择的异源信号序列优选为由宿主细胞识别和加工(即，被信号肽酶切割)的序列。在哺乳动物细胞表达中，可以获得哺乳动物信号序列以及病毒分泌先导物，例如，单纯疱疹gD信号，都是有用的。该前体区域的DNA在阅读框中与编码本申请的蛋白质构建体的DNA连接。
b)复制起始点
一般来说，哺乳动物表达载体不需要复制起始点元件(SV40起点通常仅因其包含早期启动子而使用)。
c)选择基因元件
表达和克隆载体可包含选择基因，也称为选择标记。典型的选择基因编码以下蛋白质：(a)对抗生素或其它毒素具有抗药性的蛋白质，例如，氨苄青霉素、新霉素、甲氨蝶呤或四环素、(b)补体营养缺陷蛋白质或(c)提供复杂培养基无法提供的关键营养素的蛋白质，如编码杆菌D-丙氨酸消旋酶的基因。
选择方案的一个示例是利用药物来阻止宿主细胞的生长。那些异源基因成功转化的细胞产生一种具有抗药性的蛋白质，从而在选择方案中存活下来。这种优势选择的示例使用药物新霉素、霉酚酸和潮霉素。
适用于哺乳动物细胞的可选择的标记的另一个示例是，那些能够识别有能力携带编码本申请所述蛋白质构建体核酸的细胞的可选择的标记，如DHFR、胸苷激酶、金属硫蛋白-I和-II，优选为灵长类金属硫蛋白基因、腺苷脱氨酶、鸟氨酸脱羧酶等。
例如，用DHFR选择基因转化的细胞，首先将所有转化子培养在含有甲氨蝶呤(Mtx)的培养基中进行鉴定，甲氨蝶呤是DHFR的竞争性拮抗剂。当使用野生型DHFR时，适用的宿主细胞为缺乏DHFR活性的中国仓鼠卵巢(CHO)细胞系(例如，ATCC CRL-9096)。
或者，用编码多肽的DNA序列、野生型DHFR蛋白和另一选择标记，如氨基糖苷3′-磷酸转移酶(APH)转化或共转化的宿主细胞(尤其是含有内源性DHFR的野生型宿主)，可通过在含有选择标记物的培养基中的细胞生长来选择，所述选择标记物例如氨基糖苷类抗生素，例如，卡那霉素、新霉素或G418。参见U.S.Pat.No.4,965,199。
d)启动子元件
表达和克隆载体通常包含一个启动子，所述启动子被宿主识别，并可操作地连接到编码所需多肽序列的核酸上。几乎所有真核基因都有一个富含AT的区域，位于转录起始点上游约25到30个碱基。在许多基因转录起始点上游70到80个碱基处发现的另一个序列为CNCAAT区域，其中N可以是任何核苷酸。在大多数真核生物的3′端有一个AATAAA序列，该序列可能是编码序列3′端增加多聚A尾的信号。所有这些序列都可以插入到真核表达载体。
哺乳动物宿主细胞载体中的多肽转录受启动子控制，例如，通过从病毒基因组获得的启动子，如多瘤病毒、鸡痘病毒、腺病毒(如腺病毒2)、牛乳头状瘤病毒、禽肉瘤病毒、巨细胞病毒、逆转录病毒、乙型肝炎病毒和最优选的猿猴病毒40(SV40)，来自异源哺乳动物的启动子，例如，肌动蛋白启动子或免疫球蛋白启动子，来自热休克启动子，前提是此类启动子与宿主细胞系统兼容。
SV40病毒早期和晚期启动子作为SV40限制性片段便于获得，该限制性片段也包含SV40病毒复制起始点。人类巨细胞病毒的即刻早期启动子作为HindIII E限制性片段便于获得。U.S.Pat.No.4,419,446中公开了一种使用牛乳头瘤病毒作为载体在哺乳动物宿主中表达DNA的系统。U.S.Pat.No.4,601,978中详述了对该系统的改良。参见Reyes et al.,Nature 297:598-601(1982)，关于在单纯疱疹病毒胸苷激酶启动子控制下人类干扰素cDNA在小鼠细胞中的表达。或者，可以使用劳斯肉瘤病毒长末端重复序列作为启动子。
e)增强子元件
高等真核生物对编码本申请蛋白质构建体DNA的转录通常通过在载体中插入增强子序列而增加。在哺乳动物基因中已经发现了许多增强子序列(珠蛋白、弹性蛋白酶、白蛋白、α-甲胎蛋白和胰岛素)。然而，通常使用的是真核细胞病毒的增强子。示例包括复制起始点末端(100-270bp)的SV40增强子、巨细胞病毒早期启动子增强子、复制起始点末端的多瘤病毒增强子和腺病毒增强子。参见Yaniv,Nature 297:17-18(1982)，关于激活真核生物启动子的增强元件。增强子可在多肽编码序列的5′或3′处拼接到载体上，但优选位于启动子的5′处。
f)转录终止元件
真核宿主细胞(酵母、真菌、昆虫、植物、动物、人类或其它多细胞生物的有核细胞)中使用的表达载体也包含转录终止和稳定mRNA所必需的序列。这些序列通常可从真核或病毒DNA或cDNA的5′端非翻译区获得，偶尔为3′端。这些区域包含核酸片段，所述核酸片段在编码多肽的mRNA的未翻译部分作为多聚腺苷酸片段被转录。一个合适的转录终止元件为牛生长激素多聚腺苷酸区域。参见WO94/11026和其中所公开的表达载体。
g)宿主细胞的选择和转化
用于克隆或表达本文所述载体中的DNA的合适宿主细胞包括本文所述的高等真核细胞，包括脊椎动物宿主细胞。脊椎动物细胞的培养繁殖(组织培养)已成为常规程 序。有用的哺乳动物宿主细胞系示例为SV40转化的猴肾CV1系(COS-7，ATCC CRL 1651)；来源于猴肾组织的COS成纤维细胞样细胞系；人类胚胎肾系(293或293细胞亚克隆，用于悬浮培养生长，Graham et al.,J.Gen Virol.36:59(1977))；乳仓鼠肾细胞(BHK，ATCC CCL 10)；中国仓鼠卵巢细胞/-DHFR(CHO，Urlaub et al.,Proc.Natl.Acad.Sci.USA 77:4216(1980))；小鼠塞尔托力氏细胞(TM4，Mather,Biol.Reprod.23:243-251(1980))；猴肾细胞(CV1ATCC CCL 70)；非洲绿猴肾细胞(VERO-76，ATCC CRL-1587)；人宫颈癌细胞(HELA，ATCC-ccl2)；犬肾细胞(MDCK，ATCC-ccl34)；水牛-大鼠肝细胞(BRL 3A，ATCC CRL 1442)；人类肺细胞(W138，ATCC CCL 75)；人类肝细胞(Hep G2，HB 8065)；小鼠乳腺肿瘤(MMT 060562，ATCC CCL51)；TR1细胞(Mather et al.,Annals N.Y.Acad.Sci.383:44-68(1982))；MRC5细胞；FS4细胞和人类肝癌细胞系(Hep G2)。
用上述表达载体或克隆载体转化宿主细胞以产生蛋白质结构，并在经适当改良的常规营养培养基中培养，以诱导启动子、选择转化子或扩增编码所需序列的基因。
h)培养宿主细胞
用于生产本申请的蛋白质构建体的宿主细胞可在多种培养基中培养。商用培养基，如Ham's F10(Sigma)、最小基本培养基((MEM)，Sigma)、RPMI-1640(Sigma)和Dulbecco's修饰的Eagle's Medium((DMEM)，Sigma)适合培养宿主细胞。此外，Ham et al.,Meth.Enz.58:44(1979)、Barnes et al.,Anal.Biochem.102:255(1980)、U.S.Pat.No.4,767,704；4,657,866；4,927,762；4,560,655或5,122,469、WO 90/03430、WO 87/00195或U.S.Pat.Re.30,985所述的任何培养基都可用作宿主细胞的培养基。这些培养基中的任何一种都可以根据需要补充激素和/或其它生长因子(如胰岛素、转铁蛋白或表皮生长因子)、盐(如氯化钠、钙、镁和磷酸盐)、缓冲液(如HEPE)、核苷酸(如腺苷和胸苷)、抗生素(如庆大霉素TM药物)、微量元素(定义为通常最终浓度在微摩尔范围内存在的无机化合物)和葡萄糖或等效能源。任何其它必要的补充剂也可在本领域技术人员已知的适当浓度下加入。培养条件，如温度、pH等，是那些先前宿主细胞表达所使用过的条件，并且对于普通技术人员来说显而易见。
VI.药物组合物
本申请进一步涉及包含本文所述的任何IL-2突变体或包含IL-2突变体的融合蛋白的药物组合物，或包含编码本文所述的任何IL-2突变体或包含IL-2突变体的融合蛋白 的核酸的药物组合物，或包含编码本文所述的任何IL-2突变体或包含IL-2突变体的融合蛋白的核酸的载体的药物组合物。这些组合物可以任选的进一步包含药学上可接受的载体和/或辅料。在一些实施例中，所述辅料包括赋形剂和/或稳定剂。药物组合物可以通过将本文所述的具有所需纯度的IL-2突变体或包含IL-2突变体的融合蛋白与可选的药学上可接受的载体、赋形剂或稳定剂(Remington's Pharmaceutical Sciences 16th edition,Osol,A.Ed.(1980))混合制备成冻干制剂或水溶液的形式。
VII.疾病的治疗方法
本申请所述的IL-2突变体或包含IL-2突变体的融合蛋白或包含其的组合物(例如，药物组合物)可用于多种用途，例如诊断、分子检测和治疗。在一些实施例中，本申请涉及一种治疗个体(例如，人类)疾病的方法，包括向个体施用有效剂量的如本文所述的任何IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。在一些优选实施例中，包含IL-2突变体的融合蛋白为IL-2突变体与Fc的融合蛋白。在一些实施例中，所述疾病与受试者中Treg细胞的调节相关。在一些实施例中，所述疾病包括炎性疾病或自身免疫性疾病。在一些实施例中，IL-2突变体或包含IL-2突变体的融合蛋白或包含IL-2突变体或其融合蛋白的药物组合物通过静脉注射、肌肉注射或皮下注射给药。
在一些实施中，所述疾病包括狼疮、移植物抗宿主病、C型肝炎诱发的血管炎、Ⅰ型糖尿病、Ⅱ型糖尿病、多发性硬化、类风湿性关节炎、特应性疾病、哮喘、炎症性肠病、自身免疫性肝炎、溶血性贫血、风湿热、甲状腺炎、克罗恩病、重症肌无力、肾小球肾炎、斑秃、牛皮癣、白癜风、营养不良性大疱性表皮松解症和白塞病。
例如，在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的缺失或突变。在一些实施例中，所述人野生型IL-2的氨基酸序列如SEQ ID NO:1所示。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和 宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的全部缺失。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和N71的突变。在一些实施例中，所述E67和N71的突变分别为E67K和N71S。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的全部缺失，以及相对于人野生型IL-2氨基酸序列的E67K和N71S突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67的突变。在一些实施例中，所述E67的突变为E67A。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，以及相对于人野生型IL-2氨基酸序列的E67A突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，且进一步包含相对于人野生型IL-2氨基酸序列的N71和S75的突变。在一些实施例中，所述N71和S75的突变分别为N71W和S75I。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL，以及相对于人野生型IL-2氨基酸序列的N71W和S75I突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和/或V91的突变。在一 些实施例中，所述E67的突变为E67T。在一些实施例中，所述V91的突变为V91K。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的E67T和/或V91K突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，且进一步包含相对于人野生型IL-2氨基酸序列的L70和S75的突变。在一些实施例中，所述L70和S75的突变分别为L70I和S75F。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的L70I和S75F突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，且进一步包含相对于人野生型IL-2氨基酸序列的N71的突变。在一些实施例中，所述N71的突变为N71I。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL，以及相对于人野生型IL-2氨基酸序列的N71I突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为SPL。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和N71的突变。在一些实施例中，所述E67和N71的突变分别为E67M和N71S。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为SPL，以及相对于人野生型IL-2氨基酸序列的E67M和N71S突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列的第75位氨基酸残基S(S75)和第83位氨基酸残基R(R83)的突变。在一些实施例中，所述人野生型IL-2的氨基酸序列如SEQ ID NO:1所示。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体包含相对于人野生型IL-2氨基酸序列的第75位氨基酸残基S(S75)和第83位氨基酸残基R(R83)的突变。在一些实施例中，其中所述IL-2突变体中S75被突变为不带电荷的氨基酸残基。在一些实施例中，其中S75的突变为S75P、S75F、S75Y、S75G、S75A、S75V、S75T、S75I或S75L。在一些实施例中，其中R83的突变为R83N、R83F、R83Y、R83P、R83L、R83A、R83K或R83E。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75P，R83的突变为R83Y。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和D84的突变。在一些实施例中，其中E67和D84的突变分别为E67A和D84N。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P、R83Y、E67A和D84N突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75P，R83的突变为R83A。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16、L70和N71的突变。在一些实施例中，其中H16、L70 和N71的突变分别为H16D、L70I和N71G。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75P、R83A、H16D、L70I和N71G突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75F，R83的突变为R83P。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和/或N71的突变。在一些实施例中，其中H16的突变为H16Q或H16N。在一些实施例中，其中N71的突变为N71S。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83P和H16Q突变。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83P、H16N和N71S突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75F，R83的突变为R83L。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变。在一些实施例中，其中E67和L70的突变分别为E67R和L70V。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75F、R83L、E67R和L70V突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75I，R83的突变为R83L。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变。在一些实施例中，其中E67和L70的突变分别为E67G和L70V。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75I、R83L、E67G和L70V突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含 IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75I，R83的突变为R83K。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变。在一些实施例中，其中E67和L70的突变分别为E67S和L70V。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75I、R83K、E67S和L70V突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75T，R83的突变为R83A或R83F。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和E67的突变。在一些实施例中，其中H16和E67的突变分别为H16N和E67K。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75T、R83A、H16N和E67K突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75V，R83的突变为R83F。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E15和H16的突变。在一些实施例中，其中E15和H16的突变分别为E15K和H16Q。在一些实施例中，所述的IL-2突变体或其融合蛋白，其包含相对于人野生型IL-2氨基酸序列的S75V、R83F、E15K和H16Q突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75V，R83的突变为R83N。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L70和V91的突变。在一些实施例中，其中L70和V91的突变分别为L70F和V91K。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75V、R83N、L70F和V91K突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75G，R83的突变为R83L。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17、V91和I92的突变。在一些实施例中，L17、V91和I92的突变分别为L17I、V91E和I92L。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75G、R83L、L17I、V91E和I92L突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75Y，R83的突变为R83N。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17和V91的突变。在一些实施例中，L17和V91的突变分别为L17I和V91K。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75Y、R83N、L17I和V91K突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75A，R83的突变为R83K。在一些实施例中，所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L17的突变。在一些实施例中，L17的突变为L17I。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75A、R83K和L17I突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。其中所述IL-2突变体中，S75的突变为S75L，R83的突变为R83E。在一些实施例中，本申请所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L63和E67的突变。在一些实施例中，L63和E67的突 变分别为L63V和E67F。在一些实施例中，所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的S75L、R83E、L63V和E67F突变。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体，或者包含IL-2突变体的融合蛋白。其中所述IL-2突变体或其融合蛋白，除上述突变外，还进一步包含相对于人野生型IL-2氨基酸序列C125的突变。在一些实施例中，所述C125的突变为C125S或C125A。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体，或者包含IL-2突变体的融合蛋白。所述IL-2突变体包含SEQ ID NOs:5-34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:5-34中任一所示的氨基酸序列具有至少约90％(例如至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。在一些实施例中，所述包含IL-2突变体的融合蛋白为IL-2突变体与Fc的融合蛋白。在一些实施例中，所述IL-2突变体与Fc的融合蛋白为二价形式。在一些实施例中，所述IL-2突变体与Fc的二价融合蛋白包含SEQ ID NOs:76-105中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:76-105中任一所示的氨基酸序列具有至少约80％(例如至少80％、85％、88％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同源性。在一些实施例中，所述IL-2突变体与Fc的融合蛋白为单价形式。在一些实施例中，所述疾病或病症选自狼疮、移植物抗宿主病、C型肝炎诱发的血管炎、Ⅰ型糖尿病、Ⅱ型糖尿病、多发性硬化、类风湿性关节炎、特应性疾病、哮喘、炎症性肠病、自身免疫性肝炎、溶血性贫血、风湿热、甲状腺炎、克罗恩病、重症肌无力、肾小球肾炎、斑秃、牛皮癣、白癜风、营养不良性大疱性表皮松解症和白塞病。
在一些实施例中，本申请涉及一种治疗患有炎性疾病或自身免疫性疾病(例如，狼疮)的个体的方法，包括向所述个体施用有效量的本申请所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物，还进一步包括向个体施用其他的一种或多种其它活性成分，所述活性成分是治疗的特定适应症所需的，优选具有不会影响彼此之间治疗效果的活性成分。例如，当用于治疗自身免疫性疾病或缓解或预防器官移植之后的 自身免疫反应时，可以与免疫抑制剂联用。在一些实施例中，免疫抑制剂选自：糖皮质激素；硫唑嘌呤；环孢素A；甲氨蝶呤；抗CD3抗体；或抗TNF-α抗体等。
本文所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物的给药方式可以以任何已知和方便的方式进行，包括通过注射或输液。给药途径依照已知和公认的方法，如通过单次或多次推注或以适当方式长时间输液。
本申请的药物组合物的剂量和所需药物浓度可根据特定用途而变化。确定合适的给药剂量或给药途径完全属于普通技术人员的技术范围。动物实验为确定人类治疗的有效剂量提供了可靠的指导。可以依据Mordenti,J.和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中的原则进行有效剂量的种间类推。
当使用IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物在体内给药时，治疗有效量可取决于例如治疗情境和目标。本领域普通技术人员将理解，用于治疗的适当剂量水平将取决于被递送的分子、应用IL-2突变体进行治疗的适应症、施用途径和患者的体型(体重，体表面或器官的大小)和/或状况(年龄和一般健康状况)而变化。在本申请的范围内，不同的制剂将对不同的治疗和不同的疾病有效，并且旨在治疗特定器官或组织的给药方式可能与针对另一器官或组织的方式不同。此外，剂量可通过一次或多次单独给药或持续输注给药。对于几天或更长时间的重复给药，根据病情，治疗持续到疾病症状达到预期的抑制程度为止。或者，其它剂量方案可能有用。这种治疗的进展很容易通过常规技术和分析进行监测。
VIII.制品及试剂盒
本申请进一步涉及包含本文所述任何IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物的试剂盒、单位剂量和制品。在一些实施例中，涉及包含本文所述的任一种药物组合物的试剂盒，并且优选地提供其使用说明，如用于治疗本文所述的疾病(例如，自身免疫性疾病)。
本申请的试剂盒包括一个或多个包含本文所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物的容器，例如，用于治疗疾病。例如，包含描述施用IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物以治疗疾病(如自身免疫性疾病)的说明书。试剂盒可能进一步包含基于识别个体是否患有疾病和疾病阶段来选择适合治疗的个体(例如，人类)的描述。与IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物的使用相关的说明通常包括关于预期治疗的剂量、给药计划和给药途径的信息。容器可以是单位剂量、散装包装(例如，多剂量包装)或亚单位剂量。本申请的试剂盒中提供的说明通常是标签或药品说明书上的书面说明(例如，试剂盒中包括的纸张)，但机器可读说明(例如，存储在磁盘或光盘上的说明)也是可以接受的。本申请的试剂盒采用合适的包装。合适的包装包括但不限于小瓶、瓶子、罐子、软包装(例如，密封的聚酯薄膜或塑料袋)等。还考虑与特定装置结合使用的包装，如输液装置如微型泵。试剂盒可具有无菌接入端口(例如，容器可为静脉注射溶液袋或具有可被皮下注射针刺穿的塞子的小瓶)。该组合物中的至少一种活性剂是如本文所述的IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物。容器可进一步包含第二种医药活性剂。试剂盒可选择性地提供附加组份，如缓冲液和解释信息。一般来说，试剂盒包含一个容器和容器上或与容器相关的标签或药品说明书。
例如，在一些实施例中，试剂盒包括：a)包含本文所述的任一种IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物，和b)至少一种有效量的其它药剂，其能够增强IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物的效果(如治疗效果)。
在一些实施例中，试剂盒包括：a)包含本文所述的任一种IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物，和b)向个体施用包含IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物用于治疗自身免疫性疾病(例如，狼疮)的使用说明书。
在一些实施例中，试剂盒包括：a)包含本文所述的任一种IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物，和b)至少一种有效量的其它药剂，其能够增强IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物的效果(如治疗效果)和c)向个体施用IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体、或包含其的药物组合物和其它物质用于治疗自身免疫性疾病(例如，狼疮)的使用说明书。所述IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物和其他物质可以存在于独立的容器或同一个容器中。例如，该试剂盒可以包含一种特定组合物或两种或更多种组合物，其中一种组合物包括IL-2突变体、包含IL-2突变体的融合蛋白、编码IL-2突变体或其融合蛋白的核酸、包含此核酸的载体和宿主细胞、或包含其的药物组合物，另一种组合物包括另一种药剂。
在一些实施例中，试剂盒包含一种编码IL-2突变体或其融合蛋白(例如IL-2突变体与Fc的融合蛋白)的核酸(或核酸组)。在一些实施例中，试剂盒包含：a)一种编码IL-2突变体或其融合蛋白(例如IL-2突变体与Fc的融合蛋白)的核酸(或一组核酸)，和b)一种表达核酸(或一组核酸)的宿主细胞。在一些实施例中，试剂盒包含：a)一种编码IL-2突变体或其融合蛋白(例如IL-2突变体与Fc的融合蛋白)的核酸(或一组核酸)，和b)使用说明书，适用于：i)在宿主细胞中表达IL-2突变体或其融合蛋白，ii)制备包含IL-2突变体或其融合蛋白的组合物，和iii)向个体施用包含IL-2突变体或其融合蛋白的组合物来治疗自身免疫性疾病(例如，狼疮)。在一些实施例中，试剂盒包括：a)一种(或一组)编码IL-2突变体或其融合蛋白(例如IL-2突变体与Fc的融合蛋白)的核酸，b)一种表达核酸(或核酸组)的宿主细胞，和c)使用说明书，适用于：i)在宿主细胞中表达IL-2突变体或其融合蛋白，ii)制备包含IL-2突变体或其融合蛋白的组合物，和iii)向个体施用包含IL-2突变体或其融合蛋白组合物来治疗自身免疫性疾病(例如，狼疮)。
另一方面，本申请还涉及制品，包括小瓶(如密封小瓶)、瓶、罐、软包装等。该制品包含容器和容器上或与容器相关的标签或药品说明书。合适的容器包括，例如，瓶子、小瓶、注射器等。容器可以由多种材料制成，如玻璃或塑料。一般来说，容器容纳的组合物可有效治疗本文所述疾病或紊乱(如自身免疫性疾病)，并且可具有无 菌接入端口(例如，容器可为静脉注射溶液袋或具有可被皮下注射针刺穿的塞子的小瓶)。标签或药品说明书表明该组合物用于治疗个体的特定病症。标签或药品说明书进一步包含向个体施用组合物的说明。标签可能会注明重构和/或使用的说明。容纳药物组合物的容器可以是多次使用的小瓶，允许重构制剂重复施用(例如，2-6次施用)。药品说明书是指通常包含在治疗产品商业包装中的说明书，其中包含有关使用此类治疗产品的适应症、用法、剂量、给药、禁忌症和/或警告信息。在一些实施例中，说明书标明可治疗的疾病包括狼疮、移植物抗宿主病、C型肝炎诱发的血管炎、Ⅰ型糖尿病、Ⅱ型糖尿病、多发性硬化、类风湿性关节炎、特应性疾病、哮喘、炎症性肠病、自身免疫性肝炎、溶血性贫血、风湿热、甲状腺炎、克罗恩病、重症肌无力、肾小球肾炎、斑秃、牛皮癣、白癜风、营养不良性大疱性表皮松解症和白塞病。此外，制品可能进一步包含第二容器，包含药学可接受的缓冲液，如注射用抑菌水(BWFI)、磷酸盐缓冲盐水、林格溶液和葡萄糖溶液。从商业和用户角度来看，可能进一步包括其它需要的材料，包括其它缓冲液、稀释剂、过滤器、针头和注射器。
试剂盒或制品包括多个单位剂量的药物组合物和使用说明书，包装数量足以在药房中储存和使用，例如，医院药房和复方药房。
具体实施方式
本领域的技术人员将认识到在本申请的范围和宗旨内可能的若干实施例。现在将通过参考以下非限制性实施例来更详细地描述本申请。以下实施例进一步阐明本申请，但不应解释为以任何方式进行限制其范围。
实施例1：人野生型IL-2以及IL-2突变体的设计和表达
野生型IL-2的构建和表达：
编码人野生型IL-2(氨基酸序列如SEQ ID NO:1所示)的核酸序列由南京金斯瑞生物科技有限公司合成，并在合成的野生型IL-2核酸序列的5’端和3’端分别引入酶切位点。将合成的核酸片段酶切后，与经过同样双酶切的载体片段连接，即得野生型IL-2的表达载体。另外，在此表达载体的基础上，在野生型IL-2的第125位引入C125S突变(氨基酸序列如SEQ ID NO:2所示，该突变可以防止半胱氨酸错配或聚集)，即获得IL-2 C125S突变体的表达载体；在野生型IL-2的第125位引入C125A突变(氨基酸序列如SEQ ID NO:3所示，该突变可以促进有效折叠，改善表达)，即获得IL-2  C125A突变体的表达载体。上述两种IL-2突变体与野生型IL-2相比，并未改变其与IL-2受体的结合作用。
IL-2突变体的设计、筛选和表达：
通过研究、设计和FACS筛选，最终获得了一系列的IL-2突变体，筛选过程如下：
首先制备筛选过程中所需要的生物素化标记的IL-2Rα及IL-2Rβγ：
IL-2Rα的制备：IL-2Rα(Uniprot ID:P01589)的胞外区编码序列经密码子优化后由南京金斯瑞生物科技有限公司进行全基因合成。将合成的基因序列构建到携带有His标签基因的表达载体中，并在293F细胞中进行表达。根据操作说明书，应用(Ni)镍柱对IL-2Rα进行纯化。简言之，采用QIAGEN公司的Ni-NTA进行固定化金属亲和层析(IMAC)。首先采用缓冲液A1(50mM Na3PO4，0.15M NaCl，pH 7.2)平衡镍柱，流速150cm/h。将培养液上清的pH调整至7.2，室温上样，流速150cm/h。随后，采用6倍柱体积的A1缓冲液再次平衡该柱，流速150cm/h。最后，采用10倍柱体积的50mM的PB溶液(包含0.15M NaCl和0.2M咪唑，pH 7.2)进行洗脱，收集洗脱液。
IL-2Rβγ的制备：本申请实施例中所使用的IL-2Rβγ是通过KIH(Knob-into-Hole)技术构建的Fc异源二聚体(IL-2Rβγ-Fc异源二聚体)，IL-2Rβ(Uniprot ID:P14784)和IL-2Rγ(Uniprot ID:P31785)的胞外区编码序列被分别克隆、融合到Fc hole分子(SEQ ID NO:109)和Fc knob分子(SEQ ID NO:108)的N末端，用于制备IL-2Rβγ-Fc异源二聚体。IL-2Rβ-Fc hole和IL-2Rγ-Fc knob被分别构建到pcDNA3.1真核表达载体中，然后共转染293F细胞，收集细胞培养液。IL-2Rβγ-Fc异源二聚体先后应用蛋白A亲和层析和凝胶过滤Superdex 200柱层析进行纯化。简言之，用蛋白A亲和层析柱纯化293F细胞的培养液，首先用含有0.15M NaCl(pH7.2)的50mM PBS缓冲液平衡蛋白A柱，流速为150cm/h，体积为柱体积的6倍。培养基的上清液(pH值调至7.2)，以150cm/h的流速通过柱子。进一步平衡后，使用50mM柠檬酸钠(pH3.5)洗涤柱子并收集洗脱液。然后通过凝胶过滤Superdex 200柱层析进行精纯后，获得IL-2Rβγ-Fc异源二聚体。
IL-2Rα以及IL-2Rβγ的生物素化标记：按照Biotin-Protein Ligase/BirA Enzyme(GeneCopoeia cat#BI001)试剂盒操作说明书，应用生物素化连接酶对IL-2Rα以及IL-2Rβγ进行生物素化标记。简言之，分别向IL-2Rα和IL-2Rβγ蛋白溶液中加入相应的Biotin Ligase BufferA、Buffer B和BirA连接酶后在30℃下孵育2小时。通过ELISA方法检测生物素化效率，确认生物素标记成功。
FACS筛选：将设计的IL-2突变体文库构建到酵母展示体系，应用流式细胞荧光分选技术(FACS)，从文库中筛选与IL-2Rβγ亲和力降低，但与IL-2Rα亲和力基本维持不变或未明显减弱的突变体。FACS筛选方法已为本领域技术人员所熟知，首先应用生物素化标记的IL-2Rβγ(Biotin-IL-2Rβγ)进行4轮FACS筛选，富集与IL-2Rβγ亲和力减弱的酵母阳性群。简言之，对SGCAA培养基中诱导的酵母细胞进行沉淀后加入1mL PBSM，14000g离心30sec，弃上清，洗涤细胞沉淀。加入含有Biotin-IL-2Rβγ的PBSM缓冲液重悬酵母细胞，室温孵育1h。洗涤后，用Streptavidin-PE(BD Biosciences，554061)和抗V5标签抗体[iFluor 647](GenScript，A01805-100)对细胞进行染色，进行细胞分选。然后将富集得到的与Biotin-IL-2Rβγ结合减弱的阳性酵母群，继续应用FACS方法筛选与生物素化标记的IL-2Rα(Biotin-IL-2Rα)亲和力基本维持不变或未明显减弱的单克隆。最后，对筛选得到的单克隆进行测序、构建于携带His标签基因的表达载体、制备样品并进行相应的生物学评价。
通过对单克隆的测序结果进行分析，发现这些突变体大致可以分为两组：
第一组IL-2突变体在相对于人野生型IL-2氨基酸序列中第81-85位的氨基酸残基发生了突变，包括但不限于缺失和/或取代。该组别中各突变体的具体信息示于表9中。表中的“(81-85)RPL”、“(81-85)RHL”或“(81-85)SPL”表示将IL-2的第81-85位氨基酸残基RPRDL分别替换为氨基酸残基RPL，RHL或SPL。“(81-85)RPRDL全部缺失”表示IL-2的第81-85位氨基酸残基RPRDL全部缺失。“+”代表同时突变。
表9

第二组IL-2突变体在相对于人野生型IL-2氨基酸序列的S75和R83同时发生氨基酸突变。该组别中，各突变体的具体信息示于表10中，“+”代表同时突变。
表10
IL-2突变体的表达和纯化：通过PCR获取上述IL-2突变体的基因编码序列，经过酶切后，连接至经同样双酶切的携带有His标签基因的表达载体中，得到IL-2各个突变体的重组表达载体。将上述重组表达载体转染293F细胞，并在37℃、5％CO2、 120rpm条件下培养5天后，收集细胞培养液上清液，按照(Ni)镍柱纯化说明书进行操作，完成IL-2突变体的纯化。具体操作步骤如上所述。
通过下述实验中进一步检验最终获得的两组IL-2突变体是否能够选择性地活化Treg细胞。野生型IL-2或IL-2突变体“活化Treg细胞”的能力可以通过本领域公知的方法或通过本申请实施例中公开的方法来进行测定，包括但不限于，例如，通过比较IL-2突变体与IL-2Rβγ或IL-2Rαβγ结合的EC50值，二者之间的差值越大(例如，在IL-2突变体保持与IL-2Rαβγ结合的同时，其与IL-2Rβγ结合的EC50值越大)，则说明IL-2突变体对Treg细胞的选择性越好；再例如，也可以通过在IL-2突变体刺激后Treg细胞的数量变化情况进行判断，例如通过流式细胞术测量混合细胞群中Treg细胞数量的变化等。
实施例2：IL-2突变体与IL-2Rαβγ和IL-2Rβγ结合亲和力的测定
HEK-BLUETM细胞系检测的实验原理：
HEK-BlueTM IL-2细胞系(InvivoGen,cat#hkb-il2)是稳定转染了人IL-2Rα(CD25)、IL-2Rβ(CD122)和IL-2Rγ(CD132)基因的HEK293细胞系。另一种细胞系HEK-BlueTM CD122/CD132(InvivoGen,cat#hkb-il2bg)则只表达IL-2Rβ(CD122)和IL-2Rγ(CD132)，而不表达IL-2Rα(CD25)。另外，这两种细胞系均稳定表达人JAK3和STAT5基因，具有完整功能的IL-2信号通路；此外还引入了由STAT5诱导SEAP报告基因的表达，在应用IL-2刺激后，可以使用QUANTI-BlueTM溶液(InvivoGen,cat#rep-qbs)检测STAT5诱导的SEAP表达水平。其中HEK-BlueTM IL-2细胞系用于检测IL-2或其突变体和IL-2Rαβγ的结合。HEK-BlueTM CD122/CD132细胞系用于检测IL-2或其突变体和IL-2Rβγ的结合。当向上述细胞系加入IL-2或其突变体后，IL-2或其突变体能够与IL-2高亲和力受体IL-2Rαβγ或中等亲和力受体IL-2Rβγ结合，激活细胞内Janus家族酪氨酸激酶信号级联反应(JAK1和JAK3)，启始信号转导和转录激活因子5(STAT5)的磷酸化，进而诱导SEAP的表达。使用底物QUANTI-BlueTM检测SEAP的表达水平，即在630nm处读取OD值，计算相应EC50值，以此来反映IL-2或其突变体与IL-2Rαβγ或IL-2Rβγ结合后激活STAT5的情况。在本实验中分别采用野生型人IL-2以及AMG592(AMGEN，SEQ ID NO:107)作为对照。
采用HEK-BlueTM CD122/CD132细胞系来测定IL-2突变体与IL-2Rβγ结合的EC50值，采用HEK-BlueTM IL-2细胞系来测定IL-2突变体与IL-2Rαβγ结合的EC50值，将 二者之间的比值，即HEK-IL-2Rβγ的EC50/HEK-IL-2Rαβγ的EC50，定义为IL-2选择激活Treg细胞的治疗安全窗。安全窗数值可以反映IL-2突变体对Treg细胞的选择靶向性，该数值越高，说明IL-2突变体对Treg细胞的选择靶向性越强。
HEK-BLUETM细胞系检测实验：
先将待检测IL-2突变体用检测培养液稀释至800μg/mL，然后4倍梯度稀释至96孔U型板(Coring)中，共12个浓度，备用。将消化好的细胞(HEK-BlueTM IL-2细胞系或HEK-BlueTM CD122/CD132细胞系)悬液调整至2.8×105cells/mL后，按照180μL细胞悬液/每孔接种于96-孔平底细胞培养板，每孔加入20μL稀释好的IL-2或IL-2突变体，与细胞悬液轻轻震荡混匀，然后放置在37℃、5％CO2培养箱中培养24h。从该细胞板中吸取20μL/孔的细胞培养上清液转移到另一新的96-孔平底细胞培养板中，每孔加入180μL QUANTI-BlueTM检测液混匀，37℃孵育1h，读取620nm处吸光度值。使用GraphPad Prism 8.0.1统计并计算待测样品的EC50值，拟合方法为先将浓度值转换为对数即X＝log(X)，然后按照log(agonist)vs.response-Variable slope(four parameters)拟合曲线，计算待检测样品的EC50值。
HEK-BLUETM细胞系检测的实验结果：
第一组和第二组IL-2突变体的检测结果如表11和表12所示。结果显示，与野生型IL-2相比，两组IL-2突变体均能够与IL-2Rαβγ结合，并且其与IL-2Rβγ结合的EC50值提高，亲和力降低，其治疗的安全窗显著提高，表明这些突变体具有优异的Treg细胞选择靶向性。
表11第一组IL-2突变体HEK-BLUETM检测结果

(注：*标记的数值是由于这些突变体对IL-2Rβγ细胞的激活能力较低，在实验所用浓度范围内无法通过拟合得到准确的EC50值)
表12第二组IL-2突变体HEK-BLUETM检测结果

(注：*标记的数值是由于这些突变体对IL-2Rβγ细胞的激活能力较低，在实验所用浓度范围内无法通过拟合得到准确的EC50值)
实施例3：IL-2突变体与Fc融合蛋白的表达及纯化
将上述的IL-2突变体通过连接肽与IgG1 Fc LALA进行连接，形成的IL-2与Fc的融合蛋白包含两种构建形式，分别是二价形式和单价形式。其中IgG1 Fc LALA是指具有L234A和L235A突变的IgG1 Fc，所述编号按照EU Kabat编号体系进行编号(Kabat et al.,J.Biol.Chem.252:6609-6616(1977)；Kabat et al.,U.S.Dept.of Health and Human Services,“Sequences of proteins of immunological interest”(1991))。
IL-2突变体与Fc的融合蛋白的二价形式，共包含两个IL-2突变体分子：其包含两个相同的单体，每个单体中IL-2突变体连接至Fc的N端或C端。一种此类形式的示例性结构如图1A所示(IL-2-Fc)，其由两个相同的单体组成，每个单体从N端到C端包含：IL-2突变体(SEQ ID NOs:5-34)、连接肽(SEQ ID NO:52)和IgG1 Fc LALA(SEQ ID NO:35)。另一种此类形式的示例性结构如图1B所示(Fc-IL-2)，其由两个相同的单体组成，每个单体从N端到C端包含：IgG1 Fc LALA(SEQ ID NO:35)、连接肽(SEQ ID NO:52)和IL-2突变体(SEQ ID NOs:5-34)。
IL-2突变体与Fc融合蛋白的单价形式，共包含一个IL-2突变体分子：其包含两个不同的单体，在其中一个单体中，IL-2突变体融合于Fc其中一个亚基的N端或C端，另一个单体为另一个Fc亚基，两个不对称的Fc亚基之间通过knob和hole突变来促进相互配对。在一个优选的形式中，其包含两个不同的单体，在其中一个单体中，IL-2突变体通过连接肽融合于Fc其中一个亚基的C端，该单体从N端到C端包含IgG1 Fc LALA knob(SEQ ID NO:41)、连接肽(SEQ ID NO:52)和IL-2突变体(SEQ ID NOs:5-34)；以及另一单体为IgG1 Fc LALA hole(SEQ ID NO:42)。该融合蛋白的示例性结构如图2所示。
IL-2突变体与Fc融合蛋白的具体构建过程(以IL-2突变体与Fc的二价融合蛋白形式Fc-IL-2为例)：通过PCR引入限制性内切酶HindIII和XhoI的识别位点，将编码IgG1 Fc LALA的核酸序列通过编码GGGGS的寡核苷酸序列与编码IL-2突变体的核酸序列连接，并构建到pcDNA3.1真核表达载体中，转染293F细胞，37℃、5％CO2、 120rpm培养5天后，收集培养液，先后应用蛋白A亲和层析和凝胶过滤Superdex 200柱层析纯化Fc-IL-2突变体融合蛋白。具体操作步骤如实施例1中所述。
IL-2突变体与Fc融合蛋白的不同形式的活性检测：
应用HEK-BLUETM细胞系实验分别检测验证不同的融合形式是否会对IL-2突变体活性产生影响。HEK-BLUETM具体实验步骤如实施例2中所述。
以第一组IL-2突变体中的(81-85)-mut8和(81-85)-mut10以及第二组IL-2突变体中的S75R83-mut16与Fc融合蛋白的二价形式：Fc-(81-85)-mut8、Fc-(81-85)-mut10和Fc-S75R83-mut16作为示例性融合蛋白进行检测，其包含两个相同的单体，每个单体中IL-2突变体通过连接肽GGGGS融合于Fc LALA的C末端。
同时以上述突变体与Fc融合蛋白的单价形式：Fc knob-(81-85)-mut8/Fc-hole、Fc knob-(81-85)-mut10/Fc-hole，和Fc knob-S75R83-mut16/Fc-hole作为示例性融合蛋白进行检测，其包含两个不同的单体，其中一个单体中包含一个IL-2突变体分子通过连接肽GGGGS融合于Fc LALA knob的C末端，另一个单体为Fc LALA hole。
具体检测结果如表13和表14所示：该结果证明了IL-2突变体与Fc的融合蛋白，无论是二价形式还是单价形式，均没有影响IL-2突变体与IL-2Rβγ或IL-2Rαβγ的亲和力，IL-2突变体与Fc的融合蛋白仍然具有对不同受体结合的偏好性。
表13

(注：*标记的数值是由于这些突变体对IL-2Rβγ细胞的激活能力较低，在实验所用浓
度范围内无法通过拟合得到准确的EC50值)
表14

实施例4：IL-2突变体BIACORE亲和力测定
应用Biacore实验来测定示例性的Fc-IL-2突变体二价融合蛋白Fc-(81-85)-mut8或Fc-S75R83-mut16分别与人IL-2Rβγ和人IL-2Rαβγ的亲和力。其中IL-2Rβγ采用的是由实施例1中制备的IL-2Rβγ-Fc异源二聚体，IL-2Rαβγ采用的是IL-2Rαγ-Fc knob单体与IL-2Rβ-Fc hole单体构成的异源二聚体融合蛋白(IL-2Rαβγ-Fc)。Fc-野生型IL-2二价融合蛋白在本实验中作为对照。具体操作步骤如下所述：用Biacore仪器(Biacore T200，GE)的Protein A传感芯片捕获IL-2Rβγ-Fc或IL-2Rαβγ-Fc，其中IL-2Rβγ-Fc或IL-2Rαβγ-Fc稀释至1μg/mL,以10μL/min的流速持续30秒。然后于芯片表面以30μL/min的流速流经一系列浓度梯度的野生型IL-2或其突变体二价融合蛋白，结合持续120秒，解离持续360秒，利用Biacore仪器实时检测反应信号从而获得结合和解离曲线。Kon、Koff和Kd值如表15所示。
表15
实施例5：IL-2突变体体外生物学活性测定
当IL-2与其细胞表面受体结合时，将激活JAK-STAT信号通路，使STAT5磷酸化。STAT5磷酸化是IL-2信号转导途径中的必要步骤，STAT5被磷酸化后进入细胞核内参与下游的信号转导过程。因此，细胞中磷酸化的STAT5信号能够反映IL-2的活化情况。
CD8+T淋巴细胞表面表达IL-2Rβγ，但不表达IL-2Rα。Treg细胞表面同时表达IL-2Rβγ和IL-2Rα。通过流式细胞术分别测定CD8+T淋巴细胞和Treg细胞中STAT5磷酸化(pSTAT5)的水平，以此间接反应IL-2突变体激活CD8+T淋巴细胞和Treg细胞的活性(例如，可参见文献Peterson LB,et al.J Autoimmun.2018 Dec；95:1-14)。
具体操作步骤如下：PBMC细胞(上海秒顺生物科技有限公司，cat#PB050C)提前一天进行复苏，过夜收集PBMC，用PBS清洗细胞，离心收集。用2％完全培养基混匀后计数，每孔加入60μL细胞悬液，约5～6×105个细胞接种于细胞培养板，放入细胞培养箱，待用。待测样品包括示例性的第一组IL-2突变体与Fc的二价融合蛋白Fc-(81-85)-mut8、Fc-(81-85)-mut10、Fc-(81-85)-mut2、Fc-(81-85)-mut3、Fc-(81-85)-mut9和Fc-(81-85)-mut14以及示例性的第二组IL-2突变体与Fc的二价融合蛋白Fc-S75R83-mut7和Fc-S75R83-mut16。将这些样品分别进行梯度稀释后，加入细胞中刺激30min，加入细胞固定液，清洗细胞后，加入抗CD4抗体(BD cat#562424)、抗CD25抗体(Biolegend cat#356128)和抗CD8抗体(Biolegend cat#300932)，4℃避光孵育30min，清洗细胞后加入150μL/孔预冷的PhosflowTM Perm Buffer III(BD cat#558050)，冰上孵育30min，用FACS缓冲液清洗细胞后，加入100μL/孔用FACS缓冲液稀释的PE Mouse Anti-Stat5(pY694)(BD cat#612567)和抗Foxp3抗体(Biolegend cat#320112)，4℃避光孵育30min。用FACS缓冲液清洗细胞，离心后弃去上清。用100μL/孔的FACS缓冲液重悬细胞后，上机检测。CD8+T淋巴细胞的表面标记物为CD8，在该实验中将CD4-CD8+的细胞定义为CD8+T淋巴细胞。Treg细胞的表面标记物通常包括CD4，CD25和Foxp3，在该实验中将CD4+CD25+Foxp3+的细胞定义为Treg细胞。数据处理:对上述两个细胞群在不同浓度下的IL-2突变体处理后的pSTAT5荧光值(MFI)，使用GraphPad Prism 8.0.1进行统计，并计算EC50值。拟合方法为先将浓度值转换为对数即X＝log(X)，然后按照log(agonist)vs.response-Variable slope(four parameters)拟合曲线，计算Treg细胞和CD8+T淋巴细胞的EC50值。
图3A、3C、3E、3G所示为示例性的第一组IL-2突变体与Fc的二价融合蛋白在Treg细胞中诱导STAT5磷酸化的结果；图3B、3D、3F、3H所示为示例性的第一组IL-2突变体与Fc的二价融合蛋白在CD8+T淋巴细胞中诱导STAT5磷酸化的结果。
图3I所示为示例性的第二组IL-2突变体与Fc的二价融合蛋白在Treg细胞中诱导STAT5磷酸化的结果；图3J所示为示例性的第二组IL-2突变体与Fc的二价融合蛋白在CD8+T细胞中诱导STAT5磷酸化的结果。
由上述结果可以看出：IL-2突变体与Fc二价融合蛋白在Treg细胞中均能够诱导STAT5磷酸化，而在CD8+T淋巴细胞中，IL-2突变体与Fc二价融合蛋白未明显诱导STAT5磷酸化。上述结果进一步说明本发明的IL-2突变体能够选择性地激活Treg细胞，同时不会激活CD8+T淋巴细胞，具有良好的Treg细胞靶向性，相对于野生型IL-2，在人体治疗过程中具有更高的安全性。
实施例6：IL-2突变体在小鼠体内活性测定
为了证明IL-2突变体在啮齿类动物体内对Treg细胞的选择性，通过在小鼠体内单次给予Fc-IL-2突变体二价融合蛋白后，分别检测血液中Treg细胞和CD8+T淋巴细胞的增殖情况。
具体操作如下：C57LB/6小鼠购买自维通利华并饲养在无特殊病原体的环境中。所有动物实验都是依照研究机构动物护理和使用委员会(IACUC)的方案和指导进行的。
实验动物处理：C57BL/6小鼠21只，SPF级，雌性，分成7组，每组3只，每组小鼠分别皮下注射示例性的IL-2突变体与Fc的二价融合蛋白：Fc-S75R83-mut5、Fc-S75R83-mut9、Fc-S75R83-mut16、Fc-(81-85)-mut8、Fc-(81-85)-mut10、或Fc-(81-85)-mut12、Fc-AMG592(SEQ ID NO:106)为本实验中的对照样品。
首先在0天进行单次给药，每只小鼠给药剂量为1mg/kg。在0天给药前、给药后3天分别静脉取血100μL，在EDTA抗凝采血管上标记好动物编号。
应用Trye-Nulear Transcription Factor Buffer Set(Biolegend cat#424401)进行检测。简言之，取80μL血样加入到1mL的1×Lyse/Fix缓冲液(BD Cat#558049)中，混匀后置于37℃恒温箱中30min，然后400g×8min离心后弃上清液。用流式细胞仪检测：首先固定细胞，加入1000μL/管的1×Transcription Factor Fix working solution，室温下避光孵育40min。然后进行细胞膜打孔，400g×8min离心后弃上清液，加2mL/管的1×Perm缓冲液，进行重悬，400g×8min离心后弃上清液；重复细胞膜打孔步骤一次。将管子中的细胞转到V型96孔板中。用1×Perm缓冲液重悬细胞，分别加入抗CD4抗体(Biolegend cat#317429)、抗CD25抗体(Biolegend cat#356128)、抗Foxp3抗体(Biolegend cat#320112)和抗CD8抗体(Biolegend cat#344742)。室温孵育1h后，600g×8min离心，弃上清液。用200μL/孔1％BSA清洗两遍，离心弃上清后用100μL FACS缓冲液重悬，上机检测。
使用流式软件CytExpert进行统计，淋巴细胞群(P1)门内计数30000，分别计算Treg细胞占CD4+细胞群的百分比，以及CD8+T淋巴细胞占总淋巴细胞的百分比。CD8+T淋巴细胞的表面标记物为CD8，将CD8+的细胞定义为CD8+T淋巴细胞。Treg细胞的表面标记物为CD4，CD25和Foxp3，将CD4+CD25+Foxp3+的细胞定义为Treg细胞。
结果如图4A所示，第一组IL-2突变体与Fc的二价融合蛋白：Fc-(81-85)-mut8、Fc-(81-85)-mut10和Fc-(81-85)-mut12在给药剂量为1mg/kg时，均能够在小鼠体内刺激Treg细胞的增殖，并且Fc-(81-85)-mut8、Fc-(81-85)-mut10和Fc-(81-85)-mut12与对照Fc-AMG592(SEQ ID NO:106)相比，其刺激Treg细胞增殖的活性与Fc-AMG592相当或更高。另外，如图4B所示，Fc-(81-85)-mut8、Fc-(81-85)-mut10和Fc-(81-85)-mut12均不会刺激CD8+T淋巴细胞的增殖。这表明其促进体内Treg细胞扩增的特异性。
结果如图4C所示，第二组IL-2突变体与Fc的二价融合蛋白Fc-S75R83-mut5、Fc-S75R83-mut9和Fc-S75R83-mut16在给药剂量为1mg/kg时，能够在小鼠体内刺激Treg细胞的增殖，并且与对照Fc-AMG592相比，Fc-S75R83-mut5、Fc-S75R83-mut9刺激Treg细胞增殖的活性与Fc-AMG592相当，而Fc-S75R83-mut16刺激Treg细胞增殖的活性更高。另外，如图4D所示，Fc-S75R83-mut5、Fc-S75R83-mut9和Fc-S75R83-mut16在刺激Treg细胞增殖的同时，并不会刺激CD8+T淋巴细胞的增殖。
上述结果表明，本发明的IL-2突变体分子在啮齿类动物体内能够特异性地激活Treg细胞，而不会对CD8+T细胞带来影响。
实施例7：IL-2突变体在食蟹猴体内活性测定
为了证明IL-2突变体在灵长类动物体内对Treg细胞的选择性，通过在食蟹猴体内单次给予Fc-IL-2突变体二价融合蛋白后，分别检测血液中Treg细胞和CD8+T淋巴细胞的增殖情况。
具体操作如下：食蟹猴6只，普通级，2.5-3kg，购买自北京中科灵瑞生物技术股份有限公司，饲养在无特殊病原体的环境中。所有动物实验都是依照研究机构动物护理和使用委员会(IACUC)方案和指导进行的。
首先在0天进行单次给药，每只食蟹猴皮下注射示例性的Fc-IL-2突变体二价融合蛋白：Fc-(81-85)-mut8、Fc-(81-85)-mut10、Fc-S75R83-mut5、Fc-S75R83-mut16或对照药物Fc-AMG592(AMGEN,SEQ ID NO:106)，注射剂量分为低剂量组0.3mg/kg，以 及高剂量组1.5mg/kg。其中Fc-(81-85)-mut8、Fc-(81-85)-mut10、Fc-S75R83-mut5和对照药物Fc-AMG592只进行了低剂量组实验，Fc-S75R83-mut16同时进行了低剂量组和高剂量组实验。在给药前0天和给药后7天分别静脉取血1mL。
应用Trye-Nulear Transcription Factor Buffer Set(Biolegend cat#424401)进行检测，具体操作方法如实施例6所述。
结果如图5A-5B所示，第一组IL-2突变体与Fc的二价融合蛋白Fc-(81-85)-mut8和Fc-(81-85)-mut10在低剂量0.3mg/kg施用时，均能够在食蟹猴体内刺激Treg细胞的增殖(图5A)，且与对照药物Fc-AMG592相比，Fc-(81-85)-mut8和Fc-(81-85)-mut10刺激Treg细胞增殖的活性更高。同时并不会刺激CD8+T淋巴细胞增殖，其细胞数量无明显变化(图5B)。
结果如图5C-5D所示，第二组IL-2突变体与Fc的二价融合蛋白Fc-S75R83-mut5在低剂量组0.3mg/kg能够在食蟹猴体内刺激Treg细胞的增殖(图5C)，同时在食蟹猴体内均不会刺激CD8+T淋巴细胞增殖，其细胞的数量无明显变化(图5D)。
Fc-S75R83-mut16无论是在低剂量组0.3mg/kg或高剂量组1.5mg/kg，均能够在食蟹猴体内刺激Treg细胞的增殖，且随着给药剂量的增加，其促进Treg细胞增殖的作用增强(图5C)，同时，无论是低剂量还是高剂量，Fc-S75R83-mut16在食蟹猴体内均不会刺激CD8+T淋巴细胞增殖，其细胞的数量无明显变化(图5D)。
并且与对照Fc-AMG592相比，在相同剂量下，Fc-S75R83-mut5和Fc-S75R83-mut16刺激Treg细胞增殖的活性更高(图5C)。
上述结果表明，本发明的IL-2突变体在灵长类动物体内能够特异性地激活Treg细胞，而不会对CD8+T细胞带来影响，其效果与作为对照的Fc-AMG592相当，甚至更优。
实施例8：IL-2突变体在小鼠体内的PK实验
C57LB/6小鼠购买自维通利华并饲养在无特殊病原体的环境中。所有动物实验都是依照研究机构动物护理和使用委员会(IACUC)方案和指导进行的。
小鼠体内PK实验具体操作如下：将24只C57LB/6小鼠随机分成4组，每组6只，雌雄各半。每组小鼠分别单次尾静脉注射示例性的IL-2突变体与Fc的二价融合蛋白Fc-(81-85)-mut8、Fc-(81-85)-mut10、Fc-S75R83-mut5或Fc-S75R83-mut16，注射剂量均为1mg/kg。分别于给药前0小时、给药后15分钟、5小时、24小时、72小时、168小 时、240小时、336小时、432小时、600小时采集抗凝全血，分离血浆。用ELISA法测定各样本中血浆药物浓度。采用WinNonlin 6.0非房室模型(NCA)计算各动物的药代动力学参数。
示例性的IL-2突变体与Fc的二价融合蛋白Fc-(81-85)-mut8、Fc-(81-85)-mut10、Fc-S75R83-mut5和Fc-S75R83-mut16以1mg/kg静脉注射小鼠后，血药浓度随时间延长逐渐下降。PK实验的结果如图6所示，表明本发明的IL-2突变体与Fc的二价融合蛋白在小鼠体内均具有良好的药代动力学特性(其中Fc-(81-85)-mut8和Fc-S75R83-mut5的PK曲线未显示)。

Claims (63)

  1. 一种IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的缺失或突变。
  2. 根据权利要求1中所述的IL-2突变体，其中所述人野生型IL-2氨基酸序列如SEQ ID NO:1所示。
  3. 根据权利要求1或2中所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL的全部缺失。
  4. 根据权利要求1或2中所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RPL。
  5. 根据权利要求1或2中所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为RHL。
  6. 根据权利要求1或2中所述的IL-2突变体，其包含相对于人野生型IL-2氨基酸序列第81-85位的氨基酸残基RPRDL突变为SPL。
  7. 根据权利要求3中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和N71的突变。
  8. 根据权利要求7中所述的IL-2突变体，其中所述E67和N71的突变分别为E67K和N71S。
  9. 根据权利要求4中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67的突变。
  10. 根据权利要求9中所述的IL-2突变体，其中所述E67的突变为E67A。
  11. 根据权利要求4中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的N71和S75的突变。
  12. 根据权利要求11中所述的IL-2突变体，其中所述N71和S75的突变分别为N71W和S75I。
  13. 根据权利要求5中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和/或V91的突变。
  14. 根据权利要求13中所述的IL-2突变体，其中所述E67的突变为E67T。
  15. 根据权利要求13中所述的IL-2突变体，其中所述V91的突变为V91K。
  16. 根据权利要求13中所述的IL-2突变体，其中所述E67和V91的突变分别为E67T和V91K。
  17. 根据权利要求5中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L70和S75的突变。
  18. 根据权利要求17中所述的IL-2突变体，其中所述L70和S75的突变分别为L70I和S75F。
  19. 根据权利要求5中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的N71的突变。
  20. 根据权利要求19中所述的IL-2突变体，其中所述N71的突变为N71I。
  21. 根据权利要求6中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和N71的突变。
  22. 根据权利要求21中所述的IL-2突变体，其中所述E67和N71的突变分别为E67M和N71S。
  23. 根据权利要求1-22中任一项所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的C125的突变；优选地，所述C125的突变为C125S或C125A。
  24. 一种IL-2突变体，其特征在于，所述突变体包含SEQ ID NOs:5-18中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:5-18中任一所示的氨基酸序列具有至少约90％序列同源性。
  25. 一种IL-2突变体，其包含相对于人野生型IL-2氨基酸序列的第75位氨基酸残基S(S75)和第83位氨基酸残基R(R83)的突变。
  26. 根据权利要求25中所述的IL-2突变体，其中所述人野生型IL-2氨基酸序列如SEQ ID NO:1所示。
  27. 根据权利要求25或26中所述的IL-2突变体，其中S75被突变为不带电荷的氨基酸残基。
  28. 根据权利要求25-27中任一项所述的IL-2突变体，其中所述S75的突变为S75P、S75F、S75Y、S75G、S75A、S75V、S75T、S75I或S75L，R83的突变为R83N、R83F、R83Y、R83P、R83L、R83A、R83K或R83E。
  29. 根据权利要求28中所述的IL-2突变体，其中所述S75的突变为S75P，R83的突变为R83Y或R83A。
  30. 根据权利要求29中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和D84的突变；优选地，所述E67和D84的突变分别为E67A和D84N；更优选的，所述突变体包含突变S75P、R83Y、E67A和D84N。
  31. 根据权利要求29中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16、L70和N71的突变；优选地，所述H16、L70和N71的突变分别为H16D、L70I和N71G；更优选地，所述突变体包含突变S75P、R83A、H16D、L70I和N71G。
  32. 根据权利要求28中所述的IL-2突变体，其中所述S75的突变为S75F，R83的突变为R83P或R83L。
  33. 根据权利要求32中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和/或N71的突变。
  34. 根据权利要求33中所述的IL-2突变体，其中所述H16的突变为H16Q；优选地，所述突变体包含突变S75F、R83P和H16Q。
  35. 根据权利要求33中所述的IL-2突变体，其中所述H16和N71的突变分别为H16N和N71S；优选地，所述突变体包含突变S75F、R83P、H16N和N71S。
  36. 根据权利要求32中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变；优选地，其中所述E67和L70的突变分别为E67R和L70V；更优选地，所述突变体包含突变S75F、R83L、E67R和L70V。
  37. 根据权利要求28中所述的IL-2突变体，其中所述S75的突变为S75I，R83的突变为R83L或R83K。
  38. 根据权利要求37中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E67和L70的突变；优选地，所述E67和L70的突变分别为E67G和L70V，或者E67S和L70V；更优选地，所述突变体包含突变S75I、R83L、E67G和L70V，或包含突变S75I、R83K、E67S和L70V。
  39. 根据权利要求28中所述的IL-2突变体，其中所述S75的突变为S75T，R83的突变为R83A或R83F。
  40. 根据权利要求39中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的H16和E67的突变；优选地，所述H16和E67的突变分别为 H16N和E67K；更优选地，所述突变体包含突变S75T、R83A、H16N和E67K。
  41. 根据权利要求28中所述的IL-2突变体，其中所述S75的突变为S75V，R83的突变为R83N或R83F。
  42. 根据权利要求41中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的E15和H16的突变；优选地，所述E15和H16的突变分别为E15K和H16Q；更优选地，所述突变体包含突变S75V、R83F、E15K和H16Q。
  43. 根据权利要求41中所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的L70和V91的突变；优选地，所述L70和V91的突变分别为L70F和V91K；更优选地，所述突变体包含突变S75V、R83N、L70F和V91K。
  44. 根据权利要求25或26中所述的IL-2突变体，其包含如下突变：
    S75G、R83L、L17I、V91E和I92L；或
    S75Y、R83N、L17I和V91K；或
    S75A、R83K和L17I；或
    S75L、R83E、L63V和E67F。
  45. 根据权利要求25-44中任一项所述的IL-2突变体，其进一步包含相对于人野生型IL-2氨基酸序列的C125的突变；优选地，所述C125的突变为C125S或C125A。
  46. 一种IL-2突变体，其特征在于，所述突变体包含SEQ ID NOs:19-34中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:19-34中任一所示的氨基酸序列具有至少约90％序列同源性。
  47. 根据权利要求1-46中任一项所述的IL-2突变体，与人野生型IL-2相比，所述IL-2突变体与IL-2Rβγ的结合亲和力降低，并且保持与IL-2Rα的结合。
  48. 一种包含权利要求1-47中任一项所述的IL-2突变体的融合蛋白。
  49. 根据权利要求48中所述的融合蛋白，其包含Fc；优选地，其中Fc包含能够改变效应功能的突变，和/或能够延长半衰期的突变，和/或能够促进异源多肽形成二聚体的突变；更优选地，所述Fc来源于人IgG1或人IgG4。
  50. 根据权利要求49中所述的融合蛋白，其中所述Fc包含：
    (i)L234A和L235A突变；和/或
    (ii)N297G突变；和/或
    (iii)N297A突变；和/或
    (iv)L234A、L235A和P331S突变；和/或
    (v)L234A、L235E、G237A、A330S和P331S突变，其中所述编号为EU编号系统。
  51. 根据权利要求49中所述的融合蛋白，其中所述Fc包含S228P、F234A和L235A突变，其中所述编号为EU编号系统。
  52. 根据权利要求49-51中任一项所述的融合蛋白，其中所述Fc进一步包含：
    (i)S354C和T366W突变；和/或
    (ii)Y349C、T366S、L368A、和Y407V突变，其中所述编号为EU编号系统。
  53. 根据权利要求49-52中任一项所述的融合蛋白，其中所述IL-2突变体与Fc连接；优选地，所述IL-2突变体与Fc通过连接肽连接；优选地，所述连接肽包含SEQ ID NOs:43-74中任一所示的氨基酸序列；更优选地，所述连接肽包含氨基酸序列GGGGS(SEQ ID NO:52)。
  54. 根据权利要求49-53中任一项所述的融合蛋白，其中IL-2突变体位于Fc的N端和/或C端。
  55. 一种包含IL-2突变体的融合蛋白，其特征在于，所述融合蛋白包含SEQ ID NOs:76-105中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:76-105中任一所示的氨基酸序列具有至少约80％序列同源性。
  56. 一种编码权利要求1-47中任一项所述的IL-2突变体或权利要求48-55中任一项所述的融合蛋白的核酸。
  57. 一种包含权利要求56中所述核酸的载体。
  58. 一种包含权利要求57中所述载体的宿主细胞。
  59. 一种制备IL-2突变体或包含IL-2突变体的融合蛋白的方法，包括：
    a)在能有效表达IL-2突变体或包含IL-2突变体的融合蛋白的条件下培养权利要求58中所述的宿主细胞；并且
    b)从宿主细胞中获得所表达的IL-2突变体或包含IL-2突变体的融合蛋白。
  60. 一种药物组合物，包含权利要求1-47中任一项所述的IL-2突变体，权利要求48-55中任一项所述的融合蛋白，权利要求56中所述的核酸，权利要求57中所述的载体，权利要求58中所述的宿主细胞，或应用权利要求59中所述方法 制备的IL-2突变体或包含IL-2突变体的融合蛋白以及药学上可接受的载体和/或辅料。
  61. 一种在有需要的个体中治疗和/或预防疾病或病症的方法，包括向个体施用有效量的权利要求1-47中任一项所述的IL-2突变体，或48-55中任一项所述的融合蛋白，或权利要求56中所述的核酸，或权利要求57中所述的载体，或权利要求58中所述的宿主细胞，或应用权利要求59中所述方法制备的IL-2突变体或包含IL-2突变体的融合蛋白，或权利要求60中所述的药物组合物。
  62. 根据权利要求61中所述的方法，其中所述疾病或病症包括炎性疾病或自身免疫性疾病。
  63. 根据权利要求61或62中所述的方法，其中所述疾病或病症包括狼疮、移植物抗宿主病、C型肝炎诱发的血管炎、Ⅰ型糖尿病、Ⅱ型糖尿病、多发性硬化、类风湿性关节炎、特应性疾病、哮喘、炎症性肠病、自身免疫性肝炎、溶血性贫血、风湿热、甲状腺炎、克罗恩病、重症肌无力、肾小球肾炎、斑秃、牛皮癣、白癜风、营养不良性大疱性表皮松解症和白塞病。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020057645A1 (zh) * 2018-09-21 2020-03-26 信达生物制药(苏州)有限公司 新型白介素2及其用途
CN111183149A (zh) * 2017-08-03 2020-05-19 辛索克斯公司 用于治疗自身免疫疾病的细胞因子缀合物
WO2021050554A1 (en) * 2019-09-10 2021-03-18 Synthorx, Inc. Il-2 conjugates and methods of use to treat autoimmune diseases
CN112638406A (zh) * 2018-06-22 2021-04-09 科优基因公司 白介素-2变体及其使用方法
WO2021185361A1 (zh) * 2020-03-19 2021-09-23 信达生物制药(苏州)有限公司 白介素2突变体及其用途
WO2022048640A1 (zh) * 2020-09-04 2022-03-10 江苏先声药业有限公司 Il-2突变体及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111183149A (zh) * 2017-08-03 2020-05-19 辛索克斯公司 用于治疗自身免疫疾病的细胞因子缀合物
CN112638406A (zh) * 2018-06-22 2021-04-09 科优基因公司 白介素-2变体及其使用方法
WO2020057645A1 (zh) * 2018-09-21 2020-03-26 信达生物制药(苏州)有限公司 新型白介素2及其用途
WO2021050554A1 (en) * 2019-09-10 2021-03-18 Synthorx, Inc. Il-2 conjugates and methods of use to treat autoimmune diseases
WO2021185361A1 (zh) * 2020-03-19 2021-09-23 信达生物制药(苏州)有限公司 白介素2突变体及其用途
WO2022048640A1 (zh) * 2020-09-04 2022-03-10 江苏先声药业有限公司 Il-2突变体及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMERSON, S. D. ET AL.: "NMR characterization of interleukin-2 in complexes with the IL-2Ra receptor component, and with low molecular weight compounds that inhibit the IL-2/IL-Ra interaction", PROTEIN SCIENCE, vol. 12, 31 December 2003 (2003-12-31), pages 811 - 822, XP055906902, DOI: 10.1110/ps.0232803 *
MEI, LONGCAN ET AL.: "Site-Mutation of Hydrophobic Core Residues Synchronically Poise Super Interleukin 2 for Signaling: Identifying Distant Structural Effects through Affordable Computations", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 19, 20 March 2018 (2018-03-20), XP055906909, DOI: 10.3390/ijms19030916 *

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