WO2021228253A1 - Protéine mutante pour la prolifération de lymphocytes t régulateurs - Google Patents

Protéine mutante pour la prolifération de lymphocytes t régulateurs Download PDF

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WO2021228253A1
WO2021228253A1 PCT/CN2021/093944 CN2021093944W WO2021228253A1 WO 2021228253 A1 WO2021228253 A1 WO 2021228253A1 CN 2021093944 W CN2021093944 W CN 2021093944W WO 2021228253 A1 WO2021228253 A1 WO 2021228253A1
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mutant
wild
type
cells
amino acid
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胡辉
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上海盖浦生物科技有限公司
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    • 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
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    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • A61P31/12—Antivirals
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P37/02—Immunomodulators
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    • C07—ORGANIC CHEMISTRY
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • 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
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to the field of protein engineering. Specifically, the present invention relates to a novel interleukin-2 (IL-2) mutant and its preparation method. Compared with wild-type IL-2, the interleukin-2 (IL-2) mutant and its binding partner, IL The binding ability of -2 receptor ⁇ subunit and IL-2 receptor ⁇ subunit is decreased, and the corresponding biological activity is maintained, which can better stimulate the proliferation of regulatory T cells.
  • IL-2 interleukin-2
  • IL-2 is a member of the interleukin family produced by activated T cells. It can stimulate the proliferation, development and differentiation of T cells by binding to interleukin 2 receptors on the cell surface.
  • IL-2 receptor IL-2R is composed of three subunits: ⁇ , ⁇ and ⁇ chains. According to its affinity for IL-2, IL-2R can be divided into high affinity receptors ( ⁇ chain complex), medium affinity receptors ( ⁇ chain complex), low affinity receptors (only ⁇ chain or ⁇ chain complex) ) And pseudo-high affinity receptors ( ⁇ chain complex) 4 types. IL-2 can trigger intracellular signal transduction only after binding to high-affinity receptors or medium-affinity receptors.
  • Treg cells regulatory T cells
  • IL-2R ⁇ can be continuously expressed on the surface of Treg cells for a long time
  • Treg cells are more sensitive to IL-2 than NK, Teff and other cells when the body is not stimulated by foreign antigens.
  • IL-2 low-dose IL-2 therapy has been applied to type 1 diabetes (T1D), systemic lupus erythematosus (SLE), chronic graft-versus-host disease (GVHD) And other autoimmune diseases.
  • T1D type 1 diabetes
  • SLE systemic lupus erythematosus
  • GVHD chronic graft-versus-host disease
  • IL-2 has short in vivo half-life, poor stability, short injection treatment window period, and difficult to grasp the safe dose and treatment course for immunosuppression and inflammation at the administration site.
  • Treg Regulatory T cells
  • IL-2 can stimulate T cell proliferation by binding to medium-affinity ⁇ dimers, and it can also stimulate T cell proliferation by binding to high-affinity ⁇ trimers.
  • IL-2 mutants will preferentially stimulate Treg cells with ⁇ receptors than wild-type IL-2, while avoiding stimulation of other types of T cells that mainly express ⁇ receptors Of proliferation.
  • reducing the ability of IL-2 to bind to the intermediate affinity receptor ( ⁇ chain complex) can reduce the stimulating effect of IL-2 on effector T cells, thereby increasing the ratio of Treg cells to effector T cells, and improving the effect of Treg or Treg. Autoimmune response caused by functional impairment.
  • IL-2 interleukin-2
  • the purpose of the present invention is to provide a novel IL-2 mutant.
  • the IL-2 mutant of the present invention can reduce the binding ability of its binding partner IL-2 receptor ⁇ subunit and IL-2 receptor ⁇ subunit, and maintain the corresponding biological activity , Can better stimulate the proliferation of regulatory T cells.
  • the present invention provides an IL-2 mutant. Compared with wild-type IL-2, the amino acid residues of the IL-2 mutant are mutated, which reduces the protein pair of the mutant IL-2. Affinity The affinity of the IL-2 receptor.
  • the medium-affinity IL-2 receptor contains only the IL-2 receptor ⁇ subunit and the IL-2 receptor ⁇ subunit without the IL-2 receptor ⁇ subunit.
  • the IL-2 mutant can increase the ratio of CD3+CD4+FoxP3+ cells to CD3+CD4+FoxP3-.
  • the IL-2 mutant can increase the ratio of CD3+FoxP3+ cells to CD3+FoxP3-.
  • the IL-2 mutant is mutated at the amino acid residue at position 90 corresponding to wild-type IL-2.
  • the amino acid residues of the IL-2 mutant are mutated, thereby increasing artificial glycosylation sites.
  • the glycosylation site is an N sugar site or an O sugar site; preferably an N sugar site.
  • amino acid residues of the IL-2 mutant are mutated compared to wild-type IL-2.
  • the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to the wild-type IL-2 protein: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R , N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C;
  • the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L;
  • the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S;
  • the IL-2 mutant has the following amino acid residue mutation at position 90 corresponding to wild-type IL-2: N90T.
  • the IL-2 mutant has the following amino acid residue mutations at position 3 corresponding to the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P ; Preferred T3A.
  • the IL-2 mutant is mutated at position 125 cys: C125L, C125S, C125A; preferably C125S.
  • the present invention provides a fusion protein or conjugate comprising the IL-2 mutant described in the first aspect and a non-IL-2 functional part.
  • non-IL-2 functional part is selected from the following group:
  • Fc fragments including but not limited to: Fc fragments of human IgG1, IgG2, IgG3, IgG4, and Fc fragment mutants with a homology of more than 90%;
  • HSA Human serum albumin
  • Anti-albumin polypeptide or antibody Anti-albumin polypeptide or antibody
  • CTP Human chorionic gonadotropin ⁇ subunit carboxy terminal peptide
  • Elastin-like peptide ELP
  • the antigen binding portion is:
  • Antibodies or active antibody fragments are provided.
  • the IL-2 mutant and the non-IL-2 functional part in the fusion protein can be directly connected or connected via a linker;
  • the linker can be a repeating sequence of AAA or GS, including But it is not limited to the repetitive sequence of G3S or the repetitive sequence of G4S; for example, (G3S)4.
  • the IL-2 mutant or fusion protein can be further modified as follows to form a conjugate:
  • Hyaluronic acid modification (Hyaluronic acid, HA);
  • PAS Polyamino acid modification
  • the present invention provides a polynucleotide encoding the IL-2 mutant described in the first aspect or the fusion protein or conjugate described in the second aspect.
  • the polynucleotide is DNA or RNA.
  • the present invention provides an expression vector comprising the polynucleotide of the third aspect.
  • the present invention provides a host cell, which contains the expression vector of the fourth aspect, or the genome of the host cell integrates the polynucleotide of the third aspect.
  • the host cell is a eukaryotic cell; preferably yeast, insect cell, animal cell; more preferably animal cell; most preferably mammalian cell, such as Chinese hamster ovary cell.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the IL-2 mutant protein of the first aspect or the fusion protein or conjugate of the second aspect and a pharmaceutically acceptable Of accessories.
  • the present invention provides the use of the IL-2 mutant described in the first aspect or the fusion protein described in the second aspect in the preparation of drugs for autoimmune diseases.
  • the disease is a disease in which IL-2 is used for immunotherapy.
  • the disease is immune disease, human immunodeficiency virus HIV infection, hepatitis C virus HCV infection, rheumatoid arthritis, atopic dermatitis and the like.
  • the immune disease human immunodeficiency virus HIV infection, hepatitis C virus HCV infection, rheumatoid arthritis, atopic dermatitis, etc. are treated by stimulating the immune system or by proliferating immune cells.
  • the present invention provides an IL-2 mutant that has a mutation at the 90th amino acid residue and the 39th amino acid residue corresponding to wild-type IL-2.
  • the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R, N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C;
  • the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L;
  • the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S;
  • the IL-2 mutant has the following amino acid residue mutation at position 90 corresponding to wild-type IL-2: N90T.
  • the IL-2 mutant has the following amino acid residue mutations at position 39 corresponding to wild-type IL-2: M39I, M39L, M39Q, M39A, M39V;
  • the IL-2 mutant has the following amino acid residue mutations at position 39 corresponding to wild-type IL-2: M39I, M39L.
  • the IL-2 mutant preferentially stimulates T regulatory cells compared to wild-type IL-2.
  • the T regulatory cell is a CD25 + T regulatory cell or a T regulatory cell that highly expresses CD25.
  • the present invention provides a fusion protein or conjugate comprising the IL-2 mutant described in the eighth aspect and a non-IL-2 functional part.
  • the non-IL-2 functional part is an Fc fragment.
  • the present invention provides a polynucleotide encoding the IL-2 mutant of the seventh aspect or the fusion protein or conjugate of the eighth aspect; preferably, the The polynucleotide is DNA or RNA.
  • the present invention provides an expression vector comprising the polynucleotide of the tenth aspect.
  • the present invention provides a host cell comprising the expression vector of the eleventh aspect, or the genome of the host cell integrates the polynucleotide of the tenth aspect.
  • the present invention provides a pharmaceutical composition comprising the IL-2 mutant of the seventh aspect or the fusion protein or conjugate of the eighth aspect, and a pharmaceutical composition Accepted excipients.
  • the present invention provides the use of the IL-2 mutant described in the seventh aspect or the fusion protein or conjugate described in the eighth aspect in the preparation of a medicament for the treatment of a disease in an individual.
  • Figure 1 shows the binding capacity of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc to IL-2R ⁇ detected by Biacore;
  • Figure 2 shows the binding capacity of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc to IL-2R ⁇ dimer detected by Biacore;
  • Figure 3 shows how the interleukin-2 mutant and wild-type IL-2 of the present invention stimulate the proliferation of NK92 cells
  • Figure 4 shows the sequence SEQ ID NO: 1-5 used in the present invention
  • Figure 5 shows the proliferation of NK92 cells in response to IL-2-HSA and mutant IL-2
  • Figure 6 shows the phosphorylation level of p-STAT5 in different cell populations
  • Figure 7 shows the ratio of p-STAT5 levels in the FOXP3+/FOXP3-cell population
  • Figure 8 shows the expansion of induced Treg cells (limited to CD3+CD4+CD25+Foxp3+) in mice
  • Figure 9 shows the levels of induced Treg cells (limited to CD3+CD4+CD25+Foxp3+) in mice;
  • Figure 10 shows the expansion of induced Treg cells (limited to CD3+CD4+Foxp3+) in mice
  • Figure 11 shows the levels of induced Treg cells (limited to CD3+CD4+Foxp3+) in mice
  • Figure 12 shows the amplification of induced CD8+Tregs (limited to CD3+CD8+Foxp3+) in mice.
  • Figure 13 shows the sequence SEQ ID NO: 6-18 used in the present invention.
  • the inventors unexpectedly discovered that a new type of IL-2 mutant after site-directed mutation of IL-2 polypeptide can reduce the binding to IL-2R ⁇ dimer, retain its biological activity, and preferentially Stimulates the proliferation of regulatory T cells. Therefore, the IL-2 mutant of the present invention can be used for the treatment of autoimmune diseases, but it does not have various side effects caused by immunotherapy with natural IL-2. The present invention has been completed on this basis.
  • site-directed mutagenesis changes the amino acid residues of the IL-2 polypeptide, thereby changing the binding ability or affinity of IL-2 to its receptor, but can retain biological activity.
  • the IL-2 mutant of the present invention can stimulate the proliferation of regulatory T cells (Treg), and compared with wild-type IL-2, its side effects are also significantly reduced, thereby achieving better therapeutic purposes.
  • the IL-2 mutant of the present invention is preferably expressed in eukaryotic cells and obtained by cell culture. You can choose yeast, insect cells, animal cells, or transgenic animals.
  • the host cells are eukaryotic cells; preferably yeast, insect cells, animal cells; animal cells are preferably mammalian cells, including but not limited to CHO cells, 293 cells, SP/20 cells, and NS0 cells.
  • the IL-2 mutant of the present invention can be obtained by cell-free expression, in vitro synthesis and other technical means.
  • the glycoform of the IL-2 mutant that may be obtained is non-human. Those skilled in the art know that non-human glycoforms can be further transformed into adult glycoforms.
  • prokaryotic expression fermentation or in vitro cell-free synthesis can also be used to obtain IL-2 mutants.
  • the IL-2 mutant of the present invention is mutated at position 90 corresponding to wild-type IL-2. Therefore, in a specific embodiment, the IL-2 mutant of the present invention has the following amino acid residue mutations at position 90 corresponding to the wild-type IL-2 protein: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R, N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C; preferably N90T, N90S, N90V, N90I, N90M, N90L; more preferably N90T, N90S; most preferably N90T.
  • the original O-sugar sites in IL-2 polypeptides can also be eliminated.
  • the removal of O-sugar does not affect the biological activity of IL-2.
  • the structure of O-sugar is complex and analysis is difficult.
  • genetic engineering mutation technology can usually be used to eliminate the glycosylation site. Therefore, the IL-2 mutant of the present invention may have the following amino acid residue mutations at position 3 corresponding to the wild-type IL-2 protein: T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K and T3P; preferably T3A .
  • the IL-2 mutant of the present invention can have the following amino acid residue mutations at position 125 corresponding to the wild-type IL-2 protein: C125L, C125A, C125S; preferably C125S.
  • the present inventors further found that the amino acid residue at position 90 and amino acid 39 corresponding to wild-type IL-2 The residues are mutated.
  • the thus obtained IL-2 mutant preferentially stimulates T regulatory cells; especially CD25 + T regulatory cells or T regulatory cells with high CD25 expression.
  • the IL-2 mutant has the following amino acid residue mutations at position 90 corresponding to wild-type IL-2: N90T, N90S, N90V, N90I, N90M, N90L, N90Y, N90W, N90R, N90A, N90G, N90F, N90H, N90K, N90Q, N90D, N90E, N90P, N90C; preferably N90T, N90S, N90V, N90I, N90M, N90L; more preferably N90T, N90S; most preferably N90T; at the same time, corresponding to the wild type
  • the following amino acid residue mutations occurred at position 39 of IL-2: M39I, M39L, M39Q, M39A, M39V; preferably M39I, M39L.
  • corresponding to has the meaning commonly understood by those of ordinary skill in the art. Specifically, “corresponding to” means that after two sequences are aligned for homology or sequence identity, one sequence corresponds to a designated position in the other sequence. Therefore, for example, “corresponding to wild-type IL-2” means to align a certain amino acid sequence with the amino acid sequence of wild-type IL-2, and find a site on the amino acid sequence that corresponds to wild-type IL-2.
  • conjugate refers to a water-soluble polymer covalently linked to the residues of the mutant IL-2 polypeptide.
  • the non-IL-2 functional part includes but is not limited to: Fc fragment, human serum albumin (HSA), anti-HSA antibodies and fragments, transferrin, human chorionic gonadotropin ⁇ subunit C-terminal peptide (CTP), elastin-like peptide (ELP) and antigen-binding portion, including cytokine or cytokine antibody, specifically interleukin, interferon, tumor necrosis factor superfamily, colony stimulation Factors, chemokines, growth factors, etc.
  • HSA human serum albumin
  • CTP human chorionic gonadotropin ⁇ subunit C-terminal peptide
  • ELP elastin-like peptide
  • antigen-binding portion including cytokine or cytokine antibody, specifically interleukin, interferon, tumor necrosis factor superfamily, colony stimulation Factors, chemokines, growth factors, etc.
  • the IL-2 mutant of the present invention can be directly connected to other non-IL-2 functional parts, or can be connected through a linker.
  • the linker may be a repetitive sequence of AAA or GS, including but not limited to a repetitive sequence of G 3 S or a repetitive sequence of G4S; for example, (G 3 S) 4 .
  • the IL-2 mutant or fusion protein conjugate can also be modified with polyethylene glycol (PEGylation), polysialylated (PSA), saturated fatty acid, and hyaluronic acid (Hyaluronic acid, HA) or polyamino acid modification (proline-alamine-serine polymer, PAS) to form conjugates.
  • PEGylation polyethylene glycol
  • PSA polysialylated
  • saturated fatty acid saturated fatty acid
  • hyaluronic acid Hyaluronic acid, HA
  • PAS polyamino acid modification
  • bispecific antibodies have emerged.
  • Tumor immunotherapy is currently a new direction in the treatment of tumors.
  • Bispecific antibodies can bind two different antigens, so the development prospects in the field of tumor therapy are very broad.
  • Bispecific antibodies were originally prepared by chemical coupling or hybridoma hybridization.
  • IgG type containing Fc region and non-IgG type without Fc region.
  • the structure of IgG-type bispecific antibodies is similar to that of monoclonal antibodies.
  • the relative molecular weight of the protein is relatively large, and the plasma half-life is long.
  • the structure of non-IgG bispecific antibodies is more diverse, the relative molecular weight of the protein is smaller, the tissue permeability is stronger, but the plasma half-life is shorter.
  • the IL-2 mutant of the present invention can be covalently linked to the antibody domain.
  • the antibody domain includes, but is not limited to: IgG type antibodies and non-IgG type antibodies.
  • the antibody domain may be an antibody or active antibody fragments thereof, Fab molecules, scFv molecules and VHH molecules, immunoglobulin molecules, receptor protein molecules or ligand protein molecules; the immunoglobulin The molecule can be an IgG molecule.
  • the IL-2 mutant of the present invention can be directly connected to other non-IL-2 functional parts, or can be connected through a linker.
  • the linker may be a repetitive sequence of AAA or GS, including but not limited to a repetitive sequence of G 3 S or a repetitive sequence of G 4 S; for example, (G 3 S) 4 .
  • the mutants of the present invention can be coupled with T cell surface antigen antibodies, and can also be coupled with tumor cell surface antigen antibodies.
  • the mutants of the present invention can be coupled with T cell surface antigen antibodies.
  • the mutants of the present invention can be coupled with T cell surface antigen antibodies to form bispecific antibodies, and can also be coupled with tumor cell surface antigen antibodies to form bispecific antibodies.
  • the mutants of the present invention can be coupled with T cell surface antigen antibodies to form trispecific antibodies, and can also be coupled with tumor cell surface antigen antibodies to form trispecific antibodies.
  • the mutants of the present invention can be coupled with T cell surface antigen antibodies or tumor cell surface antigen antibodies to form trispecific antibodies.
  • the pharmaceutical composition of the present invention and its administration method
  • the present invention also provides a pharmaceutical composition.
  • the pharmaceutical composition of the present invention comprises the IL-2 mutant of the present invention or the fusion protein or conjugate of claim 5 or the bispecific antibody or trispecific antibody of claim 7 Antibody and optional pharmaceutically acceptable excipients.
  • composition of the present invention further comprises a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipients can be added to the IL-2 mutant polypeptide, fusion protein or conjugate, bispecific antibody or trispecific antibody of the present invention to form a composition.
  • the IL-2 mutant of the present invention can reduce the affinity of the mutant IL-2 protein to the medium-affinity IL-2 receptor, while retaining the biological activity of IL-2, thereby better stimulating regulatory T Cells (Treg) proliferate. Therefore, the IL-2 mutant, fusion protein, conjugate, bispecific antibody or trispecific antibody, and pharmaceutical composition of the present invention can be prepared into corresponding drugs.
  • the drug can be used to expand regulatory T cells (Treg) in vitro or treat diseases that use IL-2 for immunotherapy.
  • the disease is systemic lupus erythematosus (SLE), autoimmune disease, diabetes, human immunodeficiency virus HIV infection, hepatitis C virus HCV infection, rheumatoid arthritis, atopic dermatitis, etc. .
  • SLE systemic lupus erythematosus
  • the IL-2 mutant protein of the present invention reduces the binding to IL-2R ⁇ dimer.
  • the structure of the IL-2 mutant of the present invention is closer to that of natural IL-2, avoiding the influence of mutation on other structural sites of the protein, and retaining biological activity;
  • the interleukin 2 mutant protein of the present invention can be used for the treatment of autoimmune diseases, but it does not have various side effects caused by immunotherapy with natural IL-2.
  • the IL-2 mutant of the present invention has lower immunogenicity
  • the IL-2 mutant of the present invention is convenient for production and quality control, and generally does not require an in vitro re-modification process, reducing steps to improve production efficiency;
  • the IL-2 mutant of the present invention is convenient to form a bifunctional or multifunctional fusion protein or immune composition with other molecules;
  • the IL-2 mutant of the present invention can be used for immunotherapy, but will not cause vascular (or capillary) leakage syndrome (VLS) caused by natural IL-2; and
  • the IL-2 mutants of the present invention preferentially stimulate T regulatory cells; especially CD25 + T regulatory cells or T regulatory cells with high CD25 expression cell.
  • IL-2 mutant IL-2-gmB2-hIgG4Fc SEQ ID NO: 1
  • wild-type IL2-N-hIgG4Fc SEQ ID NO: 2
  • IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc expression vectors 293E cells cultured in Freestyle medium were used for transient transfection and expression of IL-2 mutant molecules.
  • the above-mentioned diluted transfection reagent and plasmid were mixed thoroughly, incubated at room temperature for 15 minutes, then all the mixture was added to the cells, mixed, and incubated in a 37°C 5% CO 2 incubator with a shaker at 120 rpm for 7 days. Collect the cell culture supernatant, filter the supernatant with a 0.22 micron filter membrane, and then purify it with a Q-HP ion exchange chromatography column (GE), using 20mM Tris 0-500mM NaCl, pH 8.0 linear elution, continuous by volume Collect samples. Use 4-20% gradient gel (GenScript) to detect the collected components by SDS-PAGE, and combine the samples according to the electrophoresis purity.
  • GE Q-HP ion exchange chromatography column
  • human IL-2R ⁇ receptor and IL-2R ⁇ heterodimerization receptor were prepared protein.
  • the design of the human IL-2R ⁇ receptor is to link the coding sequence of the extracellular domain of IL-2R ⁇ to the 6 ⁇ His Tag coding sequence (SEQ ID NO: 3) and clone it into a eukaryotic expression vector.
  • 293E cells cultured in Freestyle medium were used for transient transfection and expression of IL-2R ⁇ receptor. Twenty-four hours before transfection, 150 ml of 0.5 ⁇ 10 6 cells/ml 293E cells were inoculated in a 1L cell culture flask and cultured in a 37°C 5% CO2 incubator with a shaker at 120 rpm.
  • the cell culture supernatant was collected, and the supernatant was filtered with a 0.22 micron filter membrane, and then purified using a Ni-NTA affinity chromatography column (GE), and eluted under the condition of 20mM PB-0.5M NaCl-100mM imidazole.
  • the purified protein was detected by SDS-PAGE using 4-20% gradient gel (GenScript).
  • IL-2R ⁇ heterodimerization receptor utilizes the pairing characteristics of CH1 and CL to complete the heterologous pairing.
  • hIL2R ⁇ SEQ ID NO: 4
  • hIL2R ⁇ SEQ ID NO: 5
  • 293E cells cultured in Freestyle medium were used for transient transfection and expression of IL-2R ⁇ heterodimerization receptor. Twenty-four hours before transfection, 150 ml of 0.5 ⁇ 10 6 cells/ml 293E cells were inoculated in a 1L cell culture flask and cultured in a 37°C 5% CO2 incubator with a shaker at 120 rpm.
  • telomeres IL-2R ⁇ heterodimerization receptor
  • hIL2R ⁇ IL-2R ⁇ heterodimerization receptor
  • the recombinant monomer IL-2R ⁇ subunit was used to determine the IL-2 mutant molecule IL-2-gmB2-hIgG4Fc by Biacore 8K (GE) under the following conditions
  • the affinity of wild-type IL2-N-hIgG4Fc to human IL-2R ⁇ subunit Immobilize human IL-2R ⁇ subunit on CM5 chip (190RU).
  • IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc were used as analytes in HBS-EP buffer at 25°C.
  • IL-2R ⁇ the analyte concentration was reduced from 200 nM to 1.526 nM (1:2 dilution), and the flow was 30 ⁇ l/min (binding time 180 seconds, dissociation time 300 seconds).
  • IL-2R ⁇ regeneration is performed with 20mM NaOH, 30ul/min for 10 seconds.
  • RI ⁇ 0, Rmax global fit data.
  • recombinant hIL2R ⁇ , ⁇ ECD-His heterodimer was used to determine the heterodimerization of IL-2 mutant molecules IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc to human IL-2R ⁇ by Biacore 8K (GE) Affinity of the aggregate: IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc were immobilized on the Protein A chip (100RU). Recombinant hIL2R ⁇ , ⁇ ECD-His heterodimer was used as analyte in HBS-EP buffer at 25°C.
  • the analyte concentration is reduced from 100nM to 0.78nM (1:2 dilution), and the flow rate is 30 ⁇ l/min (binding time 180 seconds, dissociation time 300 seconds) .
  • the flow rate is 30 ⁇ l/min (binding time 180 seconds, dissociation time 300 seconds) .
  • 10mM Glycine (pH 1.5) for regeneration 30ul/min, 30 seconds.
  • IL-2-gmB2-hIgG4Fc has a reduced affinity for IL-2R ⁇ dimer compared to wild-type IL2-N-hIgG4Fc. Therefore, IL-2-gmB2-hIgG4Fc retains its affinity with IL-2R ⁇ and decreases its affinity with IL-2R ⁇ dimer relative to wild-type IL2-N-hIgG4Fc.
  • NK92 cells are an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old white male with rapidly progressive non-Hodgkin's lymphoma.
  • the cell surface expresses IL-2 receptor ⁇ , ⁇ and ⁇ chains, and the cell surface markers are the same as those of regulatory T cells. Therefore, the present inventors used NK92 cells to evaluate the activities of IL-2-gmB2-hIgG4Fc and wild-type IL2-N-hIgG4Fc in cell proliferation analysis.
  • the coding sequences of IL-2 mutant IL-2-gmB5-hIgG4Fc (SEQ ID NO: 6) and IL-2-gmB6-hIgG4Fc (SEQ ID NO: 7) including the human IgG4Fc coding sequence were constructed by molecular cloning methods.
  • IL-2 mutants IL-2-gmB2-HSA (SEQ ID NO: 8), IL-2-gmB5-HSA (SEQ ID NO: 9), IL-2 including the human HSA coding sequence were separately obtained by molecular cloning.
  • -2-gmB6-HSA (SEQ ID NO: 10), IL-2-gmB7-HSA (SEQ ID NO: 11), IL-2-gmB8-HSA (SEQ ID NO: 12), IL-2-gmB9- HSA (SEQ ID NO: 13), IL-2-gmB10-HSA (SEQ ID NO: 14), IL-2-HSA wild type (SEQ ID NO: 15), IL-2-gmB11-HSA (SEQ ID NO :16), IL-2-gmB12-HSA (SEQ ID NO: 17), IL-2-gmB13-HSA (SEQ ID NO: 18) coding sequence was constructed into eukaryotic expression vector to prepare IL-2 mutant Expression vector.
  • 293E cells cultured in Freestyle medium were used for transient transfection and expression of IL-2 mutant molecules. Twenty-four hours before transfection, 150 ml of 0.5 ⁇ 10 6 cells/ml 293E cells were inoculated in a 1L cell culture flask, and cultured on a shaker at 120 rpm in a 37°C 5% CO 2 incubator. During transfection, 150 ⁇ l of 293fectin was added to 2.85ml of OptiMEM, mixed thoroughly, and incubated for 2 minutes at room temperature; meanwhile, 150 ⁇ g of plasmids used to express IL-2 molecules were diluted to 3ml with OptiMEM.
  • the above-mentioned diluted transfection reagent and plasmid were mixed thoroughly, incubated at room temperature for 15 minutes, then all the mixture was added to the cells, mixed, and incubated in a 37°C 5% CO 2 incubator with a shaker at 120 rpm for 7 days. Collect the cell culture supernatant, filter the supernatant with a 0.22 micron filter membrane, and then purify it with a Q-HP ion exchange chromatography column (GE), using 20mM Tris 0-500mM NaCl, pH 8.0 linear elution, continuous by volume Collect samples. Use 4-20% gradient gel (GenScript) to detect the collected components by SDS-PAGE, and combine the samples according to the electrophoresis purity.
  • GE Q-HP ion exchange chromatography column
  • NK92 cells are an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of a 50-year-old white male with rapidly progressive non-Hodgkin's lymphoma.
  • the cell surface expresses IL-2 receptor ⁇ , ⁇ and ⁇ chains, and the cell surface markers are the same as those of regulatory T cells. Therefore, the inventors used NK92 cells to evaluate the activity of IL-2 mutants and wild-type IL2-HSA in cell proliferation analysis.
  • Example 4 The method is the same as in Example 4. The results are shown in Figure 5; cell proliferation analysis was used to measure the activity of IL-2 mutant and wild-type IL2-HSA, and it was found that all test products induced the growth of NK92 cells in a dose-dependent manner. Therefore, compared to wild-type IL2-HSA, IL-2gmB2-HSA, IL-2gmB5-HSA, IL-2gmB6-HSA, IL-2gmB7-HSA, IL-2gmB8-HSA, IL-2gmB9-HSA, IL-2gmB10- HSA retains biological activity.
  • PBMCs Collect blood from healthy people into blood collection tubes containing heparin and separate PBMCs. Resuspend PBMCs in complete medium (RPMI-1640 containing 10% FBS) and inoculate them in 96 wells with a cell number of 1*10 6 /50 ⁇ l/well In the board. Dilute wild-type IL2-HSA, IL-2gmB2-HSA, IL-2gmB5-HSA, IL-2gmB6-HSA with complete medium, starting at a final concentration of 2000nM, 10 times dilution, 6 gradients, 50 ⁇ l/well to stimulate PBMC.
  • complete medium RPMI-1640 containing 10% FBS
  • Treg cells are defined as CD3 + CD4 + CD25 + Foxp3 +
  • CD4 + T cells are defined as CD3 + CD4 +
  • CD8 + T cells are defined as CD3 + CD8 +
  • NK cells are defined as CD3 ⁇ /CD56 + .
  • IL-2gmB2-HSA, IL-2gmB5-HSA and IL-2gmB6-HSA compared to the wild-type IL-2-HSA protein on CD4 + T, CD8 + T and The signal strength of NK is weakened. This is because the three mutant proteins in this experiment reduced their affinity with IL-2R ⁇ dimer.
  • the signal intensity of IL-2gmB2-HSA, IL-2gmB5-HSA and IL-2gmB6-HSA relative to the wild-type IL-2-HSA protein to Treg is equivalent at 2-200nM.
  • the signal intensity of the three mutants at 2000 nM was significantly higher than that of wild-type IL-2-HSA. This indicates that the mutant IL-2 has a stronger ability to stimulate Treg proliferation than the wild-type IL-2-HSA.
  • the MFI of CD3 + CD4 + Foxp3 + in CD4 + T cells was compared with the MFI of CD3 + CD4 + Foxp3 - .
  • IL-2gmB2-HSA, IL-2gmB5-HSA and IL-2gmB6-HSA stimulate the FoxP3 activation signal in CD4 + T cells to be stronger.
  • IL-2gmB2-HSA and IL-2gmB5 are higher than 0.2nM.
  • -HSA and IL-2gmB6-HSA stimulate the FoxP3 activation signal in T cells to be stronger.
  • mutant IL-2-HSA is more inclined to stimulate the activation of Foxp3 + cell population than the wild-type IL-2-HSA.
  • mice In order to determine whether IL-2 mutants affect T cell responses in vivo, the IL-2 mutant fusion protein was administered to mice and different T cell subpopulations were monitored.
  • the mice were administered by subcutaneous injection on day 0 and day 7. On the 10th day, the T cell subsets in the blood were determined by flow cytometry.
  • the cells were then fixed with 1 ⁇ Foxp3 Fixation/Permeabilization Solution (invitrogen) at room temperature for 30 minutes, and then washed with 1 ⁇ Permeabilization Buffer (invitrogen) for permeabilization, and centrifuged to discard the supernatant; use FOXP3 (BD) flow cytometry antibody and isotype control antibody (Invitrogen) Perform nuclear molecular staining, wash and centrifuge, discard the supernatant, resuspend the cells in Staining buffer, and finally use flow cytometry to detect and analyze different T cell subpopulations.
  • 1 ⁇ Foxp3 Fixation/Permeabilization Solution invitrogen
  • 1 ⁇ Permeabilization Buffer invitrogen
  • the Treg cells of the G2 group and the G3 group (defined as CD3 + CD4 + CD25 + Foxp3 + ) accounted for the proportion of CD3 + CD4
  • the proportion of + is higher, and the proportion of G3 is higher than that of G2.
  • the ratio of Treg cells (limited to CD3 + CD4 + CD25 + Foxp3 + ) in the G4 group to CD3 + CD4 + was slightly lower than that in the G1 group.
  • CD3 + CD4 + CD25 + Foxp3 + can inhibit the function and migration of DCs, effector T cells (Th1, Th2, Th17 cells) in allergic reactions, and can inhibit CD8 + T cells that mediate inflammation.
  • the Treg cells (defined as CD3 + CD4 + Foxp3 + ) in the G2 group, G3 group and G4 group accounted for the proportion of CD3 + CD4 + has a higher proportion, and the proportion of G3 is higher than that of G2, and the proportion of G2 is higher than that of G4.
  • CD8 + Tregs (limited to CD3 + CD8 + Foxp3 + ).
  • This type of T cell is produced by naive CD8 + T cells in the presence of IL-4 and IL-12.
  • the main role of the reaction is to inhibit the activation of naive and effector T cells, inhibit the IgG/IgE antibody response, and inhibit the expression of IL-4.
  • the proportion of CD8 + Tregs (limited to CD3 + CD8 + Foxp3 + ) in G2
  • G3 and G4 groups to CD8 + T cells (limited to CD3 + CD8 + ) Higher, and G4>G3>G2>G1. It shows that the higher the proportion of CD3 + CD8 + Foxp3 + /CD3 + CD8 + , the stronger the ability to inhibit the activation of naive and effector T cells.
  • mice In vivo evaluation in mice showed that the mutant IL-2gmB2 group preferentially amplifies Treg proliferation, which is better than the IL-2 wild-type group, showing a good ability to directionally amplify Treg.

Abstract

La présente invention concerne un mutant d'IL-2, une protéine de fusion ou un conjugué comprenant le mutant d'IL-2, et une composition pharmaceutique comprenant le mutant d'IL-2, la protéine de fusion ou le conjugué. Par comparaison avec l'IL-2 de type sauvage, le mutant d'IL-2 a une liaison réduite au dimère d'IL-2Rβγ, conserve son activité biologique, et stimule de préférence la prolifération de lymphocytes T régulateurs. Le mutant d'IL-2 peut être utilisé pour traiter des maladies auto-immunes, et ne présente pas plusieurs effets secondaires provoqués par l'immunothérapie par utilisation de l'IL-2 naturelle.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990010070A1 (fr) * 1989-02-28 1990-09-07 The Du Pont Merck Pharmaceutical Company Analogues d'il-2 contenant des sites de glycosylation a liaison n
WO2003015697A2 (fr) * 2001-08-13 2003-02-27 University Of Southern California Mutants d'interleukine-2 a toxicite reduite
WO2020057645A1 (fr) * 2018-09-21 2020-03-26 信达生物制药(苏州)有限公司 Nouvelle interleukine 2 et son utilisation
CN111944036A (zh) * 2019-05-14 2020-11-17 上海盖浦生物科技有限公司 一种增殖免疫细胞的突变体蛋白
CN111944008A (zh) * 2019-05-14 2020-11-17 上海盖浦生物科技有限公司 一种突变蛋白的方法以及得到的突变体蛋白

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990010070A1 (fr) * 1989-02-28 1990-09-07 The Du Pont Merck Pharmaceutical Company Analogues d'il-2 contenant des sites de glycosylation a liaison n
WO2003015697A2 (fr) * 2001-08-13 2003-02-27 University Of Southern California Mutants d'interleukine-2 a toxicite reduite
WO2020057645A1 (fr) * 2018-09-21 2020-03-26 信达生物制药(苏州)有限公司 Nouvelle interleukine 2 et son utilisation
CN111944036A (zh) * 2019-05-14 2020-11-17 上海盖浦生物科技有限公司 一种增殖免疫细胞的突变体蛋白
CN111944008A (zh) * 2019-05-14 2020-11-17 上海盖浦生物科技有限公司 一种突变蛋白的方法以及得到的突变体蛋白

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