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WO2025148785A1 - Variants d'il-2 modifiés et leurs procédés d'utilisation - Google Patents

Variants d'il-2 modifiés et leurs procédés d'utilisation

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Publication number
WO2025148785A1
WO2025148785A1 PCT/CN2025/070316 CN2025070316W WO2025148785A1 WO 2025148785 A1 WO2025148785 A1 WO 2025148785A1 CN 2025070316 W CN2025070316 W CN 2025070316W WO 2025148785 A1 WO2025148785 A1 WO 2025148785A1
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WIPO (PCT)
Prior art keywords
seq
amino acid
corresponds
engineered
polypeptide
Prior art date
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Pending
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PCT/CN2025/070316
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English (en)
Inventor
Chiu-Yueh SHIH
I-Yin CHEN
Yi-Shin LIAO
I-Chun Chen
Ya-Ti FAN
Yi-Hsuan Lu
Yi-Jing Chen
Tsai-Kuei Shen
Yung-Chu Lin
Yen-Cheng Li
Pan-Hsien KUO
Yu Chen Wu
Ying-Shao Lu
Chieh-Hsin HO
Yu-Chien Chen
Tzu-Lin KUO
Rui-ling LIANG
Ting-Yu Kuo
Yun-Chih CHENG
Chi-Ling Tseng
Zong Sean Juo
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Fbd Biologics Ltd
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Fbd Biologics Ltd
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Application filed by Fbd Biologics Ltd filed Critical Fbd Biologics Ltd
Publication of WO2025148785A1 publication Critical patent/WO2025148785A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7023—(Hyper)proliferation
    • G01N2800/7028—Cancer

Definitions

  • Tregs Regulatory T cells
  • Tregs suppress activation of the immune system and thereby regulate the self-tolerance of the immune system.
  • therapies that can enhance Treg activity may be useful in the treatment of autoimmune diseases.
  • cytokine e.g., IL-2
  • methods for screening a cytokine that can selectively activate Treg cells while minimizing the activation of Tconv cells.
  • the amino acid that corresponds to position 3 of SEQ ID NO: 2 is A
  • the engineered IL-2 polypeptide comprises one or more of the following:
  • the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any one of SEQ ID NOs: 3-17 and 35-51.
  • the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 90%identical to any one of SEQ ID NOs: 39, 40, 46, 48, 50, and 51.
  • the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 90%identical to SEQ ID NO: 43.
  • the engineered IL-2 polypeptide can selectively activate Treg cells while minimizing the activation of Tconv cells.
  • the disclosure is related to an engineered IL-2 polypeptide, wherein the engineered IL-2 polypeptide comprises one or more non-native disulfide bonds.
  • the engineered IL-2 polypeptide comprises one or more of the following:
  • the disclosure is related to a fusion protein comprising the engineered IL-2 polypeptide described herein.
  • the fusion protein comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any one of SEQ ID NOs: 20-34 and 52-68.
  • the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 71.
  • the engineered IL-2 polypeptide is linked to either the hole chain or the knob chain. In some embodiments, the engineered IL-2 polypeptide is linked to the hole chain.
  • the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 87 or SEQ ID NO: 88.
  • the fusion protein comprises a hinge region.
  • the hinge region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 70.
  • the engineered IL-2 polypeptide is linked to the C-terminus of the Fc region via a linker peptide. In some embodiments, the engineered IL-2 polypeptide is linked to the N-terminus of the Fc region via a linker peptide.
  • the hinge region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 70; wherein the Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 71; wherein the linker peptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 72; and/or wherein the engineered IL-2 polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any one of SEQ ID NOs: 3-17 and 35-51.
  • the fusion protein can increase Treg activation (e.g., phosphorylation of STAT5) .
  • the fusion protein can increase Treg-mediated immune suppression.
  • the disclosure is related to a pharmaceutical composition
  • a pharmaceutical composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein; and a pharmaceutically acceptable carrier.
  • the disclosure is related to a vector comprising the nucleic acid described herein.
  • the disclosure is related to a cell comprising the nucleic acid described herein or the vector described herein.
  • the disclosure is related to a method of producing an engineered IL-2 polypeptide or a fusion protein comprising the engineered IL-2 polypeptide, the method comprising culturing the cell described herein under conditions sufficient for the cell to produce the engineered IL-2 polypeptide or the fusion protein; and collecting the engineered IL-2 polypeptide or the fusion protein produced by the cell.
  • the disclosure is related to a method of treating a subject having an immune disease, the method comprising administering a therapeutically effective amount of a composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein, to the subject.
  • the subject has an autoimmune disease.
  • the autoimmune disease is rheumatoid arthritis, Crohn’s disease, diabetes (e.g., Type I) , graft versus host disease (GvHD) , systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel diseases (IBD) , ulcerative colitis, scleroderma, allergy, asthma, or atopic dermatitis.
  • diabetes e.g., Type I
  • GvHD graft versus host disease
  • IBD inflammatory bowel diseases
  • ulcerative colitis scleroderma
  • allergy asthma
  • asthma or atopic dermatitis.
  • the fusion protein described herein further comprises a His tag.
  • the His tag comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 18.
  • the engineered IL-2 polypeptide is linked to the C-terminus of the His tag.
  • the disclosure is related to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein, to the subject.
  • the subject has a solid tumor or a hematologic cancer.
  • the cancer is breast cancer, ovarian cancer, glioma, colon cancer, hepatocellular carcinoma, prostate cancer, renal cell carcinoma, melanoma, lung cancer, pancreatic cancer, or hepatoma.
  • the disclosure is related to a method of decreasing the rate of tumor growth, the method comprising contacting a tumor cell with an effective amount of a composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein.
  • the disclosure is related to a method of killing a tumor cell, the method comprising contacting a tumor cell with an effective amount of a composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein.
  • the disclosure is related to a method of increasing regulatory T cell proliferation in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein.
  • the disclosure is related to a method of activating regulatory T cells in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the engineered IL-2 polypeptide described herein or the fusion protein described herein.
  • the non-native cysteine and/or the one or more non-native disulfide bonds stabilize the internal core of the IL-2 polypeptide or the fusion protein described herein.
  • the non-native cysteine and/or the one or more non-native disulfide bonds increase the thermal stability of the IL-2 polypeptide or the fusion protein described herein.
  • the non-native cysteine and/or the one or more non-native disulfide bonds increase the serum concentration of the IL-2 polypeptide or the fusion protein. In some embodiments, the non-native cysteine and/or the one or more non-native disulfide bonds increase the pharmacokinetics of the IL-2 polypeptide or the fusion protein.
  • the non-native cysteine and/or the one or more non-native disulfide bonds increase the aggregation (T agg ) and/or onset (T onset ) temperatures by at least at least 5°C.
  • the non-native cysteine and/or the one or more non-native disulfide bonds increase the serum concentration by at least 10 times. In some embodiments, the non-native cysteine and/or the one or more non-native disulfide bonds increase the pharmacokinetics by at least 10 times.
  • the disclosure is related to a method of improving the stability of IL-2, comprising
  • the mutating the two selected amino acid residues to cysteines does not substantially change the 3D structure of IL-2.
  • the method further comprises:
  • non-native cysteine refers to a cysteine mutation, wherein a cysteine amino acid residue substitutes a non-cysteine amino acid residue in a wildtype protein, or is inserted at a position wherein a cysteine amino acid residue does not naturally exist in that position.
  • the non-native cysteine is introduced by substitution or insertion.
  • at least one or two cysteine residues are introduced by substitution.
  • the protein or the polypeptide may have additional mutations.
  • non-native disulfide bond refers to a disulfide bond that does not naturally exist in a wild-type protein.
  • the non-native disulfide bond is formed by two cysteine residues, wherein at least one of them is a mutation. In some embodiments, two of them are mutations. In some embodiments, at least one or two cysteine residues are introduced by insertion. In some embodiments, a deletion changes the distance between two existing cysteine residues, which then forms a disulfide bond that does not exist in a wild-type protein.
  • FIG. 1C shows a summary of production, purification and analysis of proteins H8-IL-2_C21, H8-IL-2_C22, H8-IL-2_C23, and H8-IL-2_C24 in Expi293 cells.
  • FIGS. 4A-4B show IL-2-pSTAT5 reporter signaling activities induced by H8-IL-2_WT_T3A, H8-IL-2_C01_T3A, H8-IL-2_C01#1, and H8-IL-2_C01#2 in HEK-Blue TM IL-2 Cells (FIG. 4A) and HEK-Blue TM CD122/CD132 Cells (FIG. 4B) , respectively.
  • FIGS. 4C-4D show IL-2-pSTAT5 reporter signaling activities induced by H8-IL-2_C01_T3A, H8-IL-2_C01#3, H8-IL-2_C01#4, H8-IL-2_C01#5, H8-IL-2_C01#6, and H8-IL-2_C01#7 in HEK-Blue TM IL-2 Cells (FIG. 4C) and HEK-Blue TM CD122/CD132 Cells (FIG. 4D) , respectively.
  • FIGS. 4E-4F show IL-2-pSTAT5 reporter signaling activities induced by H8-IL-2_C01_T3A, H8-IL-2_C01#8, H8-IL-2_C01#9, H8-IL-2_C01#10, H8-IL-2_C01#11, and H8-IL-2_C01#12 in HEK-Blue TM IL-2 Cells (FIG. 4E) and HEK-Blue TM CD122/CD132 Cells (FIG. 4F) , respectively.
  • FIGS. 4G-4H show IL-2-pSTAT5 reporter signaling activities induced by H8-IL-2_C01_T3A, H8-IL-2_C01#13, H8-IL-2_C01#14, and H8-IL-2_C01#15 in HEK-Blue TM IL-2 Cells (FIG. 4G) and HEK-Blue TM CD122/CD132 Cells (FIG. 4H) , respectively.
  • FIG. 5 shows a summary of IL-2-pSTAT5 reporter signaling activities induced by H8-IL-2_C01_T3A-derived variants in HEK-Blue TM IL-2 Cells and HEK-Blue TM CD122/CD132 Cells, respectively.
  • FIGS. 6A-6B show STAT5 phosphorylation induced by H8-IL-2_WT_T3A, H8-IL-2_C01_T3A, H8-IL-2_C01#1, H8-IL-2_C01#6, H8-IL-2_C01#7, and H8-IL-2_C01#9 in Treg cells (FIG. 6A) and Tconv cells (FIG. 6B) , respectively.
  • FIGS. 6E-6F show STAT5 phosphorylation induced by H8-IL-2_WT_T3A, H8-IL-2_C01_T3A, H8-IL-2_C01#2, H8-IL-2_C01#5, H8-IL-2_C01#8, H8-IL-2_C01#11, and H8-IL-2_C01#13 in Treg cells (FIG. 6E) and Tconv cells (FIG. 6F) , respectively.
  • FIG. 7 shows a summary of IL-2-pSTAT5 reporter signaling and STAT5 phosphorylation induced by H8-IL-2_C01_T3A and H8-IL-2_C01_T3A-derived variants.
  • FIG. 8 shows a summary of production, purification and analysis of G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, and G1LALAFc-IL-2_C01_T3A-derived variants in Expi293 cells.
  • FIGS. 9E-9F show IL-2-pSTAT5 reporter signaling activities induced by G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, G1LALAFc-IL-2_C01#12, G1LALAFc-IL-2_C01#14, and G1LALAFc-IL-2_C01#15 in HEK-Blue TM IL-2 Cells (FIG. 9E) and HEK-Blue TM CD122/CD132 Cells (FIG. 9F) , respectively.
  • FIG. 10 shows a summary of IL-2-pSTAT5 reporter signaling induced by IL-2_C01_T3A and its variants (in H8 format and G1LALAFc format) .
  • FIGS. 11A-11B show STAT5 phosphorylation induced by G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, G1LALAFc-IL-2_C01#1, G1LALAFc-IL-2_C01#6, G1LALAFc-IL-2_C01#7, and G1LALAFc-IL-2_C01#9 in Treg cells (FIG. 11A) and Tconv cells (FIG. 11B) , respectively.
  • FIGS. 11C-11D show STAT5 phosphorylation induced by G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, G1LALAFc-IL-2_C01#3, G1LALAFc-IL-2_C01#4, G1LALAFc-IL-2_C01#10, G1LALAFc-IL-2_C01#12, G1LALAFc-IL-2_C01#14, and G1LALAFc-IL-2_C01#15 in Treg cells (FIG. 11C) and Tconv cells (FIG. 11D) , respectively.
  • FIGS. 12A-12C show the stress stability of the engineered IL-2 variants and protein constructs thereof as determined by SEC-HPLC.
  • FIG. 13 shows the thermal stability of the engineered IL-2 variants and protein constructs thereof as determined by Dynamic Light Scattering (DLS) analysis.
  • DLS Dynamic Light Scattering
  • FIG. 14 shows the thermal stability of the engineered IL-2 variants and protein constructs thereof as determined by Differential Scanning Fluorimetry (DSF) analysis.
  • DSF Differential Scanning Fluorimetry
  • FIGS. 15A-15B show the treatment plan and dosing schedule (FIG. 15A) and the results (FIG. 15B) of the autoimmune efficacy of the engineered IL-2 variants and protein constructs thereof as determined by an NPG expansion model.
  • FIG. 16A is a table showing treatment plan and dosing schedule of the engineered IL-2 variants and protein constructs thereof in a C57BL/6 mice model.
  • FIG. 16B shows average body weight change of C57BL/6 mice that were treated with engineered IL-2 variants and protein constructs thereof.
  • FIGS. 17A-17B show the liver damage of the engineered IL-2 variants and protein constructs thereof as determined by AST (FIG. 17A) and the ALT (FIG. 17B) .
  • FIGS. 18A-18D show the percentages of different T cell populations induced by the engineered IL-2 variants and protein constructs thereof, as determined by flow cytometry.
  • FIG. 19 lists relevant amino acid sequences.
  • Human IL-2 (NCBI reference sequence: NP_000577.2; UniProt ID: Q0GK43; SEQ ID NO: 1) is a 153 amino acid protein that includes, from N-terminus to C-terminus, a signal peptide (corresponding to amino acids 1-20 of SEQ ID NO: 1) , and a soluble chain (corresponding to amino acids 21-153 of SEQ ID NO: 1) .
  • IL-2 is a member of a cytokine family, each member of which has a four-alpha helix bundle.
  • the family also includes IL-4, IL-7, IL-9, IL-15 and IL-21.
  • IL-2 signals through the IL-2 receptor, a complex having three chains, i.e., interleukin-2 receptor alpha chain (or IL-2RA, CD25) , Interleukin-2 receptor subunit beta (or IL-2RB, CD122) and interleukin-2 receptor subunit gamma (or IL-2RG, common gamma chain, CD132) .
  • the gamma chain is shared by all family members.
  • IL-2 has essential roles in key functions of the immune system, tolerance and immunity, primarily via its direct effects on T cells. For example, in the thymus, where T cells mature, it prevents autoimmune diseases by promoting the differentiation of certain immature T cells into regulatory T cells, which suppress other T cells that are otherwise primed to attack normal healthy cells in the body. IL-2 also promotes the differentiation of T cells into effector T cells and into memory T cells when the initial T cell is also stimulated by an antigen, thus helping the body fight off infections.
  • IL-2 can stimulate immune response by regulating effector T cells (primarily expressing IL-2RB and IL-2RG) , and dampen immune response by regulating regulatory T cells (primarily expressing IL-2RA, IL-2RB, and IL-2RG) .
  • the trimeric IL-2 receptor complex IL-2R ⁇ comprises three subunits: ⁇ (CD25) , ⁇ (CD122) , and ⁇ ( ⁇ common) .
  • the dimeric IL-2R ⁇ comprises two subunits: ⁇ (CD122) , and ⁇ ( ⁇ common) .
  • the trimeric IL-2 receptor complex IL-2R ⁇ is typically expressed at high levels by Treg cells, whereas the dimeric IL-2R ⁇ is expressed mostly on activated CD8+ T cells and NK cells. Low-dose IL-2 therapy preferentially activates Treg cells because of the constitutive high expression of IL-2R ⁇ .
  • the present disclosure provides engineered IL-2 variants having at least one non-native cysteine.
  • one or two amino acid residues in a wild-type human IL-2 are selectively mutated to cysteines.
  • the mutations do not substantially change the overall structure of IL-2, e.g., the relative position of the four alpha helices of IL-2.
  • the engineered IL-2 variants described herein can bind to the IL-2 receptor, and induce downstream signaling (e.g., STAT5 phosphorylation) .
  • the engineered IL-2 variants can selectively activate Treg cells while minimizing the activation of effector T cells.
  • the engineered IL-2 variants comprise or consist of any of the engineered IL-2 polypeptides described herein.
  • the engineered IL-2 variants further comprise a fragment crystallizable region (Fc region) .
  • the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.
  • effector function of a Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • effector function of a Fc region is phagocytosis.
  • the engineered IL-2 variants as described herein have an Fc region without effector function.
  • the Fc is a human IgG1 Fc. In some embodiments, the Fc does not have a functional Fc region.
  • the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .
  • the LALA mutation can reduce binding to the IgG Fc receptors Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII as well as to complement component C1q.
  • the engineered IL-2 variants comprise G1LALAFc (e.g., SEQ ID NO: 71) , an IgG1 Fc containing the LALA mutation.
  • the Fc region can be further linked to a hinge region (e.g., SEQ ID NO: 70) .
  • the Fc region comprises a hole chain comprising G1LA-HoleFc (e.g., SEQ ID NO: 93) and a hinge region (e.g., SEQ ID NO: 70) .
  • the Fc region comprises an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 94.
  • the Fc region comprises a knob chain comprising G1LA-KnobFc (e.g., SEQ ID NO: 95) and a hinge region (e.g., SEQ ID NO: 70) .
  • the Fc region comprises an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 96.
  • G1LA-KIHFc-IL-2_C01#6 which contains a hole chain comprising G1LA-HoleFc-IL-2_C01#6 (e.g., SEQ ID NO: 98) and a knob chain comprising G1 Hinge + G1LA-KnobFc (e.g., SEQ ID NO: 96) .
  • the engineered IL-2 variant further comprises a His tag (e.g., SEQ ID NO: 18) .
  • IL-2 is a typical four– ⁇ helix cytokine, tightly regulated at the mRNA level by signals from the T cell receptor and CD28.
  • IL-2 binds to and signals through a receptor complex consisting of three distinct subunits designated IL-2R ⁇ (CD25) , IL-2R ⁇ (CD122) , and common ⁇ chain ( ⁇ c; CD132) .
  • IL-2R ⁇ CD25
  • IL-2R ⁇ CD122
  • ⁇ c common ⁇ chain
  • the high affinity of the IL-2/IL-2R ⁇ / ⁇ trimeric complex clearly indicates that the complex is more stable than IL-2 bound to either the ⁇ chain alone or to the ⁇ chain alone.
  • the IL-2/ ⁇ / ⁇ trimer recruits the ⁇ chain into a quaternary complex, leading to intracellular signaling.
  • binding of IL-2 may induce conformational changes in the ⁇ chain that would further promote recruitment of the ⁇ c chain.
  • the ⁇ chain does not participate in signaling, whereas both the ⁇ and ⁇ c chains are necessary for signaling.
  • IL-2R ⁇ and ⁇ c can form an intermediate affinity receptor that is fully competent to signal.
  • the high-affinity receptor is the most physiologically relevant form of the IL-2R because CD25-deficient mice (which express only the intermediate-affinity IL-2R) are phenotypically indistinguishable from IL-2-deficient mice, as noted above.
  • CD25-deficient mice which express only the intermediate-affinity IL-2R
  • the structure of IL-2 and its receptor is flexible and can naturally exist in different conformations that appear to favor either the high-affinity trimeric IL-2R or the intermediate-affinity dimeric IL-2R, respectively, resulting in the activation of different immune cells.
  • distance of one or more amino acid residues in the 3D structure can be determined.
  • two amino acid residues whose distance is within 3.0-8.0 angstroms can be selected.
  • the two selected amino acid residues have a distance of to each other.
  • neither of the two selected amino acid residues is a cysteine, and the two selected amino acid residues can be mutated to cysteines without interfering the overall structure (e.g., the core structure formed by the four-helical bundle) of IL-2.
  • the newly introduced cysteines can form a disulfide bond non-native to the wild-type IL-2, which can stabilize IL-2 and/or improve the functional potencies of IL-2.
  • only one of the two selected amino acid residues is a cysteine, and the other selected amino acid residue (the non-cysteine amino acid residue) can be mutated to a cysteine without interfering the overall structure (e.g., the core structure formed by the four-helical bundle) of IL-2.
  • the newly introduced cysteine can form a disulfide bond non-native to the wild-type IL-2, which can stabilize IL-2 and/or improve the functional potencies of IL-2.
  • the engineered IL-2 variant (e.g., engineered IL-2 polypeptide) includes a first cysteine mutation and a second cysteine mutation.
  • the first cysteine mutation occurs at an amino acid residue that corresponds to position 7, 11, 40, 43, 45, 46, 48, 53, or 56 of SEQ ID NO: 2.
  • the second cysteine mutation occurs at an amino acid residue that corresponds to position 129, 113, 111, 108, 106, 96, 97, or 132 of SEQ ID NO: 2.
  • the engineered IL-2 variant (e.g., engineered IL-2 polypeptide) includes a third cysteine mutation, such that the third introduced cysteine can form a disulfide bond with either an introduced cysteine or a naturally existing cysteine.
  • the third cysteine mutation occurs at an amino acid residue that corresponds to position 11, 18, 129, 7, or 132 of SEQ ID NO: 2.
  • the engineered IL-2 polypeptides described herein can also include a mutation at a position corresponding to position 95 (e.g., E95) of SEQ ID NO: 2.
  • the engineered IL-2 polypeptides described herein can also include a E95Q or E95N mutation.
  • an engineered IL-2 polypeptide IL-2_C01#13 which includes an alanine mutation at a position corresponding to position 3 (e.g., T3) of SEQ ID NO: 2, an I92M mutation with respect to SEQ ID NO: 2, a first cysteine residue at a position corresponding to position 11 (e.g., Q11) of SEQ ID NO: 2, and a second cysteine residue at a position corresponding to position 129 (I129) of SEQ ID NO: 2.
  • the sequence of IL-2_C01#13 is set forth in SEQ ID NO: 49.
  • an engineered IL-2 polypeptide IL-2_C01#14 which includes an alanine mutation at a position corresponding to position 3 (e.g., T3) of SEQ ID NO: 2, an E95Q mutation with respect to SEQ ID NO: 2, a first cysteine residue at a position corresponding to position 11 (e.g., Q11) of SEQ ID NO: 2, and a second cysteine residue at a position corresponding to position 129 (I129) of SEQ ID NO: 2.
  • the sequence of IL-2_C01#14 is set forth in SEQ ID NO: 50.
  • the engineered IL-2 polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any one of SEQ ID NOs: 35-51.
  • the disclosure provides protein constructs comprising the engineered IL-2 variants described herein (e.g., any of the engineered IL-2 polypeptides described herein) .
  • the protein constructs described herein have a N-terminal His-tag.
  • the protein constructs described herein have a C-terminal His-tag.
  • the protein construct described herein includes, from N-terminus to C-terminus: (a) a His-tag; and (b) an engineered IL-2 polypeptide (e.g., any of the engineered IL-2 polypeptides described herein) .
  • the His-tag includes at least 6, at least 7, or at least 8 contiguous His residues.
  • the His-tag comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%to SEQ ID NO: 18.
  • the engineered IL-2 polypeptide comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17.
  • the engineered IL-2 polypeptide described herein includes at least 1, at least 2, at least 3, or at least 4 of the cysteine mutations described in Table 1.
  • the engineered IL-2 protein constructs can comprise any engineered IL-2 variant as described herein.
  • the protein construct described herein comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to any one of SEQ ID NOs: 20-34 and 52-68.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the engineered IL-2 variants can further comprise an Fc region of an antibody.
  • These antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE1, IgE2) .
  • the Fc region is derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4) .
  • the Fc region is an IgG1 Fc region (e.g., human IgG1 Fc region) .
  • the Fc region is linked to an antibody hinge region (e.g., IgG, IgE hinge region) .
  • the Fc region can be modified to provide desired effector functions or serum half-life.
  • the protein constructs as described herein include a Fc region.
  • the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.
  • effector function of a Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • effector function of a Fc region is phagocytosis.
  • effector function of a Fc region is ADCC and phagocytosis.
  • the protein constructs as described herein have an Fc region without effector function.
  • the Fc is a human IgG1 Fc.
  • the Fc does not have a functional Fc region.
  • the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .
  • the Fc region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 72. In some embodiments, the Fc region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 87. In some embodiments, the Fc region comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 88.
  • the engineered IL-2 variant (e.g., any of the engineered IL-2 variants described herein) is linked to the N-terminus or C-terminus of the Fc region (e.g., SEQ ID NO: 71) .
  • the engineered IL-2 variant is linked to the N-terminus of the Fc region.
  • the engineered IL-2 variant is linked to the C-terminus of the Fc region.
  • the Fc region is linked to a hinge region (e.g., SEQ ID NO: 70) .
  • the engineered IL-2 variant is linked to the Fc region via a linker peptide (e.g., SEQ ID NO: 72) .
  • the linker peptide is a flexible linker. Details of flexible linkers can be found, e.g., Chen, X., et al. “Fusion protein linkers: property, design and functionality. ” Advanced Drug Delivery Reviews 65.10 (2013) : 1357-1369, which is incorporated herein by reference in its entirety.
  • the linker peptide comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 72.
  • the linker peptide comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 92.
  • Various linker sequences can be used, including, without limitation, glycine serine (GS) linkers such as (GS) n, (SG) n, (GGS) n, (GSG) n, (GSGGS) n and (GGGS) n, where n represents an integer of at least 1.
  • n is 5-8.
  • n is 3-5.
  • Exemplary linker sequences can comprise an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence of any one of SEQ ID NOS: 85, 92, and 99-105.
  • protein constructs that include, from N-terminus to C-terminus, a human IgG1 hinge region and Fc region (e.g., SEQ ID NO: 69) , a linker peptide (e.g., any of the linker peptides described herein) , and an engineered IL-2 polypeptide (e.g., any of the engineered IL-2 polypeptides described herein) .
  • the protein construct described herein comprises or consists of an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NOs: 73-84.
  • the Fc region has S228P mutation (EU numbering) .
  • the S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange.
  • Fc regions are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such Fc region composition may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • the engineered IL-2 variants or protein constructs thereof described herein induce no or minimal downstream signaling (e.g., STAT5 phosphorylation) in Tconv cells (e.g., by binding to the dimeric IL-2 receptor complex IL-2R ⁇ ) .
  • STAT5 phosphorylation e.g., STAT5 phosphorylation
  • the engineered IL-2 variants e.g., any of the engineered IL-2 polypeptides described herein
  • protein constructs thereof described herein e.g., any of the G1LALAFc-fused IL-2 variants described herein
  • STAT5 signaling e.g., STAT5 phosphorylation
  • immune cells e.g., Treg cells
  • the engineered IL-2 variants or protein constructs thereof can induce STAT5 phosphorylation with a potency that is higher than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140%as compared to that of a wild-type IL-2 or protein construct thereof.
  • the engineered IL-2 variants or protein constructs thereof can preferentially induce STAT5 phosphorylation in Treg cells as compared to Tconv cells.
  • the engineered IL-2 variants or protein constructs thereof in Treg cells, can induce STAT5 phosphorylation with a potency that is higher than about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140%as compared to that of a wild-type IL-2 or protein construct thereof.
  • the engineered IL-2 variants or protein constructs thereof can induce STAT5 phosphorylation with a potency that is less than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%as compared to that of a wild-type IL-2 or protein construct thereof.
  • the engineered IL-2 variants or protein constructs thereof described herein can induce activation of Treg cells (e.g., HEK-Blue TM IL-2 Cells) with a potency that is higher than about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140%as compared to that of a wild-type IL-2 or protein construct thereof.
  • Treg cells e.g., HEK-Blue TM IL-2 Cells
  • KD is less than 1 ⁇ 10 -6 M, less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, or less than 1 ⁇ 10 -10 M. In some embodiments, the KD is less than 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM.
  • KD is greater than 1 ⁇ 10 -7 M, greater than 1 ⁇ 10 -8 M, greater than 1 ⁇ 10 -9 M, greater than 1 ⁇ 10 -10 M, greater than 1 ⁇ 10 - 11 M, or greater than 1 ⁇ 10 -12 M.
  • the engineered IL-2 variants or protein constructs thereof can bind to the dimeric IL-2 receptor complex IL-2R ⁇ .
  • the dissociation rate (k off ) is less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 , or less than 0.00001 s -1 .
  • the dissociation rate (k off ) is greater than 0.01 s -1 , greater than 0.001 s -1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 , or greater than 0.000001 s -1 .
  • KD is greater than 1 ⁇ 10 -7 M, greater than 1 ⁇ 10 -8 M, greater than 1 ⁇ 10 -9 M, greater than 1 ⁇ 10 -10 M, greater than 1 ⁇ 10 - 11 M, or greater than 1 ⁇ 10 -12 M.
  • the engineered IL-2 variants or protein constructs thereof can bind to the dimeric IL-2 receptor complex IL-2R ⁇ with a lower affinity than the wildtype IL-2.
  • the binding affinity between the engineered IL-2 variants or protein constructs thereof and the dimeric IL-2 receptor complex IL-2R ⁇ is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 400%, about 500%, or about 1000%lower than the binding affinity between the wild type IL-2 and the trimeric IL-2 receptor complex IL-2R ⁇ .
  • engineered IL-2 variants or protein constructs thereof described herein with a good expression/purification profile and desirable T cell response potency can be selected, potentially to further evaluate their immune suppression efficacy and in vivo toxicity (e.g., in a mouse model) .
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) .
  • vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • a polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus, retrovirus, or adenovirus e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus, retrovirus, or adenovirus e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art.
  • the DNA may also be “naked. ”
  • the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.
  • the DNA insert comprising a polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter e.g., a heterologous promoter
  • a heterologous promoter such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • a promoter e.g., a heterologous promoter
  • the promoter is a cytomegalovirus (CMV) promoter.
  • the promoter is a human promoter, e.g.,
  • the human promoters can improve expression of proteins derived from human. Details of such human promoters can be found, e.g., in Antoniou, M., et al. “Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing. ” Genomics 82.3 (2003) : 269-279; and Zhang, F., et al. “Aubiquitous chromatin opening element (UCOE) confers resistance to DNA methylation–mediated silencing of lentiviral vectors. ” Molecular Therapy 18.9 (2010) : 1640-1649; each of which is incorporated herein by reference in its entirety.
  • UCOE Aubiquitous chromatin opening element
  • the expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors can include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HEK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.
  • Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • Introduction of the construct into the host cell can be affected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods.
  • Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) , which is incorporated herein by reference in its entirety.
  • the host cell is a human cell suitable for protein expression, e.g., HEK 293 cells or CHO cells (e.g., CHO-Scells) .
  • the host cells are Expi293 TM cells and ExpiCHO TM cells.
  • the Expi293 Expression System is designed to deliver up to 6 ⁇ more protein in just one week, compared with other transient 293 expression systems that can take two weeks or more.
  • Expi293F cells are adapted to achieve higher pg/cell/day productivity than standard HEK 293 cells, and the Expifectamine TM 293 Transfection Reagent and enhancers enable high-efficiency transfection and expression levels of high-density HEK 293 cultures. Additionally, the Expi293 Expression System requires less plasticware, which means less waste and more incubator space.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • H8-IL-2_C01 SEQ ID NO: 20
  • H8-IL-2_C03 SEQ ID NO: 22
  • H8-IL-2_C05 SEQ ID NO: 24
  • H8-IL-2_C21 SEQ ID NO: 31
  • H8-IL-2_C22 SEQ ID NO: 32
  • H8-IL-2_C23 SEQ ID NO: 33
  • H8-IL-2_C24 SEQ ID NO: 34
  • H8-IL-2_C01, H8-IL-2_C03, H8-IL-2_C05, H8-IL-2_C21, H8-IL-2_C22, H8-IL-2_C23, H8-IL-2_C24 and H8-IL-2_WT were subjected to the subsequent in vitro tests.
  • IL-2 reporter assays were performed to test the IL-2-phospho STAT5 (pSTAT5) -signaling activities induced by the designed IL-2 molecules that were described in Example 1.2.8 x 10 5 cells/mL of HEK-Blue TM IL-2 Cells (InvivoGen, Cat. No.: hkb-il2) and HEK-Blue TM CD122/CD132 Cells (InvivoGen, Cat. No.: hkb-il2bg) were resuspended respectively in pre-warmed Dulbecco’s Modified Eagle’s Medium (DMEM) (CORNING, Cat.
  • DMEM Modified Eagle’s Medium
  • the activity of secreted alkaline phosphatase was assessed by measuring absorbance at 630 nm using a Thermo Scientific TM Varioskan TM LUK spectrophotometer (Thermo Fisher Scientific, Type 3020) .
  • the IL-2-pSTAT5 reporter signaling activities induced by the tested H8-IL-2 variants, H8-IL-2_C01, C03, C05, C21, C22, C23, and C24 was similar to activities induced by H8-IL-2_WT in HEK-Blue TM IL-2 Cells (cells overexpressing CD25, CD122, and CD132) .
  • H8-IL-2 variants induced differential IL-2-pSTAT5 reporter signaling activities as shown in FIGS. 2B and 2D.
  • H8-IL-2_C01 induced the weakest activity as compared to H8-IL-2_WT, H8-IL-2_C03, and H8-IL-2_C05.
  • the order of reporter activity from high to low is H8-IL-2_WT, C05 >> H8-IL-2_C03 > H8-IL-2_C01 in HEK-Blue TM CD122/CD132 Cells.
  • the order of reporter activity from high to low is H8-IL-2_WT, C22 >> H8-IL-2_C23, C24 > H8-IL-2_C21 in HEK-Blue TM CD122/CD132 Cells.
  • H8-IL-2_C01 and H8-IL-2_C21 exhibited a greater capability to induce IL-2 signaling activities in cells overexpressing CD25, CD122, and CD132 (like Treg cells) compared to cells overexpressing only CD122 and CD132 (like Teff cells) . Therefore, IL-2_C01 and its variants may possess the potential for autoimmune treatment.
  • IL-2_WT_T3A amino acid sequences of IL-2_WT_T3A (SEQ ID NO: 35) , IL-2_C01_T3A (SEQ ID NO: 36) , and IL-2_C01_T3A-derived variants consisting of IL-2_C01#1 to #15 (SEQ ID NOs: 37-51) are listed in FIG. 12.
  • the mutations in IL-2_C01_T3A and its variants are summarized in Table 2 as shown below.
  • Each IL-2_C01_T3A-derived variant carries an additional mutation, along with a T3A replacement, a Q11C replacement, and an I129C replacement on the IL-2_WT backbone.
  • IL-2_WT_3TA, IL-2_C01_T3A, and IL-2_C01_T3A-derived variants were fused with His tags, leading to the generation of H8-IL-2_WT_T3A (SEQ ID NO: 52) , H8-IL-2_C01_T3A (SEQ ID NO: 53) , and H8-IL-2_C01_T3A-derived variants, consisting of H8-IL-2_C01#1 to #15 (SEQ ID NOs: 54-68) .
  • IL-2_C01_T3A and its variants, fused with a His tag, were expressed in Expi293 and ExpiCHO cells following the same procedure described in Example 2.
  • H8-IL-2_C01_T3A and all 15 designed H8-IL-2_C01_T3A-derived variants were successfully produced and purified in Expi293 cells. These newly designed H8-IL-2_C01_T3A-derived variants were subjected to following in vitro tests.
  • IL-2 reporter assay was performed following the same procedure as described in Example 3.
  • the abundance of IL-2-pSTAT5 signaling activity was determined using the following formula: [ (Optical Density (OD) value at 630 nm (OD630) of each tested molecule at 600 pM –OD630 of the cell only control) / (OD630 of H8-IL-2_C01_T3A at 600 pM –OD630 of the cell only control) ] ⁇ 100%.
  • the IL-2-pSTAT5 signaling activity induced by H8-IL-2_C01_T3A at 600 pM was used as a 100%control.
  • Five “+” symbols represent 81-100%of IL-2-pSTAT5 signaling activity, four “+” symbols represent 61-80%, three “+” symbols represent 41-60%, two “+” symbols represent 21-40%, and one “+” symbol represents 11-20%.
  • the “-” symbol represents 0-10%activity.
  • the designed H8-IL-2_C01_T3A-derived variants can be categorized into five groups based on the IL-2-pSTAT5 reporter signaling-inducing activities.
  • Group 1 consisting of H8-IL-2_C01#1 (FIGS. 4A-4B) and H8-IL-2_C01#6 (FIGS. 4C-4D) , exhibited a reduced capability of inducing IL-2-pSTAT5 reporter signaling activities in HEK-Blue TM IL-2 Cells and lacked such effect in HEK-Blue TM CD122/CD132 Cells.
  • Group 2 consisting of H8-IL-2_C01#7 (FIGS.
  • H8-IL-2_C01#9 showed a moderate capability to induce IL-2-pSTAT5 reporter signaling activity in HEK-Blue TM IL-2 Cells and lacked such effect in HEK-Blue TM CD122/CD132 Cells.
  • Group 3 including H8-IL-2_C01#2 (FIGS. 4A-4B) , exhibited a moderate capability to induce IL-2-pSTAT5 reporter signaling activity in HEK-Blue TM IL-2 Cells, but a weak effect in HEK-Blue TM CD122/CD132 Cells.
  • Group 4 consisting of H8-IL-2_C01#3 (FIGS.
  • H8-IL-2_C01#8 (FIGS. 4E-4F) , H8-IL-2_C01#11 (FIGS. 4E-4F) , and H8-IL-2_C01#13 (FIGS. 4G-4H) , showed a robust induction of IL-2-pSTAT5 reporter signaling activity in HEK-Blue TM IL-2 Cells, as well as a relatively strong induction of IL-2-pSTAT5 reporter signaling activity in HEK-Blue TM CD122/CD132 Cells.
  • Treg and Tconv cells were isolated from human peripheral blood mononuclear cells (PBMCs) with EasySep TM Human CD4 + CD127 low CD25 + Regulatory T Cell Isolation kit (STEMCELL TM Technologies, Cat. No.: 18063) . Treg cells were incubated with Dynabeads TM Human T-Activator CD3/CD28 beads (Thermo Fisher Scientific, Cat. No.: 11131D) in Treg culture medium, consisting of X-VIVO TM 15 Media (Lonza, Cat.
  • Tconv cells were cultured in a Tconv culture medium supplemented with 50 IU/mL of recombinant human IL-2 (BioLegend, Cat. No.: 589106) .
  • the Tconv culture medium consists of RPMI-1640 Medium (Fisher Scientific, Cat.
  • Treg or Tconv cells were incubated with 100 ⁇ L of serially diluted designed H8-IL-2_C01_T3A-derived variants and corresponding control proteins in 96 well round bottom TC-treated microplates (SPL Life Sciences, Cat. No.: 34096) at 37°C for 30 minutes. After incubation, cells were harvested and fixed with 4%paraformaldehyde (PFA) solution (Sigma-Aldrich, Cat. No.: P6148) at 37 °C for 15 minutes. Then, cells were washed with FACS buffer (PBS supplemented with 4%FBS) and permeabilized with BD Phosflow TM Perm Buffer III (BD Biosciences, Cat.
  • FACS buffer PBS supplemented with 4%FBS
  • BD Phosflow TM Perm Buffer III BD Biosciences, Cat.
  • the designed H8-IL-2_C01_T3A-derived variants can be categorized into five groups based on the STAT5 phosphorylation levels.
  • Group 1 consisting of H8-IL-2_C01#6, exhibited a weaker STAT5 phosphorylation-inducing activity in Treg cells and no STAT5 phosphorylation-inducing activity in Tconv cells.
  • Group 2 consisting of H8-IL-2_C01#1, H8-IL-2_C01#3, H8-IL-2_C01#4, H8-IL-2_C01#9, H8-IL-2_C01#10, and H8-IL-2_C01#12, showed weaker STAT5 phosphorylation-inducing activities in both Treg cells and Tconv cells.
  • Group 3 consisting of H8-IL-2_C01#7, showed a relatively greater capability to induce STAT5 phosphorylation in Treg cells, while exhibiting a weaker effect in Tconv cells.
  • H8-IL-2_C01_T3A-derived variants could be categorized into different groups with varying properties.
  • H8-IL-2_C01#2, H8-IL-2_C01#5, H8-IL-2_C01#8, H8-IL-2_C01#11, and H8-IL-2_C01#13 showed a relatively stronger IL-2-pSTAT5 signaling-inducing activity in HEK-Blue TM CD122/CD132 Cells, as well as a stronger effect in Tconv cells compared to other variants. As a result, these five molecules were not subjected to further studies.
  • the remaining 10 clones of H8-IL-2_C01_T3A-derived variants were selected for the following G1LALAFc-IL-2 construction and in vitro tests.
  • G1LALAFc-IL-2_WT_T3A SEQ ID NO: 73
  • G1LALAFc -IL-2_C01_T3A SEQ ID NO: 74
  • G1LALAFc-IL-2_C01_T3A-derived variants including G1LALAFc-IL-2_C01#1, G1LALAFc-IL-2_C01#3, G1LALAFc-IL-2_C01#4, G1LALAFc-IL-2_C01#6, G1LALAFc-IL-2_C01#7, G1LALAFc-IL-2_C01#9, G1LALAFc-IL-2_C01#10, G1LALAFc-IL-2_C01#12, G1LALAFc-IL-2_C01#14, and G1LALAFc-IL-2_C01#15 (SEQ ID NOs: 75-84) are listed in FIG.
  • Each G1LALAFc-IL-2_C01_T3A-derived variant comprises a hinge region (SEQ ID NO: 70) , a G1LALAFc region (SEQ ID NO: 71) , and a linker region (SEQ ID NO: 72) .
  • the expressed proteins were purified by affinity chromatography using a column packed with MabSelect PrismA TM protein A chromatography resin (Cytiva, Cat. No.: 17549803) , and size exclusive chromatography (SEC) using a 200 Increase 10/300 GL column (Cytiva, Cat. No.: 28990944) .
  • the purified proteins were also analyzed by SDS-PAGE and HPLC-SEC.
  • G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A and 10 variants of G1LALAFc-IL-2_C01_T3A were successfully produced and purified. These proteins were subjected to the following in vitro tests.
  • Example 10 IL-2 reporter signaling detection in HEK-Blue TM IL-2 Cells and HEK-Blue TM CD122/CD132 Cells
  • IL-2 reporter assay was performed following the same procedure as described in Examples 3 and 6.
  • the abundance of IL-2-pSTAT5 signaling activity is determined using the following formula: [ (Optical Density (OD) value at 630 nm (OD630) of each tested molecule at 600 pM –OD630 of the cell only control) / (OD630 of H8-IL-2_C01_T3A or G1LALAFc-IL-2_C01_T3A at 600 pM –OD630 of the cell only control) ] ⁇ 100%.
  • the IL-2-pSTAT5 signaling activity induced by H8-IL-2_C01_T3A or G1LALAFc-IL-2_C01_T3A at 600 pM was used as a 100%control.
  • Five “+” symbols represent 81-100%of IL-2-pSTAT5 signaling activity, four “+” symbols represent 61-80%, three “+” symbols represent 41-60%, two “+” symbols represent 21-40%, and one “+” symbol represents 11-20%.
  • the “-” symbol represents 0-10%of IL-2-pSTAT5 signaling activity.
  • the designed G1LALAFc-IL-2_C01_T3A-derived variants can be categorized into five groups based on the IL-2-pSTAT5 reporter signaling activities.
  • Group 1 consisting of G1LALAFc-IL-2_C01#1, showed moderate IL-2-pSTAT5 reporter signaling-inducing activity in both HEK-Blue TM IL-2 Cells and HEK-Blue TM CD122/CD132 Cells.
  • Group 2 consisting of G1LALAFc-IL-2_C01#3, #4, #6, #9, and #10, showed a stronger IL-2-pSTAT5 reporter signaling-inducing activity in HEK-Blue TM IL-2 Cells but lacked such activity in HEK-Blue TM CD122/CD132 Cells.
  • Group 3 consisting of G1LALAFc-IL-2_C01#7 and #14, showed a stronger IL-2-pSTAT5 reporter signaling-inducing activity in HEK-Blue TM IL-2 Cells, while exhibiting moderate such activity in HEK-Blue TM CD122/CD132 Cells.
  • Group 4 consisting of G1LALAFc-IL-2_C01#15, showed a stronger IL-2-pSTAT5 reporter signaling-inducing activity in HEK-Blue TM IL-2 Cells and a relatively stronger such activity in HEK-Blue TM CD122/CD132 Cells.
  • G1LALAFc format-IL-2 variants exhibited a stronger potency than H8 format-IL-2 variants in inducing the IL-2-pSTAT5 reporter signaling activity.
  • the relative ranking of the IL-2-pSTAT5 reporter signaling-inducing activities shown as “+” or “-” , was similar for both IL-2 formats.
  • the STAT5 phosphorylation activity induced by designed IL-2 molecules in Treg and Tconv cells was determined as described in Example 7. As shown in FIGS. 11A-11D, based on the STAT5 phosphorylation levels, G1LALAFc-IL-2_WT_T3A, G1LALAFc-IL-2_C01_T3A, and G1LALAFc-IL-2_C01_T3A-derived variants, including G1LALAFc-IL-2_C01#1, G1LALAFc-IL-2_C01#3, G1LALAFc-IL-2_C01#4, G1LALAFc-IL-2_C01#7, G1LALAFc-IL-2_C01#9, G1LALAFc-IL-2_C01#10, G1LALAFc-IL-2_C01#12, G1LALAFc-IL-2_C01#14, and G1LALAFc-IL-2_C01#15, were able to induce STAT5
  • the stress stability of G1LALAFc-IL-2_WT, G1LALAFc-IL-2_C01#6 and a MK-6194 analog was evaluated at 40°C for 1, 2 and 4 weeks.
  • the MK-6194 analog is an engineered IL-2 variant fused to a protein backbone designed to selectively activate and expand regulatory T cells (Tregs) for the potential treatment of ulcerative colitis and other autoimmune diseases.
  • the MK-6194 analog contains a human IL-2 variant with L118I, N88D, V69A, Q74P, and C125S mutations, fused via a G4Sx4 linker (SEQ ID NO: 92) to the N-terminal of a human IgG1 Fc domain with L234A, L235A, and G237A mutations.
  • each sample was placed in 150 ⁇ L protein solution in a new eppendorf and incubated in a Constant Climate Chamber HPP110eco (Memmert GmbH) with 40°C ⁇ 2°C /75% ⁇ 5%RH condition for 1, 2 and 4 weeks.
  • a Constant Climate Chamber HPP110eco Memmert GmbH
  • an eppendorf with 150 ⁇ L protein solution was removed from the Constant Climate Chamber and stored at 4°Crefrigerator to the end of experiment.
  • all samples were filtered by 0.2 ⁇ m filters before analysis and analyzed by ACQUITY UPLC H-Class (Waters Corporation. )
  • FIGS. 12A-12C The results were shown in FIGS. 12A-12C. According to the results, after 40°Cincubation for 4 weeks, the SEC-HPLC profiles of G1LALAFc-IL-2_WT (FIG. 12A) and G1LALAFc-IL-2_C01#6 (FIG. 12B) were similar. But the HMW (%) of the MK-6194 analog (FIG. 12C) increased by over 40%. The results indicated that G1LALAFc-IL-2_C01#6 showed better stress stability than the MK-6194 analog.
  • the thermal stability of G1LALAFc-IL-2_WT, G1LALAFc-IL-2_C01#6 and a MK-6194 analog was determined by Dynamic Light Scattering (DLS) analysis. Specifically, samples were centrifuged under 12,000 rpm for 10 minutes at 8°C. 26 ⁇ L of each supernatant and 10 ⁇ L of Silicone Oil (Alfa Aesar, Cat. No.: A12728) were added into a well in a 384LV plate (AURORA, Cat. No.: P8806-38403-10 .
  • DLS Dynamic Light Scattering
  • the plate was centrifuged under 1,500 rpm for 10 minutes at 25°C to remove bubbles and sealed with UltraClear Real Time PCR Pressured Sealing Film (SMBIO, Cat. No.: P4F51-C) .
  • the plate was analyzed with DynaPro Plate Reader III (WYATT Technology) using Continuous Temperature Ramp Measure through Dynamic Light Scattering. Raw data were analyzed and fitted by DYNAMICS 7.10.1.21 to determine the value of T onset .
  • the results were shown in FIG. 13. According to the results, the onset temperature (T onset ) of G1LALAFc-IL-2_C01#6 was approximately 62°C; the T onset of G1LALAFc-IL-2_WT was 53°C; and the T onset of the MK-6194 analog was 45°C. Comparing to G1LALAFc-IL-2_WT and the MK-6194 analog, the G1LALAFc-IL-2_C01#6 showed a significantly increased T onset . The results indicated that G1LALAFc-IL-2_C01#6 showed better stability than G1LALAFc-IL-2_WT and MK-6194 analog.
  • the thermal stability of G1LALAFc-IL-2_WT, G1LALAFc-IL-2_C01#6 and a MK-6194 analog was determined by Differential Scanning Fluorimetry (DSF) analysis. Specifically, the protein of choice was resuspended in DI water to an appropriate concentration, and a 50x stock solution of Orange Protein Gel Stain (Sigma-Aldrich, Cat. No.: S5692) was prepared by diluting 2.5 ⁇ L of 5,000x concentrate into 250 ⁇ L of DI water. 10 ⁇ L of protein solution was aliquoted into each well of a Hard-Shell PCR Plate (Bio-Rad, Cat. No: HSP9601) .
  • DSF Differential Scanning Fluorimetry
  • the melting temperature (Tm) of G1LALAFc-IL-2_C01#6 was approximately 65°C; the Tm of G1LALAFc-IL-2_WT was 56°C; and the Tm of the MK-6194 analog was approximately 45°C. Comparing to G1LALAFc-IL-2_WT and the MK-6194 analog, G1LALAFc-IL-2_C01#6 showed a significantly increased melting temperature. The results indicated that G1LALAFc-IL-2_C01#6 showed better stability than G1LALAFc-IL-2_WT and the MK-6194 analog.
  • the autoimmune efficacy of G1LALAFc-IL-2_C01#6 was evaluated in an NPG expansion model.
  • the treatment plan and dosing schedule were shown in FIG. 15A. Specifically, 8-week old NPG mice were irradiated to remove endogenous immune cells and intravenous (i. v. ) injected with 5 ⁇ 10 6 human PBMCs. The treatment group mice were intraperitoneally (i. p. ) injected with 3 ⁇ g of G1LALAFc-IL-2_WT (G2) , 1 ⁇ g of G1LALAFc-IL-2_C01#6 (G3) , or 1 ⁇ g of MK-6194 analog (G4) per mouse.
  • mice The control group mice (G1) were injected with 100 ⁇ L of vehicle (isotonic sodium chloride solution) per mouse. The treatment started at 7 days after the irradiation and human PBMCs transplantation, and the injections were performed once a week for a total of 3 weeks.
  • vehicle isotonic sodium chloride solution
  • the mice were individually scored based on five clinical parameters (body weight loss, posture, activity, fur, and skin) on a scale of 0 to 2 and one additional clinical parameter (diarrhea) on a scale of 0 to 1.
  • the maximum total GvHD score is 11. A mouse was sacrificed when its total GvHD score exceeds 5 or when it has a GvHD score of 2 on a single clinical parameter (body weight loss, posture, activity, fur, or skin) .
  • Example 16 Evaluation of the PK, drug-related liver toxicity and T cell population inducing ability of G1LALAFc-IL-2_C01#6
  • the T cell population inducing ability of G1LALAFc-IL-2_C01#6 was evaluated in an C57BL/6 model.
  • the treatment plan and dosing schedule were shown in FIG. 16A. Specifically, 6-8 weeks old C57BL/6 mouse were intraperitoneally (i. p.
  • mice (G1) were injected with 10 mL/kg of vehicle (isotonic sodium chloride solution) .
  • G1LA-KIHFc-IL-2_WT contains a hole chain comprising G1LA-HoleFc-IL-2_WT (e.g., SEQ ID NO: 97) and a knob chain comprising G1 Hinge + G1LA-KnobFc (e.g., SEQ ID NO: 96) .
  • G1LA-KIHFc-IL-2_C01#6 contains a hole chain comprising G1LA-HoleFc-IL-2_C01#6 (e.g., SEQ ID NO: 98) and a knob chain comprising G1 Hinge + G1LA-KnobFc (e.g., SEQ ID NO: 96) .
  • PK Pharmacokinetics
  • IL-2 serum concentration was detected by ELISA MAX TM Deluxe Set Human IL-2 (cat. No.: 431816) .
  • AST Aspartate transaminase
  • ALT Alanine transaminase
  • FIGS. 16C-16L The serum concentration analysis showed (1) the G1LA-KIHFc-IL-2 format exhibited better PK result than the G1LALAFc-IL-2 format and (2) IL-2_C01#6 exhibited better PK result than IL-2_WT (see FIGS. 16C-16D) .
  • the data also indicated that high dose (3 mg/kg) of IL-2_WT showed significant decline at Day 3, but IL-2_C01#6 was more stable and still detectable at Day 7 (see FIGS. 16E-16F for the G1LALAFc-IL-2 format and FIGS. 16I-16J for the G1LA-KIHFc-IL-2 format) .
  • the low dose (0.3 mg/kg) of IL-2_WT also showed significant decline at Day 3, but IL-2_C01#6 was more stable and still detectable at Day 7 (see FIGS. 16G-16H for the G1LALAFc-IL-2 format and FIGS. 16K-16L for the G1LA-KIHFc-IL-2 format) .
  • the dotted line showed value of the Limit of detection (LoD) by ELISA kit and the serum concentration of tested samples below the LoD was excluded.
  • the Limit of detection (LoD) of the ELISA kit was 7.8 pg/mL.
  • the T cell population inducing ability of G1LALAFc-IL-2_C01#6 was evaluated in an C57BL/6 model.
  • the spleen cells were isolated from the spleen and processed with 1X RBC Lysis Buffer to remove red blood cells.
  • the cells were stained using: Zombie Aqua TM Fixable Viability Kit ( Cat. No.: 423102) , PE anti-mouse CD45 Antibody ( Cat. No.: 103106) , FITC anti-mouse CD3 Antibody ( Cat. No.: 100204) , PE/Dazzle TM 594 anti-mouse CD4 Antibody ( Cat. No.: 100456) , Brilliant Violet 605 TM anti-mouse CD8a Antibody ( Cat.
  • FIGS. 18A-18D The results were shown in FIGS. 18A-18D. According to the result of T cell population in mice spleen, the percentage of CD3 + T cells (FIG. 18A) was not significantly affected by IL-2 treatment at Day 3 and Day 7 post treatment. The percentages of different T cell populations including CD8 + T cells (FIG. 18B) , Treg cells (FIG. 18C) and NK cells (FIG. 18D) returned to basal level like vehicle group at Day 7 post treatment. At Day 3 post treatment, high dose (3 mg/kg) of IL-2_C01#6 significantly induced Treg population in the spleen (FIG. 18C) and high dose (3 mg/kg) of IL-2_WT induced CD8 + T cell (FIG. 18B) and NK cell (FIG. 18D) populations in the spleen. The results indicated that IL-2_C01#6 could induce the Treg population but not CD8 + T cell or NK cell populations.

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Abstract

L'invention concerne des variants d'IL-2 modifiés, et leurs procédés d'utilisation. Les variants d'IL-2 modifiés comprennent une ou plusieurs cystéines non natives.
PCT/CN2025/070316 2024-01-08 2025-01-03 Variants d'il-2 modifiés et leurs procédés d'utilisation Pending WO2025148785A1 (fr)

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CN114349843A (zh) * 2022-01-18 2022-04-15 浙江博锐生物制药有限公司 白细胞介素-2衍生物及其制备方法和应用
WO2022081728A2 (fr) * 2020-10-14 2022-04-21 Cytimm Therapeutics, Inc. Polypeptides d'interleukine 2 (il-2) modifiés, et leurs procédés de fabrication et d'utilisation
CN117247443A (zh) * 2022-06-17 2023-12-19 舒泰神（北京）生物制药股份有限公司 白介素-2(il-2)突变体及其用途

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WO2003014308A2 (fr) * 2001-08-07 2003-02-20 Sunesis Pharmaceuticals, Inc. Mutants de cysteine et procedes de detection de liaison de ligands a des molecules biologiques
CN101426916A (zh) * 2004-03-05 2009-05-06 诺华疫苗和诊断公司 改进的白介素-2突变蛋白
CN111868079A (zh) * 2017-12-27 2020-10-30 协和麒麟株式会社 Il-2变体
WO2020056066A1 (fr) * 2018-09-11 2020-03-19 Ambrx, Inc. Conjugués polypeptidiques d'interleukine-2 et leurs utilisations
WO2020103777A1 (fr) * 2018-11-22 2020-05-28 海珂分子（北京）科技有限责任公司 Mutant de la protéine interleukine 21 (il21) et son utilisation
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CN114349843A (zh) * 2022-01-18 2022-04-15 浙江博锐生物制药有限公司 白细胞介素-2衍生物及其制备方法和应用
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