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WO2025165594A1 - Protéine bifonctionnelle promédicament anti-pd-l1 et il-15 et utilisations associées - Google Patents

Protéine bifonctionnelle promédicament anti-pd-l1 et il-15 et utilisations associées

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Publication number
WO2025165594A1
WO2025165594A1 PCT/US2025/012251 US2025012251W WO2025165594A1 WO 2025165594 A1 WO2025165594 A1 WO 2025165594A1 US 2025012251 W US2025012251 W US 2025012251W WO 2025165594 A1 WO2025165594 A1 WO 2025165594A1
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Prior art keywords
amino acid
seq
acid sequence
variant
monomer
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Inventor
Wenwu Zhai
Yong Yin
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Staidson Biopharma Inc
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Staidson Biopharma Inc
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Publication of WO2025165594A1 publication Critical patent/WO2025165594A1/fr
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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]
    • C07K14/5443—IL-15
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • ANTI-PD-L1 AND IL-15 PRODRUG BIFUNCTIONAL PROTEIN AND USES THEREOF SUBMISSION OF SEQUENCE LISTING [0001] The contents of the electronic sequence listing (anti-PD-L1 and IL-15 prodrug bifunctional protein and uses thereof SEQ-240125.xml; Size: 175 KB; and Date of Creation: January 25, 2024) is herein incorporated by reference in its entirety.
  • TECHNICAL FIELD [0002] The present application relates to the IL-15 prodrugs and bifunctional proteins comprising an antibody that binds PD-L1 and IL-15 prodrug, methods of making, and using thereof.
  • This invention also relates to cleavage products of said activatable IL-15 prodrugs and methods of using thereof.
  • Cytokines are potent immune agonists, which lead to them being considered as promising therapeutic agents for oncology.
  • IL-15 interleukin- 15
  • Antibodies have been viewed as ideal candidates for use in therapy in the fields of cancer, autoimmunity, and chronic inflammatory disorders, but sometimes the antibody therapy is limited by their cross-reactivity to healthy tissue.
  • Several approaches have been described for overcoming these “off-target” effects by engineering antibodies to improve tumor targeting, for example, by generating masked antibodies that are selectively activated in the tumor microenvironment (see, e.g., W02003/068934, W02004/009638, WO 2009/025846, W02101/081173 and WO2014103973).
  • Anti-PD-L1 antibody has been a promising immunotherapy for tumor, but there remains a need for unitizing the therapeutic agents to more effectively modulate tumor.
  • Cytokines such as IL-2 and IL-15 function in aiding the proliferation and differentiation of B cells, T cells, and NK cells, which make them being considered as promising therapeutic agents for oncology.
  • the present application refers to conditionally activatable prodrugs (e.g., cytokine prodrug or antibody prodrug) that have a cleavable moiety linked to a masking polypeptide (MP) for use in the treatment of cancer or other diseases.
  • Masking polypeptides (MP) can act via steric hindrance to the biologically active moiety.
  • the cleavable moiety can be designed to be cleaved by proteases that are specific to certain tissues or pathologies, thus enabling the prodrugs to be preferentially activated in desired locations (e.g., a tumor) to overcome the dosing amounts limitation of the cytokines or the “off-target” effects of the antibody.
  • the present application also provides bifunctional proteins comprising an anti-PD-L1 antibody and an IL-15 prodrug, which is capable of providing a combined therapeutic effect of blocking immune regulatory effects mediated by PD-L1 binding to PD1 and providing immunostimulatory effects mediated by IL-15.
  • the present application provides an IL-15 prodrug, wherein the IL-15 prodrug comprises: (i) one or more IL-15 cytokine (I), (ii) one or more cleavable moiety (CM), and (iii) one or more masking polypeptide (MP).
  • the IL-15 prodrug provided herein wherein the IL-15 cytokine (I) and the masking polypeptide (MP) are linked through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the IL-15 prodrug further comprises an IL-15R ⁇ or a functional fragment thereof (S), and wherein the IL-15R ⁇ or a functional fragment thereof is selected from an extracellular domain of IL-15R ⁇ or a sushi domain or functional analogs.
  • the IL-15 prodrug provided herein wherein the IL-15R ⁇ or a functional fragment thereof (S) and masking polypeptide (MP) are linked through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the IL-15 prodrug further comprises one or more half-life extension moiety (C).
  • the IL-15 prodrug provided herein wherein the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C).
  • the IL-15 prodrug provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S) is linked to the IL-15 cytokine (I), and the masking polypeptide (MP) is linked to the IL- 15R ⁇ or a functional fragment thereof (S) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C), and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein wherein the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C), the IL-15 cytokine (I) is linked to the IL-15R ⁇ or a functional fragment thereof (S), and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C), and the masking polypeptide (MP) is linked to the IL-15R ⁇ or a functional fragment thereof (S) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the masking polypeptide (MP) is linked to the half-life extension moiety (C) through the cleavable moiety (CM), the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C), and the IL-15 cytokine (I) is linked to the IL-15R ⁇ or a functional fragment thereof (S).
  • the IL-15 prodrug provided herein wherein the masking polypeptide (MP) is linked to the half-life extension moiety (C) through the cleavable moiety (CM), the IL-15 cytokine (I) is linked to the half-life extension moiety (C), and the IL-15R ⁇ or a functional fragment thereof (S) is linked to the IL-15 cytokine (I).
  • the IL-15 prodrug provided herein is a monomer or a dimer.
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the first half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S), the IL-15 cytokine (I); and the other monomer comprises the second half-life extension moiety (C), the masking polypeptide (MP) and the cleavable moiety (CM), wherein the masking polypeptide (MP) is linked to the second half-life extension moiety (C) through the cleavable moiety (CM).
  • one monomer comprises the first half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S), the IL-15 cytokine (I); and the other monomer comprises the second half-life extension moiety (C), the masking polypeptide (MP) and the cleavable moiety (CM), wherein the masking polypeptide (MP) is linked to the second half-life extension moiety (C) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the first half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S), the masking polypeptide (MP) and the cleavable moiety (CM); and the other monomer comprises the second half-life extension moiety (C) and the IL-15 cytokine (I).
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the first half-life extension moiety (C), the IL-15 cytokine (I), the masking polypeptide (MP) and a cleavable moiety (CM); and the other monomer comprises the second half-life extension moiety (C) and the IL-15R ⁇ or a functional fragment thereof (S).
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer and the other monomer each comprises the half-life extension moiety (C), the IL-15 cytokine (I), the IL-15R ⁇ or a functional fragment thereof (S), the cleavable moiety (CM) and the masking polypeptide (MP).
  • the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: both the IL-15 cytokine (I) and the IL-15R ⁇ or a functional fragment thereof (S) are linked to the first half-life extension moiety (C); and in the other monomer: the masking polypeptide (MP) is linked to the second half-life extension moiety (C) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C) and the IL-15R ⁇ or a functional fragment thereof (S) is linked to the IL-15 cytokine (I); and in the other monomer: the masking polypeptide (MP) is linked to the second half-life extension moiety (C) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15R ⁇ or a functional fragment thereof (S) is linked to the first half-life extension moiety (C) and the IL-15 cytokine (I) is linked to the IL-15R ⁇ or a functional fragment thereof (S); and in the other monomer: the masking polypeptide (MP) is linked to the second half-life extension moiety (C) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15R ⁇ or a functional fragment thereof (S) is linked to the first half-life extension moiety (C) and the masking polypeptide (MP) is linked to the first half-life extension moiety (C) through the cleavable moiety (CM); and in the other monomer: the IL-15 cytokine (I) is linked to the second half-life extension moiety (C).
  • the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15R ⁇ or a functional fragment thereof (S) is linked to the first half-life extension moiety (C) and the masking polypeptide (MP) is linked to the IL-15R ⁇ or a functional fragment thereof (S) through the cleavable moiety (CM); and in the other monomer: the IL-15 cytokine (I) is linked to the second half-life extension moiety (C).
  • the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C) and the masking polypeptide (MP) is linked to the first half-life extension moiety (C) through the cleavable moiety (CM); and in the other monomer: the IL-15R ⁇ or a functional fragment thereof (S) is linked to the second half-life extension moiety (C).
  • the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C) and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM); and in the other monomer: the IL-15R ⁇ or a functional fragment thereof (S) is linked to the second half-life extension moiety (C).
  • the IL-15 prodrug provided herein wherein the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are covalently linked; or the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are non-covalently linked, and form an IL-15/IL-15R ⁇ complex.
  • the IL-15 prodrug provided herein wherein the IL-15 cytokine (I) comprises the one or more amino acids mutations selected from the group consisting of L45D, L45E, Q48K, S51D, L52D, E64K, I67D, I67E, I68D and N72D.
  • the IL-15 prodrug provided herein wherein the IL-15 cytokine (I) comprises the amino acid sequence of any one of SEQ ID NOs: 22-23 and 67-76, or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 22-23 and 67-76.
  • the IL-15 prodrug provided herein wherein the IL-15R ⁇ or a functional fragment thereof (S) comprises the amino acid sequence of any one of SEQ ID NOs: 24-26, or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 24-26.
  • the IL-15 prodrug provided herein, wherein the half-life extension moiety (C) comprises an Fc domain; preferably, the Fc domain is selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgA Fc domain, an IgD Fc domain, an IgE Fc domain, and an IgM Fc domain; more preferably, the Fc domain is a human IgG1 Fc domain.
  • the IL-15 prodrug provided herein wherein the Fc domain is a human IgG1 Fc domain having L234A and L235A mutations, according to EU Numbering system.
  • the IL-15 prodrug provided herein, wherein the Fc domains comprise knobs-into-holes mutations (Fc knob and Fc hole).
  • the IL-15 prodrug provided herein, wherein the Fc knob comprises a T366W mutation in the Fc domain, and the Fc hole comprises T366S, L368A, and Y407V mutations in the Fc domain, according to EU Numbering system.
  • the IL-15 prodrug provided herein, wherein the masking polypeptide (MP) is composed of five types of amino acids G, S, P, E, and A, and further wherein the percentage of amino acid residue G in the masking polypeptide is about 15%-30%, preferably about 20%; the percentage of amino acid residue S in the masking polypeptide is about 20%-40%, preferably about 40%; the percentage of amino acid residue P in the masking polypeptide is about 15%-40%, preferably about 20%; the percentage of amino acid residue E in the masking polypeptide is about 1%-20%, preferably about 10%; and the percentage of amino acid residue A in the masking polypeptide is about 5%-20%, preferably about 10%; and when the number of amino acids is not an integer, take the integer value.
  • the masking polypeptide (MP) is composed of five types of amino acids G, S, P, E, and A, and further wherein the percentage of amino acid residue G in the masking polypeptide is about 15%-30%, preferably about 20%; the percentage of amino acid
  • the IL-15 prodrug provided herein, wherein the masking polypeptide (MP) is composed of four types of amino acids S, P, E, and G, and further wherein the percentage of amino acid residue S in the masking polypeptide is about 20%-40%, preferably about 23%; the percentage of amino acid residue P in the masking polypeptide is about 15%-40%, preferably about 29%, the percentage of amino acid residue E in the masking polypeptide is about 1%-20%, preferably about 18%, and the percentage of amino acid residue G in the masking polypeptide is about 15%-30%, preferably about 30%; and when the number of amino acids is not an integer, take the integer value.
  • the masking polypeptide (MP) is composed of four types of amino acids S, P, E, and G, and further wherein the percentage of amino acid residue S in the masking polypeptide is about 20%-40%, preferably about 23%; the percentage of amino acid residue P in the masking polypeptide is about 15%-40%, preferably about 29%, the percentage of amino acid
  • the IL-15 prodrug provided herein, wherein the masking polypeptide (MP) comprises about 40 to 720 amino acid residues; preferably comprises 80 to 320 amino acid residues; and more preferably comprises 80 to 240 amino acid residues. [0047] In some embodiments, the IL-15 prodrug provided herein, wherein the masking polypeptide (MP) comprises the amino acid sequence of SEQ ID NO: 6. [0048] In some embodiments, the IL-15 prodrug provided herein, wherein the masking polypeptide (MP) comprises the amino acid sequence of SEQ ID NO: 1.
  • the IL-15 prodrug provided herein wherein the masking polypeptide (MP) comprises the amino acid sequence of any one of SEQ ID NOs: 1-5, or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-5, or a variant thereof comprising one or more amino acid substitutions, additions and/or deletions.
  • MP masking polypeptide
  • the IL-15 prodrug provided herein wherein the cleavable moiety (CM) comprises the amino acid sequence MVX 1 X 2 AX 3 TX 4 SG (SEQ ID NO: 49), wherein X1 is selected from P, L, V, or A, X2 is selected from L or S, X3 is selected from L V, P, or Y and X 4 is selected from A or V.
  • CM cleavable moiety
  • the IL-15 prodrug provided herein, wherein the cleavable moiety (CM) is a substrate of urokinase-type plasminogen activator(uPA), matrix metallopeptidase(MMP) 1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, fibroblast activation protein (FAP), matriptase, cathepsin, caspase, thrombin, metalloprotease, serine protease, cysteine protease, aspartic acid protease, Legumain, Kallikrein, Cathepsin A, Cathepsin B, chymase, protease located at a tumor site or its surrounding environment or any combination thereof.
  • uPA urokinase-type plasminogen activator
  • MMP matrix metallopeptidase
  • MMP2 matrix
  • the IL-15 prodrug provided herein wherein the cleavable moiety (CM) comprises the amino acid sequence of any one of SEQ ID NOs: 8-16, or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 8-16.
  • the IL-15 prodrug provided herein comprises two monomers, wherein in one monomer, the IL-15R ⁇ or a functional fragment thereof (S) is linked to the first Fc domain, and in the other monomer, the IL-15 cytokine (I) is linked to the second Fc domain, and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33, and the other monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 34, and the other monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 36, and the other monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 37, and the other monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 38, and the other monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 39, and the other monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 45 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45, and the other monomer comprises the amino acid sequence of SEQ ID NO: 46 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 46.
  • the IL-15 prodrug provided herein comprises two monomers, wherein one monomer comprises the amino acid sequence of SEQ ID NO: 47 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47, and the other monomer comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 40.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33, and the second monomer comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 40.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 62, and the second monomer comprises the amino acid sequence of SEQ ID NO: 63 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 63.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 63 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 63.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 64 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 64, and the second monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 65.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 62, and the second monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 65.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 65.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 66 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 66, and the second monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 65.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43, and the second monomer comprises the amino acid sequence of SEQ ID NO: 46 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 46.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 39, and the second monomer comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 40.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 87 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 87.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 91 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 91.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 93 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 93.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 94 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 94.
  • the present application provides an anti-PD-L1/IL-15 prodrug bifunctional protein comprising an anti-PD-L1 antibody and an IL-15 prodrug of any one described herein, wherein the IL-15 prodrug is linked to anti-PD-L1 antibody.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the anti-PD-L1 antibody comprises: (i) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 97, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 99; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 100, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 101, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 102, or (ii) a V H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 133, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 134, and an HC-CDR3 comprising the amino acid
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the anti-PD-L1 antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 104; and a VL comprising the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 107; or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 139 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 139; and a VL comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 140.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the anti-PD-L1 antibody comprises an Fc domain.
  • the anti-PD-L1 antibody is a full-length IgG antibody.
  • the anti-PD-L1 antibody is a full-length IgG1 or IgG4 antibody.
  • the anti-PD-L1 antibody is chimeric, human or humanized.
  • the anti-PD-L1 antibody is an antigen-binding fragment selected from the group consisting of a Fab, a Fab’, a F(ab)’2, a Fab’-SH, a single-chain Fv (scFv), an Fv fragment, a dAb, a Fd, a nanobody, a diabody, and a linear antibody.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the IL-15 prodrug is linked to the C-terminus or N-terminus of the anti-PD-L1 antibody with or without a linker.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the IL-15 prodrug is linked to the C-terminus or N-terminus of the anti-PD-L1 antibody heavy chain with or without a linker.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the IL-15 prodrug is linked to the C-terminus or N-terminus of the anti-PD-L1 antibody light chain with or without a linker.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein comprises two monomers, each monomer comprises two polypeptides, wherein: (i) one monomer comprises a polypeptide comprising the structure of V H -C H 1-hinge-C H 2-C H 3-IL-15- CM-MP, and the other polypeptide comprises the structure of VL-CL; and (ii) the other monomer comprises a polypeptide comprising the structure of VH-CH1-hinge-CH2-CH3-IL- 15R ⁇ _sushi, and the other polypeptide comprising the structure of V L -C L , wherein the V H is a heavy chain variable domain of the anti-PD-L1 antibody, VL is a light chain variable domain of the anti-PD-L1 antibody.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 113, 116-125 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 113, 116-125; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 115 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 115; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 114 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 114, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 115 or a variant thereof having at least about 80% sequence identity to the amino acid sequence
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 127, 141-150 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 127, 141-150; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 129 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 129; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 128 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 128, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 129 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 130 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 130; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 132; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 131 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 131, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 132.
  • the IL-15 drug comprises two monomers, wherein in one monomer, the IL-15R ⁇ or a functional fragment thereof (S) is linked to the first Fc domain, and in the other monomer, the IL-15 cytokine (I) is linked to the second Fc domain.
  • the IL-15 drug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43, and the second monomer comprises the amino acid sequence of SEQ ID NO: 83 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 83.
  • the IL-15 drug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43, and the second monomer comprises the amino acid sequence of SEQ ID NO: 84 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 84.
  • the IL-15 drug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43, and the second monomer comprises the amino acid sequence of SEQ ID NO: 84 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 84.
  • the present application provides an anti-PD-L1/IL-15 drug bifunctional protein comprising an anti-PD-L1 antibody and an IL-15 drug of any one described herein, wherein the IL-15 drug is linked to anti-PD-L1 antibody.
  • the anti-PD-L1/IL-15 drug bifunctional protein comprises: (i) a V H comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 97, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 99; and a V L comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 100, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 101, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 102, or (ii) a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 133, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 134, and an HC-CDR3 comprising the amino acid sequence of
  • the anti-PD-L1/IL-15 drug bifunctional protein provided herein, wherein the anti-PD-L1 antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 104; and a VL comprising the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 107; or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 139 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 139; and a V L comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 140.
  • the anti-PD-L1/IL-15 drug bifunctional protein provided herein comprises two monomers, each monomer comprises two polypeptides, wherein: (i) one monomer comprises a polypeptide comprising the structure of V H -C H 1-hinge-C H 2-C H 3-IL-15, and the other polypeptide comprises the structure of VL-CL; and (ii) the other monomer comprises a polypeptide comprising the structure of V H -C H 1-hinge-C H 2-C H 3-IL-15R ⁇ _sushi, and the other polypeptide comprising the structure of VL-CL, wherein the VH is a heavy chain variable domain of the anti-PD-L1 antibody, VL is a light chain variable domain of the anti-PD- L1 antibody.
  • the anti-PD-L1/IL-15 drug bifunctional protein provided herein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 126 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 126; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 115 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 115; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 114 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 114, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 115 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 115.
  • the anti-PD-L1/IL-15 drug bifunctional protein provided herein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 151 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 151; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 129 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 129; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 128 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 128, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 129 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 129.
  • the anti-PD-L1/IL-15 drug bifunctional protein provided herein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 152 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 152; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 132; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 131 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 131, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 132.
  • isolated nucleic acid molecule encoding any of the IL-15 prodrugs, anti-PD-L1 antibody/IL-15 prodrug bifunctional protein, IL-15 drugs or anti-PD-L1 antibody/IL-15 drug bifunctional protein provided herein, vectors comprising such nucleic acid molecule, host cell (e.g., CHO cells, HEK 293 cells, Hela cells, or COS cells) comprising such nucleic acids or vectors, compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising any of the masking polypeptides, cleavable moieties, prodrugs, drugs, or bifunctional protein provided herein.
  • host cell e.g., CHO cells, HEK 293 cells, Hela cells, or COS cells
  • compositions e.g., pharmaceutical compositions
  • kits, and articles of manufacture comprising any of the masking polypeptides, cleavable moieties, prodrugs, drugs, or bifunctional protein provided herein.
  • Fig. 1A depicts the alignment of the amino acid sequences of MP80 and MP100, the amino acid sequence marked in the box (the amino acid sequence marked in the first box is SEQ ID NO: 6) are identical between MP80 and MP100. [0099] Fig.
  • FIG. 1B depicts the alignment of the amino acid sequences of two repeats of MP80 and MP163, the amino acid sequence marked in the first box was the sequence of MP80, i.e., the MP163 comprises the amino acid sequence of MP80.
  • Fig. 1C depicts the alignment of the amino acid sequences of triple repeats of MP80 and MP240, the amino acid sequence marked in the first box was the sequence of MP80, the MP240 comprises 3 copies of the amino acid sequence of MP80.
  • Fig.2A depicts an exemplary IL-15 prodrug with an Fc domain as a half-life extension moiety, showing that an IL-15R ⁇ _sushi domain linked to the C-terminus of one Fc domain, optionally through a non-cleavable linker.
  • An IL-15 is linked to the C-terminus of the other Fc domain, optionally through a non-cleavable linker.
  • a masking polypeptide (MP) is linked to the IL-15 through a cleavable moiety (CM).
  • Fig.2B is an exemplary schematic drawing illustrating the activation process of IL-15 prodrug by released off the masking polypeptide (MP) at the target tissue (e.g., tumor with high levels of MMPs).
  • Fig.3 depicts photograph of non-reduced and reduced SDS-PAGE gels analyzing the purity of exemplary prodrugs SB1902-C2 and SB1902-C7, and drug SB1902-C1(without masking polypeptide).
  • Fig.4 depicts graphs of SEC-HPLC analyzing the homogeneity of exemplary prodrug SB1902-C2, and drug SB1902-C1(without masking polypeptide).
  • Fig.3 depicts photograph of non-reduced and reduced SDS-PAGE gels analyzing the purity of exemplary prodrugs SB1902-C2 and SB1902-C7, and drug SB1902-C1(without masking polypeptide).
  • Fig.4 depicts graphs of SEC-HPLC analyzing the homogeneity of exemplary prod
  • FIG. 5A shows the photograph of polyacrylamide gel analyzing the exemplary cleavable moiety in prodrug SB1902-C2 which is sensitive to the MMP2 enzyme.
  • Fig. 5B shows the photograph of polyacrylamide gel analyzing the exemplary cleavable moiety in prodrug SB1902-C2 which is sensitive to the MMP9 enzyme.
  • Fig. 5C shows the photograph of polyacrylamide gel analyzing the SB1902-C4 with a non-cleavable (G4S)2 linker instead of cleavable moiety, which is kept intact after enzyme MMP2 digestion.
  • Fig. 5A shows the photograph of polyacrylamide gel analyzing the exemplary cleavable moiety in prodrug SB1902-C2 which is sensitive to the MMP9 enzyme.
  • Fig. 5C shows the photograph of polyacrylamide gel analyzing the SB1902-C4 with a non-cleavable (G4S)2 linker instead of cleavable moiety, which is kept intact after enzyme MMP
  • FIG. 5D shows the photograph of polyacrylamide gel analyzing SB1902-C4 with a non-cleavable (G4S)2 linker instead of cleavable moiety, which is kept intact after enzyme MMP9 digestion.
  • Fig. 5E shows the photograph of polyacrylamide gel analyzing the exemplary cleavable moiety in prodrug SB1902-C5 which is sensitive to the MMP9 enzyme.
  • Fig. 5F shows the photograph of polyacrylamide gel analyzing the exemplary cleavable moiety in prodrug SB1902-C5 which is sensitive to the MMP2 enzyme.
  • Fig.6A depicts that the drug SB1902-C1 (without masking polypeptide and cleavable moiety), prodrug SB1902-C2, prodrug SB1902-C3, and SB1902-C4 (without cleavable moiety) show no non-specific binding with human serum protein.
  • Fig. 6B depicts that the drug SB1902-C1 (without masking polypeptide), prodrug SB1902-C2, prodrug SB1902-C3, and SB1902-C4 (without cleavable moiety) show no non- specific binding with cynomolgus monkey serum protein.
  • Fig. 6A depicts that the drug SB1902-C1 (without masking polypeptide and cleavable moiety), prodrug SB1902-C2, prodrug SB1902-C3, and SB1902-C4 (without cleavable moiety) show no non- specific binding with cynomolgus monkey serum protein.
  • FIG. 6C depicts that the drug SB1902-C1 (without masking polypeptide), prodrug SB1902-C2, prodrug SB1902-C3, and SB1902-C4 (without cleavable moiety) show no non- specific binding with rat serum protein.
  • Fig.6D depicts the bands of prodrug SB1902-C2, prodrug SB1902-C3, and SB1902- C4 (without cleavable moiety) on WB membrane before incubation with plasma or in PBS buffer.
  • Fig.6E shows no detectable degradation bands on the WB membrane for both prodrug SB1902-C2, prodrug SB1902-C3, and SB1902-C4 (without cleavable moiety) after incubated with human plasma (abbreviated as plas in the figure) or in PBS buffer.
  • Fig. 7A shows the binding affinity of the exemplary prodrug SB1902-C2 and the exemplary drug SB1902-C1 to IL-2/IL-15R ⁇ receptor
  • Figs.7B-7D show the binding affinity of the exemplary IL-15 drug SB1902-C1-variant3 with wild-type IL-15 and IL-15 variants to IL-2/IL-15R ⁇ receptor.
  • Figs. 7A shows the binding affinity of the exemplary prodrug SB1902-C2 and the exemplary drug SB1902-C1 to IL-2/IL-15R ⁇ receptor
  • Figs.7B-7D show the binding affinity of the exemplary IL-15 drug SB1902-C1-variant3 with wild-type IL
  • FIG. 8A-8B show the binding affinity of the exemplary IL-15 prodrug SB1902-C9- variant2 with wild-type IL-15 and IL-15 variants to IL-2/IL-15R ⁇ receptor.
  • Fig.9 depicts the results of the Mo7e cell proliferation assay, showing that the prodrug SB1902-C2, SB1902-C6, and SB1902-C7 significantly reduced the IL-15 function in stimulating Mo7e cell proliferation as compared to the drug SB1902-C1.
  • Figs.10A-10Q depict the results of the exemplary IL-15 drugs and IL-15 prodrugs in CD8+ T cell activation assay.
  • Fig.10A-10Q depict the results of the exemplary IL-15 drugs and IL-15 prodrugs in CD8+ T cell activation assay.
  • FIG. 11 shows the result of the exemplary prodrug SB1902-C2 and MMP-digested SB1902-C2 of which the masking polypeptide was removed off, in CD8+ T cell activation assay.
  • Fig.12A depicts the results of the IFN- ⁇ production assay in PBMCs
  • Fig.12B depicts the results of the Granzyme B production assay in PBMCs
  • Fig. 12C and Fig.12D depict the results of the IL-15 drug and IL-15 prodrug with wild-type IL-15 and IL-15 variants in IFN- ⁇ production assay.
  • Fig.12A depicts the results of the IFN- ⁇ production assay in PBMCs
  • Fig.12B depicts the results of the Granzyme B production assay in PBMCs
  • Fig. 12C and Fig.12D depict the results of the IL-15 drug and IL-15 prodrug with wild-type IL-15 and IL-15 variants in IFN- ⁇ production assay
  • Fig. 12E and Fig.12F depict the results of the IL-15 drug and IL-15 prodrug with wild-type IL-15 and IL-15 variants in Granzyme B production assay.
  • Fig. 13 depicts the results of the IFN- ⁇ production assay in Balb/c mouse, showing that mice treated with the prodrug SB1902-C2 have less IFN- ⁇ production compared to the drug SB1902-C1.
  • Figs. 14A-14B show that the animals with WEHI-164 tumor were treated with IgG1 isotype control antibody MOPC-21 (Fig. 14A) or prodrug SB1902-C2 (Fig.
  • Figs. 14C-14H show the anti-tumor activity of the different compounds in animals with WEHI-164 tumor that were treated with IgG1 isotype control antibody MOPC-21 (Fig. 14C), SB1902-C4 without cleavable moiety (Fig. 14D), drug SB1902-C1 (Fig.
  • Fig.15A and 15B show the results of the humanized anti-PD-L1 antibodies Hum5, Hum6 and Hum7 in the receptor cell assay that retain their full activity when comparing with the Ref antibody and the parent chimeric antibody Mab 3-16.
  • Fig.16A and 16B show the results of the humanized anti-PD-L1 antibodies Hum5, Hum6 and Hum7 in human PBMC assay that promote the IL-2 secretion of T cells in PBMC.
  • Fig. 17 shows the schematic structure diagram of the exemplary anti-PD-L1/IL-15 prodrug bifunctional protein.
  • Fig. 18 shows the detection results of the function of anti-PD-L1 in the exemplary bifunctional protein SB-1903 in cell line-based assay, that SB-1903 has similar activity in promoting IL-2 production as the control anti-PD-L1 antibody.
  • Fig.19 shows the detection results of the function of IL-15 prodrug in the exemplary bifunctional protein SB-1903 in CD8+ T cell activation assay, that SB-1903 has lower activity in stimulating T cell activation compared to Bis-drug.
  • Fig. 20A shows in vivo result of the inhibition activity on WEHI164 tumor of the exemplary anti-PD-L1/ IL-15 prodrug bifunctional protein (SB-1903-x1), IL-15 prodrug and anti-PD-L1 antibody.
  • Fig. 20B shows the changes of mice body weights with time of all treatment group.
  • FIG. 21A shows in vivo results of the inhibition activity on B16 F10 tumor of the exemplary anti-PD-L1/ IL-15 prodrug bifunctional protein (SB-1903-x2), IL-15 prodrug, anti- PD-L1 antibody and the combination of IL-15 prodrug with anti-PD-L1 antibody.
  • Fig. 21B shows the changes of mice body weights with time of all treatment group.
  • DETAILED DESCRIPTION [0132] Disclosed herein are IL-15 prodrugs with masking polypeptides (MP) and the cleavable moiety (CM).
  • the IL-15 prodrugs overcome the toxicity that have severely limited the clinical use of IL-15.
  • the activity of IL-15 in the prodrugs is attenuated.
  • the cleavable moiety in the prodrug includes protease cleave sites, the masking polypeptides in the prodrug is cleaved off by proteases that are associated with the desired site, such as in a tumor or tumor microenvironment, to recover the activity of IL-15.
  • an anti-PD- L1 antibody/IL-15 prodrug bifunctional protein comprising an anti-PD-L1 antibody and an IL- 15 prodrug described herein, wherein the IL-15 prodrug is linked to the anti-PD-L1 antibody.
  • treatment is an approach for obtaining beneficial or desired results including clinical results.
  • beneficial or desired results including clinical results are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival.
  • treatment is a reduction of a pathological consequence of the disease.
  • the methods of the application contemplate any one or more of these aspects of treatment.
  • an individual is successfully “treated” if one or more symptoms associated with the disease are mitigated or eliminated, including, but are not limited to, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging survival of individuals.
  • the term “prevent,” and similar words such as “prevented,” “preventing” etc. indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition.
  • prevention and similar words also include reducing the intensity, effect, symptoms, and/or burden of a disease or condition prior to recurrence of the disease or condition.
  • delaying the development of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
  • a method that “delays” development of a disease is a method that reduces probability of disease development in a given time frame and/or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of individuals.
  • the term “effective amount” used herein refers to an amount of an agent or a combination of agents, sufficient to treat a specified disorder, condition, or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms. In some embodiments, an effective amount is an amount sufficient to delay disease development. In some embodiments, an effective amount is an amount sufficient to prevent or delay disease recurrence.
  • an effective amount can be administered in one or more administrations.
  • an effective amount may be an amount sufficient to delay cancer development or progression (e.g., decrease tumor growth rate, and/or delay or prevent tumor angiogenesis, metastasis, or infiltration of cancer cells into peripheral organs), reduce the number of epithelioid cells, cause cancer regression (e.g., shrink or eradicate a tumor), and/or prevent or delay cancer occurrence or recurrence.
  • An effective amount can be administered in one or more administrations.
  • an “individual” or a “subject” refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate.
  • the individual is a human.
  • the term “antibody” includes full-length antibodies and antigen-binding fragments thereof.
  • a full-length antibody comprises two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC- CDR3).
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • antigen-binding fragment includes an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a VHH, a single-chain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragments that bind to an antigen but do not comprise a complete antibody structure.
  • an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv
  • An antigen-binding fragment also includes a fusion protein comprising the antibody fragment described above.
  • An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds.
  • an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • CDR or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem.
  • the constant domain contains the C H 1, C H 2, and C H 3 domains (collectively, C H ) of the heavy chain and the CL domain of the light chain.
  • C H constant domain
  • immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
  • the ⁇ and ⁇ classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.
  • the term “Fc”, “Fc region,” “fragment crystallizable region,” “Fc domain,” or “Fc moiety” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230 to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the protein, or by recombinantly engineering the nucleic acid encoding the protein.
  • Suitable native-sequence Fc regions for use in the constructs described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.
  • hinge region is a short sequence of the heavy chains of antibodies linking the Fab (Fragment antigen binding) region to the Fc (Fragment crystallizable region), which allows the Fab a large degree of conformation flexibility relative to the Fc and is helpful for antibody in binding to antigens of different shapes and sizes.
  • hinge region for use in the constructs described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.
  • the hinge regions are different in length and sequence.
  • IgG isotype or “subclass” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • Fc receptor or “FcR” describes a receptor that binds the Fc region of an Fc- containing construct (e.g., antibody, or protein containing Fc region, referred to as Fc fusion protein hereafter).
  • Fc fusion protein e.g., antibody, or protein containing Fc region, referred to as Fc fusion protein hereafter.
  • the preferred FcR is a native sequence of human FcR.
  • a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors, Fc ⁇ RII receptors include Fc ⁇ RIIA (an “activating receptor”) and Fc ⁇ RIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Inhibiting receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • Fc receptor or “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus.
  • FcRn the neonatal receptor
  • Methods of measuring binding to FcRn are known (see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-8 (1997); Ghetie et al., Nature Biotechnology 15 (7): 637-40 (1997); Hinton et al., J. Biol. Chem.
  • Binding to FcRn in vivo and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, e.g., in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having a variant Fc region are administered.
  • WO 2004/42072 (Presta) describes antibody variants which improved or diminished binding to FcRs. See also, e.g., Shields et al., J. Biol. Chem.9(2): 6591- 6604 (2001).
  • Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an Fc- containing construct (e.g., antibody or Fc fusion protein), and vary with Fc isotype.
  • Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
  • “Reduced or minimized” antibody effector function means that which is reduced by at least 50% (alternatively 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) from the wild type or unmodified Fc-containing construct (e.g., antibody or Fc fusion protein).
  • the determination of antibody effector function is readily determinable and measurable by one of ordinary skill in the art.
  • the antibody effector functions of complement binding, complement dependent cytotoxicity and antibody dependent cytotoxicity are affected.
  • effector function is eliminated through a mutation in the constant region that eliminated glycosylation, e.g., “effectless mutation.”
  • the effectless mutation is an N297A or DANA mutation (D265A+N297A) in the C H 2 region. Shields et al., J. Biol. Chem. 276 (9): 6591-6604 (2001).
  • additional mutations resulting in reduced or eliminated effector function include K322A and L234A/L235A (LALA).
  • effector function can be reduced or eliminated through production techniques, such as expression in host cells that do not glycosylate (e.g., E.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., natural killer (NK) cells, neutrophils, and macrophages
  • NK natural killer
  • Fc-containing constructs “arm” the cytotoxic cells and are required for killing the target cell by this mechanism.
  • NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • Fc expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991).
  • an in vitro ADCC assay such as that described in U.S. Pat. No.5,500,362 or 5,821,337 may be performed.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., PNAS USA 95:652-656 (1998).
  • “Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to Fc-containing constructs (of the appropriate subclass) which are bound to their cognate receptor through the ligand fused to Fc.
  • C1q the first component of the complement system
  • Fc-containing constructs of the appropriate subclass
  • a ligand that specifically binds a receptor is a ligand that binds this receptor with greater affinity, avidity, more readily, and/or with greater duration than it binds other receptors.
  • the extent of binding of a ligand to an unrelated receptor is less than about 10% of the binding of the ligand to the target receptor as measured, e.g., by a radioimmunoassay (RIA).
  • a ligand that specifically binds a target receptor has an equilibrium dissociation constant (Kd) of ⁇ 10 -5 M, ⁇ 10 -6 M, ⁇ 10 -7 M, ⁇ 10 -8 M, ⁇ 10 -9 M, ⁇ 10 -10 M, ⁇ 10 -11 M, or ⁇ 10 -12 M.
  • Kd equilibrium dissociation constant
  • a ligand specifically binds a receptor that is conserved among the receptors from different species.
  • specific binding can include, but does not require exclusive binding. Binding specificity of a ligand can be determined experimentally by methods known in the art.
  • Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORE TM -tests and peptide scans.
  • substrate when used in reference to a protease (e.g., metalloproteinase) is intended to mean any material or substance on which the protease (e.g., metalloproteinase) acts.
  • the material or substance can be, for example, a naturally or non- naturally occurring organic chemical, or a macromolecule such as a polypeptide or peptidomimetic.
  • a metalloproteinase substrate specifically interacts with one or more metalloproteinases, and is cleaved by the metalloproteinase. At least one molecule of the substrate is cleaved by the metalloproteinase using appropriate conditions within the time frame of an experiment. In some embodiments, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the substrate can be cleaved by the metalloproteinase [0153]
  • the term “functional analog” refers to a molecule that has the same biological specificity (e.g., binding to the same ligand) and/or activity (e.g., activating or inhibiting a target cell) as a reference molecule.
  • prodrug refers to a therapeutic molecule that is not active until being activated in vivo.
  • modulate includes “increase”, “enhance” or “stimulate” as well as “decrease” or “reduce”, typically in a statistically or physiologically significant amount or degree relative to a control.
  • variant comprises one or more substitutions, additions, deletions and/or insertions relative to a reference polypeptide or polynucleotide.
  • a variant of a polypeptide or polynucleotide comprises an amino acid or nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity or homology to a reference sequence, as described herein, and substantially retains the activity of the reference sequence.
  • sequences that consist of or differ from a reference sequence by the addition, deletion, insertion or substitution of 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides and that substantially retain at least one activity of the reference sequence.
  • addition or deletion includes C-terminal and/or N-terminal addition and/or deletion.
  • wild-type refers to a gene or gene product (e.g., a polypeptide) that is most often observed in a population, and is thus set as the “normal” or “wild-type” form of the gene.
  • linked included covalently linked or non-covalently linked, referring to a first moiety, e.g., a first amino acid sequence or nucleotide sequence, covalently or non- covalently joined to a second moiety, e.g., a second amino acid sequence or nucleotide sequence, respectively.
  • the first moiety can be directly joined or juxtaposed to the second moiety (referred to as directly linked, e.g., through peptide bond in the case of polypeptides) or, alternatively, intervening moiety (e.g., peptide linker) can be used to join the first moiety to the second moiety (referred to as indirectly linked), which can be said that the first moiety is linked to the second moiety through intervening moiety.
  • intervening moiety e.g., peptide linker
  • indirectly linked can be used to join the first moiety to the second moiety through intervening moiety.
  • the term “linked” not only includes a linkage of a first moiety (or a second moiety) at the C-terminus and/or the N-terminus, but also includes the linkage of the whole first moiety (or the second moiety) to any positions (e.g.
  • the first moiety is linked to a second moiety by a peptide bond or a linker.
  • the first moiety can be linked to a second moiety by a phosphodiester bond or a linker.
  • the term “linker” is recognized as and refers to a molecule (including but not limited to unmodified or modified nucleic acids or amino acids) or group of molecules (for example, 2 or more, e.g., 2, 3, 4, 10, 30, 50, 100 or more) or any chemical moiety connecting two moieties, such as two polypeptides.
  • Covalent bond refers to a stable bond between two atoms sharing one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, “peptide bond” refers to a covalent bond formed between a carboxyl group of an amino acid and an amine group of an adjacent amino acid. A “disulfide bond” as used herein refers to a covalent bond formed between two sulfur atoms, such as a combination of two Fc fragments by one or more disulfide bonds. One or more disulfide bonds may be formed between the two fragments by linking the thiol groups in the two fragments.
  • one or more disulfide bonds can be formed between one or more cysteines of two Fc fragments. Disulfide bonds can be formed by oxidation of two thiol groups.
  • the covalent linkage is directly linked by a covalent bond. In some embodiments, the covalent linkage is directly linked by a peptide bond or a disulfide bond. [0160]
  • the term “fused” or “fusion” in reference to two polypeptide sequences refers to the joining of the two polypeptide sequences through a backbone peptide bond. Two polypeptides may be fused directly or through a peptide linker that comprises one or more amino acids.
  • Fusion proteins are polypeptides that comprise two or more regions derived from different or heterologous, proteins or peptides. Fusion proteins are prepared using conventional techniques of enzyme cutting and ligation of fragments from, desired sequences. PCR techniques employing synthetic oligonucleotides may be used to prepare and/or amplify the desired fragments. Overlapping synthetic oligonucleotide representing the desired sequences can also be used to prepare DNA constructs encoding fusion proteins. Fusion proteins can comprise several sequences, including a leader (or signal peptide) sequence, linker sequence, a leucine zipper sequence, or other oligomer-forming sequences, and sequences encoding highly antigenic moieties that provide a means for facile purification or rapid detection of a fusion protein.
  • a fusion protein may be made by recombinant technology from a coding sequence containing the respective coding sequences for the two fusion partners, with or without a coding sequence for a peptide linker in between.
  • fusion encompasses chemical conjugation.
  • the term “IL-15/IL-15R ⁇ complex” provided herein refers to a complex in which the IL-15 cytokine and the IL-15R ⁇ or a functional fragment are non-covalently linked with each other.
  • Half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a substance (e.g., ligand) in inhibiting a specific biological or biochemical function.
  • IC50 is comparable to an “EC50” for agonist drug or other substance (e.g., ligand). EC50 also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo. As used herein, an “IC50” is used to indicate the effective concentration of a ligand needed to neutralize 50% of the receptor bioactivity in vitro.
  • IC50 or EC50 can be measured by bioassays such as inhibition of ligand binding by FACS analysis (competition binding assay), cell-based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA) [0163] “Percent (%) amino acid sequence identity” and “homology” with respect to a peptide or polypeptide sequence are defined as the percentage of amino acid residues in a candidate sequence that is identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the “C-terminus” of a polypeptide refers to the last amino acid residue of the polypeptide which donates its amine group to form a peptide bond with the carboxyl group of its adjacent amino acid residue.
  • N-terminus of a polypeptide as used herein refers to the first amino acid of the polypeptide which donates its carboxyl group to form a peptide bond with the amine group of its adjacent amino acid residue.
  • the term “moiety” refers to a portion of a molecule that has a distinct function within that molecule, and that function may be performed by that moiety in the context of another molecule. A moiety may be a chemical entity with a particular function or a portion of a biological molecule with a particular function.
  • polypeptide polypeptide
  • peptide and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non- amino acids.
  • the terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
  • An “isolated” polypeptide is one that has been identified, separated and/or recovered from a component of its production environment (e.g., natural or recombinant).
  • a component of its production environment e.g., natural or recombinant
  • the isolated polypeptide is free of association with all other components from its production environment.
  • Contaminant components of its production environment such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.
  • the polypeptide will be purified: (1) to greater than 95% by weight of polypeptides as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Blue or, preferably, silver stain.
  • Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide’s natural environment will not be present. Ordinarily, however, an isolated polypeptide will be prepared by at least one purification step.
  • polynucleotides As used herein, the terms “polynucleotides”, “nucleic acids”, “nucleotides” and “oligonucleotides” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotide structure may be imparted before or after assembly of the polymer.
  • sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • An “isolated” nucleic acid molecule encoding a construct is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced.
  • the isolated nucleic acid is free of association with all components associated with the production environment.
  • isolated nucleic acid molecules encoding the polypeptides described herein are in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides described herein existing naturally in cells.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked.
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • host cell or “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • pharmaceutical formulation or “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile.
  • a “sterile” formulation is aseptic or free from all living microorganisms and their spores.
  • embodiments of the application described herein include “consisting of” and/or “consisting essentially of” embodiments.
  • Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
  • reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat disease of type X means the method is used to treat disease of types other than X.
  • masking Polypeptide (MP) refers to a moiety capable of blocking the activity of the biologically active moiety (B). In some embodiments, the masking polypeptide (MP) can inhibit the ability of the cytokine to bind and/or activate its receptor. In some embodiments, the masking polypeptide (MP) can inhibit the ability of the antibody or antigen-binding fragment to bind to its target.
  • the masking polypeptides (MP) have a larger hydrodynamic radius than their actual molecular weight. In some embodiments, the masking polypeptides only form a random coil, without a secondary structure. In some embodiments, the masking polypeptides have a steric masking effect that typically inhibits or blocks the activity of the biologically active moiety due to its proximity to the biologically active moiety and comparative size. [0183] In some embodiments, the masking polypeptide (MP) is composed of four or five types of amino acid residues selected from a group consisting of proline (P), alanine (A), serine (S), glycine (G) and glutamic acid (E).
  • P proline
  • A alanine
  • S serine
  • G glycine
  • E glutamic acid
  • the masking polypeptide (MP) is composed of four types of amino acid residues selected from a group consisting of proline (P), alanine (A), serine (S) and glutamic acid (E). [0185] In some embodiments, the masking polypeptide (MP) is composed of four types of amino acid residues selected from a group consisting of proline (P), glycine (G), serine (S) and glutamic acid (E). [0186] In some embodiments, the masking polypeptide (MP) is composed of five types of amino acid residues selected from a group consisting of proline (P), alanine (A), serine (S), glycine (G) and glutamic acid (E).
  • the net charges in masking polypeptide (MP) are critical.
  • the negative charge is more preferred than positive charge.
  • the net negative charge of MP could avoid its potential interaction with tissues and cell membranes, which could potentially ‘sink’ it if it has a net positive charge before it reaches the destination.
  • too much negative charge could increase the interaction possibility with positively charged proteins.
  • the percentage of the net charge is also depending on the fusion partner of MP.
  • the masking polypeptide (MP) is composed of five types of amino acids G, S, P, E and A, and wherein the percentage of amino acid residue G in the masking polypeptide is about 15%-30%; the percentage of amino acid residue S in the masking polypeptide is about 20%-40%; the percentage of amino acid residue P in the masking polypeptide is about 15%-40%; the percentage of amino acid residue E in the masking polypeptide is about 1%-20%; and the percentage of amino acid residue A in the masking polypeptide is about 5%-20%; and when the number of amino acids is not an integer, take the integer value.
  • the masking polypeptide (MP) is composed of five types of amino acids G, S, P, E and A, and wherein the percentage of amino acid residue G in the masking polypeptide is about 20%; the percentage of amino acid residue S in the masking polypeptide is about 40%; the percentage of amino acid residue P in the masking polypeptide is about 20%; the percentage of amino acid residue E in the masking polypeptide is about 10%; and the percentage of amino acid residue A in the masking polypeptide is about 10%; and when the number of amino acids is not an integer, take the integer value.
  • the masking polypeptide (MP) is composed of four types of amino acids S, P, E and G, and wherein the percentage of amino acid residue S in the masking polypeptide is about 20%-40%; the percentage of amino acid residue P in the masking polypeptide is about 15%-40%; the percentage of amino acid residue E in the masking polypeptide is about 1%-20%; and the percentage of amino acid residue G in the masking polypeptide is about 15%-30%; and when the number of amino acids is not an integer, take the integer value.
  • the masking polypeptide (MP) is composed of four types of amino acids S, P, E, and G, and wherein the percentage of amino acid residue S in the masking polypeptide is about 23%; the percentage of amino acid residue P in the masking polypeptide is about 29%; the percentage of amino acid residue E in the masking polypeptide is about 18%; and the percentage of amino acid residue G in the masking polypeptide is about 30%; and when the number of amino acids is not an integer, take the integer value.
  • the masking polypeptide (MP) can be tuned by changing its amino acid chain length and its total net charge to meet certain requirements with certain partners.
  • the masking polypeptides (MP) comprises about 40 to 720 amino acid residues. In some embodiments, the masking polypeptides (MP) comprises about 80 to 320 amino acid residues. In some embodiments, the masking polypeptides (MP) comprises about 80 to 240 amino acid residues. [0194] In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence of SEQ ID NO: 6. [0195] In some embodiments, the masking polypeptide (MP) comprises the amino acid sequence of SEQ ID NO: 1.
  • the masking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-5, or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-5, or a variant thereof comprising one or more amino acid substitutions, additions and/or deletions.
  • CM Cleavable Moiety
  • the cleavable moiety (CM) is a polypeptide that comprises or is the cleavage site of an enzyme or a protease.
  • the proteases include but are not limited to urokinase-type plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, and/or MMP27); Tobacco Etch Virus (TEV) protease; plasmin; Thrombin; PSA; PSMA; ADAMS/ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM13, ADAM17/TACE, ADAMDEC1, ADAMTS1, ADAMTS4, and/or ADAMTS5); caspases (e.g., Caspase-1, Caspase-2, Caspase- 3, Caspase-4, Cas
  • the cleavable moiety comprises a substrate sequence for at least one matrix metalloprotease (MMP).
  • MMPs include MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27.
  • MMPs include MMP1; MMP2; MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15; MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27.
  • the CM comprises a substrate sequence for MMP2, MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11 and MMP19.
  • the CM comprises a substrate sequence
  • the CM comprises a substrate sequence for MMP9. In some embodiments, the CM comprises a substrate sequence for two or more MMPs. In some embodiments, the CM comprises a substrate sequence for at least MMP2 and MMP9. In some embodiments, the CM comprises two or more substrates for the same MMP. In some embodiments, the CM comprises at least two or more MMP2 substrates. In some embodiments, the CM comprises at least two or more MMP9 substrates.
  • the cleavable moiety comprises the amino acid sequence MVX1X2AX3TX4SG (SEQ ID NO: 49), wherein X1 is selected from P, L, V, or A, X2 is selected from L or S, X3 is selected from L V, P, or Y and X4 is selected from A or V.
  • SEQ ID NO: 49 amino acid sequence MVX1X2AX3TX4SG
  • the CM comprises the amino acid sequence of any one of SEQ ID NOs: 8-16, or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 8-16.
  • the biologically active moiety (B) can be a chemical entity. In some embodiments, the biologically active moiety (B) can be a therapeutic protein. In some embodiments, the biologically active moiety (B) is a cytokine. In some embodiments, the biologically active moiety (B) is an antibody or antigen-binding fragment that targets the prodrugs to a site of action (e.g., sites of inflammation, or a tumor).
  • a site of action e.g., sites of inflammation, or a tumor.
  • Cytokines is a well-known term of art that refers to any of a class of immunoregulatory proteins (such as interleukin or interferon) that are secreted by cells especially of the immune system and that are modulators of the immune system.
  • the cytokine includes the functional fragment, mutant, or variant of the cytokine.
  • Examples of cytokines may include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors.
  • cytokine prodrugs while the description below exemplifies IL-15 prodrugs.
  • prodrugs for other cytokines in particular cytokines that are potent immune regulators and have strong side effects, are also contemplated in the present disclosure.
  • cytokine Upon proteolytic cleavage of the cleavable moiety at the target site, the cytokine becomes to be an activated form, which renders it capable of binding to its cognate receptor or protein with increased affinity.
  • cytokine prodrugs may be made according to the same principles as illustrated below for IL-15 prodrugs.
  • the cytokine is selected from the group consisting of IL-1 ⁇ , IL-1 ⁇ , IL-1 receptor antagonist (IL-1RA), IL-18, IL-33, IL-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ , IL-36 receptor antagonist (IL-36RA), IL-37, and IL-38, or mutants of the cytokines.
  • the cytokine is selected from the group consisting of IL-2, IL- 3, IL-4, IL-5, IL-7, IL-9, IL-13, IL-15, IL-21, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), IL-6, IL-11, IL-12, growth hormone (GH), erythropoietin (EPO), prolactin (PRL), leukemia inhibitory factor (LIF), oncostatin (OSM), and thrombopoietin (TPO), or mutants of the cytokines.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • M-CSF macrophage colony-stimulating factor
  • IL-6 IL-11, IL-12
  • growth hormone GH
  • EPO erythropoietin
  • PRL prolactin
  • LIF leukemia
  • the cytokine is selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CCL1e, CCL2, CCL3, CCL3L1, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CX3CL1, XCL1, and XCL2, or mutants of the cytokines.
  • the cytokine is selected from the group consisting of IFN- ⁇ (alpha), IFN- ⁇ (beta), IFN- ⁇ (gamma), IFN- ⁇ (epsilon), IFN- ⁇ (kappa), IFN-( ⁇ ) (omega), IFN- ⁇ (tau), IFN- ⁇ (zeta), IFN- ⁇ (delta), and IFN- ⁇ , (lambda), or mutants of the cytokines.
  • the cytokine is selected from the group consisting of IL-1, IL- 2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL- 17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL- 29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, and IL-37.
  • the cytokine is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony- stimulating factor (M-CSF), tumor necrosis factor-alpha (TNF- ⁇ ), transforming growth factor- beta (TGF- ⁇ ), IFN- ⁇ (gamma), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, and IL-12.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • M-CSF macrophage colony- stimulating factor
  • TGF- ⁇ tumor necrosis factor-alpha
  • TGF- ⁇ transforming growth factor- beta
  • IFN- ⁇ gamma
  • the cytokine is selected from the group consisting of TNF- ⁇ (alpha), TNF- ⁇ (beta), TNF- ⁇ (gamma), CD252, CD154, CD178, CD70, CD153, 4-1BB-L, TRAIL, RANKL, APO3L, CD256, CD257, CD258, TL1, AITRL, and EDA1.
  • the cytokine disclosed herein are mutated or engineered to alter the properties of the naturally occurring cytokine, including receptor binding affinity and specificity or serum half-life.
  • Non-Cleavable Linker (L) [0212]
  • the linker is a non-cleavable linker.
  • Example of a non- cleavable linker is stable under physiological conditions and at a diseased site such as a tumor site or an inflammatory disease site.
  • the non-cleavable linker is rich in amino acid residues G and S.
  • the non-cleavable linker includes a “G4S” repeat.
  • the non-cleavable linker is a polypeptide chain comprising at least 3 residues. Portions of such linkers may be flexible, hydrophilic, and have little or no secondary structure of their own (linker portions or flexible linker portions). Linkers of at least 3 amino acids may be used to join domains and/or regions that are positioned near to one another after the molecule has assembled.
  • linkers may be about any one of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues.
  • the linkers may be the same or different (e.g., the same or different length and/or amino acid sequence).
  • the non-cleavable linker comprises or consists of a Gly-Ser linker.
  • Gly-Ser linker refers to a peptide that consists of glycine and serine residues.
  • an exemplary Gly-Ser linker comprises an amino acid sequence of GSG (SEQ ID NO: 17). In some embodiments, an exemplary Gly-Ser linker comprises an amino acid sequence of the formula (Gly 4 Ser) n , wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, a preferred Gly-Ser linker is (Gly4Ser)1. In some embodiments, a preferred Gly-Ser linker is (Gly4Ser)2. In some embodiments, a preferred Gly-Ser linker is (Gly 4 Ser) 3 . In some embodiments, a preferred Gly- Ser linker is (Gly4Ser)4.
  • a preferred Gly-Ser linker is (Gly4Ser)5. In yet other aspects, two or more Gly-Ser linkers are incorporated in series in a polypeptide linker.
  • a non-cleavable linker used in the prodrug described herein comprises an immunoglobulin (Ig) / antibody hinge region. In one embodiment, the hinge region is obtained from an IgGl antibody.
  • Ig "hinge” region refers to a polypeptide comprising an amino acid sequence that shares sequence identity, or similarity, with a portion of a naturally-occurring Ig hinge region sequence, which includes the cysteine residues at which the disulfide bonds link the two heavy chains of the immunoglobulin.
  • a non-cleavable linker is used to link any of the components of a prodrug provided herein.
  • the non-cleavable linker comprises the amino acid sequence of any one of SEQ ID NOs: 17-21 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-21.
  • Half-Life Extension Moiety C
  • the prodrug comprises an in vivo half-life extension moiety (C).
  • the term half-life extension moiety refers to a moiety that extends the half-life of the target component in serum.
  • a long half-life in vivo is important for therapeutic molecules, for example, cytokines that are administered to a subject generally have a short half-life since they are normally cleared rapidly from the subject by mechanisms including clearance by the kidney and endocytic degradation. Increasing the in vivo half-life of therapeutic molecules with naturally short half- lives allows for a more acceptable and manageable dosing regimen without sacrificing effectiveness.
  • a half-life extension moiety is linked to the biologically active moiety for the purpose of extending the half-life in vivo.
  • a “half-life extension moiety” increases the in vivo half-life and improve PK, for example, by altering its size (e.g., to be above the kidney filtration cutoff), shape, hydrodynamic radius, charge, or parameters of absorption, biodistribution, metabolism, and elimination.
  • An exemplary way to improve the PK of a polypeptide is by expression of an element in the polypeptide chain that binds to receptors that are recycled to the plasma membrane of cells rather than degraded in the lysosomes, such as the FcRn receptor on endothelial cells and transferrin receptor.
  • the half-life extension moiety (C) can also be an antibody or antigen-binding fragment that binds to a protein with a long serum half-life such as serum albumin, transferrin, and the like.
  • antibodies or antigen-binding fragments thereof include a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single-chain variable fragment (scFv), a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain of camelid-type nanobody (V HH ), a dAb and the like.
  • the half-life extension moiety (C) could also be functioned as a linker, optionally as a non-cleavable linker (L).
  • the half-life extension moiety is an antibody Fc domain (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc) or fragment thereof that is capable of FcRn-mediated recycling, such as any heavy chain polypeptide or portion thereof that is capable of FcRn- mediated recycling.
  • the Fc domain is a monomer.
  • the Fc domain is a dimer, comprising a first Fc domain and a second Fc domain.
  • the Fc domain is derived from any of IgA, IgD, IgE, IgG, and IgM, and subtypes thereof.
  • the Fc domain has the highest serum content and longest serum half-life among all immunoglobulins. Unlike other immunoglobulins, IgG is effectively recycled after binding to Fc receptors (FcRs).
  • the Fc domain is derived from an IgG (e.g., IgG1, IgG2, IgG3, or IgG4).
  • the Fc domain is derived from a human IgG.
  • the Fc domain comprises CH2 and CH3 domains.
  • the Fc domain further comprises full or part of the hinge region.
  • the Fc domain is derived from a human IgG1 or human IgG4.
  • the two subunits of the Fc domain dimerize via one or more (e.g., 1, 2, 3, 4, or more) disulfide bonds.
  • each subunit of the Fc domain comprises a full-length Fc sequence.
  • each subunit of the Fc domain comprises an N-terminus truncated Fc sequence, such as a truncated Fc domain with fewer N-terminal cysteines in order to reduce disulfide bond mispairing during dimerization.
  • the Fc domain is truncated at the N- terminus, e.g., lacks the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of a complete immunoglobulin Fc domain.
  • the Fc domain contains one or more mutations, such as insertion, deletion, and/or substitution.
  • the Fc domain contains one or more amino acid mutations altering effector function, the Fc domain is engineered (e.g., comprises one or more amino acid mutations) to have altered binding to an FcR, specifically altered binding to an Fc ⁇ receptor (responsible for ADCC), and/or altered effector function, such as altered antibody-dependent cell-mediated cytotoxicity (ADCC), Antibody-Dependent Cellular Phagocytosis (ADCP), and/or Complement-Dependent Cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP Antibody-Dependent Cellular Phagocytosis
  • CDC Complement-Dependent Cytotoxicity
  • Fc domain e.g., human IgG1 Fc
  • effectless or “almost effectless” Fc
  • the Fc is an effectless human IgG1 Fc comprising one or more of the following mutations (such as in each of Fc subunits): L234A, L235E, G237A, A330S, and P331S.
  • the Fc domain comprises L234A and L235A (“LALA”) mutations.
  • L234A and L235A (“LALA”) mutations.
  • the combinations of K322A, L234A, and L235A in IgG1 Fc are sufficient to almost completely abolish Fc ⁇ R and C1q binding (Hezareh et al. J Virol 75, 12161– 12168, 2001).
  • MedImmune identified that a set of three mutations L234F/L235E/P331S have a very similar effect (Oganesyan et al., Acta Crystallographica 64, 700–704, 2008).
  • the Fc moiety comprises a modification of the glycosylation on N297 of the IgG1 Fc domain, which is known to be required for optimal FcR interaction.
  • the Fc domain modification can be any suitable IgG Fc engineering mentioned in Wang et al. (“IgG Fc engineering to modulate antibody effector functions,” Protein Cell.2018 Jan; 9(1): 63–73), the content of which is incorporated herein by reference in its entirety.
  • Glycosylation variants [0226]
  • the Fc domain is altered to increase or decrease the extent to which the construct is glycosylated.
  • glycosylation sites to an Fc domain may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.
  • Native Fc-containing proteins produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the C H 2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an Fc moiety may be made in order to create certain improved properties.
  • the Fc domain described herein is provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc domain.
  • the amount of fucose in such Fc domain may be from about 1% to about 80%, from about 1% to about 65%, from about 5% to about 65%, or from about 20% to about 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 Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc domain (EU numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in Fc domains. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.
  • Examples of cell lines capable of producing defucosylated Fc- containing proteins include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y.
  • the present application contemplates an Fc domain that possesses some but not all Fc effector functions, which makes it a desirable candidate for applications in which the half-life in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious.
  • effector functions such as CDC and ADCC
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the Fc domain lacks Fc ⁇ R binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • the primary cells for mediating ADCC Natural Killer (NK) cells, express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Patent No.5,500,362 see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)).
  • non- radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc.
  • PBMC peripheral blood mononuclear cells
  • NK cells Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998).
  • C1q binding assays may also be carried out to confirm that the Fc domain is unable to bind C1q and hence lacks CDC activity.
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B.
  • Fc domain with reduced effector function includes those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No.6,737,056).
  • Such Fc mutants include substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • alterations are made in the Fc domain that results in altered (i.e., either improved or diminished) C1q binding and/or CDC, e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol.164: 4178-4184 (2000).
  • the Fc domain comprises one or more amino acid substitutions, which increase the half-life and/or improve binding to the neonatal Fc receptor (FcRn).
  • Antibodies with increased half-lives and improved binding to the neonatal FcRn, which is responsible for the transfer of maternal IgGs to the fetus are described in US2005/0014934A1 (Hinton et al.).
  • Those antibodies comprise an Fc domain with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Such Fc variants include those with substitutions at one or more of Fc region residues, e.g., substitution of Fc region residue 434 (US Patent No.7,371,826).
  • Cysteine-engineered variants it may be desirable to create a cysteine-engineered Fc domain, in which one or more residues of an Fc domain are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the Fc domain.
  • any one or more of the following residues may be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc domain.
  • Cysteine engineered molecules may be generated as described, e.g., in U.S. Patent No. 7,521,541.
  • the Fc domain is derived from an IgG1 Fc.
  • the Fc domain is derived from a human IgG1 Fc.
  • the Fc moiety is a wildtype IgG1 Fc (IGHG1*05).
  • the Fc domain is a natural variant of IgG1 (e.g., IGHG1*03, which comprises D239E and L241M double mutations relative to IGHG1*05).
  • the Fc domain does not comprise the hinge region of an IgG1 Fc.
  • the Fc domain comprises at most about 5 amino acids truncated from the N-terminus of an IgG1 Fc, such as truncating the first, the first two, the first three, the first four, or the first five amino acids from the N-terminus of the IgG1 Fc. In some embodiments, the Fc domain comprises one or more ineffective mutations and/or deglycosylation mutation(s). [0235] In some embodiments, the Fc domain is derived from an IgG4 Fc. In some embodiments, the Fc domain is derived from a human IgG4 Fc. In some embodiments, the Fc domain is a wildtype IgG4 Fc.
  • the Fc domain is a natural variant of IgG4. In some embodiments, the Fc domain does not comprise the hinge region of an IgG4 Fc. In some embodiments, the Fc domain comprises at most about 5 amino acids truncated from the N-terminus of an IgG4 Fc, such as truncating the first, the first two, the first three, the first four, or the first five amino acids from the N-terminus of the IgG4 Fc. In some embodiments, the Fc domain comprises one or more ineffective mutations and/or deglycosylation mutation(s).
  • the first and/ or second polypeptide chain of Fc domain each contain one or more modifications that promote heterodimerization of the first and the second Fc domain.
  • one or more amino acid modifications can be made to the first Fc domain and one or more amino acid modifications can be made to the second Fc domain using any strategy available in the art, including any strategy as described in Klein et al. (2012), MAbs, 4(6): 653- 663.
  • Exemplary strategies and modifications are the “knob into holes” approach.
  • the first Fc domain comprising a CH3 domain is a heavy chain polypeptide or a fragment thereof.
  • the CH3 domains of the two Fc domains can be altered by the “knobs-into- holes” technology (Fc knob and Fc hole), which is described in detail with several examples in, e.g., WO 1996/027011; Ridgway, J.B. et al. Protein Eng (1996) 9(7): 617-621; Merchant, A.M., et al, Nat. Biotechnoi. (1998) 16(7): 677-681. See also Klein et al. (2012), MAbs, 4(6): 653- 663.
  • the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of the two moieties containing the two altered CH3 domains. This occurs by introducing a bulky residue into the CH3 domain of one of the Fc domains, which acts as the “knob.” Then, in order to accommodate the bulky residue, a “hole” is formed in the other Fc domain that can accommodate the knob. Either of the altered CH3 domains can be the “knob” while the other can be the “hole.” The introduction of a disulfide bridge further stabilizes the heterodimers (Merchant, A.M., et al, Nat. Biotechnoi (1998) 16(7); Atwell, S., et al, J.
  • heterodimerization can be achieved by introducing the T366W and/or S354C mutations in a heavy chain to create the “knob” and by introducing the T366S, L368A, Y407V and/or Y349C mutations in a heavy chain to create the “hole” (numbering of the residues according to the Kabat EU numbering system). Carter et al. (2001), J. Immunol. Methods, 248: 7-15; Klein et al. (2012), MAbs, 4(6): 653-663.
  • the Fc domain or fragment thereof comprises the mutations of T366S, L368A, and Y407V to form a ‘hole’. In some embodiments, the Fc domain or fragment thereof comprises the mutation of T366W to form a ‘knob’. In some embodiments, the Fc domain or fragment thereof includes the mutations Y349C, T366S, L368A, and Y407V to form a ‘hole’. In some embodiments, the Fc domain or fragment thereof includes the mutations S354C and T366W to form a ‘knob’. In some embodiments, the first Fc domain or fragment thereof includes the hole mutations, and the second Fc domain or fragment thereof includes the knob mutation.
  • the first Fc domain or fragment thereof includes the knob mutations
  • the second Fc domain or fragment thereof includes the hole mutation, numbering of the residues according to the EU numbering system.
  • the knobs-into-holes mutation are present in the Fc domains in addition to the LALA (L234A and L235A) mutation.
  • the first Fc domain comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28.
  • the first Fc domain comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27.
  • the first Fc domain comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30.
  • the first Fc domain comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29.
  • Prodrugs [0243] One aspect of the present application provides activatable prodrugs that are metabolized in vivo to become active therapeutics and have fewer side effects and better target specificity.
  • the prodrugs comprise one or more biologically active moiety (B), one or more cleavable moiety (CM), and one or more masking polypeptide (MP).
  • the masking polypeptide (MP) in the prodrugs inhibits the biological functions of the biologically active moiety (B).
  • the prodrugs may be activated at a target site (e.g., at a tumor site or the surrounding environment) in the patient by protease digestion of the cleavable moiety (CM) and the consequent release of the masking polypeptide (MP) from the prodrug, exposing the previously masked biologically active moiety (B), and allowing the biologically active moiety (B) to exert its biological functions on the target cell.
  • the prodrugs further comprise one or more non-cleavable linker (L).
  • the prodrug provided herein, wherein the masking polypeptide (MP) and the biologically active moiety (B) are linked through the cleavable moiety (CM).
  • the prodrug provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably through a non-cleavable linker (L).
  • the prodrug provided herein, wherein the cleavable moiety (CM) is linked to the biologically active moiety (B), preferably through a non-cleavable linker.
  • the prodrugs further comprise one or more half-life extension moiety (C).
  • the prodrug provided herein, wherein the biologically active moiety (B) is linked to the half-life extension moiety (C).
  • the prodrug provided herein wherein the biologically active moiety (B) is linked to the half-life extension moiety (C), preferably through a non-cleavable linker (L).
  • the prodrug provided herein, wherein the cleavable moiety (CM) is linked to the half-life extension moiety (C), preferably through a non-cleavable linker (L).
  • the prodrug provided herein wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably through a non-cleavable linker (L).
  • the prodrug provided herein, wherein the prodrug comprises the construct in an N to C or in a C to N-terminal direction: B-C-CM-MP, wherein the “-” represents covalent bond with or without non-cleavable linker (L).
  • the prodrug provided herein wherein the prodrug comprises the construct in an N to C or in a C to N-terminal direction: C-B-CM-MP, wherein the “-” represents covalent bond with or without non-cleavable linker (L).
  • the prodrug provided herein is a monomer.
  • the prodrug provided herein is a dimer.
  • the dimer is monovalent.
  • the dimer is bivalent.
  • the dimer is a homodimer.
  • the dimer is a heterodimer.
  • the prodrug provided herein is a dimer, wherein one monomer comprises the construct in an N to C or in a C to N-terminal direction: C-B, the other monomer comprises the construct in an N to C or in a C to N-terminal direction: C-CM- MP, wherein the “-” represents covalent bond with or without non-cleavable linker (L).
  • C-B the construct in an N to C or in a C to N-terminal direction
  • C-CM- MP C-CM- MP
  • the “-” represents covalent bond with or without non-cleavable linker (L).
  • L non-cleavable linker
  • the IL-15 prodrugs have better PK profiles in vivo (e.g., longer half-life).
  • the illustrative structure of the exemplary IL-15 cytokine prodrug is shown in Fig. 2A.
  • the IL-15 prodrug provided herein comprises (i) one or more IL-15 cytokine(I), (ii) one or more masking polypeptide (MP) of the disclosure and (iii) one or more cleavable moiety (CM) of the disclosure.
  • the IL-15 prodrug provided herein wherein the IL-15 cytokine (I) and the masking polypeptide (MP) are linked through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein comprises the construct in an N to C-terminal or in a C to N- terminal direction: I-CM-MP, wherein the "-" represents covalent bond with or without non-cleavable linker (L).
  • the IL-15 prodrug provided herein further comprise one or more half-life extension moiety (C).
  • the IL-15 prodrug provided herein wherein the IL-15 cytokine is linked to the half-life extension moiety (C), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP) through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein wherein the cleavable moiety (CM) is linked to half-life extension moiety (C), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein comprises the construct in an N to C-terminal or in a C to N- terminal direction: C-I-CM-MP, wherein the "-" represents covalent bond with or without non-cleavable linker (L).
  • the IL-15 prodrug provided herein comprises the construct in an N to C-terminal or in a C to N- terminal direction: I-C-CM-MP, wherein the "-" represents covalent bond with or without non-cleavable linker (L)
  • the IL-15 prodrug provided herein comprises the construct in an N to C-terminal or in a C to N- terminal direction: C-CM-MP-I, wherein the "-" represents covalent bond with or without non-cleavable linker (L).
  • the IL-15 prodrug provided herein is a dimer, wherein one monomer comprises the construct in an N to C-terminal or in a C to N-terminal direction: C-I; and the other monomer comprises the construct in an N to C-terminal or in a C to N-terminal direction: C-CM-MP, and wherein the "-" represents covalent bond with or without non- cleavable linker (L).
  • the IL-15 prodrug provided herein comprises (i) one or more IL-15 cytokine (I), (ii) one or more IL-15R ⁇ or a functional fragment thereof (S), (iii) one or more masking polypeptide (MP) of the disclosure and (iv) one or more cleavable moiety (CM) of the disclosure.
  • the IL-15 prodrug provided herein wherein the cleavable moiety (CM) is linked to the IL-15 cytokine (I), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein, wherein the IL-15R ⁇ or a functional fragment thereof (S) is linked to the IL-15 cytokine (I).
  • the IL-15 prodrug provided herein, wherein the IL-15R ⁇ or a functional fragment thereof (S) and the masking polypeptide (MP) are linked through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the cleavable moiety (CM) is linked to the IL-15R ⁇ or a functional fragment thereof (S), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein, wherein the cleavable moiety (CM) is linked to the masking polypeptide (MP), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein, wherein the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are covalently linked.
  • the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are linked through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein, wherein the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are non-covalently linked, and form an IL-15/ IL-15R ⁇ complex.
  • IL-15 prodrug provided herein wherein the IL-15 R ⁇ or a functional fragment and IL-15 cytokine are transfected separately and an IL-15/IL-15R ⁇ complex is formed.
  • the IL-15 prodrug provided herein further comprises one or more life-extension moiety (C).
  • the IL-15 prodrug provided herein wherein the IL-15 cytokine (I) is linked to the half-life extension moiety (C), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein, wherein the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C).
  • the IL-15 prodrug provided herein, wherein the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein wherein the masking polypeptide (MP) is linked to the half-life extension moiety (C) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the cleavable moiety (CM) is linked to the half-life extension moiety (C), preferably through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S) is linked to the IL-15 cytokine (I), and the masking polypeptide (MP) is linked to the IL- 15R ⁇ or a functional fragment thereof (S) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C), and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein wherein the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C), the IL-15 cytokine (I) is linked to the IL-15R ⁇ or a functional fragment thereof (S), and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the IL-15 cytokine (I) is linked to the half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C), and the masking polypeptide (MP) is linked to the IL-15R ⁇ or a functional fragment thereof (S) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein, wherein the masking polypeptide (MP) is linked to the half-life extension moiety (C) through the cleavable moiety (CM), the IL-15R ⁇ or a functional fragment thereof (S) is linked to the half-life extension moiety (C), and the IL-15 cytokine (I) is linked to the IL-15R ⁇ or a functional fragment thereof (S).
  • the IL-15 prodrug provided herein wherein the masking polypeptide (MP) is linked to the half-life extension moiety (C) through the cleavable moiety (CM), the IL-15 cytokine (I) is linked to the half-life extension moiety (C), and the IL-15R ⁇ or a functional fragment thereof (S) is linked to the IL-15 cytokine (I).
  • the IL-15 prodrug provided herein, wherein the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are covalently linked.
  • the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are linked through a non-cleavable linker (L).
  • the IL-15 prodrug provided herein, wherein the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are non-covalently linked, and form an IL-15/ IL-15R ⁇ complex.
  • the IL-15 prodrug provided herein comprises the construct in an N to C or in a C to N-terminal direction: C-I-S-CM-MP, wherein the “-” represents covalent bond with or without non-cleavable linker (L).
  • the IL-15 prodrug provided herein comprises the construct in an N to C or in a C to N-terminal direction: C-S-I-CM-MP, wherein the “-” represents covalent bond with or without non-cleavable linker (L).
  • the IL-15 prodrug provided herein comprises the construct in an N to C or in a C to N-terminal direction: I-C-S-CM-MP, wherein the “-” represents covalent bond with or without non-cleavable linker (L).
  • the IL-15 prodrug provided herein comprises the construct in an N to C or in a C to N-terminal direction: S-C-I-CM-MP, wherein the “-” represents covalent bond with or without non-cleavable linker (L).
  • the IL-15 prodrug provided herein comprises the construct in an N to C or in a C to N-terminal direction: S-I-C-CM-MP, wherein the “-” represents covalent bond with or without non-cleavable linker (L).
  • the IL-15 prodrug provided herein comprises the construct in an N to C or in a C to N-terminal direction: I-S-C-CM-MP, wherein the “-” represents covalent bond with or without non-cleavable linker (L).
  • the IL-15 prodrug provided herein is a monomer.
  • the IL-15 prodrug provided herein is a dimer. In some embodiments, the dimer is monovalent. In some embodiments, the dimer is bivalent. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.
  • the IL-15 prodrug provided herein is a dimer, wherein one monomer comprises the first half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S), the IL-15 cytokine (I); and the other monomer comprises the second half- life extension moiety (C), the masking polypeptide (MP) and the cleavable moiety (CM), wherein the masking polypeptide (MP) is linked to the second half-life extension moiety (C) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein is a dimer, wherein one monomer comprises the first half-life extension moiety (C), the IL-15R ⁇ or a functional fragment thereof (S), the masking polypeptide (MP) and the cleavable moiety (CM); and the other monomer comprises the second half-life extension moiety (C) and the IL-15 cytokine (I).
  • the IL-15 prodrug provided herein is a dimer, wherein one monomer comprises the first half-life extension moiety (C), the IL-15 cytokine (I), the masking polypeptide (MP) and a cleavable moiety (CM); and the other monomer comprises the second half-life extension moiety (C) and the IL-15R ⁇ or a functional fragment thereof (S).
  • the IL-15 prodrug provided herein is a dimer, wherein one monomer and the other monomer each comprises the half-life extension moiety (C), the IL-15 cytokine (I), the IL-15R ⁇ or a functional fragment thereof (S), the cleavable moiety (CM) and the masking polypeptide (MP).
  • the IL-15 prodrug provided herein is a dimer, wherein in one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C) and the IL- 15R ⁇ or a functional fragment thereof (S) is linked to the first half-life extension moiety (C); and in the other monomer: the masking polypeptide (MP) is linked to the second half-life extension moiety (C) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein is a dimer, wherein in one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C) and the IL- 15R ⁇ or a functional fragment thereof (S) is linked to the IL-15 cytokine (I); and in the other monomer: the masking polypeptide (MP) is linked to the second half-life extension moiety (C) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein is a dimer, wherein in one monomer: the IL-15R ⁇ or a functional fragment thereof (S) is linked to the first half-life extension moiety (C) and the masking polypeptide (MP) is linked to the first half-life extension moiety (C) through the cleavable moiety (CM); and in the other monomer: the IL-15 cytokine (I) is linked to the second half-life extension moiety (C).
  • the IL-15 prodrug provided herein is a dimer, wherein in one monomer: the IL-15R ⁇ or a functional fragment thereof (S) is linked to the first half-life extension moiety (C) and the masking polypeptide (MP) is linked to the IL-15R ⁇ or a functional fragment thereof (S) through the cleavable moiety (CM); and in the other monomer: the IL-15 cytokine (I) is linked to the second half-life extension moiety (C).
  • the IL-15 prodrug provided herein is a dimer, wherein in one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C) and the masking polypeptide (MP) is linked to the first half-life extension moiety (C) through the cleavable moiety (CM); and in the other monomer: the IL-15R ⁇ or a functional fragment thereof (S) is linked to the second half-life extension moiety (C).
  • the IL-15 prodrug provided herein is a dimer, wherein in one monomer: the IL-15 cytokine (I) is linked to the first half-life extension moiety (C) and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM); and in the other monomer: the IL-15R ⁇ or a functional fragment thereof (S) is linked to the second half-life extension moiety (C).
  • the IL-15 prodrug provided herein, wherein the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are covalently linked.
  • the IL-15R ⁇ or a functional fragment thereof (S) and the IL-15 cytokine (I) are linked through a non-cleavable linker (L).
  • IL-15 is essential for natural killer cells (NK), natural killer T cells (NKT), and memory CD8+ T cells development and function.
  • IL-15 is a cytokine which is like IL-2, having originally been described as a T cell growth factor.
  • cytokines exert their cell signaling function through binding to a trimeric complex consisting of two shared receptors, the common gamma chain ( ⁇ c; CD132) and IL-2 receptor beta-chain (IL-2R ⁇ ; CD122), as well as an alpha chain receptor unique to each cytokine: IL-2 receptor alpha (IL-2R ⁇ ; CD25) or IL-15 receptor alpha (IL-15R ⁇ ; CD215).
  • IL-15 shares components of the receptor for IL-2, the alpha chain of the IL-2 receptor (IL-2R) is not required, but both beta and common gamma chains are needed for IL-15 mediated bioactivities.
  • IL-15 a novel T cell growth factor that shares activities and receptor components with IL-2. J Leukoc Biol. 1995 May;57(5):763-6.).
  • IL-15R consists of three subunits IL-15R ⁇ , IL-2/IL-15R ⁇ , and ⁇ chain, IL-15R ⁇ is required for high-affinity binding but not signaling by IL-15.
  • IL-15 functions mainly via trans-presentation (TP), during which an APC expressing IL-15 bound to IL-15R ⁇ presents the ligand to the ⁇ receptor- heterodimer on a neighboring T/NK cell (Kenesei ⁇ , Volkó J, et al. IL-15 Trans-Presentation Is an Autonomous, Antigen-Independent Process.
  • TP trans-presentation
  • IL-15 cytokine includes wild-type IL-15 or variants thereof, also includes functional fragment thereof.
  • the IL-15 or IL-15 cytokine of the present application is a wild-type IL-15.
  • the IL- 15 or IL-15 cytokine of the present application is an IL-15 variant.
  • wild- type IL-15 is synthesized as a precursor polypeptide of 162 amino acids, which is then processed into mature IL-15 by the removal of amino acid residues 1-48.
  • the IL-15 cytokine comprises the amino acid sequence SEQ ID NO: 22 or a variant thereof having at least 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22.
  • the IL-15 cytokine comprises the amino acid sequence SEQ ID NO: 23 or a variant thereof having at least 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 23.
  • the IL-15 cytokine also is an IL-15 variant or functional fragment thereof.
  • the IL-15 cytokine is any naturally occurring interleukin-15 (IL-15) protein.
  • the IL-15 cytokine is a variant thereof capable of binding to, or otherwise exhibiting improved or decreased affinity for, an interleukin- 15 receptor (IL-15R) or component thereof (e.g., the IL-15R ⁇ , IL-2/IL-15R ⁇ , and/or ⁇ chain).
  • IL-15R interleukin- 15 receptor
  • the IL-15 cytokine is an IL-15 variant comprising an amino acid sequence produced by at least one amino acid modification to the amino acid sequence of wild-type IL-15 (e.g., SEQ ID NO: 22).
  • Each at least one amino acid modification can be any amino acid modification, such as a mutation, insertion, or deletion.
  • the IL-15 cytokine comprises an amino acid sequence produced by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions in the amino acid sequence of SEQ ID NO: 22.
  • the IL-15 cytokine is an IL-15 variant that is well-known in the art, which comprises a sequence that is derived from the human mature wild type IL-15 by at least one mutations, including substitution, deletion or addition, at the residue 45, 48, 51, 52, 64, 67, 68 and/or 72 (see US patent 9,493,533 B2, and Han KP, et al., IL-15:IL-15 receptor alpha superagonist complex: high-level co-expression in recombinant mammalian cells, purification and characterization. Cytokine. 2011 Dec;56(3):804-10).
  • the IL-15 variant comprise one or more amino acid mutations selected from the group consisting of L45D, L45E, Q48K, S51D, L52D, E64K, I67D, I67E, I68D and N72D, herein the residues are numbering is corresponding to the human mature wild type IL-15 (e.g., SEQ ID NO: 22).
  • the IL-15 cytokine comprises any one of the amino acid sequence of SEQ ID NOs: 67-76.
  • IL-15R ⁇ or a functional fragment thereof [0332]
  • the IL-15R ⁇ or a functional fragment thereof according to the present application can be any species of IL-15R ⁇ or a functional fragment thereof.
  • the IL-15R ⁇ or a functional fragment thereof is selected from an extracellular region of human IL-15R ⁇ or a sushi domain or functional analogs.
  • Extracellular region of IL-15R ⁇ [0335] The extracellular region of IL-15R ⁇ is usually defined as the region of an IL-15R ⁇ sequence that extends from its first N-terminal amino acid, to the last amino acid of the tail region (or region rich in glycosylation sites). The tail region of an IL-15R ⁇ sequence can be determined by the skilled person, e.g., through the help of software.
  • IL-15R ⁇ _sushi domain [0337] The extracellular region of IL-15R ⁇ contains a domain, which is known as the sushi domain (Wei et al. 2001, J. Immunol. 167:277-282). The IL-15R ⁇ _sushi domain has a beta sheet conformation. [0338] The IL-15R ⁇ _sushi domain, bears most of the binding affinity for IL-15, and behaves as a potent IL-15 agonist by enhancing its binding and biological effects (proliferation and protection from apoptosis) through the IL-15R ⁇ heterodimer, whereas it does not affect IL-15 binding and function (Mortier E, et al.
  • the sushi domain of IL-15R ⁇ can be defined as beginning at the first cysteine residue (C1) after the signal peptide, and ending at the fourth cysteine residue (C4) after the signal peptide. Residues C1 and C4 are both included in the sushi sequence.
  • the IL-15R ⁇ sushi domain can also be determined by analysis of the amino-acid sequence of IL-15R ⁇ with appropriate software such as: Prosite (http://us.expasy.org/prosite/), (http://www.ebi.ac.uk/lnterProScan/), SMART (http://elm.eu.org/).
  • the IL-15R ⁇ _sushi domain comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the SEQ ID NO: 24.
  • the IL-15R ⁇ _sushi domain comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the SEQ ID NO: 25.
  • the IL-15R ⁇ _sushi domain comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the SEQ ID NO: 26.
  • the IL-15 prodrug wherein the IL-15 cytokine or the IL-15R ⁇ or a functional fragment thereof (e.g., IL-15R ⁇ _sushi domain) has one or more conservative amino acid substitutions.
  • Consservative substitution refers to the substitution of another amino acid with the same net charge and approximately the same size and shape as the substituted amino acid.
  • amino acids with aliphatic or substituted aliphatic amino acid side chains are roughly the same size.
  • amino acids having a phenyl or substituted phenyl group on the side chain can be considered to be approximately the same in size and shape.
  • natural amino acids are preferably used for conservative substitutions.
  • amino acid is used herein in its broadest sense, including both naturally occurring amino acids and non-naturally occurring amino acids, including amino acid analogs and derivatives. The latter includes molecules that contain amino acid moieties.
  • amino acids herein include, for example, naturally occurring L-amino acids that form proteins; D-amino acids; chemically modified amino acids, such as amino acid analogs and derivatives; naturally occurring amino acids that do not form protein, such as norleucine, ⁇ -alanine, ornithine, GABA, etc.; and chemically synthesized compounds with amino acid characteristics known in the art.
  • protein- forming refers to amino acids that can be incorporated into peptides, polypeptides, or proteins of cells through metabolic pathways.
  • Insertion of non-naturally occurring amino acids, including synthetic non-natural amino acids, substituted amino acids, or one or more D-amino acids, into the peptides can have multiple benefits.
  • D-amino acid-containing peptides and the like exhibit increased stability in vitro or in vivo compared to their counterparts containing L-amino acid. Therefore, when greater intracellular stability is desired, the construction of peptides, such as by incorporation of D-amino acids, is particularly useful.
  • D-peptide and the like are resistant to endogenous peptidase and protease activity, thereby improving the bioavailability of the molecule and extending the lifespan in vivo when needed.
  • D-peptide and the like cannot be effectively processed for limited presentation by type II major histocompatibility complexes (MHC) to T helper cells, so less prone to induce humoral immune responses in the subject.
  • MHC major histocompatibility complexes
  • Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Amino acid substitutions may be introduced into the protein constructs and the products screened for a desired activity mentioned above. Table A.
  • the binding affinity is reduced by between about 200-fold and about 1500-fold compared to IL-15 or functional fragment thereof without the MP.
  • the EC50 value increased by at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times.
  • the MP comprises the amino acid sequence of any one of SEQ ID NOs: 1-5 or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-5.
  • the cleavable moiety (CM) comprising a protease cleavage site can be located between the masking polypeptide (MP) and the IL-15 cytokine.
  • the cleavable moiety (CM) as used herein can be cleaved under certain conditions, thereby separating its N- terminal fragment from its C-terminal fragment.
  • the selection of a suitable cleavable moiety would depend on the desired action site of the IL-15 cytokine. For example, when a tumor site is the desired action site, a cleavage site of a protease specific to the tumor is used for constructing an IL-15 prodrug intended to act at the tumor site.
  • a protease specific to a tumor refers to any protease that has an elevated level and/or activity at the tumor site relative to normal tissues.
  • the protease cleavage site can be a cleavage site of a matrix metalloproteinase (MMP).
  • MMP matrix metalloproteinase
  • the protease cleavage site can be a cleavage site of MMP2. In some embodiments, the protease cleavage site can be a cleavage site of MMP9. In some embodiments, the protease cleavage site can be a cleavage site of MMP2 and MMP9. Additional information regarding tumor-specific proteases and corresponding cleavage sites is known in the art, for example, disclosed in Vasiljeva et al., Scientific Reports, 10:5894, 2020, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein.
  • the cleavable moiety comprises the amino acid sequence of any one of SEQ ID NOs: 8-16 or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 8-16.
  • the IL-15 prodrug provided herein, wherein the half-life extension moiety (C) comprises an Fc domain; preferably, the Fc domain is selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgA Fc domain, an IgD Fc domain, an IgE Fc domain, and an IgM Fc domain; more preferably, the Fc domain is a human IgG1 Fc domain.
  • the IL-15 prodrug provided herein wherein the half-life extension moiety (C) comprises a human IgG1 Fc domain having L234A and L235A (LALA) mutations, according to EU Numbering system.
  • the IL-15 prodrug provided herein, wherein the half-life extension moiety (C) further comprises knobs-into-holes mutations (Fc knob and Fc hole).
  • the IL-15 prodrug provided herein wherein the Fc knob comprises a T366W mutation in the Fc domain, and the Fc hole comprises T366S, L368A, and Y407V mutations in the Fc domain, according to EU Numbering system.
  • the IL-15 prodrug provided herein, wherein the Fc knob comprises the amino acid sequence of SEQ ID NO: 28 or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 28.
  • the IL-15 prodrug provided herein, wherein the Fc hole comprises the amino acid sequence of SEQ ID NO: 27 or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 27.
  • the IL-15 prodrug provided herein, wherein the Fc knob and Fc hole further comprises LALA mutation.
  • the IL-15 prodrug provided herein, wherein the Fc knob LALA comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30.
  • the IL-15 prodrug provided herein, wherein the Fc hole LALA comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least about 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29.
  • the IL-15 prodrug provided herein comprises two monomers, and wherein in one monomer, the IL-15R ⁇ or a functional fragment thereof is linked to the Fc domain, and in the other monomer, the IL-15 cytokine (I) is linked to the Fc domain, and the masking polypeptide (MP) is linked to the IL-15 cytokine (I) through the cleavable moiety (CM).
  • the IL-15 prodrug provided herein comprises the constructs showing in Table 2.
  • the IL-15 drug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31, and the second monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33, and the second monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 34, and the second monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 36 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 36, and the second monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 37, and the second monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 38, and the second monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 39, and the second monomer comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 32.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 45 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45, and the second monomer comprises the amino acid sequence of SEQ ID NO: 46 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 46.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 47 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47, and the second monomer comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 40.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33, and the second monomer comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 40.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 62, and the second monomer comprises the amino acid sequence of SEQ ID NO: 63 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 63.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 63 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 63.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 64 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 64, and the second monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 65.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 62 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 62, and the second monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 65.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 65.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 66 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 66, and the second monomer comprises the amino acid sequence of SEQ ID NO: 65 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 65.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43, and the second monomer comprises the amino acid sequence of SEQ ID NO: 46 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 46.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 39, and the second monomer comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 40.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 87 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 87.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 88 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 88.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 89 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 89.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 90 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 90.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 91 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 91.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 92 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 92.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 93 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 93.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 94 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 94.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 95 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 95.
  • the IL-15 prodrug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41, and the second monomer comprises the amino acid sequence of SEQ ID NO: 96 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 96.
  • the application also provided an IL-15 drug without masking polypeptide (MP) and cleavable moiety (CM) which comprises two monomers, wherein in one monomer, the IL-15R ⁇ or a functional fragment thereof (S) is linked to the first Fc domain, and in the other monomer, the IL-15 cytokine (I) is linked to the second Fc domain.
  • MP masking polypeptide
  • CM cleavable moiety
  • the IL-15 drug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43, and the second monomer comprises the amino acid sequence of SEQ ID NO: 77 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 77.
  • the IL-15 drug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43, and the second monomer comprises the amino acid sequence of SEQ ID NO: 81 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 81.
  • the IL-15 drug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43, and the second monomer comprises the amino acid sequence of SEQ ID NO: 83 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 83.
  • the IL-15 drug provided herein comprises two monomers, wherein the first monomer comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43, and the second monomer comprises the amino acid sequence of SEQ ID NO: 84 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence SEQ ID NO: 84.
  • the application provides an anti-PD-L1/IL-15 prodrug bifunctional protein, comprising an anti-PD-L1 antibody; and an IL-15 prodrug described above which comprises (i) one or more IL-15 cytokine (I), (ii) one or more cleavable moiety (CM), and (iii) one or more masking polypeptide (MP), wherein the IL-15 prodrug is linked to the anti-PD-L1 antibody.
  • an anti-PD-L1/IL-15 prodrug bifunctional protein comprising an anti-PD-L1 antibody
  • an IL-15 prodrug described above which comprises (i) one or more IL-15 cytokine (I), (ii) one or more cleavable moiety (CM), and (iii) one or more masking polypeptide (MP), wherein the IL-15 prodrug is linked to the anti-PD-L1 antibody.
  • the application provides an anti-PD-L1/IL-15 drug bifunctional protein, comprising an anti-PD-L1 antibody; and an IL-15 drug described above wherein the IL-15 drug is linked to the anti-PD-L1 antibody.
  • the IL-15 drug comprises two monomers, and wherein in one monomer, the IL-15R ⁇ or a functional fragment thereof (S) is linked to the first Fc domain, and in the other monomer, the IL-15 cytokine (I) is linked to the second Fc domain.
  • the anti-PD-L1 antibody includes full-length antibody and antigen-binding fragment thereof.
  • the anti-PD-L1 antibodies that specifically bind to human and/or cynomolgus monkey PD-L1.
  • Anti-PD-L1 antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and/or light chain CDRs discussed herein.
  • the contemplated anti-PD-L1 antibodies include, for example, full-length anti- PD-L1 antibodies (e.g., full-length IgG1 or IgG4), anti-PD-L1 scFvs, anti-PD-L1 Fc fusion proteins, multi-specific (such as bispecific) anti-PD-L1 antibodies, anti-PD-L1 immunoconjugates, and the like.
  • the anti-PD-L1 antibody is a Fab, a Fab', a F(ab)'2, a Fab'-SH, a single-chain Fv (scFv), an Fv fragment, a dAb, a Fd, a nanobody, a diabody, or a linear antibody.
  • reference to an antibody that specifically binds to PD-L1 means that the antibody binds to PD-L1 with an affinity that is at least about 10 times (including for example at least about any one of 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 times) more tightly than its binding affinity for a non-target.
  • the non-target is an antigen that is not PD-L1.
  • Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA).
  • Kd can be determined by methods known in the art, such as surface plasmon resonance (SPR) assay or biolayer interferometry (BLI).
  • SPR surface plasmon resonance
  • BLI biolayer interferometry
  • the anti-PD-L1 antibody comprises an antibody heavy chain constant region and an antibody light chain constant region.
  • the anti-PD-L1 antibody comprises an IgG1 heavy chain constant region.
  • the anti-PD-L1 antibody comprises an IgG2 heavy chain constant region. In some embodiments, the anti-PD-L1 antibody comprises an IgG3 heavy chain constant region. In some embodiments, the anti-PD- L1 antibody comprises an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region comprises (including consisting of or consisting essentially of) the amino acid sequence of SEQ ID NO: 109. In some embodiments, the heavy chain constant region comprises (including consisting of or consisting essentially of) the amino acid sequence of SEQ ID NO: 110. In some embodiments, the anti-PD-L1 antibody comprises a kappa light chain constant region.
  • the light chain constant region comprises (including consisting of or consisting essentially of) the amino acid sequence of SEQ ID NO: 111.
  • the anti-PD-L1 antibody comprises a lambda light chain constant region.
  • the light chain constant region comprises (including consisting of or consisting essentially of) the amino acid sequence of SEQ ID NO: 112.
  • the anti- PD-L1 antibody comprises an antibody heavy chain variable domain and an antibody light chain variable domain.
  • the anti-PD-L1 antibody comprises a V H comprising: an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 97, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 98, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 99; and a VL comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 100, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 101, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 102.
  • the anti-PD-L1 antibody comprises a VH comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 133, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 134, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 135; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 136, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 137, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 138.
  • the anti-PD-L1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 104, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity, and a VL comprising the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the anti-PD-L1 antibody comprises a V H comprising the amino acid sequence of SEQ ID NO: 104 and a VL comprising the amino acid sequence of SEQ ID NO: 107.
  • the anti-PD-L1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 139 or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 139; and a V L comprising the amino acid sequence of SEQ ID NO: 140, or a variant thereof having at least about 80% (such as at least about any of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 140.
  • Exemplary anti-PD-L1 antibody sequences are shown in Tables B and C, wherein the CDR numbering is according to the EU index of IMGT. Those skilled in the art will recognize that many algorithms are known for prediction of CDR positions and for delimitation of antibody heavy chain and light chain variable regions. Anti-PD-L1 antibodies comprising CDRs, V H and/or V L sequences from antibodies described herein, but based on prediction algorithms other than those exemplified in the tables below, are within the scope of this application. Table B. Exemplary anti-PD-L1 antibody CDR sequences. Antibody Name HC-CDR1 HC-CDR2 HC-CDR3 ab e C. xemp ary sequences.
  • the IL-15 cytokine comprises the amino acid sequence SEQ ID NO: 22 or a variant thereof having at least 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 22.
  • the IL-15 cytokine comprises the amino acid sequence SEQ ID NO: 23 or a variant thereof having at least 90% (such as at least about any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 23.
  • the IL-15 variant comprise one or more amino acid mutations selected from the group consisting of L45D, L45E, Q48K, S51D, L52D, E64K, I67D, I67E, I68D and N72D, herein the residues are numbering is corresponding to the human mature wild type IL-15 (e.g., SEQ ID NO: 22).
  • the IL-15 cytokine comprises any one of the amino acid sequences of SEQ ID NOs: 67-76.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the masking polypeptide is anyone as described above.
  • the masking polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-5.
  • the cleavable moiety comprises the amino acid sequence of any one of SEQ ID NOs: 8-16.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein or the anti- PD-L1/IL-15 drug bifunctional protein provided herein wherein the IL-15R ⁇ or a functional fragment thereof is anyone as described above.
  • the IL-15R ⁇ or a functional fragment thereof is an IL-15R ⁇ _sushi domain or functional analogs.
  • the IL-15R ⁇ _sushi domain or functional analog is anyone as describe above.
  • the IL-15R ⁇ _sushi domain or functional analog comprises the amino acid sequence of SEQ ID NO: 24 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the SEQ ID NO: 24.
  • the IL-15R ⁇ _sushi domain or functional analog comprises the amino acid sequence of SEQ ID NO: 25 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the SEQ ID NO: 25.
  • the IL-15R ⁇ _sushi domain or functional analog comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the SEQ ID NO: 26.
  • the IL-15 R ⁇ _sushi domain or functional analog and the IL-15 cytokine are covalently linked, preferably through a non-cleavable linker.
  • the IL-15 R ⁇ _sushi domain or functional analog and the IL-15 cytokine are non-covalently linked, and form an IL-15/ IL-15R ⁇ complex.
  • the Fc domain has been altered or otherwise changed so that it has enhanced antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) effector function.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • the anti-PD-L1/IL-15 prodrug bifunctional protein or the anti- PD-L1/IL-15 drug bifunctional protein provided herein wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein or the anti- PD-L1/IL-15 drug bifunctional protein provided herein wherein the first Fc domain comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30, and the second Fc domain comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29 [0423] In some embodiments, the anti-PD-L1/IL-15 prodrug bifunctional protein or the anti- PD-L1/IL-15 drug bifunctional protein provided herein, wherein the antigen-binding domain that binds to PD-L
  • the full-length anti-PD-L1 antibody is an IgA, IgD, IgE, IgG, or IgM.
  • the full-length anti-PD-L1 antibody comprises IgG constant domains, such as constant domains of any one of IgG1, IgG2, IgG3, and IgG4 including variants thereof.
  • the full-length anti-PD-L1 antibody comprises a lambda light chain constant region.
  • the full-length anti-PD-L1 antibody comprises a kappa light chain constant region.
  • the full-length anti-PD-L1 antibody is a full-length human anti-PD-L1 antibody.
  • the full-length anti-PD-L1 antibody comprises an Fc sequence of a mouse immunoglobulin.
  • the anti-PD-L1 antibody is an antigen-binding fragment selected from the group consisting of a Fab, a Fab’, a F(ab)’2, a Fab’-SH, a single-chain Fv (scFv), an Fv fragment, a dAb, a Fd, a nanobody, a diabody, and a linear antibody.
  • the anti-PD-L1 antibody is chimeric, human or humanized.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the IL-15 prodrug is linked to the C-terminus or N-terminus of the anti-PD-L1 antibody with or without a linker.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the IL-15 prodrug is linked to the C-terminus or N-terminus of the anti-PD-L1 antibody heavy chain with or without a linker.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein, wherein the IL-15 prodrug is linked to the C-terminus or N-terminus of the anti-PD-L1 antibody light chain with or without a linker.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein comprises two monomers, and each monomer comprises two polypeptides, wherein: (i) one monomer comprises a polypeptide comprising the structure of V H -C H 1-hinge-C H 2-C H 3-IL- 15-CM-MP, and the other polypeptide comprising the structure of VL-CL; and (ii) the other monomer comprises a polypeptide comprising the structure of VH-CH1-hinge-CH2-CH3-IL- 15R ⁇ _sushi, and the other polypeptide comprising the structure of VL-CL; wherein the VH is a heavy chain variable domain of the anti-PD-L1 antibody, V L is a light chain variable domain of the anti-PD-L1 antibody.
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein comprises two monomers, and each monomer comprises two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 113, 116-125 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 113, 116-125, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 115 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 115; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 114 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 114, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 115 or a variant thereof having at least about 80% sequence identity to the amino acid sequence
  • the anti-PD-L1/IL-15 prodrug bifunctional protein provided herein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 127, 141-150 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 127, 141-150; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 129 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 129; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 128 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 128, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 129 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of
  • the anti-PD-L1/IL-15 prodrug bifunctional protein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 130 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 130; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 132; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 131 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 131, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 132 [0433] In some embodiments, the anti-
  • the anti-PD-L1/IL-15 drug bifunctional protein provided herein comprises two monomers, and each monomer comprises two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of SEQ ID NOs: 126 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 126, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 115 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 115; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 114 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 114, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 115 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 115.
  • the anti-PD-L1/IL-15 drug bifunctional protein provided herein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 151 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 151; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 129 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 129; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 128 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 128, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 129 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 129.
  • the anti-PD-L1/IL-15 drug bifunctional protein provided herein comprises two monomers, each monomer comprising two polypeptides, wherein: (i) In the first monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 152 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 152; and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 132; and (ii) In the second monomer, one polypeptide comprises the amino acid sequence of SEQ ID NO: 131 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 131, and the other polypeptide comprises the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 132.
  • Binding affinity of a molecule e.g., IL-15 or functional fragment thereof
  • its binding partner e.g., IL-2/IL-15R ⁇
  • binding affinity of a molecule can be determined experimentally by any suitable ligand binding assays or antibody/antigen binding assays known in the art, e.g., Western blots, sandwich enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence, bead based multiplex immunoassays (MIA), RIA, Surface Plasma Resonance (SPR), ECL, IRMA, EIA, Biacore assay, Octet analysis, peptide scans, etc.
  • IL-15 or functional fragment thereof or its receptor (e.g., IL-2/IL-15R ⁇ ) or subunits thereof marked with a variety of marker agents, as well as by using BiacoreX (Amersham Biosciences), which is an over-the-counter, measuring kit, or similar kit, according to the user’s manual and experiment operation method attached with the kit.
  • BiacoreX Anamersham Biosciences
  • protein microarray is used for analyzing the interaction, function, and activity of the IL-15 or functional fragment thereof, described herein to its receptor, on a large scale.
  • the protein chip has a support surface-bound with a range of capture proteins (e.g., IL-15 receptor or subunits thereof).
  • Fluorescently labeled probe molecules e.g., IL-15 or functional fragment thereof described herein
  • Binding affinity can also be measured using SPR (Biacore T-200).
  • SPR Biacore T-200
  • anti- human IgG antibody is coupled to the surface of a CM-5 sensor chip using EDC/NHS chemistry.
  • human IL-2/IL-15R ⁇ -Fc fusion protein is used as the captured ligand over this surface.
  • PK Pharmacokinetics
  • Pharmacokinetics refers to the absorption, distribution, metabolism, and excretion of a drug (e.g., IL-15 cytokine or IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein described herein) once it has been administered to a subject.
  • Pharmacokinetic parameters that may be useful in determining clinical utility include but are not limited to serum/plasma concentration, serum/plasma concentration over time, maximum serum/plasma concentration (C max ), time to reach maximum concentration (T max ), half-life (t 1/2 ), area under concentration time curve within the dosing interval (AUC ⁇ ), etc.
  • C max maximum serum/plasma concentration
  • T max time to reach maximum concentration
  • t 1/2 half-life
  • AUC ⁇ area under concentration time curve within the dosing interval
  • the PK curves of the IL-15 cytokine or IL-15 prodrug described herein in the individual is measured in a blood, plasma, or serum sample from the individual.
  • the PK curves of the IL- 15 cytokine or IL-15 prodrug described herein in the individual is measured using a mass spectrometry technique, such as LC-MS/MS, or ELISA.
  • PK analysis on PK curves can be conducted by any methods known in the art, such as non-compartmental analysis, e.g., using PKSolver V2 software (Zhang Y. et al., “PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel,” Comput Methods Programs Biomed. 2010; 99(3):306-1).
  • PKSolver V2 software Zhang Y. et al., “PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel,” Comput Methods Programs Biomed. 2010; 99(3):306-1).
  • C denotes the concentration of drug or prodrug (e.g., IL-15 cytokine or IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein) in blood plasma, serum, or in any appropriate body fluid or tissue of a subject, and is generally expressed as mass per unit volume, for example nanograms per milliliter.
  • drug concentration e.g., IL-15 cytokine or IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein
  • concentration of drug in serum or plasma is referred to herein as “serum concentration” or “plasma concentration.”
  • the serum/plasma concentration at any time following drug administration e.g., IL-15 cytokine or IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein, such as i.v., i.p., or s.c.
  • Ctime or Ct The maximum serum/plasma drug concentration during the dosing period is referenced as C max , while C min refers to the minimum serum/plasma drug concentration at the end of a dosing interval; and Cave refers to an average concentration during the dosing interval.
  • bioavailability refers to an extent to which and sometimes rate at which the drug or prodrug (e.g., IL-15 cytokine or IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein) enters systemic circulation, thereby gaining access to the site of action.
  • AUC is the area under the serum/plasma concentration-time curve and is considered to be the most reliable measure of bioavailability, such as area under concentration time curve within the dosing interval (AUC ⁇ ), “overall exposure” or “total drug exposure across time” (AUC0-last or AUC0-inf), area under concentration time curve at time t post-administration (AUC0-t), etc.
  • Serum/plasma concentration peak time (Tmax) is the time when peak serum/plasma concentration (Cmax) is reached after administration of a drug or prodrug (e.g., IL-15 cytokine or IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein).
  • Half-life is the amount of time required for the drug or prodrug concentration (e.g., IL-15 cytokine or IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein) measured in plasma or serum (or other biological matrices) to be reduced to exactly half of its concentration or amount at certain time point.
  • the drug or prodrug concentration e.g., IL-15 cytokine or IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein
  • plasma or serum or other biological matrices
  • the elimination half-life is determined from the terminal or elimination (dominant) phase of the plasma/serum concentration versus time curve. See, e.g., Michael Schrag and Kelly Regal, “Chapter 3 - Pharmacokinetics and Toxicokinetics” of “A Comprehensive Guide to Toxicology in Preclinical Drug Development”, 2013.
  • Vectors [0448] The present application also provides isolated nucleic acids encoding any of the masking polypeptides (MP), any of the cleavable moieties (CM), any of the non-cleavable linker (L) or any of the prodrugs (e.g., IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein) described herein, vectors comprising the nucleic acids described herein. Also provided are isolated host cells (e.g., CHO cells, HEK 293 cells, Hela cells, or COS cells) comprising nucleic acids or vectors described herein.
  • isolated host cells e.g., CHO cells, HEK 293 cells, Hela cells, or COS cells
  • Suitable nucleic acid constructs include, but are not limited to, constructs that are capable of expression in prokaryotic or eukaryotic cells. Expression constructs are generally selected so as to be compatible with the host cell in which they are to be used.
  • the vector encodes a masking polypeptide (e.g., MP80, MP96new, MP100, MP163 or MP240).
  • the vector encodes a cleavable moiety (e.g., CM1, CM2 or CM4).
  • the vector encodes a non- cleavable linker (e.g., lk, lk1, lk2, lk3 or lk5).
  • the vector encodes a protein or prodrugs (e.g., masking polypeptides or IL-15 prodrug).
  • the vector comprising a nucleic acid encoding the prodrug or any components of the prodrugs described herein is suitable for replication and integration in eukaryotic cells, such as mammalian cells (e.g., CHO cells, HEK 293 cells, Hela cells, COS cells).
  • the vector is a viral vector.
  • the vector is a non-viral vector, such as pTT5.
  • a number of viral based systems have been developed for gene transfer into mammalian cells.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, herpes simplex viral vectors, and derivatives thereof.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the engineered mammalian cell in vitro or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • self- inactivating lentiviral vectors are used.
  • self-inactivating lentiviral vectors carrying the construct protein coding sequence(s) can be packaged with protocols known in the art.
  • the resulting lentiviral vectors can be used to transduce a mammalian cell using methods known in the art.
  • Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long- term gene transfer, because they allow long-term, stable integration of a transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity, and can transduce non-proliferating cells.
  • the vector is a non-viral vector. In some embodiments, the vector is a pTT5 vector. In some embodiments, the vector is a transposon, such as a Sleeping Beauty (SB) transposon system, or a PiggyBac transposon system.
  • SB Sleeping Beauty
  • the vector is a polymer-based non-viral vector, including for example, poly (lactic-co-glycolic acid) (PLGA) and poly lactic acid (PLA), poly (ethylene imine) (PEI), and dendrimers.
  • the vector is a cationic-lipid based non-viral vector, such as cationic liposome, lipid nanoemulsion, and solid lipid nanoparticle (SLN).
  • the vector is a peptide-based gene non-viral vector, such as Poly-L-lysine. Any of the known non-viral vectors suitable for genome editing can be used for introducing the IL-15 prodrug-encoding nucleic acid(s) to the host cells.
  • any one or more of the nucleic acids or vectors encoding the prodrugs described herein is introduced to the host cells (e.g., CHO, HEK 293, Hela, or COS) by a physical method, including, but not limited to electroporation, sonoporation, photoporation, magnetofection, hydroporation.
  • the vector contains a selectable marker gene or a reporter gene to select cells expressing the prodrugs described herein from the population of host cells transfected through vectors (e.g., lentiviral vectors, pTT5 vectors). Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in the host cells.
  • the vector may contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid sequences.
  • the nucleic acid can be cloned into the vector using any known molecular cloning methods in the art, including, for example, using restriction endonuclease sites and one or more selectable markers.
  • the nucleic acid is operably linked to a promoter. Varieties of promoters have been explored for gene expression in prokaryotic cells or eukaryotic cells (e.g., mammalian cells), and any of the promoters known in the art may be used in the present application.
  • Promoters may be roughly categorized as constitutive promoters or regulated promoters, such as inducible promoters.
  • the nucleic acid encoding the prodrugs described herein is operably linked to a constitutive promoter.
  • Constitutive promoters allow heterologous genes (also referred to as transgenes) to be expressed constitutively in the host cells.
  • Exemplary promoters contemplated herein include, but are not limited to, cytomegalovirus immediate- early promoter (CMV), human elongation factors-1alpha (hEF1 ⁇ ), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), chicken ⁇ -Actin promoter coupled with CMV early enhancer (CAGG), a Rous Sarcoma Virus (RSV) promoter, a polyoma enhancer/herpes simplex thymidine kinase (MC1) promoter, a beta actin ( ⁇ -ACT) promoter, a “myeloproliferative sarcoma virus enhancer, negative control region deleted, d1587rev primer-binding site substituted (MND)” promoter.
  • CMV cytomegalovirus immediate- early promoter
  • hEF1 ⁇ human elongation factors-1alpha
  • UbiC ubiquitin
  • the nucleic acid encoding the prodrugs described herein is operably linked to CMV promoter.
  • the nucleic acid encoding the prodrugs described herein is operably linked to an inducible promoter.
  • Inducible promoters belong to the category of regulated promoters.
  • the inducible promoter can be induced by one or more conditions, such as a physical condition, microenvironment of the host cells, or the physiological state of the host cells, an inducer (i.e., an inducing agent), or a combination thereof.
  • the inducing condition does not induce the expression of endogenous genes in the host cell.
  • the inducing condition is selected from the group consisting of inducer, irradiation (such as ionizing radiation, light), temperature (such as heat), redox state, and the activation state of the host cell.
  • the inducible promoter can be an NFAT promoter, a TETON ® promoter, or an NF ⁇ B promoter.
  • Methods of preparation Also provided are methods of preparing any of the masking polypeptides (MP), the cleavable moieties (CM), the non-cleavable linkers (L) or prodrugs or anti-PD-L1/IL-15 prodrug bifunctional protein described herein.
  • a method of producing the masking polypeptides (MP), the cleavable moieties (CM), the non- cleavable linkers (L) or the prodrugs comprising: (a) culturing a host cell (e.g., CHO cell, HEK 293 cell, Hela cell, or COS cell) comprising any of the nucleic acids or vectors encoding the masking polypeptides (MP), the cleavable moieties (CM), the non-cleavable linkers (L) or the prodrugs described herein under a condition effective to express the encoded prodrug; and (b) obtaining the expressed the masking polypeptides (MP), the cleavable moieties (CM), the non- cleavable linkers (L) or prodrugs from said host cell.
  • a host cell e.g., CHO cell, HEK 293 cell, Hela cell, or COS cell
  • the method of step (a) further comprises producing a host cell comprising the nucleic acid or vector encoding the masking polypeptides (MP), the cleavable moieties (CM), the non-cleavable linkers (L) or the prodrug described herein.
  • the masking polypeptides (MP), the cleavable moieties (CM), the non-cleavable linkers (L) or the prodrugs described herein may be prepared using any methods known in the art or as described herein.
  • the masking polypeptides (MP), the cleavable moieties (CM), the non-cleavable linkers (L) or the prodrugs or anti-PD-L1/IL-15 prodrug bifunctional protein described herein are expressed with eukaryotic cells, such as mammalian cells.
  • the masking polypeptides (MP), the cleavable moieties (CM), the non-cleavable linkers (L) or the prodrugs or anti-PD-L1/IL-15 prodrug bifunctional protein described herein are expressed with prokaryotic cells.
  • the vector components generally include, but are not limited to, one or more of the following, a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • a) Signal sequence component A vector for use in a eukaryotic host may also be an insert that encodes a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available.
  • the DNA for such precursor region is ligated in reading frame to DNA encoding the protein constructs of the present application.
  • Origin of replication Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).
  • Selection gene component Expression and cloning vectors may contain a selection gene, also termed a selectable marker.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • antibiotics or other toxins e.g., ampicillin, neomycin, methotrexate, or tetracycline
  • c supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.
  • Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up nucleic acid encoding the protein constructs of the present application, such as DHFR, thymidine kinase, metallothionein-I and - II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • DHFR thymidine kinase
  • metallothionein-I and - II preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
  • DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR.
  • Mtx methotrexate
  • DHFR Chinese hamster ovary
  • ATCC CRL- 9096 Chinese hamster ovary
  • host cells transformed or co-transformed with the polypeptide encoding-DNA sequences, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3′-phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.
  • Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the desired polypeptide sequences.
  • Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 based upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of the transcription of many genes is a CNCAAT region where N may be any nucleotide.
  • N may be any nucleotide.
  • AATAAA sequence At the 3′ end of most eukaryotic is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3′ end of the coding sequence. All of these sequences may be inserted into eukaryotic expression vectors. Also see section “Vectors” above.
  • Polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment.
  • a system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978.
  • Enhancer element component Transcription of a DNA encoding the protein constructs of the present application by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ - fetoprotein, and insulin).
  • an enhancer from a eukaryotic cell virus examples include the SV40 enhancer on the late side of the replication origin (100-270 bp), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters.
  • the enhancer may be spliced into the vector at a position 5′ or 3′ to the polypeptide encoding sequence, but is preferably located at a site 5′ from the promoter.
  • Transcription termination component f) Transcription termination component
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the polypeptide-encoding mRNA.
  • One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein include higher eukaryote cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); COS fibroblast-like cell lines derived from monkey kidney tissue; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Host cells are transformed with the above-described expression or cloning vectors for protein construct production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • h) Culturing the host cells The host cells used to produce the protein constructs of the present application may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. No.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells.
  • any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCINTM drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.
  • the protein constructs of the present application can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the protein construct is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies that are secreted to the periplasmic space of E. coli.
  • cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the protein composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the Fc-containing protein construct.
  • Protein A can be used to purify Fc-containing proteins based on human immunoglobulins containing 1, 2, or 4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human 3 (Guss et al., EMBO J.
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrene-divinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the protein construct comprises a C H 3 domain, the Bakerbond ABXTMresin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • the mixture comprising the protein constructs of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).
  • compositions comprising prodrugs (e.g., IL-15 prodrug) or anti-PD-L1/IL-15 prodrug bifunctional protein described herein, and optionally a pharmaceutically acceptable carrier.
  • pharmaceutical compositions can be prepared by mixing a prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein described herein having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • a reconstituted formulation can be prepared by dissolving a lyophilized prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein in a diluent such that the protein is dispersed throughout.
  • exemplary pharmaceutically acceptable (safe and non-toxic for administration to a human) diluents suitable for use in the present application include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate- buffered saline), sterile saline solution, Ringer’s solution or dextrose solution, or aqueous solutions of salts and/or buffers.
  • the pharmaceutical composition comprises a homogeneous population of prodrugs (e.g., IL-15 prodrug) or anti-PD-L1/IL-15 prodrug bifunctional protein described herein.
  • a homogeneous population means the prodrugs are exactly the same to each other, e.g., same IL-15 prodrug configuration, same IL-15 cytokine, same IL-15R ⁇ sushi domain, same masking polypeptides, same cleavable moiety, same non- cleavable linker if any, and same Fc domain.
  • At least about 70% (such as at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the IL-15 prodrug in the pharmaceutical composition are homogeneous.
  • the pharmaceutical composition is preferably to be stable, in which the proteins contained within essentially retain their physical and chemical stability and integrity upon storage.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period.
  • the formulation may be kept at 40°C for 2 weeks to 1 month, at which time stability is measured.
  • time stability is measured.
  • the extent of aggregation during storage can be used as an indicator of protein stability.
  • the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial.
  • the pharmaceutical composition is contained in a multi-use vial.
  • the pharmaceutical composition is contained in bulk in a container.
  • the pharmaceutical composition is cryopreserved.
  • Methods of treating diseases are methods of treating a subject with or at risk of developing a disease or disorder, such as proliferative disease, a tumorous disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or graft-versus-host disease.
  • the methods administering to a subject in need thereof an effective amount of an activatable prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein as disclosed herein that is typically administered as a pharmaceutical composition, wherein the prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein is activated upon cleavage by an enzyme.
  • the method further comprises selecting a subject with or at risk of developing such a disease or disorder.
  • the prodrug is activated in a tumor microenvironment.
  • the prodrug or anti-PD- L1/IL-15 prodrug bifunctional protein is therapeutically active after it has cleaved from the masking polypeptides.
  • the active agent is the cleavage product.
  • the prodrugs can be used to treat a disease depending on the antigen bound by the antigen-binding domain.
  • a method of treating a disease comprising administering to the individual an effective amount of any of the prodrugs (e.g., IL-15 prodrug) or anti-PD-L1/IL-15 prodrug bifunctional protein described herein or pharmaceutical compositions thereof.
  • the prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein is administered intravenously, intramuscularly, or subcutaneously.
  • the method of treatment further comprises administering an additional therapeutic agent in combination with (before, after, or concurrently with) the prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein.
  • the additional agent may be an antibody or antigen-binding fragment thereof, a small molecule drug, or other types of therapeutic drug.
  • the IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein is used to treat a cancer or tumor in a subject comprises administering to the subject an effective amount of an IL-15 prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein.
  • tumor or cancer refers to all types of cancer, neoplasm or malignant tumors found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas.
  • Exemplary cancers that may be treated with a masked cytokine, pharmaceutical composition, or method provided herein, include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g.
  • ER positive triple negative
  • ER negative chemotherapy resistant
  • Herceptin resistant Herceptin resistant
  • HER2 positive Herceptin resistant
  • doxorubicin resistant doxorubicin resistant
  • tamoxifen resistant ductal carcinoma, lobular carcinoma, primary, metastatic
  • ovarian cancer pancreatic cancer
  • liver cancer e.g. hepatocellular carcinoma
  • lung cancer e.g.
  • nonsmall cell lung carcinoma nonsmall cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma.
  • squamous cell carcinoma e.g., head, neck, or esophagus
  • colorectal cancer leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma.
  • Additional examples include, cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, esophagus, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial
  • the IL-15 prodrug is used to treat a bacterial infection such as sepsis.
  • the bacteria causing the bacterial infection are drug-resistant bacteria.
  • the antigen-binding moiety binds to a bacterial antigen.
  • the IL-15 prodrug is used to treat a viral infection.
  • the virus causing the viral infection is hepatitis C (HCV), hepatitis B (HBV), human immunodeficiency vims (HIV), or human papilloma virus (HPV).
  • the antigen-binding moiety binds to a viral antigen.
  • Administration of the prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein described herein or pharmaceutical compositions thereof may be carried out in any convenient manner, including by injection or transfusion.
  • the route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a long period of time in a suitable manner.
  • the prodrug or pharmaceutical compositions thereof may be administered to a patient orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally, intraocularly, topically, transarterially, intradermally, intranodally, intraputaminally, or intramedullary, intrathecally, intraventricularly, intracerebrally, intraspinally, intrathecially, ntralesionally, or intraocularly.
  • the prodrug or pharmaceutical composition thereof is administered systemically.
  • the prodrug or pharmaceutical composition thereof is administered to an individual by infusion, such as intravenous infusion.
  • the prodrug or pharmaceutical composition thereof is administered to an individual by intradermal or subcutaneous (i.e. beneath the skin) injection.
  • the prodrug or pharmaceutical composition thereof may be injected using a syringe.
  • other devices for administration of the prodrug or pharmaceutical composition thereof are available such as injection devices; injector pens; auto-injector devices, needleless devices; and subcutaneous patch delivery systems.
  • the prodrug or pharmaceutical composition thereof is administered by intravenous injection.
  • the prodrug or pharmaceutical composition thereof is injected directly into the brain or spine. In some embodiments, the prodrug or pharmaceutical composition thereof is administered by sustained release or extended-release means.
  • Dosages and desired drug concentration of pharmaceutical compositions of the present application may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W.
  • the dosage amounts may vary depending upon the route of administration and mammal type. It is within the scope of the present application that different formulations will be effective for different treatments and different disorders, and that administration intended to treat a specific organ or tissue may necessitate delivery in a manner different from that to another organ or tissue. Moreover, dosages may be administered by one or more separate administrations, or by continuous infusion.
  • the prodrug or pharmaceutical composition thereof is administered for a single time (e.g. bolus injection). In some embodiments, the prodrug or pharmaceutical composition thereof is administered for multiple times (such as any of 2, 3, 4, 5, 6, or more times). If multiple administrations, they may be performed by the same or different routes and may take place at the same site or at alternative sites.
  • the prodrug or pharmaceutical composition thereof may be administered daily to once per year. The interval between administrations can be about any one of 24 hours to a year.
  • Intervals can also be irregular (e.g. following tumor progression). In some embodiments, there is no break in the dosing schedule.
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the prodrug or pharmaceutical composition thereof is administered in split doses, such as about any one of 2, 3, 4, 5, or more doses. In some embodiments, the split doses are administered over about a week, a month, 2 months, 3 months, or longer. In some embodiments, the dose is equally split. In some embodiments, the split doses are about 20%, about 30% and about 50% of the total dose.
  • the interval between consecutive split doses is about 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, a month, 3 months, 6 months, or longer.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • kits which contains any one of the prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein compositions described herein and preferably provides instructions for its use, such as for use in the treatment of the disorders described herein (e.g., tumor).
  • Kits of the application include one or more containers comprising a prodrug or anti- PD-L1/IL-15 prodrug bifunctional protein described herein, e.g., for treating a disease.
  • the instructions comprise a description of administration of the prodrug to treat a disease, such as a tumor.
  • the kit may further comprise a description of selecting an individual (e.g., human) suitable for treatment based on identifying whether that individual has the disease and the stage of the disease.
  • the instructions relating to the use of the prodrug or anti-PD-L1/IL- 15 prodrug bifunctional protein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the application are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
  • kits for use in combination with a specific device such as an infusion device such as a minipump.
  • a kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is a prodrug or anti-PD-L1/IL-15 prodrug bifunctional protein as described herein.
  • the container may further comprise a second pharmaceutically active agent.
  • the kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.
  • the present application thus also provides articles of manufacture, which include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating a disease or disorder (such as a tumor) described herein, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert indicates that the composition is used for treating the particular condition in an individual.
  • the label or package insert will further comprise instructions for administering the composition to the individual.
  • the label may indicate directions for reconstitution and/or use.
  • the container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation.
  • Package insert refers to instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as
  • Example 1 Generation Of Masking Polypeptide (MP) Composition of the masking polypeptide: [0497] In consideration of choosing the amino acids for a chemically stable and predominantly unstructured masking polypeptide, the masking polypeptide comprises at least 40 contiguous amino acids and is substantially incapable of non-specific binding to serum protein.
  • the final amino acids chosen for masking polypeptide was composed of four types or five types of amino acid residues selected from a group consisting of proline (P), alanine (A), serine (S), glutamic acid (E) and glycine (G).
  • the masking polypeptide sequence was arranged in the ways that there was no single amino acid repeated in three times except serine (S), and the percentage of each amino acid in the masking peptide has a certain ratio: the percentage of amino acid residue A in the masking polypeptide about 5%-20%, the percentage of amino acid residue E in the masking polypeptide is about 1%-20%,the percentage of amino acid residue G in the masking polypeptide is about 15%-30%, the percentage of amino acid residue P in the masking polypeptide is about 15%-40%, the percentage of amino acid residue S in the masking polypeptide is about 20%-40%.
  • the masking polypeptide comprises at least 40 amino acids, the length of the sequence can be extended with the non-repetitive unstructured polypeptides.
  • the exemplary masking polypeptide MP80 was first designed with the sequence SEQ ID NO: 1, the masking polypeptide MP163 and the masking polypeptide MP240 comprise the amino acid sequence of the MP80. Following the rules described above, the masking polypeptides can be designed, not limited to the masking polypeptides shown in Table 1, and tested for the masking activity.
  • Figs. 1A-1C showed the sequence alignment of MP100 with MP80, MP163 with two repeats of MP80, and MP240 with triple repeats of MP80, respectively.
  • MP80 and MP100 comprised the amino acid sequence of SEQ ID NO: 6, and MP163 and MP240 all comprised the amino acid sequence of MP80.
  • IL-15 prodrugs and drugs were shown in Table 6.
  • An exemplary schematic construct illustrating an IL-15 prodrug that includes a masking polypeptide was shown in Fig.2A.
  • Fig.2B was an exemplary schematic drawing illustrating the activation process of IL-15 prodrug by released off the masking polypeptide (MP) at the target tissue (e.g., tumor with high levels of MMPs).
  • MP masking polypeptide
  • IL-15 drug has no masking polypeptide and cleavable moiety (e.g., SB1902-C1, SB1902-C1-variant1, SB1902-C1-variant2 and SB1902-C1-variant3).
  • the drug has no masking polypeptide and cleavable moiety (e.g., SB1902-C1, SB1902-C1-variant1, SB1902-C1-variant2 and SB1902-C1-variant3).
  • IL-15_L45D, IL-15_L45E, IL-15_Q48K, IL-15_S51D, IL-15_L52D, IL-15_E64K, IL-15_I67D, IL-15_I67E, IL-15_I68D or IL-15_N72D refers to the IL-15 variant comprising mutation L45D, L45E, Q48K, S51D, L52D, E64K, I67D, I67E, I68D or N72D corresponding to human mature wild type IL-15, respectively.
  • SB1902-C1-variant3_L45D refers to the IL-15 drug comprising IL-15 L45D variant instead of human mature wild type IL-15 in SB1902-C1-variant3.
  • the non-activatable IL-15 cytokine construct that did not include a cleavable moiety was constructed as control in the following experiments, e.g., SB1902-C4.
  • Plasmid construction is exemplified with prodrug SB1902-C2 (hIgG Fc(hole)-lk1- IL15-lk2-CM1-lk2-MP80/ hIgG Fc(knob)-lk5-IL15R ⁇ _sushi). Similar methods were used for other prodrugs and drugs.
  • prodrug expression vector Cloning of prodrug expression vector was accomplished with standard molecular techniques.
  • the gene fragments of masking polypeptide e.g., MP80
  • human IL-15 human IL-15R ⁇ _sushi was synthesized commercially (Genscripts USA) and digested with restriction enzymes correspondingly.
  • the human IgG1 Fc(hole) or Fc(knob) was PCR amplified and digested with restriction enzymes correspondingly.
  • the cleavable moiety CM1 was synthesized in a single forward and a single reverse nucleotide chain with corresponding restriction enzyme sites in both the 5’ and in the 3’ ends after annealing at 50°C.
  • CM1-CM10 The exemplary sequences of the cleavable moieties (CM1-CM10) were shown in Table 2, the non-cleavable linkers (lk, lk1, lk2, lk3, and lk5) were shown in Table 3, the exemplary sequences of human wild-type IL-15 (mature form or precursor form), IL-15 variants and IL-15 R ⁇ _sushi (long version from or short version form) were shown in Table 4, the sequences of human IgG1 Fc(hole), Fc(knob), Fc(knob-LALA) and Fc(hole-LALA) were shown in Table 5, and the exemplary sequences of IL-15 prodrug constructs were shown in Table 6A.
  • the human IL-15 or IL-15R ⁇ _sushi domain is italicized, the non- cleavable linker is bolded, the cleavable moiety is underlined with single line, the masking polypeptide is underlined with double lines, and the introduced restriction enzyme recognition sites is underlined with dotted line.
  • Other IL-15 drugs or prodrugs with IL-15 variants instead of wild type IL-15 are shown in Table 6B.
  • N-terminal 8 ⁇ His tagged expression constructs of both prodrug and drug were cloned and verified accordingly.
  • CM1, CM2, or CM4 for use in the activatable cytokine of this disclosure could be cleaved by matrix metalloprotease 2 (MMP2), and matrix metalloprotease 9 (MMP9).
  • MMP2 matrix metalloprotease 2
  • MMP9 matrix metalloprotease 9
  • hIgG Fc(hole-LALA)-lk3-IL15R ⁇ _sushi 43 the vectors coding the IL-15 prodrug, IL-15 drug or non-activatable IL- 15 cytokine (i.e., SB1902-C4, without cleavable moiety) as shown in Table 6A were transiently transfected, and proteins were expressed in Expi293 cells (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The culture supernatant media were clarified by centrifugation and 0.2 ⁇ m membrane filtration.
  • the exemplary IL-15 prodrugs, the exemplary IL-15 drugs and the non-activatable IL-15 cytokine SB1902-C4 as shown in Table 2 were purified by a two-step purification process comprising a precast MabSelect SuRe pcc column (Cytiva lifescience, Cat number17549112) and size-exclusion chromatography (Superdex200, Cytiva, USA), according to the manufacturer’s protocol.
  • MBP-MP80 was a fusion protein of maltose binding protein and masking polypeptide MP80 (SEQ ID NO: 1), and was purified with a precast column with amylose resin (NEB, Cat number E8021L, USA) by following the manufacturer’s instructions.
  • SDS-PAGE Analysis The exemplary purified prodrugs SB1902-C2, SB1902-C7 and drug SB1902-C1 were analyzed with 4-20% polyacrylamide SDS-PAGE under reduced or non- reduced conditions for purity evaluation, the gel was stained using SimplyBlue SafeStain (Fig. 3).
  • SEC-HPLC Analysis the prodrug SB1902-C2 and drug SB1902-C1 were analyzed with analytic SEC-HPLC for homogeneity assessment (Fig.4). HPLC analysis was performed using TSKgel G3000SWxl column according to the manufacturer's suggested running protocol. All purified drug and prodrugs were analyzed by both SDS-PAGE and SEC-HPLC in the same way.
  • CM1- CM10 a series of substrate sequences (cleavage moiety) for both MMP2 and MMP9 with suitable sensitivity were obtained, named CM1- CM10, of which the sequences and in vitro cleavage efficiency results were shown in Table 7.
  • CM1- CM10 a series of substrate sequences (cleavage moiety) for both MMP2 and MMP9 with suitable sensitivity were obtained, named CM1- CM10, of which the sequences and in vitro cleavage efficiency results were shown in Table 7.
  • recombinant human MMP2 (rhMMP2) (R&D Systems, Cat number 902-MP-010) and recombinant human MMP9 (rhMMP9) (R&D Systems, Cat number 911-MP-010) were diluted to 100 ⁇ g/mL with Assay Buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5), and the rhMMP2 and rhMMP9 were activated by 1mM of APMA (4- aminophenylmercuric acetate, Sigma-Aldrich, USA).Briefly, one microgram of each of diluted MMP2 and MMP9 was aliquoted into Eppendorf tubes and p-aminophenylmercuric acetate (APMA) (Sigma, Catalog number A-9563) was added to a final concentration of 1 mM.
  • APMA p-aminophenylmercuric
  • the tubes were incubated at 37°C for 2 hours for rhMMP2 and overnight for rhMMP9 to activate rhMMP-2 and rhMMP9.
  • Activated rhMMP-2 or rhMMP9 was diluted to 1 ng/ ⁇ L in Assay Buffer. Load 16 ⁇ L of activated rhMMP2 and rhMMP9 respectively into the first tube, add 4 ⁇ L of Assay buffer. Start to do 2 times serial dilution from tube 1 to tube 7 to get the final enzyme concentration from 400ng/mL to 6.25ng/mL.
  • protease digestion was carried out by adding 1.5 ⁇ g of SB1902-C2, SB1902-C5 or SB1902-C4 to each Eppendorf tube with preloaded active rhMM2 or rhMM9 with different enzyme concentrations for 5 hours at 37°C. After 5 hours of incubation, the prodrugs or drug prior to and after digestion were analyzed with 4-20% polyacrylamide gel.
  • the mutated substrate CM1 was cleavable in vitro by both MMP2 and MMP9 enzymes, with an enzyme dose-dependent manner.
  • Figs. 5E-5F depicted a proper cleavage profile of CM4 in prodrug SB1902-C5 after MMP2 and MMP9 digestion in vitro according to the protocol described above.
  • the mutated substrate CM4 was cleavable in vitro by both MMP2 and MMP9 enzymes, with an enzyme dose-dependent manner.
  • the other cleavable moieties (such as CM2, CM5-CM10) was also cleavable in vitro by both MMP2 and MMP9 (data not shown).
  • a serial diluted exemplary prodrugs or drugs was loaded to the plate and incubated for 2 hours.
  • Human IgG1 isotype antibody MOPC21 abbreviated as hIgG1 in Fig.7A (see Hamlyn PH, Gait MJ, Milstein C. (1981) Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing. Nucleic Acids Res.9(18):4485-4494) was used as a negative control.
  • an anti-human IgG-Fc-AP conjugated antibody at 1:2000 dilution was added to the individual well of the plate and incubated for 45min.
  • Mo7e cells (DSMZ, Cat number ACC 104) were maintained in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin and streptomycin, and 10 ng/ml GM-CSF (Peprotech, Cat number 300-03) in the incubator at 37 °C (5% CO2). Mo7e cells were harvested in their logarithmic growth phase and washed twice with GM-CSF free medium. Cells were plated in a 384-well plate with a cell number of 1 ⁇ 10 4 /well, and a medium volume of 45 ⁇ L/well (GM-CSF free).
  • Fig.9 showed that all the exemplary prodrugs SB1902-C2, SB1902-C6 and SB1902- C7 and drug SB1902-C1 had a dose-dependent stimulation of cell proliferation. However, the prodrugs SB1902-C2, SB1902-C6 and SB1902-C7 showed significantly reduced functionality compared to the drug SB1902-C1. The results demonstrated that the masking polypeptide MP80, MP96new and MP100 prevented IL-15 prodrug from binding to its receptor IL-2/IL- 15R ⁇ and reduced its signal transduction and downstream function.
  • Example 6 In Vitro Functional Experiments: CD8+ T Cell Activation Assay [0524] It was well documented that IL-15 promotes CD8+ memory T, natural killer (NK) and NKT cells proliferation, survival, and homeostasis. IL-15 causes T cell activation indicated by upregulation of the membrane surface expression of CD69 and cytokine release including IFN- ⁇ . CD69 was an early activation marker of T cells. The percentage of CD8+ T cell activation was reflected by the percentage of CD69 surface expression.
  • the prodrug SB1902-C7 with masking polypeptide MP100 had a significantly lower activity to stimulate T cell activation compared to the drug SB1902-C1.
  • the prodrug SB1902-C2 with masking polypeptide MP80 had a significantly lower activity to stimulate T cell activation compared to the drug SB1902-C1.
  • both the prodrug SB1902-C2 with masking polypeptide MP80 and the prodrug SB1902-C6 with masking polypeptide MP96new had low activity to stimulate T cell activation.
  • SB1902-C2 showed better efficacy in inhibiting CD69 activation in CD8+ T cells than SB1902-C6.
  • the prodrug SB1902-C9-variant4 with masking polypeptide MP163 and a LALA mutation (L234A and L235A) in Fc domain had a significantly lower activity to stimulate T cell activation compared to the drug SB1902-C1-variant2 and SB1902- C1-variant3.
  • the prodrug SB1902-C10-variant1 with masking polypeptide MP240 had a significantly lower activity to stimulate T cell activation compared to the drug SB1902-C1-variant1.
  • the prodrug SB1902-C2-variant1 with masking polypeptide MP80 had a significantly lower activity to stimulate T cell activation compared to the drug SB1902-C1.
  • the prodrug SB1902-C9-variant0 with masking polypeptide MP163 had a significantly lower activity to stimulate T cell activation compared to the drug SB1902-C1-variant1.
  • the prodrug SB1902-C9-variant2 with masking polypeptide MP163 had a significantly lower activity to stimulate T cell activation compared to the drug SB1902-C1.
  • the prodrugs SB1902-C9-variant1, SB1902-C9-variant2 and SB1902-C9-variant3 with masking polypeptide MP163 had low activity to stimulate T cell activation.
  • These results above demonstrated that the activity of IL15 cytokine in different prodrug formats with different masking polypeptides in activating T cells was significantly shielded by the masking polypeptides, demonstrated that the masking polypeptides of prodrugs prevented IL-15 from activating CD8+T cells.
  • the SB1902-C9 (Fig. 10J), SB1902-C9-variant2 (Fig. 10K) or SB1902-C9-variant6 (Fig. 10L) with masking polypeptide MP163 showed better efficacy in inhibiting CD69 activation in CD8+ T cells than the SB1902- C2 (Fig.10J), SB1902-C2-variant2 (Fig.10K) or SB1902-C2-variant0 (Fig.10L) with masking polypeptide MP80 did, respectively, indicating that the masking polypeptide MP163 had better masking effect than MP80.
  • the masking polypeptides with different lengths all had a good masking effect.
  • the length of masking polypeptide in these tested lengths ranges from 80-100 amino acids, the small change in the MP length would not impact the activity much.
  • the length of masking polypeptide plays some role when within the range of 80-163, there is some enhancements on the masking efficiency as the length increases.
  • the exemplary prodrug with wild-type IL-15 (SB1902-C9-variant2) and the prodrugs with IL-15 variants (SB1902-C9-variant2_L45D, SB1902-C9-variant2_L52D, SB1902-C9- variant2_I67D and SB1902-C9-variant2_I67E) were also detected in this assay.
  • SB1902-C9-variant2_L45D, SB1902-C9-variant2_L52D, SB1902-C9- variant2_I67D and SB1902-C9-variant2_I67E were also detected in this assay.
  • the recombinant human MMP-2 (rhMMP2) (R&D Systems, Cat number 902-MP-010) was activated by following the manufacturer’s instructions. Then, one microgram of activated MMP2 was added to an Eppendorf tube containing 250 ⁇ g of purified prodrug SB1902-C2, and the digestion volume was added to 0.5mL with digestion buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5). The tube containing enzyme/prodrug mix was incubated at 37°C for 5 hrs. After enzyme digestion, the sample was taken for evaluation of the digestion efficiency by SDS-PAGE.
  • digestion buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij 35, pH 7.5.
  • T cell-derived IFN- ⁇ is a key cytokine that stimulates innate immune responses.
  • IL- 15 has a significant role as an activator of T cell functions. It has been shown that IL-15 induces the expression of IFN- ⁇ (Strengell M, et al. IL-21 in synergy with IL-15 or IL-18 enhances IFN- gamma production in human NK and T cells. J Immunol. 2003 Jun 1;170(11):5464-9). Overproduction of the proinflammatory cytokine IFN- ⁇ systemically could cause unwanted side effects/toxicity in vivo.
  • Granzymes are serine proteases that are released by cytoplasmic granules within cytotoxic T cells and natural killer cells.
  • Human Granzyme B is one of the five members of the human granzyme family which includes Granzymes A, B, G, H, and K.
  • the IL-15 treatment resulted in increased NK and CD8+T cell activation, the activation of NK and CD8+T cell with features of enhanced IFN- ⁇ production, proliferation (Ki67+), cytotoxic potential (Granzyme B production) and expression of the survival factor Bcl-2.
  • Heterodimeric IL-15 delays tumor growth and promotes intratumoral CTL and dendritic cell accumulation by a cytokine network involving XCL1, IFN- ⁇ , CXCL9 and CXCL10. J Immunother Cancer.2020 May;8(1):e000599).
  • the activity of the prodrugs on the IL-15 cytokine-dependent IFN- ⁇ and Granzyme B production was detected essentially as follows: as mentioned above (e.g., in CD8+ T cell activation assay), Human PBMCs were stimulated with serially diluted IL-15 prodrugs or drugs. Cell plates were cultured in the incubator for 3 days. Then the cells were centrifuged at room temperature, 150 ⁇ g for 5 min.
  • the supernatant was collected for Granzyme B and IFN- ⁇ detection.
  • the human Granzyme B and IFN- ⁇ concentrations in supernatant were quantified by ELISA using LEGEND MAXTM Human Granzyme B ELISA Kit (BioLegend, cat# 439207) and Human IFN- ⁇ ELISA MAX Deluxe kit (BioLegend, cat# 430116), respectively.
  • the data was analyzed and plotted with GraphPad Prism 8 software.
  • IFN- ⁇ production assay [0562] The result of the drugs with wild-type IL-15 or IL-15 variants (Fig.12C) showed that cells incubated with the exemplary drugs with IL-15 variants all had comparable or even higher efficacy in stimulation of T cell production of IFN- ⁇ compared to the drug with wild-type IL- 15 (SB1902-C1-variant3).
  • Granzyme B production assay [0566] The result of the drugs with wild-type IL-15 or IL-15 variants (Fig. 12E) showed that cells incubated with the exemplary drugs with IL-15 variants had higher efficacy in stimulation of T cell production of Granzyme B compared to the drug with wild-type IL-15 (SB1902-C1- variant3).
  • IL-15 variants in the prodrugs exhibited more reduced binding activity to the receptor compared to prodrug with wild-type IL-15, which were also correlated with a more decrease of Granzyme B and IFN- ⁇ production. It could potentially be translated to a wider dosing window with less systemic toxicity in clinic practice when compared to the prodrug with wild-type IL-15.
  • Example 8 In Vivo IFN- ⁇ Production Assay [0571] The IFN- ⁇ production was also assayed in mouse, to measure if the masking polypeptide in the prodrug effect the IL-15 cytokine-dependent IFN- ⁇ production in vivo, the assay was performed essentially as follows: [0572] The drug SB1902-C1 or the prodrug SB-1902-C2 as an example was intravenously injected into Balb/c mice with the same molarity. Plasma was collected at 6 and 24 hours after injection. IFN- ⁇ was detected with an ELISA kit (ThermoFisher Scientific, Cat# KMC4021C) according to the instruction. [0573] As shown in Fig.
  • mice treated with the exemplary prodrug SB1902-C2 had less IFN- ⁇ production compared to the drug SB1902-C1 without masking polypeptide.
  • Example 9 In Vivo Tumor Models To Evaluate Activity Of Prodrug And Drug [0574] The ability of the IL-15 prodrug and drug without masking polypeptide to promote tumor eradication and inhibit metastasis is assessed in vivo using the mouse WEHI-164 tumor model.
  • a human IgG1 isotype antibody MOPC21 (see Hamlyn PH, Gait MJ, Milstein C. (1981) Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing. Nucleic Acids Res.9(18):4485-4494) was used as a control in this experiment.
  • A. In vivo activity of prodrug in WEHI-164 subcutaneous tumor model [0575] Animals and husbandry: Female mice (7-9 weeks of age) were used in the studies. The animals were fed irradiated Harlan 2918.15 Rodent Diet and water ad libitum.
  • WEHI-164 cells were cultured and expanded in Dulbecco’s Modified Eagles Medium (DMEM) with 2mM L-glutamine, 10% fetal bovine serum (FBS), and 1% 100 ⁇ Penicillin/Streptomycin (PS).
  • DMEM Modified Eagles Medium
  • FBS fetal bovine serum
  • PS Penicillin/Streptomycin
  • the growth environment was maintained in an incubator with a 5% CO2 atmosphere at 37°C.
  • the cells were trypsinized using a 0.25% trypsin-EDTA solution.
  • the cells were then washed and counted. Pre- implantation cell viability was >95%.
  • the cells were resuspended in Dulbecco’s Phosphate Buffered Saline (DPBS).
  • DPBS Phosphate Buffered Saline
  • Test animals were sterilized at the implantation site with an alcohol prep pad and were implanted subcutaneously on Day 0 in 0.1 mL using a 25-gauge needle and 1 mL syringe.
  • Measurements and treatment Tumors were allowed to grow at the range of 70- 150mm 3 and were then randomized into study groups. Mice were distributed to ensure that the mean body weights for all groups were within 10% of the overall mean tumor burden for the study population. Mice were intravenously injected twice weekly with a dose of 3 mg/kg of human IgG1 isotype antibody or the prodrug SB1902-C2 for 2 weeks and tumor volumes were monitored.
  • Assessment of side effects All animals were observed for clinical signs of distress or toxicity at least once daily.
  • Prodrugs were intravenously injected at a dose of 1mg/kg for all groups except the drug SB1902-C1 group which was dosed at 0.3mg/kg which was MTD based on previous dosing experiments.
  • the dosing time was at day0, day4, day7, day10 and day14. Tumor volumes were monitored.
  • Example 10 Development of anti-PD-L1 antibodies
  • This example briefly illustrates the methods of generating, screening and selecting anti-PD-L1 antibodies. Also see U.S. provisional application NO. 63/371,900, filed Aug. 19, 2022, the content of which was incorporated here in its entirety.
  • Immunizations and Hybridoma screening Mice were immunized with recombinant extracellular domains of human PD-L1 fused with mouse IgG2a Fc, or the V-domain of human PD-L1 fused with the C-domain of mouse PD-L1 and mouse IgG2a Fc.
  • mouse B cells were isolated and fused with myeloma cells SP2/0-Ag14 cells (American Type Culture Collection CRL 1581). Following standard protocols, monoclonal Hybridoma clones were picked and subjected to primary screening by ELISA.
  • Generation of chimeric antibody and humanization the recombinant chimeric antibody construct Mab 3-16 (IgG1 version) with heavy and light chain variable domain of mouse antibody and human constant regions were made by using methods well known in the field. Mab 3-16 was further humanized.
  • the human germline sequence of IGHV3-48* 01 and IGKV3-11* 01 were chosen as recipients , and three CDR loops from Mab 3-16 were grafted into the homologous human scaffold according to Kabat numbering method (Kabat et al., 1987).
  • the full-length IgG versions of humanized PD-L1 antibodies Hum5, Hum6, and Hum7 were generated and further tested.
  • the sequences of the anti- human PD-L1 antibodies employed in the examples were summarized in Table B and C.
  • Example 11 Characterization of anti-PD-L1 antibodies Characterization of binding affinity and dissociation constant (Kd) of the anti-human PD- L1antibodies [0690]
  • the binding affinity of anti-human PD-L1 antibodies were determined using FortéBio BioLayer Interferometry (BLI).
  • BBI FortéBio BioLayer Interferometry
  • the anti-human PD-L1 antibody with human IgG1 isotype was captured by the anti-human Fc sensors (AHC, Fortebio) at the loading concentration of 2 ⁇ g/mL in kinetic buffer for 300 seconds.
  • the PD-l/PD-L1 signaling pathway inhibits TCR/CD28 co-stimulatory signals, which can be read out as reduced cytokine production, such as IL-2 and IFN- ⁇ . Accordingly, inhibition of the PD-l pathway via blocking PD-1 interaction with PD-L1 by the antibody to PD-L1 can enhance T cell activation.
  • cytokine production such as IL-2 and IFN- ⁇ .
  • inhibition of the PD-l pathway via blocking PD-1 interaction with PD-L1 by the antibody to PD-L1 can enhance T cell activation.
  • Jurkat 6E-1 cell line is derived from human acute T lymphocyte leukemia. This cell is characterized by production of large amount of IL-2 after stimulation, such as with phorbol esters.
  • Jurkat 6E-1 cell is engineered with human PD-1 gene.
  • Raji cell line a human B lymphocyte-derived cell, expressing human PD-L1 (www.Invivogen.com) was used as antigen presenting cells.
  • the ability of anti PD-L1 antibodies to enhance IL-2 production can be observed after presenting T cells with super antigen such as Staphylococcal enterotoxin E (SEE) by antigen presenting cells (APCs).
  • SEE Staphylococcal enterotoxin E
  • APCs antigen presenting cells
  • the human PD-1 gene engineered Jurkat cell line was made in-house by using lentivirus mediated gene integration method. Briefly, the human PD-1 gene (NP_005009.2) was synthesized (IDT DNA company) and subcloned into the lentivirus vector.
  • the infectious virus particles were packaged by using lentivirus packaging system (Clontech, Lenti-XTM Packaging Single Shots, VSV-G). After infection, the Jurkat cell was subjected to selection by the antibiotic Blastcidin (3 ⁇ g/ml) for 3-4 weeks until the Jurkat cells were established to be almost 100% positive to PD-1 by using FACS analysis with anti-human PD-1 antibody (APC-anti- human PD-1, Biolegend). The human PD-L1-expressing Raji Cell line was purchased from www.Invivogen.com. [0696] The Jurkat and Raji cells were maintained in RPMI 1640 containing 10% FBS, 1 % L Pen/Strep in 5% CO2 at 37 o C separately.
  • the Raji B cells in 1 ⁇ 10 6 /ml were pre-incubated with 2-3 ng/ml SEE superantigen (Staphylococcal enterotoxin E, Toxin Technology #ET404) and then mixed with equal volume of the Jurkat cells in 2 ⁇ 10 6 /ml.
  • 100 ⁇ l of the Jurkat and Raji cell mixtures and 100 ⁇ l of the antibodies with 1:3 serially diluted concentration were transferred into a 96-well U-bottom plate in triplicate wells containing RPMI 1640, 10% FBS, 1 % L Glutamine and 0.1 % Pen/Strep in 5% CO 2 at 37 o C.
  • the culture supernatants were collected after 48 hours.
  • Human IL-2 was quantified by ELISA using IL-2 ELISA kit (Biolegend MAX Human IL-2 ELISA kit) according to its protocol.
  • IL-2 ELISA kit Biolegend MAX Human IL-2 ELISA kit
  • the humanized anti-PD-L1 antibodies Hum5, Hum6 and Hum7 retain their full activity in this assay when comparing with the reference antibody Atezolizumab (anti-PD-L1 antibody, Roche; referred to as “Ref” in the following) and parent antibody Mab 3-16 in the meaning of EC 50 value.
  • the chimeric Mab 3-16, the full-length IgG1 humanized anti-PD-L1 antibody, reference antibody or IgG1 control (Biolegend, catalog# 403501) at serially diluted concentration were then added into the plate in triplicate wells with the staphylococcal enterotoxin B (SEB; Toxin Technology) at the concentration of 100ng/ml in final.
  • SEB staphylococcal enterotoxin B
  • the total volume per well is 200 ⁇ l each well and the cells were cultured in for 3 days in RPMI 1640 containing 10% FBS, 1 % L Glutamine and 0.1 % Pen/Strep in 5% CO 2 at 37 o C.
  • IL-2 levels in culture supernatants were measured by ELISA analysis (Biolegend MAX human IL-2 ELISA).
  • the light chain comprises V L -C L with an amino acid sequence as shown in SEQ ID NO: 115.
  • the first heavy chains comprises the structure of VH-CH1-hinge-CH2-CH3-IL-15-CM1-MP163, with an amino acid sequence as shown in SEQ ID NO: 113, wherein the Fc domain comprising C H 2 and C H 3 domain carried T366W mutation as an Fc knob; and the second heavy chain comprises the structure of VH- C H 1-hinge-C H 2-C H 3-IL-15R ⁇ _sushi domain, with an amino acid sequence as shown in SEQ ID NO: 114, wherein the Fc domain comprising CH2 and CH3 domain carried T366S, L368A, and Y407V mutations as an Fc hole.
  • one heavy chain and one light chain constitute one monomer of the bifunctional protein.
  • the Numbering was according to the EU Numbering system.
  • Other Bis-prodrugs with IL-15 variants were also provided, in which the first heavy chains thereof comprised IL-15 variants described above instead of wild-type IL-15.
  • the first heavy chains of these Bis-prodrugs with IL-15 variants were shown in SEQ ID NO: 116-125 and in Table 9B.
  • the exemplary sequence and constructs of the anti-PD-L1/IL-15 prodrug bifunctional protein was shown in Table 9A.
  • the schematic structure diagram of the exemplary anti-PD- L1/IL-15 prodrug bifunctional protein was shown in FIG.17.
  • the plasmid DNA of heavy chain and light chain were transfected into HEK293 by following the protocol provided with transfection kit from vendor (Thermo-Fisher, USA).
  • the expressed bifunctional fusion proteins were purified with MabSelect column (Cytiva, US) on AKTA explorer following the protocol from the vendor.
  • Lentivirus carrying human PD-L1 gene (Uniprot, Q9NZQ7) was packaged by using the virus packaging kit (Lenti-XTM Packaging Single Shots, Cat# 631275, Takarabio). The cells were transduced with the lentivirus carrying human PD-L1 gene and selected in RPMI medium supplemented with 10ug/ml Blasticidin. PD-L1 expression was demonstrated by anti- PD-L1 antibody (eBioscience, catalog # 12-5983-42) staining. [00706] Jurkat cells (ATCC, Catalog #TIB-152), were maintained in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin and streptomycin Blasticidin in the incubator at 37 °C (5% CO2).
  • Lentivirus carrying human PD-1 gene (Uniprot, Q15116) was packaged by using the virus packaging kit. The cells were transduced with the lentivirus carrying human PD-1 gene and selected in RPMI medium supplemented with 10ug/ml Blasticidin. PD-1 expression was demonstrated by anti-PD-1 antibody (eBioscience, catalog # 12-2799-42) staining. [00707] Raji-PD-L1 cells and Jurkat-PD-1 cells were co-cultured at 2:1 ratio with the stimulation of 20ng/ml bispecific antibody specifically recognizing CD3 and CD20 (Creative Biolabs, catalog# SCTB-025).
  • Atezolizumab variant also abbreviated as ATZ-v, in which mutations of L234A/L235A were introduced into Fc domain of Atezolizumab that is abbreviated as ATZ, numbering of the residues according to the EU numbering system, the amino acids of CDRs or VH/VL of ATZ/ATZ-v were shown in Table B and C) or IgG1 isotype antibody (Biolegend, catalog# 403501) were serially diluted and added to the culture for 72 hours. The supernatant was collected for human IL-2 ELISA (Biolegend, catalog# 431804). Absorbance at 450 nm (OD450) was detected.
  • SB1903, anti-PD-L1/IL-15 drug bifunctional protein also referred to as Bis-drug, the bifunctional protein corresponds to SB1903 except for being without cleavable moiety and masking moiety was used as an representative example in the Example, the amino acid sequences of which were shown in Table 10) and anti-PD-L1 antibody ATZ-v were serially diluted and added to the cells for 72-hour culture. Then the cells were centrifuged at room temperature, 150 ⁇ g for 5 min.
  • the supernatant was discarded, and the cell pellet was stained with anti-CD3 antibody (Biolegend, catalog# 317305), anti-CD8 antibody (Biolegend, catalog# 980904), and anti-CD69 antibody (Biolegend, catalog# 985202) in FACS buffer for 30 minutes. They were washed twice with FACS buffer and acquired by flow cytometry with Attune (ThermoFisher Scientific). Data were analyzed with FlowJo software. The percentage of CD69+ cells in CD8+ T cells was plotted with Graphpad Prism 8 software.
  • the exemplary animo acid sequences of the anti-PD-L1/IL-15 drug bifunctional protein (Bis-Drug) used in the example was shown in Table 10.
  • Anti PD-L1 antibody ATZ-v alone has no effect on T cell activation in vitro.
  • These bioassay results described above showed that the PD-L1/IL-15 prodrug bifunctional protein maintains the activity of PD-L1 antibody and exhibits much less activity of IL-15 in vitro.
  • the PD-L1/IL-15 drug bifunctional protein exhibits the activity of CD8+ T cell activation, indicating that the PD-L1/IL-15 prodrug bifunctional protein would exhibit the activity of IL-15 once the making peptide is removed in desired circumstance.
  • Example 14 Evaluation of the efficacy of the anti-PD-L1/ IL-15 prodrug bifunctional protein in vivo
  • two more Bis-prodrugs were constructed and employed in the example, the structure and construction of which were as SB1903 (i.e, comprising two identical light chains and two different heavy chains), except that the anti-PD- L1 VH/VL domains of the two Bis-prodrugs were derived from Atezolizumab (ATZ).
  • AZA Atezolizumab
  • SB1903-x1 and SB1903-x2 Constant regions were derived from mouse instead of human
  • the cells were cultured and expanded in Dulbecco’s Modified Eagles Medium (DMEM) with 2mM L-glutamine, 10% fetal bovine serum (FBS), and 1% 100 ⁇ Penicillin/Streptomycin (PS).
  • DMEM Modified Eagles Medium
  • FBS fetal bovine serum
  • PS Penicillin/Streptomycin
  • the growth environment was maintained in an incubator with a 5% CO 2 atmosphere at 37°C.
  • the cells (passage 3) were trypsinized using a 0.25% trypsin- EDTA solution.
  • the cells were then washed and counted. Pre-implantation cell viability was more than 95%.
  • the cells were suspended at the concentration of 1 ⁇ 10 ⁇ 7 cells/ml in the solution of PBS. [00717] Cell implantation: The right flank of each mouse was shaved on Day -1.
  • IL-15 prodrug SB1902-C2-variant2 was used as example
  • Bis-prodrug SB1903-x1 was used as example
  • anti-PD-L1 antibody ATZ-v was used as example
  • placebo IgG1 isotype antibody
  • B16 F10 Tumor animal model [00722] Animals and husbandry: As described above. [00723] Cell preparation: As described above, except for that the cells were suspended at the concentration of 5 ⁇ 10 ⁇ 6 cells/ml in the solution of PBS. [00724] Cell implantation: The right flank of each mouse was shaved on Day -1. On Day 0, 100 ⁇ L of the B16F10 cell suspensions (5 ⁇ 10 6 cells/mL) were injected subcutaneously into right flank of each of the 140 mice under aseptic condition using 1 ml syringes. Mice were anesthetized with isoflurane (2-3% to effects on O2) to facilitate the implantation.
  • IL-15 prodrug SB1902-C2-variant2 was used as example
  • Bis-prodrug SB1903-x2 was used as example
  • anti-PD-L1 antibody ATZ-v was used as example
  • anti-PD-L1 antibody ATZ-v+ SB1902-C2-variant2
  • Table 13 G roup Reagent Administrated Dose(mg/kg) Dose per mouse ( ⁇ l) 1 ATZ-v 6.4 200 [ 00726] Body weights were recorded when the animals arrive at the Test Facility and once weekly thereafter.
  • the tumor volume was smaller than that in IL-15 prodrug treated group and the combinational treated group, suggesting anti-PD-L1/IL-15 prodrug bifunctional protein exhibited better activity on inhibiting B16 F10 tumor in vivo than IL-15 prodrug and the combination of IL-15 and anti- PD-L1 antibody did, although anti-PD-L1 antibody didn’t show inhibition activity.

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Abstract

La présente demande concerne des promédicaments d'IL-15 comprenant le polypeptide de masquage (MP) et le fragment clivable (CM), et des protéines bifonctionnelles comprenant un anticorps qui se lie à PD-L1 et au promédicament d'IL-15. L'invention concerne également les molécules d'acide nucléique isolées codant pour les protéines bifonctionnelles; des vecteurs comprenant les molécules d'acide nucléique; des cellules hôtes contenant les molécules ou vecteurs d'acide nucléique; des compositions pharmaceutiques contenant les protéines bifonctionnelles, les molécules d'acides nucléiques isolées, les vecteurs ou les cellules hôtes. L'invention concerne également des procédés de production et d'utilisation des protéines bifonctionnelles ou des compositions pharmaceutiques.
PCT/US2025/012251 2024-02-02 2025-01-18 Protéine bifonctionnelle promédicament anti-pd-l1 et il-15 et utilisations associées Pending WO2025165594A1 (fr)

Applications Claiming Priority (2)

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US202463548862P 2024-02-02 2024-02-02
US63/548,862 2024-02-02

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WO2025165594A1 true WO2025165594A1 (fr) 2025-08-07

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WO (1) WO2025165594A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210260163A1 (en) * 2018-03-09 2021-08-26 AskGene Pharma, Inc. Novel cytokine prodrugs
US20220002370A1 (en) * 2018-09-27 2022-01-06 Xilio Development, Inc. Masked cytokine polypeptides
US20230331799A1 (en) * 2020-04-01 2023-10-19 Xilio Development, Inc. Masked il-15 cytokines and their cleavage products

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210260163A1 (en) * 2018-03-09 2021-08-26 AskGene Pharma, Inc. Novel cytokine prodrugs
US20220002370A1 (en) * 2018-09-27 2022-01-06 Xilio Development, Inc. Masked cytokine polypeptides
US20230331799A1 (en) * 2020-04-01 2023-10-19 Xilio Development, Inc. Masked il-15 cytokines and their cleavage products

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