WO2021142476A1 - Single-chain polypeptides - Google Patents

Single-chain polypeptides Download PDF

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Publication number
WO2021142476A1
WO2021142476A1 PCT/US2021/013091 US2021013091W WO2021142476A1 WO 2021142476 A1 WO2021142476 A1 WO 2021142476A1 US 2021013091 W US2021013091 W US 2021013091W WO 2021142476 A1 WO2021142476 A1 WO 2021142476A1
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WIPO (PCT)
Prior art keywords
polypeptide
chain
terminus
fragment
carrier
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PCT/US2021/013091
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French (fr)
Inventor
Asya Grinberg
Pyae P. HEIN
Zong Sean JUO
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Dragonfly Therapeutics, Inc.
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Publication date
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Publication of WO2021142476A1 publication Critical patent/WO2021142476A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—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]
    • 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
    • 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
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • 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
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • the invention generally relates to single-chain immunoregulatory polypeptides, pharmaceutical compositions comprising such polypeptides, and methods of treating a cancer using the same.
  • Physiologically active proteins generally have the disadvantage of having a short in vivo half-life.
  • PEG polyethylene glycol
  • protein carriers such as: antibodies or fragments thereof (e.g. fragment crystallizable (Fc) domains), nanobodies (VHH), or human serum albumin (HSA) and fragments thereof.
  • Fc fragment crystallizable domains
  • VHH nanobodies
  • HSA human serum albumin
  • monomeric proteins and their cognate binding partner proteins (or portions thereof) that together form a complex to exert physiological activity can be joined to carriers to form single-chain polypeptides to increase monomeric protein stability, minimize in vivo clearance, and increase the in vivo half-life of the functional-protein complex.
  • the carrier polypeptides may be derived from IgGl, IgG2, IgG3, IgG4 antibodies (e.g, Fc, Fab, variable fragment (Fv), or VHH) or HSA.
  • inventions described in the present disclosure provide designs for improving recombinant protein stability and physiological activity, wherein a carrier is joined via a linker to a complex comprising a single- subunit cytokine or functional fragment thereof (e.g, interleukin 15 (IL- 15)) joined directly or indirectly to the cognate receptor of the single- subunit cytokine, or functional fragment thereof, to form a single-chain immunoregulatory polypeptide.
  • a carrier is joined via a linker to a complex comprising a single- subunit cytokine or functional fragment thereof (e.g, interleukin 15 (IL- 15)) joined directly or indirectly to the cognate receptor of the single- subunit cytokine, or functional fragment thereof, to form a single-chain immunoregulatory polypeptide.
  • a carrier is joined via a linker to a complex comprising a single- subunit cytokine or functional fragment thereof (e.g, interleukin 15 (IL- 15)) joined directly or indirectly to the cognate
  • the present invention provides single-chain immunoregulatory polypeptides.
  • These single-chain polypeptide constructs can have increased stability, reduced in vivo clearance, and increased half-life when used as a therapeutic.
  • the present invention provides a single-chain polypeptide comprising: (a) an effector moiety polypeptide or a functional fragment thereof; (b) a receptor polypeptide or a functional fragment thereof; (c) a multi-chain carrier or single-chain carrier; and (d) two or more linkers, wherein a linker independently joins the single or multi- chain carrier to the effector moiety polypeptide or functional fragment thereof, and/or to the receptor polypeptide or functional fragment thereof.
  • the carrier is multi-chain
  • two chains of the multi-chain carrier are joined by a linker.
  • the present invention provides a single-chain polypeptide comprising three or more units comprising an effector moiety polypeptide or a functional fragment thereof, a receptor polypeptide or a functional fragment thereof, and a single-chain or multi-chain carrier, and wherein each unit is independently joined to another unit by a linker.
  • the effector moiety polypeptide or functional fragment thereof, or the receptor polypeptide or functional fragment thereof is joined to each chain of the multi-chain carrier by a linker.
  • the effector moiety polypeptide or functional fragment thereof, or the receptor polypeptide or functional fragment thereof is independently joined to one chain of the multi-chain carrier.
  • the receptor polypeptide or functional fragment thereof binds to the effector moiety polypeptide or functional fragment thereof to form a complex.
  • the effector moiety polypeptide comprises a single- subunit cytokine polypeptide.
  • the single-subunit cytokine comprises an interleukin 15 (IL-15) polypeptide.
  • the IL-15 polypeptide is human IL-15.
  • the receptor polypeptide comprises an IL-15 receptor polypeptide or a functional fragment thereof.
  • the IL-15 receptor polypeptide is an IL-15 receptor alpha (IL-15R ⁇ ) polypeptide or a functional fragment thereof.
  • the functional fragment of IL-15R ⁇ comprises a sushi domain.
  • the IL-15R ⁇ polypeptide is human IL-15R ⁇ polypeptide.
  • the carrier is selected from: a unit comprising a fragment crystallizable (Fc) domain, an anti-HSA nanobody (VHH), a human serum albumin (HSA) polypeptide or functional fragment thereof, an anti-HSA Fab domain, and an anti-HSA single-chain variable fragment (scFv).
  • Fc fragment crystallizable
  • VHH anti-HSA nanobody
  • HSA human serum albumin
  • scFv anti-HSA single-chain variable fragment
  • the carrier is a unit comprising an Fc domain, wherein the Fc domain comprises a first Fc domain polypeptide and a second Fc domain polypeptide.
  • the Fc domain is a human Fc domain.
  • the Fc domain is an IgGl, IgG2, IgG3, or IgG4 Fc domain.
  • a linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • an additional linker joins the C-terminus of the carrier to the N-terminus of the receptor polypeptide or functional fragment thereof.
  • the IL-15 polypeptide comprises one or more than one mutation.
  • the one or more than one mutation removes one or more than one glycosylation site.
  • the one or more than one mutation does not substantially affect binding kinetics or binding affinity to the IL-2/IL-15 receptor common b subunit (IL-15R ⁇ ),
  • the one or more than one mutation comprises a mutation of the serine at position 73.
  • the mutation of the serine at position 73 is to alanine.
  • the one or more than one mutation further comprises a mutation of the histidine at position 105.
  • the mutation of the histidine at position 105 is to glutamate.
  • the description provides a single-chain polypeptide comprising: (a) a human IL-15 polypeptide or a functional fragment thereof; (b) a human IL- 15Ra polypeptide or a functional fragment thereof; and (c) a multi-chain carrier comprising an Fc domain, wherein a linker joins the N-terminus of a first fragment of an immunoglobulin heavy chain to the C-terminus of a second fragment of an immunoglobulin heavy chain, wherein an additional linker joins the C-terminus of the first fragment of an immunoglobulin heavy chain to the N-terminus of the IL-15 polypeptide or functional fragment thereof, and an additional linker joins the N-terminus of the second fragment of an immunoglobulin heavy chain to the C-terminus of the IL-15R ⁇ polypeptide or fragment thereof, and wherein the first and the second fragment of the immunoglobulin heavy chain each comprises or consists of an Fc domain polypeptide.
  • a single-chain polypeptide is provided that includes the amino acid sequence of SEQ ID NO:37. In some embodiments, a single-chain polypeptide is provided that includes the amino acid sequence of SEQ ID NO:71. In some embodiments, a single-chain polypeptide is provided that includes the amino acid sequence of SEQ ID NO:72.
  • the present invention provides a single-chain polypeptide comprising: (a) an effector moiety polypeptide or a functional fragment thereof; (b) a receptor polypeptide or a functional fragment thereof; (c) a carrier comprising (i) a multi- chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain, or (ii) a single-chain carrier; (d) a linker joining the carrier to the receptor polypeptide or functional fragment thereof; and (e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the carrier.
  • the additional linker joins the carrier to the effector moiety polypeptide or functional fragment thereof.
  • the linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the carrier, and the additional linker joins the C-terminus of the carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • additional linker joins the C-terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the carrier, and the linker joins the C-terminus of the carrier to the N-terminus of the receptor polypeptide or functional fragment thereof.
  • the additional linker joins the effector moiety polypeptide or functional fragment thereof to the receptor polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the carrier to the N-terminus of the receptor polypeptide or functional fragment thereof. In some embodiments, the additional linker joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • the linker chains, polypeptides, and/or units comprise a sequence of (GGGGS) 4 (SEQ ID NO:31), (GGGGS) 5 (SEQ ID NO:32), (GGGGS) 6 (SEQ ID NO:33), or (SGGGG) 6 (SEQ ID NO:34).
  • the multi-chain carrier is a fragment crystallizable (Fc) domain carrier comprising two Fc-domain polypeptide chains.
  • the single-chain carrier is a nanobody (VHH) carrier.
  • the present invention provides a single-chain polypeptide comprising: (a) an effector moiety polypeptide or a functional fragment thereof; (b) a receptor polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)- domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker;
  • the linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier, and the additional linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • the linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the receptor polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • the present invention provides a single-chain polypeptide comprising: (a) an effector moiety polypeptide or a functional fragment thereof; (b) a receptor polypeptide or a functional fragment thereof; (c) a nanobody (VHH) carrier; (d) a linker joining the VHH carrier to the receptor polypeptide or functional fragment thereof; and
  • the linker joins the C-terminus of the VHH carrier to the N-terminus of the receptor polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • the linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the additional linker joins the C- terminus of the VHH carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • the additional linker joins the C-terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the linker joins the C-terminus of the VHH carrier to the N-terminus of the receptor polypeptide or functional fragment thereof.
  • the present invention provides a single-chain polypeptide comprising: (a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof; (b) an IL-15 receptor alpha (IL-15R ⁇ ) polypeptide or a functional fragment thereof; (c) a carrier comprising (i) a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain, or (ii) a single-chain carrier; (d) a linker joining the carrier to the IL-15R ⁇ polypeptide or functional fragment thereof; and (e) an additional linker, wherein the additional linker joining the IL-15 polypeptide or functional fragment thereof to (i) the IL-15R ⁇ polypeptide or functional fragment thereof, or (ii) the carrier.
  • IL-15 interleukin 15
  • IL-15R ⁇ IL-15 receptor alpha
  • the additional linker joins the carrier to the IL-15 polypeptide or functional fragment thereof.
  • the linker joins the C- terminus of the IL-15R ⁇ polypeptide or functional fragment thereof to the N-terminus of the carrier, and the additional linker joins the C-terminus of the multi-chain carrier to the N- terminus of the IL-15 polypeptide or functional fragment thereof.
  • the additional linker joins the C-terminus of the IL-15 polypeptide or functional fragment thereof to the N-terminus of the carrier, and the linker joins the C-terminus of the carrier to the N- terminus of the IL-15R ⁇ polypeptide or functional fragment thereof.
  • the additional linker joins the IL-15 polypeptide or functional fragment thereof to the IL- 15Ra polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the carrier to the N-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof. In some embodiments, the additional linker joins the C-terminus of the IL- 15Ra polypeptide or functional fragment thereof, to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
  • the multi-chain carrier is a fragment crystallizable (Fc) domain carrier comprising two Fc-domain polypeptide chains. In some embodiments, the single-chain carrier is a nanobody (VHH) carrier.
  • the present invention provides a single-chain polypeptide comprising: (a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof; (b) an interleukin 15 receptor alpha (IL-15R ⁇ ) polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)-domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker; (d) a linker joining the Fc-domain carrier to the receptor polypeptide or functional fragment thereof; and (e) an additional linker joining the IL-15 polypeptide or functional fragment thereof to (i) the IL-15R ⁇ polypeptide or functional fragment thereof, or (ii) the Fc-domain carrier.
  • IL-15 interleukin 15
  • IL-15R ⁇ interleukin 15 receptor alpha
  • Fc fragment crystallizable
  • the linker joins the C-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier, and the additional linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
  • the present invention provides a single-chain polypeptide comprising: (a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof; (b) an IL-15R ⁇ polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)- domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker; (d) a linker joining the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof; and (e) an additional linker joining the C- terminus of the IL-15R ⁇ polypeptide or functional fragment thereof to the N-terminus of Fc- domain carrier.
  • IL-15 interleukin 15
  • Fc fragment crystallizable
  • the present invention provides a single-chain polypeptide comprising: (a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof; (b) an IL-15R ⁇ polypeptide or a functional fragment thereof; (c) a nanobody (VHH) carrier; (d) a linker joining the VHH carrier to the IL-15R ⁇ polypeptide or functional fragment thereof; and (e) an additional linker joining the IL-15 polypeptide or functional fragment thereof to (i) the IL-15R ⁇ polypeptide or functional fragment thereof, or (ii) the VHH carrier.
  • IL-15 interleukin 15
  • VHH nanobody
  • the linker joins the C-terminus of the VHH carrier to the N-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof, to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
  • the linker joins the C-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the additional linker joins the C-terminus of the VHH carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
  • the additional linker joins the C-terminus of the IL-15 polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the linker joins the C- terminus of the VHH carrier to the N-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof.
  • the present invention provides a single-chain polypeptide, comprising: (a) a human interleukin 15 (IL-15) polypeptide or a functional fragment thereof comprising a mutation of the serine at position 73 to alanine; (b) an IL-15R ⁇ polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)-domain carrier comprising two Fc-domain polypeptide chains joined by an inter-chain linker; (d) a linker joining the C- terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof; and (e) an additional linker joining the C-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier.
  • IL-15 human interleukin 15
  • Fc fragment crystallizable
  • the present invention provides a single-chain polypeptide, comprising: (a) a human interleukin 15 (IL-15) polypeptide or a functional fragment thereof comprising a mutation of the serine at position 73 to alanine and a mutation of the histidine at position 105 to glutamate; (b) an IL-15R ⁇ polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)-domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker; (d) a linker joining the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof; and (e) an additional linker joining the C-terminus of the IL-15R ⁇ polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier.
  • IL-15 human interleukin 15
  • Fc fragment crystallizable
  • the single-chain polypeptide has a binding affinity dissociation constant (K D ) of 20 nM to 30 nM.
  • K D binding affinity dissociation constant
  • the single-chain polypeptide induces enhanced signal transduction as compared to wild-type IL-15 when contacted with a cell expressing the IL-15R.
  • the signal transduction is measured using a HEK Blue IL-2 assay.
  • the single-chain polypeptide induces enhanced STAT5 phosphorylation as compared to wild-type IL-15 when contacted with the cell expressing the IL-15R. STAT5 phosphorylation can be measured, for example, using flow cytometry or western blot analysis.
  • the cell expressing the IL-15R can be, for example, a CD8 + T-cell, a natural killer (NK) cell, a natural killer T-cell (NKT cell), a CD4 + T-cell, or a regulatory T-cell (T reg ).
  • the single-chain polypeptide induces proliferation and/or activation of one or more than one immune cell selected from the group consisting of CD8 + T-cells, a NK cells, NKT cells, CD4 + T-cells, and Treg cells, when administered to a subject.
  • the single-chain polypeptide induces proliferation of the one or more than one immune cell after about 4 days.
  • the polypeptide induces the expression of one or more than one protein in the one or more immune cells selected from the group consisting of CD25, Ki-67, NKG2D, and/or granzyme B (GrzB).
  • the single-chain polypeptide induces the expression of the one or more than one protein after about 4 days.
  • effector moiety polypeptide or functional fragment thereof can bind to the receptor polypeptide or functional fragment thereof to form a complex.
  • effector moiety polypeptide or functional fragment thereof comprises a single- subunit cytokine polypeptide or a functional fragment thereof.
  • the single-subunit cytokine polypeptide of functional fragment thereof comprises an interleukin 15 (IL-15) polypeptide or functional fragment thereof, which can optionally be human or include one or more than one mutation.
  • IL-15 interleukin 15
  • the one or more than one mutation may remove one or more than one glycosylation site; may not substantially affect binding kinetics or binding affinity to the IL-2/IL-15 receptor common b subunit (IL-15R ⁇ ); may include a mutation of the serine at position 73 ( e.g . to alanine); and may include mutations both of the serine at position 73 and of the histidine at position 105 (e.g. to alanine and glutamate, respectively).
  • the receptor polypeptide or functional fragment thereof comprises an IL-15 receptor polypeptide or a functional fragment thereof.
  • the IL-15 receptor polypeptide or functional fragment thereof is an IL-15 receptor alpha (IL-15R ⁇ ) polypeptide or a functional fragment thereof.
  • the IL-15R ⁇ polypeptide can be human and can include a sushi domain.
  • the single-chain polypeptide has higher binding affinity for IL-15R ⁇ compared to wild-type IL-15.
  • the Fc-domain carrier comprises human or murine Fc- domain polypeptide chains.
  • the Fc-domain polypeptide chains are IgGl, IgG2, IgG3, or IgG4 Fc-domain polypeptide chains.
  • the Fc- domain polypeptide chains comprise one or more than one mutation that reduces an effector function of the Fc-domain carrier.
  • the effector function comprises the ability of the Fc-domain carrier to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC).
  • the Fc-domain polypeptide chains are human IgGl Fc- domain polypeptide chains comprising one or more than one mutation that reduces an effector function of the Fc-domain carrier at position 234, 235, 237, 329, 330, and/or 331, according to the EU numbering system.
  • the one or more than one mutation that reduces an effector function of the Fc-domain carrier is selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, numbered according to the EU numbering system.
  • the two Fc-domain polypeptide chains each comprise mutations L234A, L235A, and P329A.
  • the two Fc-domain polypeptide chains each comprise mutations L234A, and L235A. In some embodiments, the two Fc-domain polypeptide chains each comprise mutations L234A, L235A, G237A, A330S, and P331S. In some embodiments, the two Fc-domain polypeptide chains each further comprise mutation C220S.
  • the VHH carrier is an anti-human serum albumin (HSA) nanobody.
  • HSA serum albumin
  • compositions that include a single-chain polypeptide described herein and a pharmaceutically acceptable vehicle or excipient.
  • the present invention also provides methods of treating a cancer, by administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition comprising a single-chain polypeptide or a formulation described herein.
  • FIG. 1 illustrates various elements that are present in various single-chain polypeptides of the present disclosure.
  • FIG. 1A illustrates the C-terminus of a receptor polypeptide connected to the N-terminus of an effector moiety polypeptide via a linker.
  • FIG. IB illustrates the C-terminus of an effector moiety polypeptide connected to the N-terminus of a receptor polypeptide via a linker.
  • FIG. 1C illustrates a multi-chain carrier comprising a first chain of a multi-chain carrier and a second chain of a multi-chain carrier, wherein the C- terminus of the first chain is connected to the N-terminus of the second chain via a linker.
  • FIG. ID illustrates a single-chain carrier.
  • FIG. 2A illustrates an exemplary single-chain polypeptide comprising a first chain of a multi-chain carrier connected by a linker to a second chain of a multi-chain carrier, the second chain connected by a linker to the N-terminus of a receptor polypeptide, and the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide.
  • FIG. 2B illustrates an exemplary single-chain polypeptide comprising the C- terminus of a receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide, the C-terminus of the effector moiety polypeptide connected by a linker to the N-terminus of a first chain of a multi-chain carrier, and the C-terminus of the first chain connected by a linker to the N-terminus of a second chain of the multi-chain carrier.
  • FIG. 2C illustrates an exemplary single-chain polypeptide comprising a first chain of a multi-chain carrier connected by a linker to a second chain of a multi-chain carrier, the second chain connected by a linker to the N-terminus of an effector moiety polypeptide, and the C-terminus of the effector moiety polypeptide connected by a linker to the N- terminus of a receptor polypeptide.
  • FIG. 2D illustrates an exemplary single-chain polypeptide comprising the C- terminus of an effector moiety polypeptide connected by a linker to the N-terminus of a receptor polypeptide, the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of a first chain of a multi-chain carrier, and the C-terminus of the first chain connected by a linker to the N-terminus of a second chain of the multi-chain carrier.
  • 3A illustrates an exemplary single-chain polypeptide comprising a single- chain carrier (shaded) connected by a linker to the N-terminus of a receptor polypeptide, and the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide.
  • FIG. 3B illustrates an exemplary single-chain polypeptide comprising the C- terminus of a receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide, the C-terminus of the effector moiety polypeptide connected by a linker to the N-terminus of a single-chain carrier (shaded).
  • FIG. 3C illustrates an exemplary single-chain polypeptide comprising a single- chain carrier (shaded) connected by a linker to the N-terminus of an effector moiety polypeptide, and the C-terminus of the effector moiety polypeptide connected by a linker to the N-terminus of a receptor polypeptide.
  • FIG. 3D illustrates an exemplary single-chain polypeptide comprising the C- terminus of an effector moiety polypeptide connected by a linker to the N-terminus of a receptor polypeptide, the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of a single-chain carrier (shaded).
  • FIG. 4A illustrates an exemplary single-chain polypeptide comprising a first chain of a multi-chain carrier connected by a linker to the N-terminus of a receptor polypeptide, the C-terminus of the receptor polypeptide connected by a linker to the N- terminus of an effector moiety polypeptide, and the C-terminus of the effector moiety polypeptide connected by a linker to a second chain of the multi-chain carrier.
  • FIG. 4B illustrates an exemplary single-chain polypeptide comprising the C- terminus of an effector moiety polypeptide connected by a linker to a first chain of a multi- chain carrier, the C-terminus of the first chain connected by a linker to the N-terminus of a second chain of the multi-chain carrier, and the C-terminus of the second chain connected by a linker to the N-terminus of a receptor polypeptide.
  • FIG. 4C illustrates an exemplary single-chain polypeptide comprising a first chain of a multi-chain carrier connected by a linker to the N-terminus of an effector moiety polypeptide, the C-terminus of the effector moiety polypeptide connected by a linker to the N-terminus of a receptor polypeptide, and the C-terminus of the receptor polypeptide connected by a linker to a second chain of the multi-chain carrier.
  • 4D illustrates an exemplary single-chain polypeptide comprising the C- terminus of a receptor polypeptide connected by a linker to a first chain of a multi-chain carrier, the first chain connected by a linker to a second chain of the multi-chain carrier, and the second chain connected by a linker to the N-terminus of an effector moiety polypeptide.
  • FIG. 5A illustrates an exemplary single-chain polypeptide comprising the C- terminus of an effector moiety polypeptide connected by a linker to the N-terminus of a single-chain carrier (shaded), and the C-terminus of the single-chain carrier connected by a linker to the N-terminus of a receptor polypeptide.
  • FIG. 5B illustrates an exemplary single-chain polypeptide comprising the C- terminus of a receptor polypeptide connected by a linker to the N-terminus of a single-chain carrier (shaded), and the C-terminus of the single-chain carrier connected by a linker to the N-terminus of an effector moiety polypeptide.
  • FIG. 6A shows an SDS-PAGE for Single-chain Polypeptide A (A) which is an human IgGl Fc - Linker - human IgGl Fc - Linker - human IL-15R ⁇ sushi domain - Linker - human IL-15 single-chain polypeptide comprising a first chain of a multi-chain carrier (Fc) connected by a linker to a second chain of a multi-chain carrier (Fc), the second chain connected by a linker to the N-terminus of a receptor polypeptide (human IL-15R ⁇ (huIL-15R ⁇ )), and the C-terminus of the receptor polypeptide connected by a linker to the N- terminus of an effector moiety polypeptide (human IL-15 (huIL-15)) and wherein the human IgGl Fc (huFc) domain chains comprise heterodimerization mutations and effector function silencing mutations, and Single-chain Polypeptide B (B) which is an hu
  • FIG. 6B shows an SDS-PAGE for Single-chain Polypeptide A(A) and Single- chain Polypeptide (B) as described in FIG. A under non-reducing conditions.
  • FIG. 7A shows an SDS-PAGE for the VHH-RLI single-chain polypeptide (Single-chain Polypeptide C) comprising a single-chain anti-human serum albumin (HSA) carrier (VHH) connected by a linker to the N-terminus of a receptor polypeptide (huIL-15R ⁇ sushi domain), and the C-terminus of the receptor polypeptide connected by a linker to the N- terminus of an effector moiety polypeptide (huIL-15).
  • HSA serum albumin
  • FIG. 7B is a graph showing IL-2 response using a HEK-Blue IL-12 reporter assay.
  • FIGS. 8A-8C are graphs showing IL-2 response using a HEK-Blue IL-12 reporter assay. In triplicate experiments, Experiment I (FIG. 8A), Experiment II (FIG. 8B), and Experiment III (FIG.
  • FIG. 9 is a graph showing IL-2 response using a HEK-Blue IL-12 reporter assay.
  • FIGS. 10A-10C are graphs showing STAT5 phosphorylation using a pSTAT5 flow cytometry assay.
  • Experiment I FIG. 10 A
  • Experiment II FIG. 10B
  • Experiment III FIGS. 10A-10C
  • FIG. 11 is a graph showing a dose-response of STAT5 phosphorylation in KHYG-1 cells treated with single-chain IL-15R ⁇ -IL-15 Fc polypeptides.
  • the levels of STAT5 phosphorylation of KHYG-1 cells treated with three single-chain Fc polypeptides huIL-15R ⁇ sushi domain- Linker - muFc - Linker - muFc - Linker -huIL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide F), huIL-15R ⁇ sushi domain- Linker - muFc - Linker - muFc - Linker -huIL-15 comprising a S73A mutation in IL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide G), and huIL-15R ⁇ sushi domain- Linker a
  • FIGS. 12A-12E are graphs showing in vitro immune cell proliferation induced by single-chain IL-15R ⁇ - IL- 15 Fc polypeptides measured by detecting the proliferation- specific marker (Ki67) on CD8 T cells (FIG. 12A), NK cells (FIG. 12B), NKT cells (FIG. 12C), CD4 T cells (FIG. 12D), and Treg cells (FIG. 12E) by flow cytometry.
  • Ki67 proliferation- specific marker
  • FIGS. 13A-13D are graphs showing flow cytometry analysis of percent CD25 positive CD8 T cells (FIG. 13 A), NK cells (FIG. 13B), CD4 cells (FIG. 13C) and NKT T cells (FIG. 13D) as an indicator of immune cell activation.
  • FIGS. 13E-13H are graphs showing flow cytometry analysis of NKG2D mean fluorescence intensity (MFI) of CD8 T cells (FIG. 13E), NK cells (FIG. 13F), NKT cells (FIG. 13G) and ⁇ T cells (FIG. 13H) as an indicator of immune cell activation.
  • MFI mean fluorescence intensity
  • FIGS. 13I-13L are graphs showing flow cytometry analysis of Granzyme B (GrzB) MFI of CD8 T cells (FIG. 131), NK cells (FIG. 13 J), NKT cells (FIG. 13K) and ⁇ T cells (FIG. 13L) using FACs based assays.
  • GrzB Granzyme B
  • FIGS. 13I-13L are graphs showing flow cytometry analysis of Granzyme B (GrzB) MFI of CD8 T cells (FIG. 131), NK cells (FIG. 13 J), NKT cells (FIG. 13K) and ⁇ T cells (FIG. 13L) using FACs based assays.
  • FIG. 14 are graphs showing expansion of live cells induced by single-chain IL15R ⁇ -IL15 Fc polypeptides. Cells treated with huIL-15R ⁇ sushi domain - Linker - muFc
  • Single-chain Polypeptide C Single-chain Polypeptide
  • Reference Control 3 huIL-15/huIL-15R ⁇ complex in which the huIL-15 sequence has a N72D mutation for promoting binding to its receptor, and the huIL-15R ⁇ sushi domain is fused to a muIgG2A Fc-domain
  • Reference Control 4 huIL-15/huIL- 15Ra complex in which the huIL-15 sequence has a N72D mutation for promoting binding to its receptor, and huIL-15R ⁇ sushi domain is fused to an muIgG2A Fc-domain, which includes LALAPA effector silencing mutations).
  • FIGS. 15A-15C are graphs showing the percentage of live NK cells (FIG. 15 A), NKT cells (FIG. 15B), and CD8 T cells (FIG. 15C) in mice following treatment with single- chain IL-15R ⁇ -IL-15 Fc polypeptides.
  • Single-chain Polypeptide F Single-chain polypeptide
  • Reference Control 3 Reference Control 4
  • FIGS. 16A-16C are graphs showing fold changes in the population of NK cells (FIG. 16 A), NKT cells (FIG. 16B), and CD8 T cells (FIG. 16C) in mice following treatment with single-chain IL-15R ⁇ -IL-15 Fc polypeptides.
  • FIGS. 17A-17C are graphs showing flow cytometry analysis of proliferation specific marker (Ki67; FIG. 17A), Granzyme B (GrzB; FIG.
  • NK cells obtained from mice treated with huIL-15R ⁇ sushi domain - Linker - muFc - Linker - muFc - Linker - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide F), anti-HSA VHH - Linker - huIL-15R ⁇ sushi domain - Linker - huIL-15 (Single-chain Polypeptide C), Reference Control 3, and Reference Control 4.
  • FIGS. 17D-17F are graphs showing flow cytometry analysis of proliferation specific marker (Ki67; FIG. 17A), Granzyme B (GrzB; FIG. 17B), andNKG2D (FIG. 17C) in CD8 T cells obtained from mice treated with huIL-15R ⁇ sushi domain - Linker - muFc - Linker - muFc - Linker - huIL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide F), anti-HSA VHH - Linker - huIL-15R ⁇ sushi domain - Linker - huIL-15 (Single-chain Polypeptide C), Reference Control 3, and Reference Control 4.
  • the invention provides improvements on immunoregulatory polypeptides, pharmaceutical compositions comprising such polypeptides, and therapeutic methods using such proteins and pharmaceutical compositions, including for the treatment of cancer.
  • a and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate.
  • “a” can refer to one linker or more than one linker. If referring to more than one linker, “a” may be in relation to linkers having the same amino acid sequence, or linkers having different amino acid sequences.
  • carrier refers to a polypeptide.
  • the polypeptide is a full-length or a functional fragment of a protein
  • multi-chain carrier refers to a carrier that comprises at least two polypeptide chains, wherein each polypeptide chain has an N-terminal end and a C- terminal end.
  • an Fc-domain (Fc-Fc) or a Fab is a multi-chain carrier of the present invention.
  • single-chain carrier refers to a carrier comprising no more than one polypeptide chain that has a single N-terminal end and a single C-terminal end.
  • a nanobody (VHH), an scFv, or HSA is a single-chain carrier of the present invention.
  • effector moiety polypeptide refers to a single polypeptide protein that exerts a physiological/biological effect when it binds to its cognate receptor.
  • receptor polypeptide refers to a single polypeptide that binds to an effector moiety polypeptide.
  • the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g ., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.
  • the term “effective amount” refers to the amount of a compound (e.g., a compound of the present invention) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • the term “treating” includes any effect, e.g, lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.
  • composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • the term “pharmaceutically acceptable vehicle or excipient” refers to any of the standard pharmaceutical vehicles or excipients, such as a phosphate buffered saline solution, water, emulsions (e.g, such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • vehicles, excipients, stabilizers and adjuvants see e.g,
  • the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g, acid or base) of a compound of the present invention which, upon administration to a subject, is capable of providing a compound of this invention or an active metabolite or residue thereof.
  • salts of the compounds of the present invention may be derived from inorganic or organic acids and bases.
  • Exemplary acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p- sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like.
  • Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.
  • Exemplary bases include, but are not limited to, alkali metal (e.g ., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NWf, wherein W is C 1-4 alkyl, and the like.
  • alkali metal e.g ., sodium
  • alkaline earth metal e.g., magnesium
  • W is C 1-4 alkyl
  • Exemplary salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemi sulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate
  • salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable.
  • salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
  • the present invention provides single-chain polypeptides having immunoregulatory function.
  • Single-chain polypeptides described herein comprise three or more polypeptides joined by two or more linkers resulting in a single, contiguous polypeptide chain.
  • single-chain polypeptides of the present invention comprise 3, 4, 5, 6, 7, or 8 polypeptides.
  • single-chain polypeptides of the present invention comprise 2, 3, 4, 5, 6, or 7 linkers.
  • single- chain polypeptides of the present invention comprise 4 polypeptides joined by 3 linkers.
  • single-chain polypeptides of the present invention comprise 3 polypeptides joined by 2 linkers.
  • one or more than one polypeptide can form a unit, wherein a unit corresponds to a protein, a protein subunit, a protein domain, or functional fragments thereof.
  • single-chain polypeptides of the present invention comprise 3 or more units.
  • single-chain polypeptides of the present invention comprise 3, 4 or 5 units.
  • single-chain polypeptides of the present invention comprise 3 units.
  • functional fragments refer to portions or truncations of a full-length polypeptide that retain the physiological or structural function of the full-length polypeptide.
  • Single-chain polypeptides of the present invention comprise three or more units comprising an effector moiety polypeptide or a functional fragment thereof, a receptor polypeptide or a functional fragment thereof, or a single-chain or multi-chain carrier, wherein each unit is independently joined by a linker to form the contiguous single-chain polypeptides.
  • Single-chain polypeptides disclosed herein have increased stability, reduced in vivo clearance, and an increased in vivo half-life when used as a therapeutic as compared to the effector moiety polypeptide alone or the effector moiety polypeptide in complex with the receptor polypeptide.
  • single-chain polypeptides of the present invention have more potent biological activity as compared to the effector moiety polypeptide alone or the effector moiety polypeptide in complex with the receptor polypeptide.
  • single chain polypeptides of the present invention bind to receptors or receptor subunits on the surface of cells with greater affinity than the effector moiety polypeptide alone or the effector moiety polypeptide in complex with the receptor polypeptide.
  • single chain polypeptides bind to receptors on the surface of immune cells and induce greater activation and/or proliferation of the immune cells as compared to the effector moiety polypeptide alone or the effector moiety polypeptide in complex with the receptor polypeptide.
  • the effector moiety polypeptide comprises a single-subunit cytokine polypeptide or functional fragment thereof, for example, but not limited to, interleukin 15 (IL-15).
  • IL-15 interleukin 15
  • the effector moiety polypeptide comprises an IL-15 polypeptide which complexes with the receptor polypeptide comprising an IL-15 receptor a (IL-15R ⁇ ) to form a complex capable of binding to and signaling through the IL-15R ⁇ /g common chains on the surface of immune cells.
  • IL-15R ⁇ IL-15 receptor a
  • IL-15 is a 14-15 kDa monomeric protein comprised of four a-helices. IL-15 is expressed as two alternatively spliced variant isoforms differing only in their signal peptides.
  • the long signal peptide isoform of IL-15 (GenBank Accession No. NP_000576.1, set forth in SEQ ID NO: 1) is 162 amino acids in length with residues 1 to 48 comprising the long signal peptide (set forth in SEQ ID NO:4) and residues 49 to 162 comprising the mature 114 amino acid IL-15 polypeptide (set forth as SEQ ID NO:2).
  • the short signal peptide isoform of IL- 15 (GenBank Accession No.
  • NP 751915.1, set forth in SEQ ID NO:3) is 135 amino acids in length with the first 21 N-terminal amino acids corresponding to the short signal peptide (set forth in SEQ ID NO: 5) and residues 22-135 corresponding to the mature 114 amino acid IL- 15 polypeptide (SEQ ID NO:2).
  • the IL-15 polypeptide is a mammalian IL-15 polypeptide. In certain embodiments, the IL-15 polypeptide is a human IL-15 polypeptide.
  • the effector moiety polypeptide comprises a polypeptide comprising an amino acid sequence at least 90% (e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:2.
  • the effector moiety polypeptide comprises an IL-15 polypeptide fragment having an amino acid sequence at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:2 over the length of the IL-15 polypeptide fragment.
  • amino acid sequences of wild-type IL-15 e.g, human IL-15
  • SEQ ID NO:6 and SEQ ID NO:7 are amino acid sequences of substituted IL-15.
  • the substituted amino acid sequences SEQ ID NO:6 and SEQ ID NO:7 are variant sequences of SEQ ID NO:2, with SEQ ID NO:6 and SEQ ID NO:7 having at least one substitution when compared to SEQ ID NO:2.
  • one or both of the native Asn77 and Gly78 of SEQ ID NO:2 is substituted, and either or both of the native Asn71 and Asn72 of SEQ ID NO:2 may be substituted or may be unsubstituted.
  • an amino acid sequence comprising SEQ ID NO:6, wherein Xaa71 is selected from the group consisting of Ser, Ala and Asn; Xaa72 is selected from the group consisting of Ser, Ala and Asn; Xaa77 is selected from the group consisting of Gin,
  • SEQ ID NO:6 generally corresponds to the native, unsubstituted IL-15 amino acid sequence SEQ ID NO:2 with the exception that in SEQ ID NO:6, at least Asn77 is substituted, and Gly78, Asn71, and Asn72 substituted or unsubstituted.
  • the present disclosure also provides an amino acid sequence comprising SEQ ID NO:7, wherein Xaa71 is selected from the group consisting of Ser, Ala and Asn; Xaa72 is selected from the group consisting of Ser, Ala and Asn; Xaa77 is selected from the group consisting of Gin, Ser, Lys, Ala, Glu, and Asn; and Xaa78 is selected from the group consisting of Ser and Ala.
  • SEQ ID NO:7 generally corresponds to the native, unsubstituted IL-15 amino acid sequence SEQ ID NO:2 with the exception that in SEQ ID NO:7, at least Gly78 is substituted, and Asn77, Asn71, and Asn72 may be substituted or unsubstituted.
  • the IL-15 amino acid sequence is at least 90% (e.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:2.
  • the IL-15 of the present invention has mutation(s) of N71S/N72A/N77A, N77A, G78A, or N77A/G78A, the amino acid positions corresponding to SEQ ID NO:2.
  • the IL-15 in the present invention comprises the amino acid sequence of SEQ ID NO:2.
  • the IL-15 in the present invention comprises the amino acid sequence of SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, or SEQ ID NO : 11.
  • the IL-15 amino acid sequence comprises a mutation of the serine at position 73 of SEQ ID NO:2.
  • the serine at position 73 of SEQ ID NO:2 is mutated to alanine.
  • the IL-15 effector moiety polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO:69.
  • the IL-15 amino acid sequence comprises a mutation of the histidine at position 105 of SEQ ID NO:2.
  • the histidine at position 105 of SEQ ID NO:2 is mutated to glutamate.
  • the IL-15 amino acid sequence comprises mutations at both the serine at position 73 and the histidine at position 105.
  • the IL-15 effector moiety polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO:70.
  • the IL-15 amino acid sequence comprises one or mutation(s) that removes a glycosylation site.
  • mutation of one or more than one residue does not impact IL-15 function but removes heterogeneity for Chemistry, Manufacturing and Controls (CMC).
  • one or more than one mutation in the IL-15 amino acid sequence does/do not inhibit IL-15 binding affinity for the IL-15 receptor (IL-15R).
  • IL-15 comprising one or more mutation does not have impaired binding to the human IL-2R/IL- 15 ⁇ receptor.
  • one or more than one mutation in the IL-15 amino acid sequence enhances IL- 15 binding affinity for the IL-15 receptor (IL-15R).
  • IL- 15 comprising one or more than one mutation enhances binding to the human IL-2R/IL- 15 ⁇ receptor.
  • one or more than one mutation in the IL-15 amino acid sequence enhances IL-15 activation of immune cells.
  • IL- 15 comprising one or more than one mutation enhances STAT5 phosphorylation when contacted with immune cells, as compared to wild-type IL-15.
  • single-chain polypeptides of the present invention comprise receptor polypeptides.
  • receptor polypeptides comprise the cognate receptor of the single- subunit cytokine polypeptide.
  • receptor polypeptides comprise an IL-15 receptor or functional fragment thereof, or an IL-2 receptor or functional fragment thereof.
  • single-chain polypeptides disclosed herein comprise a receptor polypeptide comprising an IL-15 receptor polypeptide or functional fragment thereof.
  • the IL-15 receptor polypeptide is a mammalian IL-15 receptor polypeptide.
  • the IL-15 receptor polypeptide is a human IL- 15 polypeptide.
  • receptor polypeptides of the present invention comprise one or more than one subunit of a cytokine receptor capable of binding to its cognate cytokine.
  • single-chain polypeptides of the present invention comprise receptor polypeptides comprising an IL-15R ⁇ polypeptide or functional fragment thereof.
  • IL-15R ⁇ is a 28.2 kDa protein reported to have nine isoforms produced by alternative splicing. The canonical IL-15R ⁇ isoform (isoform 1, GenBank Accession No.
  • NP_002180.1 set forth in SEQ ID NO:12
  • SEQ ID NO: 17 residues 1 to 30 corresponding to the signal peptide
  • residues 31 to 95 corresponding with a sushi domain critical for IL-15 binding
  • residues 206 to 228 corresponding to a transmembrane region
  • residues 229 to 267 corresponding to a C-terminal tail.
  • IL-15R ⁇ may be expressed as a soluble protein (having an amino acid sequence as set forth in SEQ ID NO: 14), wherein the mature IL-15R ⁇ protein (as set forth in SEQ ID NO: 13) is truncated by removal of the transmembrane region and C- terminal tail.
  • IL-15R ⁇ may also be expressed as a soluble protein (having an amino acid sequence as set forth in SEQ ID NO: 15), wherein the 67 C-terminal amino acids of the mature IL-15R ⁇ protein (SEQ ID NO: 13) are removed, corresponding to the C-terminal region after residue G170 that is proteolytically cleaved at the surface of cells to liberate soluble IL-15R ⁇ in vivo.
  • an IL-15R ⁇ polypeptide comprises a polypeptide comprising an amino acid sequence at least 90% (e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 13.
  • the receptor polypeptide comprises an IL-15R ⁇ polypeptide fragment having an amino acid sequence at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 13 over the length of the IL-15R ⁇ polypeptide fragment.
  • the receptor polypeptide comprises an IL-15R ⁇ polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 13. In other embodiments, the receptor polypeptide comprises an IL-15R ⁇ polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 13.
  • the receptor polypeptide comprises an IL-15R ⁇ polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 14, wherein the putative IL-15R ⁇ transmembrane region and C-terminal tail region have been removed. In certain embodiments, the receptor polypeptide comprises an IL-15R ⁇ polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 14. [0118] Further contemplated are single-chain polypeptides, wherein the receptor polypeptide comprises an IL-15R ⁇ polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 15, wherein the C-terminal region after residue G170 is removed. In other embodiments, the receptor polypeptide comprises an IL-15R ⁇ polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 15.
  • the receptor polypeptide comprises an IL-15R ⁇ sushi domain having an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 16.
  • the receptor polypeptide comprises an IL-15R ⁇ sushi domain having an amino acid sequence at least 95% identical to SEQ ID NO:16.
  • the receptor polypeptide comprises a functional fragment of IL-15R ⁇ , wherein the functional fragment comprises the Sushi domain of IL-15R ⁇ .
  • the functional fragment of the receptor polypeptide further comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acid residues immediately C-terminal to the Sushi domain sequence of IL-15R ⁇ .
  • the IL-15R ⁇ or functional fragment thereof in the present invention comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
  • the receptor polypeptide comprises an IL-15R ⁇ functional fragment having an amino acid sequence at least 90% (e.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:21.
  • the receptor polypeptide comprises an IL-15R ⁇ functional fragment having an amino acid sequence at least 95% identical to SEQ ID NO:21.
  • single-chain polypeptides of the present invention comprise effector moiety polypeptides or functional fragments thereof and receptor polypeptides or functional fragments thereof that bind to form complexes.
  • Complexes of the single-chain polypeptides disclosed herein are capable of stimulating a response when exposed to immune cells.
  • a complex comprises an effector moiety polypeptide joined to a receptor polypeptide via a linker.
  • a linker can join the C-terminal residue of the receptor polypeptide comprising the IL-15R ⁇ polypeptide or functional fragment thereof or IL-15R ⁇ sushi domain polypeptide or functional fragment thereof, to the N-terminal residue of the effector moiety polypeptide comprising the IL-15 polypeptide or functional fragment thereof.
  • a linker can join the N-terminal residue of the receptor polypeptide comprising the IL-15R ⁇ polypeptide or functional fragment thereof or IL-15R ⁇ sushi domain polypeptide or functional fragment thereof, to the C-terminal residue of the effector moiety comprising the IL-15 polypeptide or functional fragment thereof.
  • an effector moiety polypeptide interacts with a receptor polypeptide via non-covalent interactions or additionally via one or more than one engineered disulfide bond.
  • a single-chain polypeptide according to the invention includes an artificial or engineered disulfide bridge between the effector moiety polypeptide and the receptor polypeptide.
  • a single-chain polypeptide according to the invention includes an artificial or engineered disulfide bridge between an IL-15 polypeptide and an IL-15R ⁇ polypeptide.
  • Such a single-chain polypeptide includes an effector moiety polypeptide comprising a substitution selected from L52C and E53C in the IL-15 polypeptide and a receptor polypeptide comprising a substitution selected from S40C, A37C, and G38C in the IL-15R ⁇ polypeptide, wherein the substituted cysteine residues form a disulfide bond.
  • a single-chain polypeptide of the present invention includes an effector moiety polypeptide comprising a L52C substitution in the IL-15 polypeptide and a receptor polypeptide comprising a S40C substitution in the IL-15R ⁇ polypeptide, wherein the substituted cysteine residues form a disulfide bond.
  • the effector moiety polypeptide comprising the IL-15 polypeptide includes one or more substitution(s) selected from L45D, L45E, Q48K, S51D, L52D, E64K, I68D, and L69R.
  • substitutions described in US Patent No. 9,493,533 and incorporated herein by reference, have been demonstrated to increase the binding affinity of IL-15 for IL-15R ⁇ .
  • the effector moiety polypeptide comprising the IL-15 polypeptide comprises an N72D substitution.
  • mutation of the asparagine residue at position 72 of IL-15 to aspartic acid increases IL-15 binding to the IL- 1511bg receptor subunits as compared to native IL-15 polypeptide.
  • the effector moiety polypeptide comprises an IL-15 polypeptide having one or more amino acid substitution(s) selected from L45D, L45E, Q48K, S51D, L52C, L52D, E53C, E64K, I68D, L69R, and N72D.
  • the receptor polypeptide comprises an IL-15R ⁇ polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or comprises an IL-15R ⁇ sushi domain having an amino acid sequence at least 90% identical to SEQ ID NO: 16, wherein the IL-15R ⁇ polypeptide or IL-15R ⁇ sushi domain further comprises one or more amino acid substitution(s) selected from S40C, A37C, and G38C.
  • any of the IL-15 polypeptides disclosed herein can be paired with any of the IL-15R ⁇ polypeptides disclosed herein (for example, but not limited to, the sequences presented in 2) to form a complex.
  • the effector moiety polypeptide comprises a polypeptide comprising an amino acid sequence at least 90% (e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:2, and the receptor polypeptide comprises an IL-15R ⁇ functional fragment having an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:21.
  • the effector moiety polypeptide comprises an IL-15 polypeptide fragment having an amino acid sequence at least 90% (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:2 over the length of the IL-15 polypeptide fragment
  • the receptor polypeptide comprises an IL-15R ⁇ functional fragment having an amino acid sequence at least 90% (e.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:21.
  • any of the linkers disclosed in Table 8 can be used to join any of the IL-15 polypeptides disclosed herein to any of the IL-15R ⁇ polypeptides disclosed herein.
  • the present disclosure provides, but is not limited to, complexes having sequences as presented in Table 3.
  • a “carrier” refers to a unit that is joined to an effector moiety polypeptide and/or a receptor polypeptide by one or more than one linker, thereby promoting increased stability, reduced in vivo clearance, and an increased in vivo half-life of an effector moiety polypeptide as compared to an effector moiety polypeptide alone, or an effector moiety polypeptide in complex with a receptor polypeptide.
  • a “multi-chain” carrier refers to two or more polypeptide chains that interact to form a unit. In some embodiments, two polypeptide chains comprising the multi-chain carrier are joined by a linker to form a unit.
  • polypeptide chains comprising the multi-chain carrier are not directly joined by linkers and interact via non-covalent interactions to form a unit.
  • multi-chain carriers that are not directly joined by linkers further comprise engineered mutations that introduce one or more than one disulfide bond between two polypeptide chains.
  • a “single-chain” carrier refers to a single polypeptide chain that forms a unit.
  • a carrier can be a multi-chain carrier.
  • the multi-chain carrier can comprise a unit comprising a fragment crystallizable (Fc) domain.
  • a multi-chain carrier comprises a first chain of a multi-chain carrier and a second chain of a multi-chain carrier.
  • the first chain of a multi-chain carrier and the second chain of a multi-chain carrier are directly joined to each other by a linker to form a unit.
  • the first chain of a multi-chain carrier and the second chain of a multi-chain carrier are not directly joined together by a linker and dimerize via non-covalent interactions to form a unit.
  • the first and second chains of a multi-chain carrier comprise IgGl, IgG2, IgG3, or IgG4 Fc domain polypeptide chains.
  • the first and second chains of a multi-chain carrier comprise mammalian Fc domain polypeptide chains.
  • the first and second Fc domain polypeptide chains are human Fc domain polypeptide chains.
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g ., IL-15) or a functional fragment thereof;
  • a receptor polypeptide e.g., IL-15R ⁇
  • a functional fragment thereof e.g, sushi domain
  • a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain
  • a linker joining the contiguous polypeptide chain to the receptor polypeptide or functional fragment thereof and (e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the contiguous polypeptide chain.
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof;
  • a receptor polypeptide e.g, IL-15R ⁇
  • a functional fragment thereof e.g, sushi domain
  • a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain;
  • an additional linker that joins the C-terminus of the contiguous polypeptide chain to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g ., IL-15) or a functional fragment thereof;
  • a receptor polypeptide e.g., IL-15R ⁇
  • a functional fragment thereof e.g, sushi domain
  • a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain
  • a linker joins the C-terminus of the contiguous polypeptide chain to the N-terminus of the receptor polypeptide or functional fragment thereof
  • an additional linker joins the C-terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the contiguous polypeptide chain.
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof;
  • a receptor polypeptide e.g, IL-15R ⁇
  • a functional fragment thereof e.g, sushi domain
  • a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain
  • a linker that joins the contiguous polypeptide chain to the receptor polypeptide or functional fragment thereof and (e) an additional linker that joins the effector moiety polypeptide or functional fragment thereof to the receptor polypeptide or functional fragment thereof.
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof;
  • a receptor polypeptide e.g, IL-15R ⁇
  • a functional fragment thereof e.g, sushi domain
  • a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain
  • a linker that joins the C- terminus of the contiguous polypeptide chain to the N-terminus of the receptor polypeptide or functional fragment thereof and (e) an additional linker that joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • a single-chain polypeptide of the present invention comprises a fragment crystallizable (Fc) domain (two Fc-domain polypeptide chains) as a multi-chain carrier.
  • a single-chain polypeptide of the present invention comprises an IgGl, IgG2, IgG3, or IgG4 fragment crystallizable (Fc) domain (two Fc-domain polypeptide chains) as a multi-chain carrier.
  • a single-chain polypeptide of the present invention comprises a multi-chain carrier, wherein the multi-chain carrier comprises a Fc domain, and wherein a first Fc domain polypeptide chain and a second Fc domain polypeptide chain are both IgGl Fc domain polypeptide chains.
  • a multi-chain carrier comprises a first IgGl heavy chain polypeptide fragment and a second IgGl heavy chain polypeptide fragment, each comprising an Fc domain polypeptide chain, are directly joined to each other by a linker to form a unit.
  • first IgGl heavy chain polypeptide fragment and the second IgGl heavy chain polypeptide fragment, each comprising an Fc domain polypeptide chain are not directly joined together by a linker and dimerize via non-covalent interactions to form a unit.
  • IL-15R ⁇ -human IgGl Fc-human IgGl Fc-IL-15 (FIG. 2A); an IL15R ⁇ -linker- Fc-linker-Fc-linker-IL15 single-chain polypeptide comprising the C-terminus of a receptor polypeptide (IL-15) connected by a linker to a first chain of a multi-chain carrier (Fc), the C- terminus of the first chain connected by a linker to the N-terminus of a second chain of the multi-chain carrier (Fc), and the C-terminus of the second chain connected by a linker to the N-terminus of an effector moiety polypeptide (IL-15); an Fc-linker-IL15R ⁇ -linker-IL15- linker-Fc single-chain polypeptide comprising a first chain of a multi-chain carrier (Fc) connected by a linker to the N-terminus of a receptor polypeptide (IL-15R ⁇ ), the C-terminus of the receptor
  • an IL15-linker-Fc- linker-Fc-linker-IL15R ⁇ single-chain polypeptide comprising the N-terminus of an effector moiety polypeptide (IL-15) connected by a linker to a first chain of a multi-chain carrier (Fc), the N-terminus of the first chain connected by a linker to the C-terminus of a second chain of the multi-chain carrier (Fc), and the N-terminus of the second chain connected by a linker to the C-terminus of a receptor polypeptide (IL-15a).
  • the first heavy chain polypeptide fragment and second heavy chain polypeptide fragment of the multi-chain carrier both comprise an amino acid sequence at least 90% (e.g at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a wild-type human IgGl sequence (SEQ ID NO:24).
  • the first heavy chain polypeptide fragment and second heavy chain polypeptide fragment of the multi-chain carrier both comprise an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:25).
  • a single-chain polypeptide of the present disclosure comprises a first IgGl heavy chain polypeptide fragment and a second IgGl heavy chain polypeptide fragment, wherein each heavy chain polypeptide fragment comprises an IgGl Fc domain polypeptide chain, and both Fc domain polypeptide chains have one or more than one mutation to reduce binding to an Fc ⁇ R (e.g., Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIB, Fc ⁇ RIIIIA, or Fc ⁇ RIIIB) or complement component (e.g, Clq).
  • Fc ⁇ R e.g., Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIB, Fc ⁇ RIIIIA, or Fc ⁇ RIIIB
  • complement component e.g, Clq
  • a single-chain polypeptide of the present disclosure may include LALA (L234A and L235A) mutations, LALAPA (L234A, L235A, and P329A) mutations, LALAPG (L234A, L235A, and P329G) mutations, or LALEGAASPS (L234A, L235E, G237A, A330S, and P331S) mutations.
  • LALA L234A and L235A
  • LALAPA L234A, L235A, and P329A
  • LALAPG L234A, L235A, and P329G mutations
  • LALEGAASPS L234A, L235E, G237A, A330S, and P331S
  • a multi-chain carrier comprises a first IgGl heavy chain polypeptide fragment and a second IgGl heavy chain polypeptide fragment, each comprising an Fc domain polypeptide chain, , wherein the first and second IgGl heavy chain polypeptide fragment are joined by a linker, and wherein the multi-chain carrier comprises a sequence according to SEQ ID NO:26.
  • a multi-chain carrier is a unit comprising an anti-HSA Fab heavy chain polypeptide and an anti-HSA light chain polypeptide.
  • the carrier is a unit comprising an anti-HSA Fab, wherein a fragment of an immunoglobulin heavy chain and an immunoglobulin light chain are joined by a linker to form a unit.
  • the fragment of an immunoglobulin heavy chain and immunoglobulin light chain are not directly joined by a linker and interact via non-covalent interactions to form a unit.
  • an anti-HSA Fab comprises a fragment of an immunoglobulin heavy chain and an immunoglobulin light chain that are not directly joined by a linker, and further comprise engineered mutations that introduce one or more than one disulfide bond between the heavy chain and light chain.
  • a multi-chain carrier comprises an anti-HSA Fab comprising a light chain having an amino acid sequence at least 80% (e.g ., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:28.
  • a multi-chain carrier comprises an anti-HSA Fab comprising a heavy chain having an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • any of the linkers provided in Table 8 below can be used to directly join the C- terminus of a light chain having a sequence according to SEQ ID NO:28 to the N-terminus of a heavy chain having a sequence according to SEQ ID NO:40.
  • any of the linkers provided in Table 8 below can be used to directly join the C-terminus of a heavy chain having a sequence according to SEQ ID NO:40 to the N-terminus of a light chain having a sequence according to SEQ ID NO:28.
  • the present disclosure provides, but is not limited to, single chain anti-HSA Fab fragments having amino acid sequences at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • a multi-chain carrier of the present invention comprises an anti-HSA single-chain variable fragment (scFv), wherein the heavy chain variable domain and light chain variable domain of the scFv are joined by a linker to form a unit.
  • scFv anti-HSA single-chain variable fragment
  • a multi-chain carrier comprises an anti-HSA scFv comprising a light chain variable domain having an amino acid sequence at least 80% (e.g. , at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:29.
  • a multi-chain carrier comprises an anti-HSA scFv comprising a heavy chain variable domain having an amino acid sequence at least 80% (e.g, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
  • any of the linkers provided in Table 8 below can be used to directly join the C- terminus of a light chain variable domain having a sequence according to SEQ ID NO:29 to the N-terminus of a heavy chain variable domain having a sequence according to SEQ ID NO:43.
  • any of the linkers provided in Table 8 below can be used to directly join the C-terminus of a heavy chain variable domain having a sequence according to SEQ ID NO:43 to the N-terminus of a light chain variable domain having a sequence according to SEQ ID NO:29.
  • the present disclosure provides, but is not limited to, anti-HSA scFv having amino acid sequences at least 80% (e.g, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:44 or SEQ ID NO:45.
  • scFv Carrier Sequences e.g, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
  • a single-chain polypeptide of the present invention comprises a single-chain carrier, wherein the single-chain carrier comprises a human serum albumin (HSA) polypeptide or functional fragment thereof, or an anti-HSA nanobody (VHH).
  • HSA human serum albumin
  • VHH anti-HSA nanobody
  • the single-chain carrier is directly joined via a linker to an effector moiety polypeptide and/or a receptor polypeptide.
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g. , IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g, IL-15R ⁇ ) or a functional fragment thereof (e.g, sushi domain); (c) a single-chain carrier; (d) a linker joining the single-chain carrier to the receptor polypeptide or functional fragment thereof; and (e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the single-chain carrier.
  • an effector moiety polypeptide e.g. , IL-15
  • a receptor polypeptide e.g, IL-15R ⁇
  • a functional fragment thereof e.g, sushi domain
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g ., IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g., IL-15R ⁇ ) or a functional fragment thereof (e.g, sushi domain); (c) a single-chain carrier; (d) a linker that joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the single-chain carrier; and (e) an additional linker that joins the C-terminus of the single-chain carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • an effector moiety polypeptide e.g IL-15
  • a receptor polypeptide e.g., IL-15R ⁇
  • a functional fragment thereof e.g, sushi domain
  • a single-chain carrier e.g., a linker that joins the C-terminus of the receptor polypeptide or functional fragment thereof to
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g, IL-15R ⁇ ) or a functional fragment thereof (e.g, sushi domain); (d) a linker joins the C-terminus of the single-chain carrier to the N-terminus of the receptor polypeptide or functional fragment thereof; and (e) an additional linker joins the C- terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the single-chain carrier
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g, IL-15R ⁇ ) or a functional fragment thereof (e.g, sushi domain); (d) a linker that joins the single-chain carrier to the receptor polypeptide or functional fragment thereof; and (e) an additional linker that joins the effector moiety polypeptide or functional fragment thereof to the receptor polypeptide or functional fragment thereof.
  • an effector moiety polypeptide e.g, IL-15
  • a receptor polypeptide e.g, IL-15R ⁇
  • a functional fragment thereof e.g, sushi domain
  • a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g, IL-15R ⁇ ) or a functional fragment thereof (e.g, sushi domain); (c) a single-chain carrier; (d) a linker that joins the C-terminus of the single-chain carrier to the N-terminus of the receptor polypeptide or functional fragment thereof; and (e) an additional linker that joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
  • an effector moiety polypeptide e.g, IL-15
  • a receptor polypeptide e.g, IL-15R ⁇
  • a functional fragment thereof e.g, sushi domain
  • a single-chain carrier e.g, a linker that joins the C-terminus of the single-chain carrier to the N-
  • a single-chain polypeptide of the present invention comprises a nanobody (VHH) as a single-chain carrier.
  • a single-chain polypeptide of the present invention comprises an anti-human serum albumin (HSA) nanobody (VHH).
  • HSA anti-human serum albumin
  • the carrier is a unit comprising an anti-HSA nanobody (VHH).
  • single-chain polypeptides include an VHH-linker-IL15R ⁇ -linker-IL15 single-chain polypeptide comprising single-chain carrier (VHH) connected by a linker to the N-terminus of a receptor polypeptide (IL-15R ⁇ ), and the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide (IL- 15) (FIG.
  • an IL15-linker- VHH-linker- IL15R ⁇ single-chain polypeptide comprising the C-terminus of an effector moiety polypeptide (IL-15) connected by a linker to the N-terminus of a single-chain carrier (VHH), the C-terminus of the single-chain carrier connected by a linker to the N-terminus of a receptor polypeptide (IL-15R ⁇ )
  • IL-15 effector moiety polypeptide
  • VHH single-chain carrier
  • IL-15R ⁇ receptor polypeptide
  • an IL15R ⁇ -linker- VHH-linker-IL15 single-chain polypeptide comprising the C-terminus of a receptor polypeptide (IL-15R ⁇ ), connected by a linker to the N-terminus of a single-chain carrier (VHH), the C-terminus of the single-chain carrier connected by a linker to the N-terminus of an effector moiety polypeptide (IL-15) (FIG. 5B).
  • a single-chain carrier comprises an HSA polypeptide comprising an amino acid sequence at least 80% (e.g ., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:27.
  • a single-chain carrier comprises an anti-HSA nanobody comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:30.
  • linker comprises, in accordance with the present invention, a polypeptide joining two units and/or two polypeptide chains. Exemplary linker sequences are provided in Table 8 below. [0179] In some embodiments presently described, linkers comprise amino acid sequences that confer flexibility and do not interfere with the binding of the effector moiety polypeptide and receptor polypeptide to each other; and resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance.
  • Linkers suitable for joining units of the single-chain polypeptides presently described include a (GS) n (SEQ ID NO: 60), (GGS) n (SEQ ID NO: 61), (GGGS) n (SEQ ID NO: 62), (GGSG) n (SEQ ID NO: 63),
  • a linker comprises (GGGGS) 4 (SEQ ID NO: 31), (GGGGS) 5 (SEQ ID NO:32), (GGGGS)6 (SEQ ID NO:33), or (SGGGG) 6 (SEQ ID NO:34). [0180] Any of the linkers provided in Table 8 below can be used to directly join any effector moiety polypeptide described in Table 1 to any receptor polypeptide described in Table 2.
  • linkers provided in Table 8 below can be used to directly join any multi- chain carrier (e.g . as described in Tables 4, 5, and 6) or single-chain carrier ( e.g ., as described in Table 7) to an effector moiety polypeptide (e.g ., as described in Table 1) and/or a receptor polypeptide (e.g., as described in Table 2).
  • single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a single-chain or multi-chain carrier, a linker, an effector moiety polypeptide or functional fragment thereof, a linker, and a receptor polypeptide, or functional fragment thereof.
  • a linker joins two chains of the carrier.
  • single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a single-chain or multi-chain carrier, a linker, a receptor polypeptide or functional fragment thereof, a linker, and an effector moiety polypeptide or functional fragment thereof.
  • a linker joins two chains of the carrier.
  • single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a receptor polypeptide or functional fragment thereof, a linker, an effector moiety polypeptide or functional fragment thereof, a linker, and a single-chain or multi-chain carrier.
  • a linker joins two chains of the carrier.
  • single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, an effector moiety polypeptide or functional fragment thereof, a linker, a receptor polypeptide or functional fragment thereof, a linker, and a single-chain or multi-chain carrier.
  • a linker joins two chains of the carrier.
  • single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a first chain of a multi-chain carrier, a linker, a receptor polypeptide or functional fragment thereof, a linker, an effector moiety polypeptide or functional fragment thereof, a linker, and a second chain of the multi-chain carrier.
  • single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a first chain of a multi-chain carrier, a linker, an effector moiety polypeptide or functional fragment thereof, a linker, a receptor polypeptide or functional fragment thereof, a linker, and a second chain of the multi-chain carrier.
  • single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, an effector moiety polypeptide or functional fragment thereof, a linker, a single-chain or multi-chain carrier, a linker, and a receptor polypeptide or functional fragment thereof.
  • a linker joins two chains of the carrier.
  • single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a receptor polypeptide or functional fragment thereof, a linker, a single-chain or multi-chain carrier, a linker, and an effector moiety polypeptide or functional fragment thereof.
  • a linker joins two chains of the carrier.
  • the effector moiety polypeptide comprises IL-15 or a functional fragment thereof.
  • the effector moiety polypeptide comprises a polypeptide at least 90% identical to SEQ ID NO:2.
  • the receptor polypeptide comprises an IL- 15Ra polypeptide or functional fragment thereof, or an IL-15R ⁇ sushi domain.
  • the receptor polypeptide comprises a polypeptide at least 90% identical to SEQ ID NO: 13, 16, or 21.
  • the multi-chain carrier comprises a unit comprising an IgGl, IgG2, IgG3, or IgG4 Fc domain.
  • the multi-chain carrier comprises a first IgGl Fc domain polypeptide and second IgGl Fc domain polypeptide having a sequence at least 90% identical to SEQ ID NO: 24 or 25.
  • the single-chain carrier comprises an HSA polypeptide or a functional fragment thereof, or an anti-HSA nanobody (VHH).
  • the single-chain carrier comprises a polypeptide having a sequence at least 90% identical to SEQ ID NO:27.
  • a linker comprises a polypeptide having a sequence according to SEQ ID NO: 31, 32, 33 or 34.
  • the present disclosure provides, but is not limited to, single-chain polypeptides as provided in Table 9 (amino acid sequences of an effector moiety (IL-15) and a receptor (IL-15R ⁇ ) are in bold; bolded and underlined amino acid residues are mutations (compared to the human wild-type IgGl sequence) that reduce effector function of the IgGl Fc domain; italicized amino acid residues represent either a section of an IgGl hinge or a linker (G 4 S) n (SEQ ID NO: 67); bolded and italicized (but not underlined) amino acid residues represent a fragment of the IL-15R ⁇ ).
  • single-chain polypeptides of the present disclosure have the sequence according to SEQ ID NO: 37, SEQ ID NO:71, or SEQ ID NO:72.
  • the proteins of the present invention can be made using recombinant DNA technology well known to a skilled person in the art.
  • a first nucleic acid sequence encoding a first polypeptide comprising a first immunoregulatory polypeptide linked to a first antibody Fc domain polypeptide can be cloned into a first expression vector;
  • a second nucleic acid sequence encoding a second polypeptide comprising a second, different immunoregulatory polypeptide linked to a second, different antibody Fc domain polypeptide can be cloned into a second expression vector; and the first and the second expression vectors can be stably transfected together into host cells to produce the single-chain polypeptide.
  • Clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the proteins of the present invention.
  • the proteins can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.
  • a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15R ⁇ or a functional fragment thereof (e.g ., sushi domain) has higher binding affinity for IL-15R ⁇ compared to wild-type IL-15.
  • a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15R ⁇ or a functional fragment thereof (e.g., sushi domain) has a binding affinity dissociation constant (K D ) is 20 nM to 30 nM.
  • a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15R ⁇ or a functional fragment thereof (e.g, sushi domain) induces enhanced signal transduction as compared to wild-type IL-15 when contacted with a cell expressing the IL-15R.
  • a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15R ⁇ or a functional fragment thereof (e.g, sushi domain) induces enhanced STAT5 phosphorylation as compared to wild- type IL-15 when contacted with the cell expressing the IL-15R.
  • a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15R ⁇ or a functional fragment thereof (e.g, sushi domain) induces enhanced STAT5 phosphorylation as compared to wild- type IL-15 when contacted with the cell expressing the IL-15R, wherein the cell is a CD8 + T- cell, a natural killer (NK) cell, a natural killer T-cell (NKT cell), a CD4 + T-cell, or a regulatory T-cell (Treg).
  • NK natural killer
  • NKT cell natural killer T-cell
  • Treg regulatory T-cell
  • a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15R ⁇ or a functional fragment thereof (e.g, sushi domain) induces proliferation and/or activation of one or more than one immune cell selected from the group consisting of CD8 + T-cells, a NK cells, NKT cells, CD4 + T-cells, and T regs , upon administered to a subject.
  • a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15R ⁇ or a functional fragment thereof (e.g ., sushi domain) induces proliferation of the one or more than one immune cell after about 4 days (e.g., 4 days, 5 days, 6 days, or 7 days).
  • a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15R ⁇ or a functional fragment thereof (e.g, sushi domain) induces the expression of one or more than one protein in the one or more immune cells selected from the group consisting of CD25, Ki-67, NKG2D, and/or granzyme B (GrzB) after about 4 days (e.g, 4 days, 5 days, 6 days, or 7 days).
  • compositions that contain a therapeutically effective amount of a single-chain polypeptide described herein.
  • the composition can be formulated for use in a variety of drug delivery systems.
  • One or more physiologically acceptable excipients or vehicles can also be included in the composition for proper formulation.
  • Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g, Langer (Science 249:1527-1533, 1990).
  • the intravenous drug delivery formulation of the present disclosure may be contained in a bag, a pen, or a syringe.
  • the bag may be connected to a channel comprising a tube and/or a needle.
  • the formulation may be a lyophilized formulation or a liquid formulation.
  • the formulation may be freeze-dried (lyophilized) and contained in about 12-60 vials.
  • the formulation may be freeze-dried and 45 mg of the freeze-dried formulation may be contained in one vial.
  • the about 40 mg - about 100 mg of freeze-dried formulation may be contained in one vial.
  • the freeze- dried formulation from 12, 27, or 45 vials are combined to obtain a therapeutic dose of the single-chain polypeptide in the intravenous drug formulation.
  • the formulation may be a liquid formulation and stored as about 250 mg/vial to about 1000 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 600 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 250 mg/vial. [0209]
  • a single-chain polypeptide of the present invention could exist in a liquid aqueous pharmaceutical formulation including a therapeutically effective amount of the protein in a buffered solution forming a formulation.
  • compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicles or excipients prior to administration.
  • the pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5.
  • the resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents.
  • composition in solid form can also be packaged in a container for a flexible quantity.
  • the present disclosure provides a formulation with an extended shelf life including the single-chain polypeptide of the present disclosure, in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.
  • an aqueous formulation is prepared including the single- chain polypeptide of the present disclosure in a pH-buffered solution.
  • the buffer of this invention may have a pH ranging from about 4 to about 8, e.g ., from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. Ranges intermediate to the above recited pH's are also intended to be part of this disclosure. For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included. Examples of buffers that will control the pH within this range include acetate (e.g, sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.
  • the formulation includes a buffer system which contains citrate and phosphate to maintain the pH in a range of about 4 to about 8.
  • the pH range may be from about 4.5 to about 6.0, or from about pH 4.8 to about 5.5, or in a pH range of about 5.0 to about 5.2.
  • the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate.
  • the buffer system includes about 1.3 mg/mL of citric acid (e.g, 1.305 mg/mL), about 0.3 mg/mL of sodium citrate (e.g, 0.305 mg/mL), about 1.5 mg/mL of disodium phosphate dihydrate (e.g., 1.53 mg/mL), about 0.9 mg/mL of sodium dihydrogen phosphate dihydrate (e.g, 0.86), and about 6.2 mg/mL of sodium chloride (e.g, 6.165 mg/mL).
  • the buffer system includes 1-
  • the pH of the formulation is adjusted with sodium hydroxide.
  • a polyol which acts as a tonicifier and may stabilize the antibody, may also be included in the formulation.
  • the polyol is added to the formulation in an amount which may vary with respect to the desired isotonicity of the formulation.
  • the aqueous formulation may be isotonic.
  • the amount of polyol added may also be altered with respect to the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g, mannitol) may be added, compared to a disaccharide (such as trehalose).
  • the polyol which may be used in the formulation as a tonicity agent is mannitol.
  • the mannitol concentration may be about 5 to about 20 mg/mL. In certain embodiments, the concentration of mannitol may be about
  • the concentration of mannitol may be about 10-14 mg/mL. In certain embodiments, the concentration of mannitol may be about 12 mg/mL. In certain embodiments, the polyol sorbitol may be included in the formulation.
  • a detergent or surfactant may also be added to the formulation.
  • exemplary detergents include nonionic detergents such as polysorbates (e.g, polysorbates 20, 80 etc.) or poloxamers (e.g, poloxamer 188).
  • the amount of detergent added is such that it reduces aggregation of the formulated single-chain polypeptide and/or minimizes the formation of particulates in the formulation and/or reduces adsorption.
  • the formulation may include a surfactant which is a polysorbate.
  • the formulation may contain the detergent polysorbate 80 or Tween 80.
  • Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996).
  • the formulation may contain between about 0.1 mg/mL and about 10 mg/mL of polysorbate 80, or between about 0.5 mg/mL and about 5 mg/mL. In certain embodiments, about 0.1% polysorbate 80 may be added in the formulation.
  • the single-chain polypeptide product of the present disclosure is formulated as a liquid formulation.
  • the liquid formulation may be presented at a 10 mg/mL concentration in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure.
  • the stopper may be made of elastomer complying with USP and Ph Eur.
  • vials may be filled with 61.2 mL of the protein product solution in order to allow an extractable volume of 60 mL.
  • the liquid formulation may be diluted with 0.9% saline solution.
  • the liquid formulation of the disclosure may be prepared as a 10 mg/mL concentration solution in combination with a sugar at stabilizing levels.
  • the liquid formulation may be prepared in an aqueous vehicles or excipients.
  • a stabilizer may be added in an amount no greater than that which may result in a viscosity undesirable or unsuitable for intravenous administration.
  • the sugar may be disaccharides, e.g ., sucrose.
  • the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.
  • the pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and/or base.
  • the pharmaceutically acceptable acid may be hydrochloric acid.
  • the base may be sodium hydroxide.
  • deamidation is a common product variant of peptides and proteins that may occur during fermentation, harvest/cell clarification, purification, drug substance/drug product storage and during sample analysis.
  • Deamidation is the loss of N3 ⁇ 4 from a protein forming a succinimide intermediate that can undergo hydrolysis.
  • the succinimide intermediate results in a 17 dalton mass decrease of the parent peptide.
  • the subsequent hydrolysis results in an 18 dalton mass increase.
  • Isolation of the succinimide intermediate is difficult due to instability under aqueous conditions. As such, deamidation is typically detectable as 1 dalton mass increase. Deamidation of an asparagine results in either aspartic or isoaspartic acid.
  • the parameters affecting the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation and tertiary structure.
  • the amino acid residues adjacent to Asn in the peptide chain affect deamidation rates. Gly and Ser following an Asn in protein sequences results in a higher susceptibility to deamidation.
  • the liquid formulation of the present disclosure may be preserved under conditions of pH and humidity to prevent deamination of the protein product.
  • the aqueous vehicle or excipient of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation.
  • Illustrative vehicles or excipients include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g ., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
  • a preservative may be optionally added to the formulations herein to reduce bacterial action.
  • the addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.
  • Intravenous (IV) formulations may be the preferred administration route in particular instances, such as when a patient is in the hospital after transplantation receiving all drugs via the IV route.
  • the liquid formulation is diluted with 0.9% sodium chloride solution before administration.
  • the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.
  • a salt or buffer components may be added in an amount of 10 mM - 200 mM.
  • the salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines.
  • the buffer may be phosphate buffer.
  • the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion.
  • a preservative may be optionally added to the formulations herein to reduce bacterial action.
  • the addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.
  • the aqueous vehicle excipient of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation.
  • Illustrative vehicles or excipients include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
  • the single-chain polypeptide of the present disclosure could exist in a lyophilized formulation including the single-chain polypeptides and a lyoprotectant.
  • the lyoprotectant may be sugar, e.g, disaccharides.
  • the lyoprotectant may be sucrose or maltose.
  • the lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and/or a preservative.
  • the amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1 :2 protein to sucrose or maltose.
  • the protein to sucrose or maltose weight ratio may be of from 1 :2 to 1:5.
  • the pH of the formulation, prior to lyophilization may be set by addition of a pharmaceutically acceptable acid and/or base.
  • the pharmaceutically acceptable acid may be hydrochloric acid.
  • the pharmaceutically acceptable base may be sodium hydroxide.
  • the pH of the solution containing the protein of the present disclosure may be adjusted between 6 to 8.
  • the pH range for the lyophilized drug product may be from 7 to 8.
  • a salt or buffer components may be added in an amount of 10 mM - 200 mM.
  • the salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines.
  • the buffer may be phosphate buffer.
  • the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion.
  • a “bulking agent” may be added.
  • a “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g ., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure).
  • Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents.
  • a preservative may be optionally added to the formulations herein to reduce bacterial action.
  • the addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.
  • the lyophilized drug product may be constituted with an aqueous vehicle or excipient.
  • the aqueous vehicle or excipient of interest herein is one which is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization.
  • Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g ., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
  • the lyophilized drug product of the current disclosure is reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% sodium chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into a solution.
  • SWFI Sterile Water for Injection
  • USP 0.9% sodium chloride Injection
  • the lyophilized protein product of the instant disclosure is constituted to about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution).
  • the specific dose can be a uniform dose for each patient, for example, 50-5000 mg of protein.
  • a patient’s dose can be tailored to the approximate body weight or surface area of the patient.
  • Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially in light of the dosage information and assays disclosed herein.
  • the dosage can also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data. An individual patient's dosage can be adjusted as the progress of the disease is monitored.
  • Blood levels of the targetable construct or complex in a patient can be measured to see if the dosage needs to be adjusted to reach or maintain an effective concentration.
  • Pharmacogenomics may be used to determine which targetable constructs and/or complexes, and dosages thereof, are most likely to be effective for a given individual (Schmitz et al., Clinica ChimicaActa 308: 43-53, 2001; Steimer et al ., Clinica ChimicaActa 308: 33-41, 2001).
  • dosages based on body weight are from about 0.01 ⁇ g to about 100 mg per kg of body weight, such as about 0.01 ⁇ g to about 100 mg/kg of body weight, about 0.01 ⁇ g to about 50 mg/kg of body weight, about 0.01 ⁇ g to about 10 mg/kg of body weight, about 0.01 ⁇ g to about 1 mg/kg of body weight, about 0.01 ⁇ g to about 100 ⁇ g/kg of body weight, about 0.01 ⁇ g to about 50 ⁇ g/kg of body weight, about 0.01 ⁇ g to about 10 ⁇ g/kg of body weight, about 0.01 ⁇ g to about 1 ⁇ g/kg of body weight, about 0.01 ⁇ g to about 0.1 ⁇ g/kg of body weight, about 0.1 ⁇ g to about 100 mg/kg of body weight, about 0.1 ⁇ g to about 50 mg/kg of body weight, about 0.1 ⁇ g to about 10 mg/kg of body weight, about 0.1 ⁇ g to about 1 mg/kg of body weight, about 0.01 ⁇ g to about
  • Doses may be given once or more times daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the heterodimer Fc- fused protein in bodily fluids or tissues. Administration of the present invention could be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter or by direct intralesional injection. This may be administered once or more times daily, once or more times weekly, once or more times monthly, and once or more times annually.
  • the invention provides methods for treating cancer using a single-chain polypeptide described herein and/or a pharmaceutical composition described herein.
  • the methods may be used to treat a variety of cancers by administering to a patient in need thereof a therapeutically effective amount of the single-chain polypeptide described herein.
  • the cancer that is treated by a method disclosed herein is a locally advanced malignancy.
  • the locally advanced malignancy can be fully resected.
  • the locally advanced malignancy has been fully resected, and the treatment is provided subsequent to the resection.
  • a single-chain polypeptide of the present invention e.g ., a single-chain polypeptide comprising 11-15 and IL-15R ⁇
  • a single-chain polypeptide of the present invention e.g., a single-chain polypeptide comprising IL-15 and IL-15R ⁇
  • a single-chain polypeptide of the present invention is used as an adjuvant for active immunotherapy of severe infectious diseases.
  • a single-chain polypeptide of the present invention e.g, a single-chain polypeptide comprising IL-15 and IL-15R ⁇
  • a single-chain polypeptide of the present invention is used for treating myelosuppression following radiotherapy.
  • the present invention also provides a method of reducing hematopoietic toxicity.
  • Hematopoietic toxicity can result from genetic, infective, or environmental causes, including but not limited to irradiation and chemotherapy.
  • the present invention provides a method of treating a disease or disorder associated with radiation (e.g, ionizing radiation, alpha radiation, beta radiation, gamma radiation, X radiation, or neutron radiation).
  • a disease or disorder associated with radiation e.g, ionizing radiation, alpha radiation, beta radiation, gamma radiation, X radiation, or neutron radiation.
  • the present invention provides a method of treating myelosuppression following a radiation therapy, the method comprising administering to a patient in need thereof a therapeutically effective amount of a single-chain polypeptide or a formulation described herein.
  • the dose of the radiation therapy is at least 1, 5, 10, 15, or 20 Gy.
  • the radiation therapy causes damage in a system, organ, or tissue selected from the group consisting of bone marrow, lymphatic system, immune system, mucosal tissue, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lung, kidney, skin and brain.
  • the method of the present invention reduces the damage.
  • the present invention provides a method of treating myelosuppression occurring in the context of an accidental exposure to radiation, the method comprising administering to a patient in need thereof a therapeutically effective amount of a single-chain polypeptide or a formulation described herein.
  • the present invention provides a method of treating acute radiation syndrome (ARS), the method comprising administering to a patient in need thereof a therapeutically effective amount of a single-chain polypeptide or a formulation described herein.
  • ARS includes but is not limited to hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, and cutaneous radiation syndrome.
  • hematopoietic radiation syndrome results from, at least in part, depletion of hematopoietic stem cell pool and shows signs of lymphopenia and granulocytopenia.
  • Gastrointestinal syndrome results from, at least in part, damage of stem cells and progenitor cells located in the crypts and failure to replace the cells in the surface of the villi and shows signs of watery diarrhea, dehydration, electrolyte loss, gastrointestinal bleeding, and perforation.
  • the method of treatment provided herein is conducted at the prodromal phase of the ARS.
  • the prodromal phase is the initial phase of acute illness, characterized by the symptoms of nausea, vomiting, anorexia, fever, headache, and/or early skin erythema, typically within 1-3 days after the exposure to radiation.
  • the method of treatment provided herein is conducted at the latent phase of the ARS.
  • the latent phase is a phase characterized by improvement of symptoms but exhibition of lymphopenia and granulocytopenia in lab tests, and may last hours to weeks depending on the dose of exposure.
  • Treatment in the prodromal phase or latent phase may mitigate the development of the syndromes in the affected systems, organs, and/or tissues.
  • the method of treatment provided herein is conducted at the manifest illness phase of the ARS. Treatment in this phase may still promote recovery from the ARS.
  • the present invention also provides a method of increasing the survival, proliferation, differentiation, and/or activity of an immune cell, the method comprising contacting the immune cell with a single-chain polypeptide or a formulation disclosed herein.
  • the immune cell is a T cell (e.g ., CD4 + T cells).
  • the immune cell is an NK cell.
  • the instant disclosure provides a method of increasing or modulating the survival, proliferation, and/or activity of an immune cell, the method comprising contacting the immune cell with a single-chain polypeptide, pharmaceutical composition, or formulation disclosed herein.
  • the immune cell is a T cell (e.g., a CD8 + T cell, a CD4 + T cell, a regulatory T cell, or an NKT cell) or an NK cell.
  • the immune cell is contacted with the single-chain polypeptide, pharmaceutical composition, or formulation in vivo for preventing or treating a disease or disorder.
  • diseases and disorders suitable for such treatment include but are not limited to cancer and infection (e.g ., viral infection, bacterial infection, or parasitic infection).
  • the invention provides methods for treating cancer using a single-chain polypeptide described herein and/or a pharmaceutical composition described herein. The methods may be used to treat a variety of cancers by administering to a patient in need thereof an effective amount of a single-chain polypeptide described herein.
  • single-chain polypeptides or compositions disclosed herein can be administered to a subject to treat a disorder associated with abnormal apoptosis or a differentiative process (e.g., cellular proliferative disorders or cellular differentiative disorders, such as cancer, by, for example, producing an active or passive immunity).
  • a disorder associated with abnormal apoptosis or a differentiative process e.g., cellular proliferative disorders or cellular differentiative disorders, such as cancer, by, for example, producing an active or passive immunity.
  • the disclosed Single-chain polypeptides comprising IL-15 and IL- 15Ra may possess advantageous properties, such as reduced vascular leak syndrome.
  • cellular proliferative and/or differentiative disorders include cancer (e.g, carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g, leukemias).
  • a metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of
  • the single-chain polypeptides comprising IL-15 and IL-15R ⁇ can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma, or any viral disease.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • the term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • proliferative disorders include hematopoietic neoplastic disorders.
  • the skin disorder may involve the aberrant activity of a cell or a group of cells or layers in the dermal, epidermal, or hypodermal layer, or an abnormality in the dermal- epidermal junction.
  • the skin disorder may involve aberrant activity of keratinocytes (e.g, hyperproliferative basal and immediately suprabasal keratinocytes), melanocytes, Langerhans cells, Merkel cells, immune cell, and other cells found in one or more of the epidermal layers, e.g, the stratum basale (stratum germinativum), stratum spinosum, stratum granulosum, stratum lucidum or stratum comeum.
  • stratum basale stratum germinativum
  • stratum spinosum stratum granulosum
  • the disorder may involve aberrant activity of a dermal cell, for example, a dermal endothelial, fibroblast, immune cell (e.g, mast cell or macrophage) found in a dermal layer, for example, the papillary layer or the reticular layer.
  • a dermal cell for example, a dermal endothelial, fibroblast, immune cell (e.g, mast cell or macrophage) found in a dermal layer, for example, the papillary layer or the reticular layer.
  • a single-chain polypeptide or composition disclosed herein can be administered to a patient who has a viral infection (e.g, influenza, herpesvirus such as Epstein-Barr virus or cytomegalovirus).
  • a viral infection e.g, influenza, herpesvirus such as Epstein-Barr virus or cytomegalovirus.
  • T cells e.g, CD8 + T cells
  • the viral infection that can be treated by a single-chain polypeptide or composition disclosed herein is a chronic (e.g, persistent or recurrent) viral infection.
  • the patient who has a chronic viral infection is an AIDS patient.
  • a single-chain polypeptide of the present invention is used for treating an advanced malignancy or a chronic viral infection as a monotherapy.
  • a single-chain polypeptide of the present invention e.g, a single-chain polypeptide comprising IL-15 and IL-15R ⁇
  • a single-chain polypeptide of the present invention is used as an adjuvant for active immunotherapy of cancer or chronic infectious diseases.
  • a single-chain polypeptide of the present invention e.g, a single-chain polypeptide comprising IL-15 and IL-15R ⁇
  • is used for treating is used as an adjuvant for prophylactic vaccination.
  • the adjuvant can be administered prior to or subsequent to the administration of a vaccine. Alternatively, the adjuvant and the vaccine can be administered together in a single composition.
  • a single-chain polypeptide described herein can be used in combination with additional therapeutic agents to treat the cancer.
  • a single-chain polypeptide of the present invention is used in treating an advanced malignancy in combination with another therapeutic agent selected from: cytotoxic chemotherapy; radiotherapy; an antibody that targets a molecule involved in an anti-tumor immune response, such as CTLA-4, PD-1, PD-L1, or TGF-b; an antibody that acts by ADCC on a tumor-associated antigen; a multispecific antibody binding NKG2D, CD 16, and a tumor- associated antigen, optionally administered in combination with an antibody that targets PD-1 or PD-L1; a personalized cancer vaccine; an oncolytic cancer vaccine; and a personalized vaccine administered in combination with an antibody that targets PD-1 or PD-L1.
  • another therapeutic agent selected from: cytotoxic chemotherapy; radiotherapy; an antibody that targets a molecule involved in an anti-tumor immune response, such as CTLA-4, PD-1, PD-L1, or TGF-b; an antibody that acts by ADCC on a tumor-associated antigen; a multispecific antibody binding NKG2D,
  • a single-chain polypeptide of the present invention e.g ., a single-chain polypeptide comprising IL-15 and IL-15R ⁇
  • a single-chain polypeptide comprising IL-15 and IL-15R ⁇ is used in treating locally advanced malignancy that can be fully resected, in combination with a cancer vaccine or an antibody that targets PD-1 or PD-L1.
  • Single-chain polypeptides of the invention can also be used as an adjunct to surgical removal of the primary lesion.
  • the amount of single-chain polypeptide and additional therapeutic agent and the relative timing of administration may be selected in order to achieve a desired combined therapeutic effect.
  • the therapeutic agents in the combination, or a pharmaceutical composition or compositions comprising the therapeutic agents may be administered in any order such as, for example, sequentially, concurrently, together, simultaneously and the like.
  • a single-chain polypeptide may be administered during a time when the additional therapeutic agent(s) exerts its prophylactic or therapeutic effect, or vice versa.
  • the single-chain polypeptides of the present invention were made using recombinant DNA technology.
  • the clones are cultured under conditions suitable for bio-reactor scale-up and maintained expression of the proteins.
  • the proteins are isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.
  • Nucleic acids comprising sequences encoding IL-15R ⁇ sushi domain (SEQ ID NO:21); human IgGl Fc domain having heterodimerization mutations and effector silencing mutations (SEQ ID NO:25), murine IgGl having heterodimerization mutations and effector silencing mutations, or anti-human serum albumin (HSA) nanobody (VHH); IL-15 (SEQ ID NO:2) or IL-15 having a S73A mutation (SEQ ID NO:69), and linkers (SEQ ID NOs: 32 or 33) can be synthesized or PCR amplified, restriction digested and/or ligated together to form a contiguous nucleic acid encoding, in sequence from N to C-terminus, the following single- chain polypeptides (the single-chain polypeptides described below include polypeptides that are human (hu or h) or murine (mu or m)):
  • Nucleic acids can be cloned into an expression vector (pET-pSURE-Puro) and stably transfected into host cells to produce the single-chain polypeptides.
  • Single-chain polypeptides were collected from host cell lysates, purified by size exclusion chromatography (SEC) (data not shown), and analyzed by SDS- polyacrylamide gel electrophoresis (PAGE) under reducing and non-reducing conditions.
  • FIG. 6 shows that despite the presence of a long linker (30 amino acids) connecting the two Fc chains, Single-chain Polypeptide A and Single-chain Polypeptide B, each resolved as a single band under reducing conditions without the presence of protein smearing or degradation products, suggesting proper protein folding (FIG. 6A).
  • the larger molecular weight bands observed under non-reducing conditions suggests dimerization, which may be an artifact of the conditions used for analysis (FIG. 6B).
  • This example describes characterization of Single-chain Polypeptide C, by SEC and SDS-PAGE.
  • PBMCs peripheral blood mononuclear cells
  • IL-2R + HEK-Blue reporter cells (InvivoGen) were harvested from culture and adjusted to lxlO 6 cells/mL in culture media. Single-chain Polypeptide C, Reference Control 1, and RLI were diluted in media. 100 ⁇ L of PBMC suspension was mixed with 100 pL of diluted test polypeptide and incubated for 48 hours. The supernatant was harvested and contacted with the HEK-Blue reporter cells. Activation of signaling pathways downstream of the IL-2 receptor was detected by measurement of secreted embryonic alkaline phosphatase from the IL-2R + HEK-Blue reporter cells following the manufacturer’s instructions.
  • the Single-chain Polypeptide C eluting in SEC peak 2 (P2) and Single-chain Polypeptide C eluting in SEC peak 1 (PI) have comparable dose responses in the IL-2 reporter assay as compared to the RLI construct without a carrier, and both have significantly increased potency in inducing IL- 2 production as compared to Reference Control 1 at the concentrations examined.
  • This example shows that single-chain polypeptides of the present invention are significantly more potent in inducing IL-2 production PBMCs as compared to Reference Control 1, and have comparable potencies as compared to RLI and human IL-15.
  • This example also demonstrates that the IL-15 mutations in Single-chain Polypeptides G and H do not significantly impact potency in inducting IL-2 production as compared to Single-chain Polypeptide F ( i.e . comprising wild-type IL-15 sequence).
  • the average EC50 for Reference Control 1 was 10.778 nM with a standard deviation of 15.289 nM
  • the average EC50 for Single-chain Polypeptide A was 0.019 nM with a standard deviation of 0.0188 nM
  • the average EC50 for Single-chain Polypeptide B was 0.012 nM with a standard deviation of 0.0008 nM
  • the average EC50 for RLI was 0.0182 nM with a standard deviation of 0.007 nM.
  • STAT5 is a signaling molecule in the IL-2 pathway. Activation of the IL-2/IL-15 common b/g receptor induces STAT5 phosphorylation. The STAT5 phosphorylation in KHYG-1 NK cells treated with IL-15R ⁇ -IL-15-Fc Single-chain Polypeptides was measured by flow cytometry using phospho-specific STAT5 antibody.
  • Single-chain Polypeptide F, Single-chain Polypeptide G, Single-chain Polypeptide H, human IL-15 (hu-IL-15), Reference Control 1, and Reference Control 2 all induced a dose-dependent increase in STAT5 phosphorylation in KHYG-1 NK cells.
  • the EC50 for Single-chain Polypeptides G and H were similar.
  • This example shows that single-chain polypeptides of the invention have potencies for inducing immune cell proliferation comparable to RLI and Reference Control 2. This example also demonstrates that single-chain polypeptides of the invention have a greater potency for inducing immune cell proliferation as compared to Reference Control 1.
  • Single-chain Polypeptide A and Single-chain Polypeptide B, RLI, Reference Control 1, and Reference Control 2 induced a dose-dependent increase in proliferation of CD8 T cells (FIG. 12A), NK cells (FIG. 12B), NKT cells (FIG. 12C), CD4 T cells (FIG. 12D), and Tregs (FIG. 12E).
  • All single-chain polypeptides tested (Single-chain Polypeptide A, Single-chain Polypeptide B, and RLI) showed significantly greater potency in inducing proliferation of NK cells, NKT cells, CD8 T cells, CD4 T cells, and Tregs cells, as compared to Reference Control 1.
  • Single-chain Polypeptide A and Single- chain Polypeptide B exhibited slightly lower potency for inducing immune cell proliferation as compared to RLI and Reference Control 2.
  • This example shows that single-chain polypeptides of the present invention induce dose-dependent increases in the expression of immune cell activation markers CD25, NKG2D and Granzyme B (GrzB) in CD8 T-cells, NK cells, NKT cells, and CD4 T-cells, and/or ⁇ T-cells.
  • the potencies of Single-chain Polypeptides A and B are significantly greater than Reference Control 1, but slightly lower than RLI and Reference control 2.
  • Table 13 Activation of CD25 Expression in Immune Cell Subsets by Single- chain Polypeptides
  • Table 14 Activation of NKG2D Expression in Immune Cell Subsets by Single- chain Polypeptides
  • Example 8 in vivo Expansion of Live Cells Induced by Single-chain Polypeptides [0294] This example shows that single-chain polypeptides of the present invention induce live cell expansion when administered to mice.
  • mice were administered with treatment polypeptides according to the groupings and amounts indicated in TABLE 16 and fold-change of live cells in a peripheral blood sample was measured and compared to untreated mice.
  • mice treated with Reference Control 3 Reference Control
  • mice [0298] This example shows that single-chain polypeptides of the invention induce NK cell, NKT cell, and CD8 T cell expansion when injected into mice. [0299] Mice were administered with treatment polypeptides and measurements of the percentage of live NK cell, NKT cell and CD8 T cells at days 1, 4, and 9 post-injection were obtained.
  • FIG. 16 As shown in FIG. 16, among the molecules that induced an expansion in live NK cells (FIG. 16A), NKT cells (FIG. 16B), and CD8 T cells (FIG. 16C) at 4 days post-injection (Single-chain Polypeptide F, Single-chain Polypeptide C, Reference Control 3, and Reference Control 4), Single-chain Polypeptide F maintained an increase in the population of NK cells and NKT cells as compared to levels in untreated mice, and exhibited even greater CD8 T cell expansion at 9 days post-injection.
  • Single-chain Polypeptide F Single-chain Polypeptide F maintained an increase in the population of NK cells and NKT cells as compared to levels in untreated mice, and exhibited even greater CD8 T cell expansion at 9 days post-injection.
  • the number of NKG2D NK cells was significantly induced in mice treated with Single-chain Polypeptide F at day 4 post-inject compared to mice treated with Single-chain Polypeptide C, Reference Control 3, and Reference Control 4, RLI, and huIL-15, and remained elevated at day 9 post-injection.
  • Single-chain Polypeptide A, Single-chain Polypeptide G, and Single-chain Polypeptide H all had about a 2-fold higher binding affinity for IL-2R ⁇ compared to WT IL-15, which had a stead state affinity of 53.3 nM.
  • the higher affinity for IL-2R ⁇ of the single-chain polypeptides is primarily driven by faster on-rate kinetics of the 1 : 1 binding.
  • IL-15 mutant single-chain polypeptides having the structure huIL-15R ⁇ -Linker-huFcIgGl-Linker-huFcIgGl-Linker-huIL-15, in which the huFcIgGl polypeptide chains each comprise heterodimerization mutations and effector function silencing mutations, and having a S73A point mutation, or S73A and H105E mutations in the human IL-15 (huIL-15) polypeptide unit, and produced, purified, and characterized according to methods described herein and known in the art.
  • Recombinant DNA techniques can be used to produce the IL-15 mutant single- chain polypeptides.
  • in vitro assays such as the HEC Blue IL-2 assay and flow cytometry-based analysis of STAT3 phosphorylation are used to characterize the signal transduction potential of the IL-15 mutant single-chain polypeptides.
  • Flow cytometry analysis is also used to assess the potency of the IL-15 mutant single-chain polypeptides to induce immune cell proliferation (e.g. KI- 67) and activation (e.g.
  • CD25, NKG2D, and GrzB markers in in vitro cultures of total PBMCs, CD8 T cells, NK cells, NKT cells, CD4 T cells and/or ⁇ T cells, or in vivo in mice injected with the mutant IL-15 single-chain polypeptides.
  • SPR is used to characterize the binding affinity of the mutant IL-15 single-chain polypeptides to measure 1 : 1 binding and steady state affinity binding kinetics.

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Abstract

The present invention provides single-chain immunoregulatory polypeptides. These single-chain polypeptides have increased stability, reduced in vivo clearance, increased in vivo half-life when used as a therapeutic, and enhanced potency in activating immune cells.

Description

SINGLE-CHAIN POLYPEPTIDES
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/960,030, filed January 12, 2020, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 11, 2021, is named DFY-082WO_SL.txt and is 198,142 bytes in size.
FIELD OF THE INVENTION
[0003] The invention generally relates to single-chain immunoregulatory polypeptides, pharmaceutical compositions comprising such polypeptides, and methods of treating a cancer using the same.
BACKGROUND
[0004] Physiologically active proteins generally have the disadvantage of having a short in vivo half-life. In order to overcome this disadvantage, there have been attempts to conjugate these proteins to PEG (polyethylene glycol) or other polymers, or to fuse them to protein carriers, such as: antibodies or fragments thereof (e.g. fragment crystallizable (Fc) domains), nanobodies (VHH), or human serum albumin (HSA) and fragments thereof.
[0005] Thus, monomeric proteins and their cognate binding partner proteins (or portions thereof) that together form a complex to exert physiological activity can be joined to carriers to form single-chain polypeptides to increase monomeric protein stability, minimize in vivo clearance, and increase the in vivo half-life of the functional-protein complex. The carrier polypeptides may be derived from IgGl, IgG2, IgG3, IgG4 antibodies (e.g, Fc, Fab, variable fragment (Fv), or VHH) or HSA.
[0006] The inventions described in the present disclosure provide designs for improving recombinant protein stability and physiological activity, wherein a carrier is joined via a linker to a complex comprising a single- subunit cytokine or functional fragment thereof (e.g, interleukin 15 (IL- 15)) joined directly or indirectly to the cognate receptor of the single- subunit cytokine, or functional fragment thereof, to form a single-chain immunoregulatory polypeptide.
SUMMARY
[0007] The present invention provides single-chain immunoregulatory polypeptides.
These single-chain polypeptide constructs can have increased stability, reduced in vivo clearance, and increased half-life when used as a therapeutic.
[0008] In one aspect the present invention provides a single-chain polypeptide comprising: (a) an effector moiety polypeptide or a functional fragment thereof; (b) a receptor polypeptide or a functional fragment thereof; (c) a multi-chain carrier or single-chain carrier; and (d) two or more linkers, wherein a linker independently joins the single or multi- chain carrier to the effector moiety polypeptide or functional fragment thereof, and/or to the receptor polypeptide or functional fragment thereof. In some embodiments, when the carrier is multi-chain, two chains of the multi-chain carrier are joined by a linker.
[0009] In one aspect the present invention provides a single-chain polypeptide comprising three or more units comprising an effector moiety polypeptide or a functional fragment thereof, a receptor polypeptide or a functional fragment thereof, and a single-chain or multi-chain carrier, and wherein each unit is independently joined to another unit by a linker.
[0010] In some embodiments, when the carrier is multi-chain, the effector moiety polypeptide or functional fragment thereof, or the receptor polypeptide or functional fragment thereof is joined to each chain of the multi-chain carrier by a linker. In some embodiments, when the carrier is multi-chain, the effector moiety polypeptide or functional fragment thereof, or the receptor polypeptide or functional fragment thereof is independently joined to one chain of the multi-chain carrier. In some embodiments, the receptor polypeptide or functional fragment thereof binds to the effector moiety polypeptide or functional fragment thereof to form a complex.
[0011] In some embodiments, the effector moiety polypeptide comprises a single- subunit cytokine polypeptide. In some embodiments, the single-subunit cytokine comprises an interleukin 15 (IL-15) polypeptide. In some embodiments, the IL-15 polypeptide is human IL-15. In some embodiments, the receptor polypeptide comprises an IL-15 receptor polypeptide or a functional fragment thereof. In some embodiments, the IL-15 receptor polypeptide is an IL-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof. In some embodiments, the functional fragment of IL-15Rα comprises a sushi domain. In some embodiments, the IL-15Rα polypeptide is human IL-15Rα polypeptide.
[0012] In some embodiments, the carrier is selected from: a unit comprising a fragment crystallizable (Fc) domain, an anti-HSA nanobody (VHH), a human serum albumin (HSA) polypeptide or functional fragment thereof, an anti-HSA Fab domain, and an anti-HSA single-chain variable fragment (scFv).
[0013] In some embodiments, the carrier is a unit comprising an Fc domain, wherein the Fc domain comprises a first Fc domain polypeptide and a second Fc domain polypeptide. In some embodiments, the Fc domain is a human Fc domain. In some embodiments, the Fc domain is an IgGl, IgG2, IgG3, or IgG4 Fc domain.
[0014] In some embodiments, a linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the effector moiety polypeptide or functional fragment thereof. In some embodiments, an additional linker joins the C-terminus of the carrier to the N-terminus of the receptor polypeptide or functional fragment thereof.
[0015] In some of the embodiments that include an IL-15 polypeptide, the IL-15 polypeptide comprises one or more than one mutation. In some embodiments, the one or more than one mutation removes one or more than one glycosylation site. In certain embodiments, the one or more than one mutation does not substantially affect binding kinetics or binding affinity to the IL-2/IL-15 receptor common b subunit (IL-15Rβ), In some embodiments, the one or more than one mutation comprises a mutation of the serine at position 73. In some embodiments, the mutation of the serine at position 73 is to alanine. In certain embodiments, the one or more than one mutation further comprises a mutation of the histidine at position 105. In some embodiments the mutation of the histidine at position 105 is to glutamate.
[0016] In some embodiments, the description provides a single-chain polypeptide comprising: (a) a human IL-15 polypeptide or a functional fragment thereof; (b) a human IL- 15Ra polypeptide or a functional fragment thereof; and (c) a multi-chain carrier comprising an Fc domain, wherein a linker joins the N-terminus of a first fragment of an immunoglobulin heavy chain to the C-terminus of a second fragment of an immunoglobulin heavy chain, wherein an additional linker joins the C-terminus of the first fragment of an immunoglobulin heavy chain to the N-terminus of the IL-15 polypeptide or functional fragment thereof, and an additional linker joins the N-terminus of the second fragment of an immunoglobulin heavy chain to the C-terminus of the IL-15Rα polypeptide or fragment thereof, and wherein the first and the second fragment of the immunoglobulin heavy chain each comprises or consists of an Fc domain polypeptide.
[0017] In some embodiments, a single-chain polypeptide is provided that includes the amino acid sequence of SEQ ID NO:37. In some embodiments, a single-chain polypeptide is provided that includes the amino acid sequence of SEQ ID NO:71. In some embodiments, a single-chain polypeptide is provided that includes the amino acid sequence of SEQ ID NO:72.
[0018] In one aspect, the present invention provides a single-chain polypeptide comprising: (a) an effector moiety polypeptide or a functional fragment thereof; (b) a receptor polypeptide or a functional fragment thereof; (c) a carrier comprising (i) a multi- chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain, or (ii) a single-chain carrier; (d) a linker joining the carrier to the receptor polypeptide or functional fragment thereof; and (e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the carrier.
[0019] In some embodiments, the additional linker joins the carrier to the effector moiety polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the carrier, and the additional linker joins the C-terminus of the carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof. In some embodiments, additional linker joins the C-terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the carrier, and the linker joins the C-terminus of the carrier to the N-terminus of the receptor polypeptide or functional fragment thereof. In some embodiments, the additional linker joins the effector moiety polypeptide or functional fragment thereof to the receptor polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the carrier to the N-terminus of the receptor polypeptide or functional fragment thereof. In some embodiments, the additional linker joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof. [0020] In some embodiments, the linker chains, polypeptides, and/or units comprise a sequence of (GGGGS)4 (SEQ ID NO:31), (GGGGS)5 (SEQ ID NO:32), (GGGGS)6 (SEQ ID NO:33), or (SGGGG)6 (SEQ ID NO:34).
[0021] In some embodiments, the multi-chain carrier is a fragment crystallizable (Fc) domain carrier comprising two Fc-domain polypeptide chains. In some embodiments, the single-chain carrier is a nanobody (VHH) carrier.
[0022] In one aspect, the present invention provides a single-chain polypeptide comprising: (a) an effector moiety polypeptide or a functional fragment thereof; (b) a receptor polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)- domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker;
(d) a linker joining the Fc-domain carrier to the receptor polypeptide or functional fragment thereof; and (e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the Fc- domain carrier.
[0023] In some embodiments, the linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier, and the additional linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the receptor polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
[0024] In one aspect, the present invention provides a single-chain polypeptide comprising: (a) an effector moiety polypeptide or a functional fragment thereof; (b) a receptor polypeptide or a functional fragment thereof; (c) a nanobody (VHH) carrier; (d) a linker joining the VHH carrier to the receptor polypeptide or functional fragment thereof; and
(e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the VHH carrier.
[0025] In some embodiments, the linker joins the C-terminus of the VHH carrier to the N-terminus of the receptor polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the additional linker joins the C- terminus of the VHH carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof. In some embodiments, the additional linker joins the C-terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the linker joins the C-terminus of the VHH carrier to the N-terminus of the receptor polypeptide or functional fragment thereof.
[0026] In one aspect, the present invention provides a single-chain polypeptide comprising: (a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof; (b) an IL-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof; (c) a carrier comprising (i) a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain, or (ii) a single-chain carrier; (d) a linker joining the carrier to the IL-15Rα polypeptide or functional fragment thereof; and (e) an additional linker, wherein the additional linker joining the IL-15 polypeptide or functional fragment thereof to (i) the IL-15Rα polypeptide or functional fragment thereof, or (ii) the carrier.
[0027] In some embodiments, the additional linker joins the carrier to the IL-15 polypeptide or functional fragment thereof. In some embodiments, the linker joins the C- terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the carrier, and the additional linker joins the C-terminus of the multi-chain carrier to the N- terminus of the IL-15 polypeptide or functional fragment thereof. In some embodiments, the additional linker joins the C-terminus of the IL-15 polypeptide or functional fragment thereof to the N-terminus of the carrier, and the linker joins the C-terminus of the carrier to the N- terminus of the IL-15Rα polypeptide or functional fragment thereof. In some embodiments, the additional linker joins the IL-15 polypeptide or functional fragment thereof to the IL- 15Ra polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the carrier to the N-terminus of the IL-15Rα polypeptide or functional fragment thereof. In some embodiments, the additional linker joins the C-terminus of the IL- 15Ra polypeptide or functional fragment thereof, to the N-terminus of the IL-15 polypeptide or functional fragment thereof. In some embodiments, the multi-chain carrier is a fragment crystallizable (Fc) domain carrier comprising two Fc-domain polypeptide chains. In some embodiments, the single-chain carrier is a nanobody (VHH) carrier. [0028] In one aspect, the present invention provides a single-chain polypeptide comprising: (a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof; (b) an interleukin 15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)-domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker; (d) a linker joining the Fc-domain carrier to the receptor polypeptide or functional fragment thereof; and (e) an additional linker joining the IL-15 polypeptide or functional fragment thereof to (i) the IL-15Rα polypeptide or functional fragment thereof, or (ii) the Fc-domain carrier.
[0029] In some embodiments, the linker joins the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier, and the additional linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15Rα polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
[0030] In one aspect, the present invention provides a single-chain polypeptide comprising: (a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof; (b) an IL-15Rα polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)- domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker; (d) a linker joining the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof; and (e) an additional linker joining the C- terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of Fc- domain carrier.
[0031] In one aspect, the present invention provides a single-chain polypeptide comprising: (a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof; (b) an IL-15Rα polypeptide or a functional fragment thereof; (c) a nanobody (VHH) carrier; (d) a linker joining the VHH carrier to the IL-15Rα polypeptide or functional fragment thereof; and (e) an additional linker joining the IL-15 polypeptide or functional fragment thereof to (i) the IL-15Rα polypeptide or functional fragment thereof, or (ii) the VHH carrier. [0032] In some embodiments, the linker joins the C-terminus of the VHH carrier to the N-terminus of the IL-15Rα polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the IL-15Rα polypeptide or functional fragment thereof, to the N-terminus of the IL-15 polypeptide or functional fragment thereof. In some embodiments, the linker joins the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the additional linker joins the C-terminus of the VHH carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof. In some embodiments, the additional linker joins the C-terminus of the IL-15 polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the linker joins the C- terminus of the VHH carrier to the N-terminus of the IL-15Rα polypeptide or functional fragment thereof.
[0033] In one aspect, the present invention provides a single-chain polypeptide, comprising: (a) a human interleukin 15 (IL-15) polypeptide or a functional fragment thereof comprising a mutation of the serine at position 73 to alanine; (b) an IL-15Rα polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)-domain carrier comprising two Fc-domain polypeptide chains joined by an inter-chain linker; (d) a linker joining the C- terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof; and (e) an additional linker joining the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier.
[0034] In one aspect, the present invention provides a single-chain polypeptide, comprising: (a) a human interleukin 15 (IL-15) polypeptide or a functional fragment thereof comprising a mutation of the serine at position 73 to alanine and a mutation of the histidine at position 105 to glutamate; (b) an IL-15Rα polypeptide or a functional fragment thereof; (c) a fragment crystallizable (Fc)-domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker; (d) a linker joining the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof; and (e) an additional linker joining the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier.
[0035] In some embodiments, the single-chain polypeptide has a binding affinity dissociation constant (KD) of 20 nM to 30 nM. In some embodiments, the single-chain polypeptide induces enhanced signal transduction as compared to wild-type IL-15 when contacted with a cell expressing the IL-15R. In some embodiments, the signal transduction is measured using a HEK Blue IL-2 assay. In some embodiments, the single-chain polypeptide induces enhanced STAT5 phosphorylation as compared to wild-type IL-15 when contacted with the cell expressing the IL-15R. STAT5 phosphorylation can be measured, for example, using flow cytometry or western blot analysis. The cell expressing the IL-15R can be, for example, a CD8+ T-cell, a natural killer (NK) cell, a natural killer T-cell (NKT cell), a CD4+ T-cell, or a regulatory T-cell (Treg). In some embodiments, the single-chain polypeptide induces proliferation and/or activation of one or more than one immune cell selected from the group consisting of CD8+ T-cells, a NK cells, NKT cells, CD4+ T-cells, and Treg cells, when administered to a subject. In some embodiments, the single-chain polypeptide induces proliferation of the one or more than one immune cell after about 4 days. In some embodiments, the polypeptide induces the expression of one or more than one protein in the one or more immune cells selected from the group consisting of CD25, Ki-67, NKG2D, and/or granzyme B (GrzB). In some embodiments, the single-chain polypeptide induces the expression of the one or more than one protein after about 4 days.
[0036] The effector moiety polypeptide or functional fragment thereof can bind to the receptor polypeptide or functional fragment thereof to form a complex. In some embodiments, effector moiety polypeptide or functional fragment thereof comprises a single- subunit cytokine polypeptide or a functional fragment thereof. In some embodiments, the single-subunit cytokine polypeptide of functional fragment thereof comprises an interleukin 15 (IL-15) polypeptide or functional fragment thereof, which can optionally be human or include one or more than one mutation. The one or more than one mutation, if present, may remove one or more than one glycosylation site; may not substantially affect binding kinetics or binding affinity to the IL-2/IL-15 receptor common b subunit (IL-15Rβ); may include a mutation of the serine at position 73 ( e.g . to alanine); and may include mutations both of the serine at position 73 and of the histidine at position 105 (e.g. to alanine and glutamate, respectively).
[0037] In some embodiments, the receptor polypeptide or functional fragment thereof comprises an IL-15 receptor polypeptide or a functional fragment thereof. In some of these embodiments, the IL-15 receptor polypeptide or functional fragment thereof is an IL-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof. The IL-15Rα polypeptide can be human and can include a sushi domain. [0038] In some embodiments, the single-chain polypeptide has higher binding affinity for IL-15Rβ compared to wild-type IL-15.
[0039] In some embodiments, the Fc-domain carrier comprises human or murine Fc- domain polypeptide chains. In some embodiments, the Fc-domain polypeptide chains are IgGl, IgG2, IgG3, or IgG4 Fc-domain polypeptide chains. In some embodiments, the Fc- domain polypeptide chains comprise one or more than one mutation that reduces an effector function of the Fc-domain carrier. In some embodiments, the effector function comprises the ability of the Fc-domain carrier to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC).
[0040] In some embodiments, the Fc-domain polypeptide chains are human IgGl Fc- domain polypeptide chains comprising one or more than one mutation that reduces an effector function of the Fc-domain carrier at position 234, 235, 237, 329, 330, and/or 331, according to the EU numbering system. In some embodiments, the one or more than one mutation that reduces an effector function of the Fc-domain carrier is selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, numbered according to the EU numbering system. In some embodiments, the two Fc-domain polypeptide chains each comprise mutations L234A, L235A, and P329A. In some embodiments, the two Fc-domain polypeptide chains each comprise mutations L234A, and L235A. In some embodiments, the two Fc-domain polypeptide chains each comprise mutations L234A, L235A, G237A, A330S, and P331S. In some embodiments, the two Fc-domain polypeptide chains each further comprise mutation C220S.
[0041] In some embodiments, the VHH carrier is an anti-human serum albumin (HSA) nanobody.
[0042] The application also describes pharmaceutical compositions that include a single-chain polypeptide described herein and a pharmaceutically acceptable vehicle or excipient.
[0043] The present invention also provides methods of treating a cancer, by administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition comprising a single-chain polypeptide or a formulation described herein. [0044] Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing Brief Description of the Drawings, Detailed Description, Examples, and Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 illustrates various elements that are present in various single-chain polypeptides of the present disclosure. FIG. 1A illustrates the C-terminus of a receptor polypeptide connected to the N-terminus of an effector moiety polypeptide via a linker. FIG. IB illustrates the C-terminus of an effector moiety polypeptide connected to the N-terminus of a receptor polypeptide via a linker. FIG. 1C illustrates a multi-chain carrier comprising a first chain of a multi-chain carrier and a second chain of a multi-chain carrier, wherein the C- terminus of the first chain is connected to the N-terminus of the second chain via a linker. FIG. ID illustrates a single-chain carrier.
[0046] FIG. 2A illustrates an exemplary single-chain polypeptide comprising a first chain of a multi-chain carrier connected by a linker to a second chain of a multi-chain carrier, the second chain connected by a linker to the N-terminus of a receptor polypeptide, and the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide.
[0047] FIG. 2B illustrates an exemplary single-chain polypeptide comprising the C- terminus of a receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide, the C-terminus of the effector moiety polypeptide connected by a linker to the N-terminus of a first chain of a multi-chain carrier, and the C-terminus of the first chain connected by a linker to the N-terminus of a second chain of the multi-chain carrier.
[0048] FIG. 2C illustrates an exemplary single-chain polypeptide comprising a first chain of a multi-chain carrier connected by a linker to a second chain of a multi-chain carrier, the second chain connected by a linker to the N-terminus of an effector moiety polypeptide, and the C-terminus of the effector moiety polypeptide connected by a linker to the N- terminus of a receptor polypeptide.
[0049] FIG. 2D illustrates an exemplary single-chain polypeptide comprising the C- terminus of an effector moiety polypeptide connected by a linker to the N-terminus of a receptor polypeptide, the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of a first chain of a multi-chain carrier, and the C-terminus of the first chain connected by a linker to the N-terminus of a second chain of the multi-chain carrier. [0050] FIG. 3A illustrates an exemplary single-chain polypeptide comprising a single- chain carrier (shaded) connected by a linker to the N-terminus of a receptor polypeptide, and the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide.
[0051] FIG. 3B illustrates an exemplary single-chain polypeptide comprising the C- terminus of a receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide, the C-terminus of the effector moiety polypeptide connected by a linker to the N-terminus of a single-chain carrier (shaded).
[0052] FIG. 3C illustrates an exemplary single-chain polypeptide comprising a single- chain carrier (shaded) connected by a linker to the N-terminus of an effector moiety polypeptide, and the C-terminus of the effector moiety polypeptide connected by a linker to the N-terminus of a receptor polypeptide.
[0053] FIG. 3D illustrates an exemplary single-chain polypeptide comprising the C- terminus of an effector moiety polypeptide connected by a linker to the N-terminus of a receptor polypeptide, the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of a single-chain carrier (shaded).
[0054] FIG. 4A illustrates an exemplary single-chain polypeptide comprising a first chain of a multi-chain carrier connected by a linker to the N-terminus of a receptor polypeptide, the C-terminus of the receptor polypeptide connected by a linker to the N- terminus of an effector moiety polypeptide, and the C-terminus of the effector moiety polypeptide connected by a linker to a second chain of the multi-chain carrier.
[0055] FIG. 4B illustrates an exemplary single-chain polypeptide comprising the C- terminus of an effector moiety polypeptide connected by a linker to a first chain of a multi- chain carrier, the C-terminus of the first chain connected by a linker to the N-terminus of a second chain of the multi-chain carrier, and the C-terminus of the second chain connected by a linker to the N-terminus of a receptor polypeptide.
[0056] FIG. 4C illustrates an exemplary single-chain polypeptide comprising a first chain of a multi-chain carrier connected by a linker to the N-terminus of an effector moiety polypeptide, the C-terminus of the effector moiety polypeptide connected by a linker to the N-terminus of a receptor polypeptide, and the C-terminus of the receptor polypeptide connected by a linker to a second chain of the multi-chain carrier. [0057] FIG. 4D illustrates an exemplary single-chain polypeptide comprising the C- terminus of a receptor polypeptide connected by a linker to a first chain of a multi-chain carrier, the first chain connected by a linker to a second chain of the multi-chain carrier, and the second chain connected by a linker to the N-terminus of an effector moiety polypeptide.
[0058] FIG. 5A illustrates an exemplary single-chain polypeptide comprising the C- terminus of an effector moiety polypeptide connected by a linker to the N-terminus of a single-chain carrier (shaded), and the C-terminus of the single-chain carrier connected by a linker to the N-terminus of a receptor polypeptide.
[0059] FIG. 5B illustrates an exemplary single-chain polypeptide comprising the C- terminus of a receptor polypeptide connected by a linker to the N-terminus of a single-chain carrier (shaded), and the C-terminus of the single-chain carrier connected by a linker to the N-terminus of an effector moiety polypeptide.
[0060] FIG. 6A shows an SDS-PAGE for Single-chain Polypeptide A (A) which is an human IgGl Fc - Linker - human IgGl Fc - Linker - human IL-15Rα sushi domain - Linker - human IL-15 single-chain polypeptide comprising a first chain of a multi-chain carrier (Fc) connected by a linker to a second chain of a multi-chain carrier (Fc), the second chain connected by a linker to the N-terminus of a receptor polypeptide (human IL-15Rα (huIL-15Rα)), and the C-terminus of the receptor polypeptide connected by a linker to the N- terminus of an effector moiety polypeptide (human IL-15 (huIL-15)) and wherein the human IgGl Fc (huFc) domain chains comprise heterodimerization mutations and effector function silencing mutations, and Single-chain Polypeptide B (B) which is an huIL-15Rα sushi domain - Linker - huFc - Linker - huFc - Linker - huIL-15 single-chain polypeptide comprising the C-terminus of a receptor polypeptide (IL-15Rα) connected by a linker to a first chain of a multi-chain carrier (Fc), the C-terminus of the first chain connected by a linker to the N-terminus of a second chain of the multi-chain carrier (Fc), and the C-terminus of the second chain connected by a linker to the N-terminus of an effector moiety polypeptide (IL- 15) and wherein the huFc domain chains comprise heterodimerization mutations and effector function silencing mutations under reducing conditions.
[0061] FIG. 6B shows an SDS-PAGE for Single-chain Polypeptide A(A) and Single- chain Polypeptide (B) as described in FIG. A under non-reducing conditions.
[0062] FIG. 7A shows an SDS-PAGE for the VHH-RLI single-chain polypeptide (Single-chain Polypeptide C) comprising a single-chain anti-human serum albumin (HSA) carrier (VHH) connected by a linker to the N-terminus of a receptor polypeptide (huIL-15Rα sushi domain), and the C-terminus of the receptor polypeptide connected by a linker to the N- terminus of an effector moiety polypeptide (huIL-15).
[0063] FIG. 7B is a graph showing IL-2 response using a HEK-Blue IL-12 reporter assay. The in vitro IL-2 inducing activity of proteins eluted from two peaks (P1 and P2) obtained from size exclusion chromatography of a single-chain anti-HSA nanobody (VHH) - huIL-15Rα sushi domain - Linker - huIL-15 polypeptide (Single-chain Polypeptide C), were compared with a single-chain huIL-15Rα sushi domain -Linker- huIL-15 polypeptide without a carrier (RLI), and Reference Control 1 (huIL-15/huIL-15Rα-Fc, which includes Fc-domain heterodimerization mutations and 3 IL-15 mutations for reducing binding to its receptor).
[0064] FIGS. 8A-8C are graphs showing IL-2 response using a HEK-Blue IL-12 reporter assay. In triplicate experiments, Experiment I (FIG. 8A), Experiment II (FIG. 8B), and Experiment III (FIG. 8C), the in vitro IL-2 inducing activity of two single-chain Fc polypeptides, huFc-Linker-huFc-Linker-huIL-15Rα sushi domain - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide A) and huIL-15Rα sushi domain - huFc - huFc - huIL- 15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide B), were compared with an huIL15Rα sushi domain - Linker - huIL-15 single-chain polypeptide without a carrier (RLI) and Reference Control 1.
[0065] FIG. 9 is a graph showing IL-2 response using a HEK-Blue IL-12 reporter assay. The in vitro IL-2 inducing activity of single-chain Fc polypeptides huIL-15Rα sushi domain - Linker - murine IgG Fc (muFc) - Linker - muFc - huIL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single- chain Polypeptide F) and huIL-15Rα sushi domain - Linker - muFc - Linker - muFc - Linker - huIL-15 having S73A wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide G) or S73A H105E (Single-chain Polypeptide H) mutations, were compared with a huIL-15Rα sushi domain - Linker - huIL-15 single chain polypeptide without a carrier (RLI) and Reference Control 1.
[0066] FIGS. 10A-10C are graphs showing STAT5 phosphorylation using a pSTAT5 flow cytometry assay. In Experiment I (FIG. 10 A), Experiment II (FIG. 10B), and Experiment III (FIG. IOC), levels of STAT5 phosphorylation in KHYG-1 cells treated with two single-chain Fc polypeptides (huFc - Linker - huFc - Linker - huIL-15Rα sushi domain - Linker - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide A) and huIL-15Rα sushi domain - Linker - huFc - Linker - huFc - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single- chain Polypeptide B)) were compared with cells treated with an huIL-15Rα sushi domain - Linker - huIL-15 single-chain polypeptide without a carrier (RLI) and Reference Control 1.
[0067] FIG. 11 is a graph showing a dose-response of STAT5 phosphorylation in KHYG-1 cells treated with single-chain IL-15Rα-IL-15 Fc polypeptides. The levels of STAT5 phosphorylation of KHYG-1 cells treated with three single-chain Fc polypeptides (huIL-15Rα sushi domain- Linker - muFc - Linker - muFc - Linker -huIL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide F), huIL-15Rα sushi domain- Linker - muFc - Linker - muFc - Linker -huIL-15 comprising a S73A mutation in IL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide G), and huIL-15Rα sushi domain- Linker - muFc - Linker - muFc - Linker -huIL-15 comprising S73A and H105E mutations in IL-15 wherein the muFc- domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide H)) were compared with an huIL-15Rα - Linker - huIL- 15 single-chain polypeptide without a carrier (RLI) and Reference Control 1.
[0068] FIGS. 12A-12E are graphs showing in vitro immune cell proliferation induced by single-chain IL-15Rα - IL- 15 Fc polypeptides measured by detecting the proliferation- specific marker (Ki67) on CD8 T cells (FIG. 12A), NK cells (FIG. 12B), NKT cells (FIG. 12C), CD4 T cells (FIG. 12D), and Treg cells (FIG. 12E) by flow cytometry. Cells treated with two single-chain Fc polypeptides (huFc - Linker - huFc Linker - huIL-15Rα sushi domain - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide A) and huIL-15Rα - Linker - huFc - Linker - huFc - Linker - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain ) were compared with an huIL-15Rα sushi domain - Linker-huIL-15 single-chain polypeptide without a carrier (RLI), Reference Control 1. [0069] FIGS. 13A-13D are graphs showing flow cytometry analysis of percent CD25 positive CD8 T cells (FIG. 13 A), NK cells (FIG. 13B), CD4 cells (FIG. 13C) and NKT T cells (FIG. 13D) as an indicator of immune cell activation. Cell treated with two single-chain Fc polypeptides (huFc Linker - huFc - Linker - huIL-15Rα sushi domain - Linker - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide A) and huIL-15Rα sushi domain - Linker - huFc - Linker - huFc-Linker huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide B) were compared with cells treated with huIL-15Rα sushi domain - Linker - huIL-15 single-chain polypeptide without a carrier (RLI), Reference Control 1 (IL-15/IL- 15Ra-Fc, which includes Fc-domain heterodimerization mutations and 3 IL-15 mutations for reducing binding to its receptor), and Reference Control 2 (huIL-15/huIL-15Rα complex in which the IL-15 sequence has a N72D mutation for promoting binding to its receptor, and the huIL-15Rα sushi domain is fused to an huIgGl Fc-domain).
[0070] FIGS. 13E-13H are graphs showing flow cytometry analysis of NKG2D mean fluorescence intensity (MFI) of CD8 T cells (FIG. 13E), NK cells (FIG. 13F), NKT cells (FIG. 13G) and γδ T cells (FIG. 13H) as an indicator of immune cell activation. Cells treated with two single-chain Fc polypeptides (huFc Linker - huFc - Linker - huIL-15Rα sushi domain - Linker - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide A) and huIL- 15Ra sushi domain - Linker - huFc - Linker - huFc-Linker - huIL-15 wherein the huFc- domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide B) were compared with cells treated with huIL-15Rα sushi domain - Linker - huIL-15 single-chain polypeptide without a carrier (RLI), Reference Control 1, and Reference Control 2.
[0071] FIGS. 13I-13L are graphs showing flow cytometry analysis of Granzyme B (GrzB) MFI of CD8 T cells (FIG. 131), NK cells (FIG. 13 J), NKT cells (FIG. 13K) and γδ T cells (FIG. 13L) using FACs based assays. Cells treated with two single-chain Fc polypeptides (huFc - Linker - huFc - Linker - huIL-15Rα sushi domain - Linker - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide A) and huIL-15Rα sushi domain - Linker - huFc - Linker - huFc-Linker huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide B) were compared with cells treated with huIL-15Rα sushi domain - Linker - huIL-15 single-chain polypeptide without a carrier (RLI), Reference Control 1, and Reference Control 2.
[0072] FIG. 14 are graphs showing expansion of live cells induced by single-chain IL15Rα-IL15 Fc polypeptides. Cells treated with huIL-15Rα sushi domain - Linker - muFc
- Linker - muFc - Linker - huIL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide F) and anti-HSA nanobody (VHH) - Linker - huIL-15Rα sushi domain - Linker
- huIL-15 single chain polypeptide (Single-chain Polypeptide C) were compared to cells treated with Reference Control 3 (huIL-15/huIL-15Rα complex in which the huIL-15 sequence has a N72D mutation for promoting binding to its receptor, and the huIL-15Rα sushi domain is fused to a muIgG2A Fc-domain), and Reference Control 4 (huIL-15/huIL- 15Ra complex in which the huIL-15 sequence has a N72D mutation for promoting binding to its receptor, and huIL-15Rα sushi domain is fused to an muIgG2A Fc-domain, which includes LALAPA effector silencing mutations).
[0073] FIGS. 15A-15C are graphs showing the percentage of live NK cells (FIG. 15 A), NKT cells (FIG. 15B), and CD8 T cells (FIG. 15C) in mice following treatment with single- chain IL-15Rα-IL-15 Fc polypeptides. Mice treated with huIL-15Rα sushi domain - Linker - muFc - Linker - muFc - Linker - huIL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide F), anti-HSA VHH - Linker - huIL-15Rα - Linker - huIL-15 single-chain polypeptide (Single-chain Polypeptide C), Reference Control 3, and Reference Control 4 were compared to cell populations in untreated mice.
[0074] FIGS. 16A-16C are graphs showing fold changes in the population of NK cells (FIG. 16 A), NKT cells (FIG. 16B), and CD8 T cells (FIG. 16C) in mice following treatment with single-chain IL-15Rα-IL-15 Fc polypeptides. Mice treated with huIL-15Rα sushi domain - Linker - muFc - Linker - muFc - Linker - IL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single- Chain Polypeptide F), anti-HSA VHH - Linker - huIL-15Rα - Linker - huIL-15 (Single- chain Polypeptide C), Reference Control 3, and Reference Control 4 were compared to NK cell populations in untreated mice. [0075] FIGS. 17A-17C are graphs showing flow cytometry analysis of proliferation specific marker (Ki67; FIG. 17A), Granzyme B (GrzB; FIG. 17B), andNKG2D (FIG. 17C) in NK cells obtained from mice treated with huIL-15Rα sushi domain - Linker - muFc - Linker - muFc - Linker - huIL-15 wherein the huFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide F), anti-HSA VHH - Linker - huIL-15Rα sushi domain - Linker - huIL-15 (Single-chain Polypeptide C), Reference Control 3, and Reference Control 4.
[0076] FIGS. 17D-17F are graphs showing flow cytometry analysis of proliferation specific marker (Ki67; FIG. 17A), Granzyme B (GrzB; FIG. 17B), andNKG2D (FIG. 17C) in CD8 T cells obtained from mice treated with huIL-15Rα sushi domain - Linker - muFc - Linker - muFc - Linker - huIL-15 wherein the muFc-domain chains comprise heterodimerization mutations and effector function silencing mutations (Single-chain Polypeptide F), anti-HSA VHH - Linker - huIL-15Rα sushi domain - Linker - huIL-15 (Single-chain Polypeptide C), Reference Control 3, and Reference Control 4.
DETAILED DESCRIPTION
[0077] The invention provides improvements on immunoregulatory polypeptides, pharmaceutical compositions comprising such polypeptides, and therapeutic methods using such proteins and pharmaceutical compositions, including for the treatment of cancer.
[0078] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.
[0079] The terms “a” and “an” as used herein mean “one or more” and include the plural unless the context is inappropriate. For example, when referring to “a linker”, “a” can refer to one linker or more than one linker. If referring to more than one linker, “a” may be in relation to linkers having the same amino acid sequence, or linkers having different amino acid sequences.
[0080] As used herein, the term “carrier” refers to a polypeptide. In some embodiments, the polypeptide is a full-length or a functional fragment of a protein
[0081] As used herein, the term “multi-chain carrier” refers to a carrier that comprises at least two polypeptide chains, wherein each polypeptide chain has an N-terminal end and a C- terminal end. For example, an Fc-domain (Fc-Fc) or a Fab is a multi-chain carrier of the present invention. [0082] As used herein, the term “single-chain carrier” refers to a carrier comprising no more than one polypeptide chain that has a single N-terminal end and a single C-terminal end. For example, a nanobody (VHH), an scFv, or HSA is a single-chain carrier of the present invention.
[0083] As used herein, the term “effector moiety polypeptide” refers to a single polypeptide protein that exerts a physiological/biological effect when it binds to its cognate receptor.
[0084] As used herein, the term “receptor polypeptide” refers to a single polypeptide that binds to an effector moiety polypeptide.
[0085] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals ( e.g ., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably include humans.
[0086] As used herein, the term “effective amount” refers to the amount of a compound (e.g., a compound of the present invention) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. As used herein, the term “treating” includes any effect, e.g, lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.
[0087] As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
[0088] As used herein, the term “pharmaceutically acceptable vehicle or excipient” refers to any of the standard pharmaceutical vehicles or excipients, such as a phosphate buffered saline solution, water, emulsions (e.g, such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of vehicles, excipients, stabilizers and adjuvants, see e.g,
Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975],
[0089] As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g, acid or base) of a compound of the present invention which, upon administration to a subject, is capable of providing a compound of this invention or an active metabolite or residue thereof. As is known to those of skill in the art, “salts” of the compounds of the present invention may be derived from inorganic or organic acids and bases. Exemplary acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p- sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.
[0090] Exemplary bases include, but are not limited to, alkali metal ( e.g ., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NWf, wherein W is C1-4 alkyl, and the like.
[0091] Exemplary salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemi sulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, NH4+, and NW4+ (wherein W is a C1-4 alkyl group), and the like.
[0092] For therapeutic use, salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
[0093] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps. [0094] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
Single-Chain Polypeptides
[0095] The present invention provides single-chain polypeptides having immunoregulatory function. Single-chain polypeptides described herein comprise three or more polypeptides joined by two or more linkers resulting in a single, contiguous polypeptide chain. In some embodiments, single-chain polypeptides of the present invention comprise 3, 4, 5, 6, 7, or 8 polypeptides. For example, in some embodiments, single-chain polypeptides of the present invention comprise 2, 3, 4, 5, 6, or 7 linkers. In some embodiments, single- chain polypeptides of the present invention comprise 4 polypeptides joined by 3 linkers. In some embodiments, single-chain polypeptides of the present invention comprise 3 polypeptides joined by 2 linkers.
[0096] As described herein, one or more than one polypeptide can form a unit, wherein a unit corresponds to a protein, a protein subunit, a protein domain, or functional fragments thereof. In some embodiments, single-chain polypeptides of the present invention comprise 3 or more units. For example, in some embodiments, single-chain polypeptides of the present invention comprise 3, 4 or 5 units. In some embodiments, single-chain polypeptides of the present invention comprise 3 units. As used herein, functional fragments refer to portions or truncations of a full-length polypeptide that retain the physiological or structural function of the full-length polypeptide.
[0097] Single-chain polypeptides of the present invention comprise three or more units comprising an effector moiety polypeptide or a functional fragment thereof, a receptor polypeptide or a functional fragment thereof, or a single-chain or multi-chain carrier, wherein each unit is independently joined by a linker to form the contiguous single-chain polypeptides. Single-chain polypeptides disclosed herein have increased stability, reduced in vivo clearance, and an increased in vivo half-life when used as a therapeutic as compared to the effector moiety polypeptide alone or the effector moiety polypeptide in complex with the receptor polypeptide. In some embodiments, single-chain polypeptides of the present invention have more potent biological activity as compared to the effector moiety polypeptide alone or the effector moiety polypeptide in complex with the receptor polypeptide. For example, in some embodiments, single chain polypeptides of the present invention bind to receptors or receptor subunits on the surface of cells with greater affinity than the effector moiety polypeptide alone or the effector moiety polypeptide in complex with the receptor polypeptide. In some embodiments, single chain polypeptides bind to receptors on the surface of immune cells and induce greater activation and/or proliferation of the immune cells as compared to the effector moiety polypeptide alone or the effector moiety polypeptide in complex with the receptor polypeptide.
1. Effector Moiety Polypeptides
[0098] In some embodiments, the effector moiety polypeptide comprises a single-subunit cytokine polypeptide or functional fragment thereof, for example, but not limited to, interleukin 15 (IL-15). As described in the present disclosure, effector moiety polypeptides complex with a receptor polypeptide to form a complex having immunoregulatory function. For example, in some embodiments, the effector moiety polypeptide comprises an IL-15 polypeptide which complexes with the receptor polypeptide comprising an IL-15 receptor a (IL-15Rα) to form a complex capable of binding to and signaling through the IL-15Rβ/g common chains on the surface of immune cells.
[0099] IL-15 is a 14-15 kDa monomeric protein comprised of four a-helices. IL-15 is expressed as two alternatively spliced variant isoforms differing only in their signal peptides. The long signal peptide isoform of IL-15 (GenBank Accession No. NP_000576.1, set forth in SEQ ID NO: 1) is 162 amino acids in length with residues 1 to 48 comprising the long signal peptide (set forth in SEQ ID NO:4) and residues 49 to 162 comprising the mature 114 amino acid IL-15 polypeptide (set forth as SEQ ID NO:2). The short signal peptide isoform of IL- 15 (GenBank Accession No. NP 751915.1, set forth in SEQ ID NO:3) is 135 amino acids in length with the first 21 N-terminal amino acids corresponding to the short signal peptide (set forth in SEQ ID NO: 5) and residues 22-135 corresponding to the mature 114 amino acid IL- 15 polypeptide (SEQ ID NO:2).
[0100] In some embodiments, the IL-15 polypeptide is a mammalian IL-15 polypeptide. In certain embodiments, the IL-15 polypeptide is a human IL-15 polypeptide.
[0101] In certain embodiments, the effector moiety polypeptide comprises a polypeptide comprising an amino acid sequence at least 90% ( e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:2. In some embodiments, the effector moiety polypeptide comprises an IL-15 polypeptide fragment having an amino acid sequence at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:2 over the length of the IL-15 polypeptide fragment.
[0102] Amino acid sequences of wild-type IL-15 (e.g, human IL-15) and its variants are known in the art (e.g, U.S. Patent No. 8,415,456). For example, SEQ ID NO:6 and SEQ ID NO:7 are amino acid sequences of substituted IL-15. In general, the substituted amino acid sequences SEQ ID NO:6 and SEQ ID NO:7 are variant sequences of SEQ ID NO:2, with SEQ ID NO:6 and SEQ ID NO:7 having at least one substitution when compared to SEQ ID NO:2. In certain embodiments, one or both of the native Asn77 and Gly78 of SEQ ID NO:2 is substituted, and either or both of the native Asn71 and Asn72 of SEQ ID NO:2 may be substituted or may be unsubstituted.
[0103] In some embodiments, an amino acid sequence comprising SEQ ID NO:6, wherein Xaa71 is selected from the group consisting of Ser, Ala and Asn; Xaa72 is selected from the group consisting of Ser, Ala and Asn; Xaa77 is selected from the group consisting of Gin,
Ser, Lys, Ala, and Glu; and Xaa78 is selected from the group consisting of Ser, Ala, and Gly. In some embodiments, SEQ ID NO:6 generally corresponds to the native, unsubstituted IL-15 amino acid sequence SEQ ID NO:2 with the exception that in SEQ ID NO:6, at least Asn77 is substituted, and Gly78, Asn71, and Asn72 substituted or unsubstituted.
[0104] The present disclosure also provides an amino acid sequence comprising SEQ ID NO:7, wherein Xaa71 is selected from the group consisting of Ser, Ala and Asn; Xaa72 is selected from the group consisting of Ser, Ala and Asn; Xaa77 is selected from the group consisting of Gin, Ser, Lys, Ala, Glu, and Asn; and Xaa78 is selected from the group consisting of Ser and Ala. In some embodiments, SEQ ID NO:7 generally corresponds to the native, unsubstituted IL-15 amino acid sequence SEQ ID NO:2 with the exception that in SEQ ID NO:7, at least Gly78 is substituted, and Asn77, Asn71, and Asn72 may be substituted or unsubstituted.
[0105] In certain embodiments, the IL-15 amino acid sequence is at least 90% (e.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:2. In certain embodiments, the IL-15 of the present invention has mutation(s) of N71S/N72A/N77A, N77A, G78A, or N77A/G78A, the amino acid positions corresponding to SEQ ID NO:2. In certain embodiments, the IL-15 in the present invention comprises the amino acid sequence of SEQ ID NO:2. In certain embodiments, the IL-15 in the present invention comprises the amino acid sequence of SEQ ID NO : 8, SEQ ID NO : 9, SEQ ID NO : 10, or SEQ ID NO : 11. [0106] In certain embodiments, the IL-15 amino acid sequence comprises a mutation of the serine at position 73 of SEQ ID NO:2. For example, in some embodiments the serine at position 73 of SEQ ID NO:2 is mutated to alanine. In some embodiments, the IL-15 effector moiety polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO:69.
[0107] In some embodiments, the IL-15 amino acid sequence comprises a mutation of the histidine at position 105 of SEQ ID NO:2. For example, in some embodiments the histidine at position 105 of SEQ ID NO:2 is mutated to glutamate. In some embodiments the IL-15 amino acid sequence comprises mutations at both the serine at position 73 and the histidine at position 105. For example, in some embodiments, the IL-15 effector moiety polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO:70.
[0108] In some embodiments, the IL-15 amino acid sequence comprises one or mutation(s) that removes a glycosylation site. For example, in some embodiments, mutation of one or more than one residue does not impact IL-15 function but removes heterogeneity for Chemistry, Manufacturing and Controls (CMC). In some embodiments, one or more than one mutation in the IL-15 amino acid sequence does/do not inhibit IL-15 binding affinity for the IL-15 receptor (IL-15R). For example, in some embodiments, IL-15 comprising one or more mutation does not have impaired binding to the human IL-2R/IL- 15β receptor. In some embodiments, one or more than one mutation in the IL-15 amino acid sequence enhances IL- 15 binding affinity for the IL-15 receptor (IL-15R). For example in some embodiments, IL- 15 comprising one or more than one mutation enhances binding to the human IL-2R/IL- 15β receptor.
[0109] In some embodiments, one or more than one mutation in the IL-15 amino acid sequence enhances IL-15 activation of immune cells. For example in some embodiments, IL- 15 comprising one or more than one mutation enhances STAT5 phosphorylation when contacted with immune cells, as compared to wild-type IL-15.
[0110] TABLE 1: Human IL-15 Amino Acid Sequences
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
2. Receptor Polypeptides
[0111] In some embodiments, single-chain polypeptides of the present invention comprise receptor polypeptides. In certain embodiments, receptor polypeptides comprise the cognate receptor of the single- subunit cytokine polypeptide. For example, in some embodiments, receptor polypeptides comprise an IL-15 receptor or functional fragment thereof, or an IL-2 receptor or functional fragment thereof.
[0112] In some embodiments, single-chain polypeptides disclosed herein comprise a receptor polypeptide comprising an IL-15 receptor polypeptide or functional fragment thereof. In some embodiments, the IL-15 receptor polypeptide is a mammalian IL-15 receptor polypeptide. In some embodiments, the IL-15 receptor polypeptide is a human IL- 15 polypeptide.
[0113] In certain embodiments, receptor polypeptides of the present invention comprise one or more than one subunit of a cytokine receptor capable of binding to its cognate cytokine. For example, in some embodiments, single-chain polypeptides of the present invention comprise receptor polypeptides comprising an IL-15Rα polypeptide or functional fragment thereof. [0114] IL-15Rα is a 28.2 kDa protein reported to have nine isoforms produced by alternative splicing. The canonical IL-15Rα isoform (isoform 1, GenBank Accession No. NP_002180.1, set forth in SEQ ID NO:12) is 267 amino acids in length with residues 1 to 30 corresponding to the signal peptide (set forth in SEQ ID NO: 17), residues 31 to 95 corresponding with a sushi domain critical for IL-15 binding (set forth in SEQ ID NO: 16), residues 206 to 228 corresponding to a transmembrane region, and residues 229 to 267 corresponding to a C-terminal tail. IL-15Rα may be expressed as a soluble protein (having an amino acid sequence as set forth in SEQ ID NO: 14), wherein the mature IL-15Rα protein (as set forth in SEQ ID NO: 13) is truncated by removal of the transmembrane region and C- terminal tail. IL-15Rα may also be expressed as a soluble protein (having an amino acid sequence as set forth in SEQ ID NO: 15), wherein the 67 C-terminal amino acids of the mature IL-15Rα protein (SEQ ID NO: 13) are removed, corresponding to the C-terminal region after residue G170 that is proteolytically cleaved at the surface of cells to liberate soluble IL-15Rα in vivo.
[0115] In certain embodiments, an IL-15Rα polypeptide comprises a polypeptide comprising an amino acid sequence at least 90% ( e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 13. In some embodiments, the receptor polypeptide comprises an IL-15Rα polypeptide fragment having an amino acid sequence at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 13 over the length of the IL-15Rα polypeptide fragment.
[0116] In certain embodiments of the single-chain polypeptide of the present invention, the receptor polypeptide comprises an IL-15Rα polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 13. In other embodiments, the receptor polypeptide comprises an IL-15Rα polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 13.
[0117] Also contemplated are single-chain polypeptides, wherein the receptor polypeptide comprises an IL-15Rα polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 14, wherein the putative IL-15Rα transmembrane region and C-terminal tail region have been removed. In certain embodiments, the receptor polypeptide comprises an IL-15Rα polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 14. [0118] Further contemplated are single-chain polypeptides, wherein the receptor polypeptide comprises an IL-15Rα polypeptide having an amino acid sequence at least 90% identical to SEQ ID NO: 15, wherein the C-terminal region after residue G170 is removed. In other embodiments, the receptor polypeptide comprises an IL-15Rα polypeptide having an amino acid sequence at least 95% identical to SEQ ID NO: 15.
[0119] In certain embodiments of the single-chain polypeptide of the present invention, the receptor polypeptide comprises an IL-15Rα sushi domain having an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 16. In certain embodiments, the receptor polypeptide comprises an IL-15Rα sushi domain having an amino acid sequence at least 95% identical to SEQ ID NO:16.
[0120] Amino acid sequences of wild-type IL-15Rα (e.g, human IL-15Rα) and its variants are also known in the art (e.g, Bouchaud et al. J. Mol. Biol. (2008) 382: 1-12). In certain embodiments, the receptor polypeptide comprises a functional fragment of IL-15Rα, wherein the functional fragment comprises the Sushi domain of IL-15Rα. In certain embodiments, the functional fragment of the receptor polypeptide further comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 consecutive amino acid residues immediately C-terminal to the Sushi domain sequence of IL-15Rα. In certain embodiments, the IL-15Rα or functional fragment thereof in the present invention comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21.
[0121] In certain embodiments of the single-chain polypeptide of the present invention, the receptor polypeptide comprises an IL-15Rα functional fragment having an amino acid sequence at least 90% (e.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:21. In certain embodiments, the receptor polypeptide comprises an IL-15Rα functional fragment having an amino acid sequence at least 95% identical to SEQ ID NO:21.
[0122] TABLE 2: Human IL-15Ra Amino Acid Sequences
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
3. Complexes
[0123] In some embodiments, single-chain polypeptides of the present invention comprise effector moiety polypeptides or functional fragments thereof and receptor polypeptides or functional fragments thereof that bind to form complexes. Complexes of the single-chain polypeptides disclosed herein are capable of stimulating a response when exposed to immune cells.
[0124] In certain embodiments, a complex comprises an effector moiety polypeptide joined to a receptor polypeptide via a linker. For example, in some embodiments, a linker can join the C-terminal residue of the receptor polypeptide comprising the IL-15Rα polypeptide or functional fragment thereof or IL-15Rα sushi domain polypeptide or functional fragment thereof, to the N-terminal residue of the effector moiety polypeptide comprising the IL-15 polypeptide or functional fragment thereof. In alternative embodiments, a linker can join the N-terminal residue of the receptor polypeptide comprising the IL-15Rα polypeptide or functional fragment thereof or IL-15Rα sushi domain polypeptide or functional fragment thereof, to the C-terminal residue of the effector moiety comprising the IL-15 polypeptide or functional fragment thereof.
[0125] In other embodiments, an effector moiety polypeptide interacts with a receptor polypeptide via non-covalent interactions or additionally via one or more than one engineered disulfide bond.
[0126] Some single-chain polypeptides of the present invention include an artificial or engineered disulfide bridge between the effector moiety polypeptide and the receptor polypeptide. For example, in some embodiments, a single-chain polypeptide according to the invention includes an artificial or engineered disulfide bridge between an IL-15 polypeptide and an IL-15Rα polypeptide. Such a single-chain polypeptide includes an effector moiety polypeptide comprising a substitution selected from L52C and E53C in the IL-15 polypeptide and a receptor polypeptide comprising a substitution selected from S40C, A37C, and G38C in the IL-15Rα polypeptide, wherein the substituted cysteine residues form a disulfide bond. The foregoing substitutions in IL-15 and IL-15Rα have been described in Hu et al ., 2018, herein incorporated by reference, and are predicted to introduce an inter-unit disulfide bond between IL-15 and IL-15Rα without disrupting the native secondary structure of the IL-15 and IL-15Rα polypeptides.
[0127] In another embodiment, a single-chain polypeptide of the present invention includes an effector moiety polypeptide comprising a L52C substitution in the IL-15 polypeptide and a receptor polypeptide comprising a S40C substitution in the IL-15Rα polypeptide, wherein the substituted cysteine residues form a disulfide bond.
[0128] In certain embodiments of the single-chain polypeptide of the present invention, the effector moiety polypeptide comprising the IL-15 polypeptide includes one or more substitution(s) selected from L45D, L45E, Q48K, S51D, L52D, E64K, I68D, and L69R. These IL-15 substitutions, described in US Patent No. 9,493,533 and incorporated herein by reference, have been demonstrated to increase the binding affinity of IL-15 for IL-15Rα.
[0129] In further embodiments of the single-chain polypeptide of the present invention, the effector moiety polypeptide comprising the IL-15 polypeptide comprises an N72D substitution. As described in US 8,507,222, herein incorporated by reference, mutation of the asparagine residue at position 72 of IL-15 to aspartic acid increases IL-15 binding to the IL- 1511bg receptor subunits as compared to native IL-15 polypeptide.
[0130] In certain embodiments, the effector moiety polypeptide comprises an IL-15 polypeptide having one or more amino acid substitution(s) selected from L45D, L45E, Q48K, S51D, L52C, L52D, E53C, E64K, I68D, L69R, and N72D.
[0131] In certain embodiments, the receptor polypeptide comprises an IL-15Rα polypeptide having an amino acid sequence at least 90% identical to any one of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, or comprises an IL-15Rα sushi domain having an amino acid sequence at least 90% identical to SEQ ID NO: 16, wherein the IL-15Rα polypeptide or IL-15Rα sushi domain further comprises one or more amino acid substitution(s) selected from S40C, A37C, and G38C.
[0132] Any of the IL-15 polypeptides disclosed herein (for example, but not limited to, the sequences presented in Table 1) can be paired with any of the IL-15Rα polypeptides disclosed herein (for example, but not limited to, the sequences presented in 2) to form a complex. In certain embodiments, the effector moiety polypeptide comprises a polypeptide comprising an amino acid sequence at least 90% ( e.g ., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:2, and the receptor polypeptide comprises an IL-15Rα functional fragment having an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:21. In some embodiments, the effector moiety polypeptide comprises an IL-15 polypeptide fragment having an amino acid sequence at least 90% (e.g, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:2 over the length of the IL-15 polypeptide fragment, and the receptor polypeptide comprises an IL-15Rα functional fragment having an amino acid sequence at least 90% (e.g, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:21.
[0133] Any of the linkers disclosed in Table 8 can be used to join any of the IL-15 polypeptides disclosed herein to any of the IL-15Rα polypeptides disclosed herein. In certain exemplary embodiments, the present disclosure provides, but is not limited to, complexes having sequences as presented in Table 3.
[0134] TABLE 3:
Figure imgf000033_0001
4. Carriers [0135] As used herein, a “carrier” refers to a unit that is joined to an effector moiety polypeptide and/or a receptor polypeptide by one or more than one linker, thereby promoting increased stability, reduced in vivo clearance, and an increased in vivo half-life of an effector moiety polypeptide as compared to an effector moiety polypeptide alone, or an effector moiety polypeptide in complex with a receptor polypeptide. [0136] As used herein, a “multi-chain” carrier refers to two or more polypeptide chains that interact to form a unit. In some embodiments, two polypeptide chains comprising the multi-chain carrier are joined by a linker to form a unit. In other embodiments, polypeptide chains comprising the multi-chain carrier are not directly joined by linkers and interact via non-covalent interactions to form a unit. In some embodiments, multi-chain carriers that are not directly joined by linkers, further comprise engineered mutations that introduce one or more than one disulfide bond between two polypeptide chains.
[0137] A “single-chain” carrier, as used herein, refers to a single polypeptide chain that forms a unit.
4.1 Multi-Chain Carriers
[0138] In some embodiments, a carrier can be a multi-chain carrier. For example, in some embodiments, the multi-chain carrier can comprise a unit comprising a fragment crystallizable (Fc) domain. In certain embodiments, a multi-chain carrier comprises a first chain of a multi-chain carrier and a second chain of a multi-chain carrier. In some embodiments, the first chain of a multi-chain carrier and the second chain of a multi-chain carrier are directly joined to each other by a linker to form a unit. In other embodiments the first chain of a multi-chain carrier and the second chain of a multi-chain carrier are not directly joined together by a linker and dimerize via non-covalent interactions to form a unit.
[0139] In certain embodiments, the first and second chains of a multi-chain carrier comprise IgGl, IgG2, IgG3, or IgG4 Fc domain polypeptide chains. In certain embodiments, the first and second chains of a multi-chain carrier comprise mammalian Fc domain polypeptide chains. In preferred embodiments, the first and second Fc domain polypeptide chains are human Fc domain polypeptide chains.
[0140] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide ( e.g ., IL-15) or a functional fragment thereof;
(b) a receptor polypeptide (e.g., IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (c) a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain; (d) a linker joining the contiguous polypeptide chain to the receptor polypeptide or functional fragment thereof; and (e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the contiguous polypeptide chain.
[0141] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof;
(b) a receptor polypeptide (e.g, IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (c) a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain; (d) a linker that joins the C- terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the contiguous polypeptide chain; and (e) an additional linker that joins the C-terminus of the contiguous polypeptide chain to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
[0142] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide ( e.g ., IL-15) or a functional fragment thereof;
(b) a receptor polypeptide (e.g., IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (c) a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain; (d) a linker joins the C-terminus of the contiguous polypeptide chain to the N-terminus of the receptor polypeptide or functional fragment thereof; and (e) an additional linker joins the C-terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the contiguous polypeptide chain.
[0143] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof;
(b) a receptor polypeptide (e.g, IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (c) a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain; (d) a linker that joins the contiguous polypeptide chain to the receptor polypeptide or functional fragment thereof; and (e) an additional linker that joins the effector moiety polypeptide or functional fragment thereof to the receptor polypeptide or functional fragment thereof.
[0144] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof;
(b) a receptor polypeptide (e.g, IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (c) a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain; (d) a linker that joins the C- terminus of the contiguous polypeptide chain to the N-terminus of the receptor polypeptide or functional fragment thereof; and (e) an additional linker that joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof. [0145] In some embodiments, a single-chain polypeptide of the present invention comprises a fragment crystallizable (Fc) domain (two Fc-domain polypeptide chains) as a multi-chain carrier. In some embodiments, a single-chain polypeptide of the present invention comprises an IgGl, IgG2, IgG3, or IgG4 fragment crystallizable (Fc) domain (two Fc-domain polypeptide chains) as a multi-chain carrier.
IgGl Fc Carriers
[0146] In some embodiments, a single-chain polypeptide of the present invention comprises a multi-chain carrier, wherein the multi-chain carrier comprises a Fc domain, and wherein a first Fc domain polypeptide chain and a second Fc domain polypeptide chain are both IgGl Fc domain polypeptide chains.
[0147] In some embodiments, a multi-chain carrier comprises a first IgGl heavy chain polypeptide fragment and a second IgGl heavy chain polypeptide fragment, each comprising an Fc domain polypeptide chain, are directly joined to each other by a linker to form a unit.
In other embodiments the first IgGl heavy chain polypeptide fragment and the second IgGl heavy chain polypeptide fragment, each comprising an Fc domain polypeptide chain, are not directly joined together by a linker and dimerize via non-covalent interactions to form a unit.
[0148] IL-15Rα-human IgGl Fc-human IgGl Fc-IL-15 (FIG. 2A); an IL15Rα-linker- Fc-linker-Fc-linker-IL15 single-chain polypeptide comprising the C-terminus of a receptor polypeptide (IL-15) connected by a linker to a first chain of a multi-chain carrier (Fc), the C- terminus of the first chain connected by a linker to the N-terminus of a second chain of the multi-chain carrier (Fc), and the C-terminus of the second chain connected by a linker to the N-terminus of an effector moiety polypeptide (IL-15); an Fc-linker-IL15Rα-linker-IL15- linker-Fc single-chain polypeptide comprising a first chain of a multi-chain carrier (Fc) connected by a linker to the N-terminus of a receptor polypeptide (IL-15Rα), the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide (IL-15), and the C-terminus of the effector moiety polypeptide connected by a linker to a second chain of the multi-chain carrier (Fc) (FIG. 4A); or an IL15-linker-Fc- linker-Fc-linker-IL15Rα single-chain polypeptide comprising the N-terminus of an effector moiety polypeptide (IL-15) connected by a linker to a first chain of a multi-chain carrier (Fc), the N-terminus of the first chain connected by a linker to the C-terminus of a second chain of the multi-chain carrier (Fc), and the N-terminus of the second chain connected by a linker to the C-terminus of a receptor polypeptide (IL-15a). [0149] In certain embodiments of the single-chain polypeptide of the present invention, the first heavy chain polypeptide fragment and second heavy chain polypeptide fragment of the multi-chain carrier both comprise an amino acid sequence at least 90% ( e.g at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to a wild-type human IgGl sequence (SEQ ID NO:24).
[0150] In certain embodiments of the single-chain polypeptide of the present invention, the first heavy chain polypeptide fragment and second heavy chain polypeptide fragment of the multi-chain carrier both comprise an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:25).
[0151] In some embodiments, a single-chain polypeptide of the present disclosure comprises a first IgGl heavy chain polypeptide fragment and a second IgGl heavy chain polypeptide fragment, wherein each heavy chain polypeptide fragment comprises an IgGl Fc domain polypeptide chain, and both Fc domain polypeptide chains have one or more than one mutation to reduce binding to an FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIIA, or FcγRIIIB) or complement component (e.g, Clq). Such mutations are useful for reducing effector functions. For example, a single-chain polypeptide of the present disclosure may include LALA (L234A and L235A) mutations, LALAPA (L234A, L235A, and P329A) mutations, LALAPG (L234A, L235A, and P329G) mutations, or LALEGAASPS (L234A, L235E, G237A, A330S, and P331S) mutations. The two Fc-domain polypeptide chains each may further comprise mutation C220S.
[0152] In certain embodiments, a multi-chain carrier comprises a first IgGl heavy chain polypeptide fragment and a second IgGl heavy chain polypeptide fragment, each comprising an Fc domain polypeptide chain, , wherein the first and second IgGl heavy chain polypeptide fragment are joined by a linker, and wherein the multi-chain carrier comprises a sequence according to SEQ ID NO:26.
[0153] TABLE 4: IgGl Heavy Chain Polypeptide Fragment Carrier Sequences
Figure imgf000037_0001
Figure imgf000038_0001
Anti-HSA Fab Carriers
[0154] In some embodiments, a multi-chain carrier is a unit comprising an anti-HSA Fab heavy chain polypeptide and an anti-HSA light chain polypeptide. In some embodiments of the present invention, the carrier is a unit comprising an anti-HSA Fab, wherein a fragment of an immunoglobulin heavy chain and an immunoglobulin light chain are joined by a linker to form a unit. In some embodiments, the fragment of an immunoglobulin heavy chain and immunoglobulin light chain are not directly joined by a linker and interact via non-covalent interactions to form a unit. In some embodiments, an anti-HSA Fab comprises a fragment of an immunoglobulin heavy chain and an immunoglobulin light chain that are not directly joined by a linker, and further comprise engineered mutations that introduce one or more than one disulfide bond between the heavy chain and light chain.
[0155] In certain embodiments, a multi-chain carrier comprises an anti-HSA Fab comprising a light chain having an amino acid sequence at least 80% ( e.g ., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:28.
[0156] In certain embodiments, a multi-chain carrier comprises an anti-HSA Fab comprising a heavy chain having an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%) identical to SEQ ID NO:40.
[0157] Any of the linkers provided in Table 8 below can be used to directly join the C- terminus of a light chain having a sequence according to SEQ ID NO:28 to the N-terminus of a heavy chain having a sequence according to SEQ ID NO:40. Alternatively, any of the linkers provided in Table 8 below can be used to directly join the C-terminus of a heavy chain having a sequence according to SEQ ID NO:40 to the N-terminus of a light chain having a sequence according to SEQ ID NO:28.
[0158] In certain exemplary embodiments, the present disclosure provides, but is not limited to, single chain anti-HSA Fab fragments having amino acid sequences at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:41 or SEQ ID NO:42.
[0159] TABLE 5: Fab Carrier Sequences
Figure imgf000039_0001
Figure imgf000040_0001
Single-Chain Variable Fragment (scFv) Carriers
[0160] In some embodiments, a multi-chain carrier of the present invention comprises an anti-HSA single-chain variable fragment (scFv), wherein the heavy chain variable domain and light chain variable domain of the scFv are joined by a linker to form a unit.
[0161] In certain embodiments, a multi-chain carrier comprises an anti-HSA scFv comprising a light chain variable domain having an amino acid sequence at least 80% ( e.g. , at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:29.
[0162] In certain embodiments, a multi-chain carrier comprises an anti-HSA scFv comprising a heavy chain variable domain having an amino acid sequence at least 80% (e.g, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:43.
[0163] Any of the linkers provided in Table 8 below can be used to directly join the C- terminus of a light chain variable domain having a sequence according to SEQ ID NO:29 to the N-terminus of a heavy chain variable domain having a sequence according to SEQ ID NO:43. Alternatively, any of the linkers provided in Table 8 below can be used to directly join the C-terminus of a heavy chain variable domain having a sequence according to SEQ ID NO:43 to the N-terminus of a light chain variable domain having a sequence according to SEQ ID NO:29.
[0164] In certain exemplary embodiments, the present disclosure provides, but is not limited to, anti-HSA scFv having amino acid sequences at least 80% (e.g, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:44 or SEQ ID NO:45. [0165] TABLE 6: scFv Carrier Sequences
Figure imgf000041_0001
4.2 Single-Chain Carriers
[0166] In some embodiments, a single-chain polypeptide of the present invention comprises a single-chain carrier, wherein the single-chain carrier comprises a human serum albumin (HSA) polypeptide or functional fragment thereof, or an anti-HSA nanobody (VHH).
[0167] In some embodiments, the single-chain carrier is directly joined via a linker to an effector moiety polypeptide and/or a receptor polypeptide.
[0168] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide ( e.g. , IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g, IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (c) a single-chain carrier; (d) a linker joining the single-chain carrier to the receptor polypeptide or functional fragment thereof; and (e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the single-chain carrier. [0169] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide ( e.g ., IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g., IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (c) a single-chain carrier; (d) a linker that joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the single-chain carrier; and (e) an additional linker that joins the C-terminus of the single-chain carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
[0170] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g, IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (d) a linker joins the C-terminus of the single-chain carrier to the N-terminus of the receptor polypeptide or functional fragment thereof; and (e) an additional linker joins the C- terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the single-chain carrier
[0171] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g, IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (d) a linker that joins the single-chain carrier to the receptor polypeptide or functional fragment thereof; and (e) an additional linker that joins the effector moiety polypeptide or functional fragment thereof to the receptor polypeptide or functional fragment thereof.
[0172] In certain embodiments, a single-chain polypeptide of the present invention comprises (a) an effector moiety polypeptide (e.g, IL-15) or a functional fragment thereof; (b) a receptor polypeptide (e.g, IL-15Rα) or a functional fragment thereof (e.g, sushi domain); (c) a single-chain carrier; (d) a linker that joins the C-terminus of the single-chain carrier to the N-terminus of the receptor polypeptide or functional fragment thereof; and (e) an additional linker that joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
[0173] In some embodiments, a single-chain polypeptide of the present invention comprises a nanobody (VHH) as a single-chain carrier. In some embodiments, a single-chain polypeptide of the present invention comprises an anti-human serum albumin (HSA) nanobody (VHH).
[0174] In some embodiments, the carrier is a unit comprising an anti-HSA nanobody (VHH). For example, single-chain polypeptides include an VHH-linker-IL15Rα-linker-IL15 single-chain polypeptide comprising single-chain carrier (VHH) connected by a linker to the N-terminus of a receptor polypeptide (IL-15Rα), and the C-terminus of the receptor polypeptide connected by a linker to the N-terminus of an effector moiety polypeptide (IL- 15) (FIG. 3 A); an IL15-linker- VHH-linker- IL15Rα single-chain polypeptide comprising the C-terminus of an effector moiety polypeptide (IL-15) connected by a linker to the N-terminus of a single-chain carrier (VHH), the C-terminus of the single-chain carrier connected by a linker to the N-terminus of a receptor polypeptide (IL-15Rα) (FIG. 5 A); or an IL15Rα-linker- VHH-linker-IL15 single-chain polypeptide comprising the C-terminus of a receptor polypeptide (IL-15Rα), connected by a linker to the N-terminus of a single-chain carrier (VHH), the C-terminus of the single-chain carrier connected by a linker to the N-terminus of an effector moiety polypeptide (IL-15) (FIG. 5B).
[0175] In certain embodiments, a single-chain carrier comprises an HSA polypeptide comprising an amino acid sequence at least 80% ( e.g ., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:27.
[0176] In certain embodiments, a single-chain carrier comprises an anti-HSA nanobody comprising an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:30.
[0177] Table 7: Single-Chain Polypeptide Carrier Sequences
Figure imgf000044_0001
5. Linkers
[0178] The term “linker” comprises, in accordance with the present invention, a polypeptide joining two units and/or two polypeptide chains. Exemplary linker sequences are provided in Table 8 below. [0179] In some embodiments presently described, linkers comprise amino acid sequences that confer flexibility and do not interfere with the binding of the effector moiety polypeptide and receptor polypeptide to each other; and resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. Linkers suitable for joining units of the single-chain polypeptides presently described include a (GS)n (SEQ ID NO: 60), (GGS)n (SEQ ID NO: 61), (GGGS)n (SEQ ID NO: 62), (GGSG)n (SEQ ID NO: 63),
(GGSGG)n (SEQ ID NO: 64), (GGGGS)n (SEQ ID NO: 65), or (SGGGG)n (SEQ ID NO: 66) sequence, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, a linker comprises (GGGGS)4 (SEQ ID NO: 31), (GGGGS)5 (SEQ ID NO:32), (GGGGS)6 (SEQ ID NO:33), or (SGGGG)6 (SEQ ID NO:34). [0180] Any of the linkers provided in Table 8 below can be used to directly join any effector moiety polypeptide described in Table 1 to any receptor polypeptide described in Table 2. Any of the linkers provided in Table 8 below can be used to directly join any multi- chain carrier ( e.g . as described in Tables 4, 5, and 6) or single-chain carrier ( e.g ., as described in Table 7) to an effector moiety polypeptide ( e.g ., as described in Table 1) and/or a receptor polypeptide (e.g., as described in Table 2).
[0181] Table 8: Linker Sequences
Figure imgf000045_0001
[0182] In certain embodiments of the present invention, single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a single-chain or multi-chain carrier, a linker, an effector moiety polypeptide or functional fragment thereof, a linker, and a receptor polypeptide, or functional fragment thereof. In embodiments where the carrier is multi-chain, a linker joins two chains of the carrier.
[0183] In certain embodiments of the present invention, single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a single-chain or multi-chain carrier, a linker, a receptor polypeptide or functional fragment thereof, a linker, and an effector moiety polypeptide or functional fragment thereof. In embodiments where the carrier is multi-chain, a linker joins two chains of the carrier.
[0184] In certain embodiments of the present invention, single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a receptor polypeptide or functional fragment thereof, a linker, an effector moiety polypeptide or functional fragment thereof, a linker, and a single-chain or multi-chain carrier. In embodiments where the carrier is multi- chain, a linker joins two chains of the carrier.
[0185] In certain embodiments of the present invention, single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, an effector moiety polypeptide or functional fragment thereof, a linker, a receptor polypeptide or functional fragment thereof, a linker, and a single-chain or multi-chain carrier. In embodiments where the carrier is multi- chain, a linker joins two chains of the carrier. [0186] In certain embodiments of the present invention, single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a first chain of a multi-chain carrier, a linker, a receptor polypeptide or functional fragment thereof, a linker, an effector moiety polypeptide or functional fragment thereof, a linker, and a second chain of the multi-chain carrier.
[0187] In certain embodiments of the present invention, single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a first chain of a multi-chain carrier, a linker, an effector moiety polypeptide or functional fragment thereof, a linker, a receptor polypeptide or functional fragment thereof, a linker, and a second chain of the multi-chain carrier.
[0188] In certain embodiments of the present invention, single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, an effector moiety polypeptide or functional fragment thereof, a linker, a single-chain or multi-chain carrier, a linker, and a receptor polypeptide or functional fragment thereof. In embodiments where the carrier is multi-chain, a linker joins two chains of the carrier.
[0189] In certain embodiments of the present invention, single-chain polypeptides comprise, in sequence from N-terminus to C-terminus, a receptor polypeptide or functional fragment thereof, a linker, a single-chain or multi-chain carrier, a linker, and an effector moiety polypeptide or functional fragment thereof. In embodiments where the carrier is multi-chain, a linker joins two chains of the carrier.
[0190] For example, in some embodiments, the effector moiety polypeptide comprises IL-15 or a functional fragment thereof. In certain examples, the effector moiety polypeptide comprises a polypeptide at least 90% identical to SEQ ID NO:2.
[0191] For example, in certain embodiments, the receptor polypeptide comprises an IL- 15Ra polypeptide or functional fragment thereof, or an IL-15Rα sushi domain. In certain examples, the receptor polypeptide comprises a polypeptide at least 90% identical to SEQ ID NO: 13, 16, or 21.
[0192] For example, in certain embodiments, the multi-chain carrier comprises a unit comprising an IgGl, IgG2, IgG3, or IgG4 Fc domain. In certain examples, the multi-chain carrier comprises a first IgGl Fc domain polypeptide and second IgGl Fc domain polypeptide having a sequence at least 90% identical to SEQ ID NO: 24 or 25. [0193] For example, in some embodiments, the single-chain carrier comprises an HSA polypeptide or a functional fragment thereof, or an anti-HSA nanobody (VHH). In certain embodiments, the single-chain carrier comprises a polypeptide having a sequence at least 90% identical to SEQ ID NO:27.
[0194] For example, in some embodiments, a linker comprises a polypeptide having a sequence according to SEQ ID NO: 31, 32, 33 or 34.
[0195] In certain exemplary embodiments, the present disclosure provides, but is not limited to, single-chain polypeptides as provided in Table 9 (amino acid sequences of an effector moiety (IL-15) and a receptor (IL-15Rα) are in bold; bolded and underlined amino acid residues are mutations (compared to the human wild-type IgGl sequence) that reduce effector function of the IgGl Fc domain; italicized amino acid residues represent either a section of an IgGl hinge or a linker (G4S)n (SEQ ID NO: 67); bolded and italicized (but not underlined) amino acid residues represent a fragment of the IL-15Rα). In certain embodiments, single-chain polypeptides of the present disclosure have the sequence according to SEQ ID NO: 37, SEQ ID NO:71, or SEQ ID NO:72.
[0197] Table 9: Exemplary Single-Chain Polypeptides
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
METHODS OF PREPARATION
[0198] The proteins of the present invention can be made using recombinant DNA technology well known to a skilled person in the art. For example, a first nucleic acid sequence encoding a first polypeptide comprising a first immunoregulatory polypeptide linked to a first antibody Fc domain polypeptide can be cloned into a first expression vector; a second nucleic acid sequence encoding a second polypeptide comprising a second, different immunoregulatory polypeptide linked to a second, different antibody Fc domain polypeptide can be cloned into a second expression vector; and the first and the second expression vectors can be stably transfected together into host cells to produce the single-chain polypeptide. [0199] To achieve the highest yield of the protein, different ratios of the first and second expression vectors can be explored to determine the optimal ratio for transfection into the host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.
Clones can be cultured under conditions suitable for bio-reactor scale-up and maintained expression of the proteins of the present invention. The proteins can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.
CHARACTERISTICS OF THE SINGLE-CHAIN POLYPEPTIDES
[0200] In certain embodiments, a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15Rα or a functional fragment thereof ( e.g ., sushi domain) has higher binding affinity for IL-15Rβ compared to wild-type IL-15. In certain embodiments, a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15Rα or a functional fragment thereof (e.g., sushi domain) has a binding affinity dissociation constant (KD) is 20 nM to 30 nM.
[0201] In certain embodiments, a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15Rα or a functional fragment thereof (e.g, sushi domain) induces enhanced signal transduction as compared to wild-type IL-15 when contacted with a cell expressing the IL-15R.
[0202] In certain embodiments, a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15Rα or a functional fragment thereof (e.g, sushi domain) induces enhanced STAT5 phosphorylation as compared to wild- type IL-15 when contacted with the cell expressing the IL-15R.
[0203] In certain embodiments, a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15Rα or a functional fragment thereof (e.g, sushi domain) induces enhanced STAT5 phosphorylation as compared to wild- type IL-15 when contacted with the cell expressing the IL-15R, wherein the cell is a CD8+ T- cell, a natural killer (NK) cell, a natural killer T-cell (NKT cell), a CD4+ T-cell, or a regulatory T-cell (Treg).
[0204] In certain embodiments, a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15Rα or a functional fragment thereof (e.g, sushi domain) induces proliferation and/or activation of one or more than one immune cell selected from the group consisting of CD8+ T-cells, a NK cells, NKT cells, CD4+ T-cells, and Tregs, upon administered to a subject.
[0205] In certain embodiments, a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15Rα or a functional fragment thereof ( e.g ., sushi domain) induces proliferation of the one or more than one immune cell after about 4 days (e.g., 4 days, 5 days, 6 days, or 7 days).
[0206] In certain embodiments, a single-chain polypeptide of the present invention comprising IL-15 or a functional fragment thereof, and IL-15Rα or a functional fragment thereof (e.g, sushi domain) induces the expression of one or more than one protein in the one or more immune cells selected from the group consisting of CD25, Ki-67, NKG2D, and/or granzyme B (GrzB) after about 4 days (e.g, 4 days, 5 days, 6 days, or 7 days).
PHARMACEUTICAL COMPOSITIONS
[0207] The present disclosure also features pharmaceutical compositions that contain a therapeutically effective amount of a single-chain polypeptide described herein. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or vehicles can also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g, Langer (Science 249:1527-1533, 1990).
[0208] The intravenous drug delivery formulation of the present disclosure may be contained in a bag, a pen, or a syringe. In certain embodiments, the bag may be connected to a channel comprising a tube and/or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and contained in about 12-60 vials. In certain embodiments, the formulation may be freeze-dried and 45 mg of the freeze-dried formulation may be contained in one vial. In certain embodiments, the about 40 mg - about 100 mg of freeze-dried formulation may be contained in one vial. In certain embodiments, the freeze- dried formulation from 12, 27, or 45 vials are combined to obtain a therapeutic dose of the single-chain polypeptide in the intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and stored as about 250 mg/vial to about 1000 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 600 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 250 mg/vial. [0209] A single-chain polypeptide of the present invention could exist in a liquid aqueous pharmaceutical formulation including a therapeutically effective amount of the protein in a buffered solution forming a formulation.
[0210] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicles or excipients prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents.
The composition in solid form can also be packaged in a container for a flexible quantity.
[0211] In certain embodiments, the present disclosure provides a formulation with an extended shelf life including the single-chain polypeptide of the present disclosure, in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.
[0212] In certain embodiments, an aqueous formulation is prepared including the single- chain polypeptide of the present disclosure in a pH-buffered solution. The buffer of this invention may have a pH ranging from about 4 to about 8, e.g ., from about 4.5 to about 6.0, or from about 4.8 to about 5.5, or may have a pH of about 5.0 to about 5.2. Ranges intermediate to the above recited pH's are also intended to be part of this disclosure. For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included. Examples of buffers that will control the pH within this range include acetate (e.g, sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.
[0213] In certain embodiments, the formulation includes a buffer system which contains citrate and phosphate to maintain the pH in a range of about 4 to about 8. In certain embodiments the pH range may be from about 4.5 to about 6.0, or from about pH 4.8 to about 5.5, or in a pH range of about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system includes about 1.3 mg/mL of citric acid (e.g, 1.305 mg/mL), about 0.3 mg/mL of sodium citrate (e.g, 0.305 mg/mL), about 1.5 mg/mL of disodium phosphate dihydrate (e.g., 1.53 mg/mL), about 0.9 mg/mL of sodium dihydrogen phosphate dihydrate (e.g, 0.86), and about 6.2 mg/mL of sodium chloride (e.g, 6.165 mg/mL). In certain embodiments, the buffer system includes 1-
1.5 mg/mL of citric acid, 0.25 to 0.5 mg/mL of sodium citrate, 1.25 to 1.75 mg/mL of disodium phosphate dihydrate, 0.7 to 1.1 mg/mL of sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg/mL of sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.
[0214] A polyol, which acts as a tonicifier and may stabilize the antibody, may also be included in the formulation. The polyol is added to the formulation in an amount which may vary with respect to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation may be isotonic. The amount of polyol added may also be altered with respect to the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g, mannitol) may be added, compared to a disaccharide (such as trehalose). In certain embodiments, the polyol which may be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the mannitol concentration may be about 5 to about 20 mg/mL. In certain embodiments, the concentration of mannitol may be about
7.5 to 15 mg/mL. In certain embodiments, the concentration of mannitol may be about 10-14 mg/mL. In certain embodiments, the concentration of mannitol may be about 12 mg/mL. In certain embodiments, the polyol sorbitol may be included in the formulation.
[0215] A detergent or surfactant may also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g, polysorbates 20, 80 etc.) or poloxamers (e.g, poloxamer 188). The amount of detergent added is such that it reduces aggregation of the formulated single-chain polypeptide and/or minimizes the formation of particulates in the formulation and/or reduces adsorption. In certain embodiments, the formulation may include a surfactant which is a polysorbate. In certain embodiments, the formulation may contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitanmonooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the formulation may contain between about 0.1 mg/mL and about 10 mg/mL of polysorbate 80, or between about 0.5 mg/mL and about 5 mg/mL. In certain embodiments, about 0.1% polysorbate 80 may be added in the formulation.
[0216] In embodiments, the single-chain polypeptide product of the present disclosure is formulated as a liquid formulation. The liquid formulation may be presented at a 10 mg/mL concentration in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of elastomer complying with USP and Ph Eur. In certain embodiments vials may be filled with 61.2 mL of the protein product solution in order to allow an extractable volume of 60 mL. In certain embodiments, the liquid formulation may be diluted with 0.9% saline solution.
[0217] In certain embodiments, the liquid formulation of the disclosure may be prepared as a 10 mg/mL concentration solution in combination with a sugar at stabilizing levels. In certain embodiments the liquid formulation may be prepared in an aqueous vehicles or excipients. In certain embodiments, a stabilizer may be added in an amount no greater than that which may result in a viscosity undesirable or unsuitable for intravenous administration. In certain embodiments, the sugar may be disaccharides, e.g ., sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffering agent, a surfactant, and a preservative.
[0218] In certain embodiments, the pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and/or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide.
[0219] In addition to aggregation, deamidation is a common product variant of peptides and proteins that may occur during fermentation, harvest/cell clarification, purification, drug substance/drug product storage and during sample analysis. Deamidation is the loss of N¾ from a protein forming a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate results in a 17 dalton mass decrease of the parent peptide. The subsequent hydrolysis results in an 18 dalton mass increase. Isolation of the succinimide intermediate is difficult due to instability under aqueous conditions. As such, deamidation is typically detectable as 1 dalton mass increase. Deamidation of an asparagine results in either aspartic or isoaspartic acid. The parameters affecting the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation and tertiary structure. The amino acid residues adjacent to Asn in the peptide chain affect deamidation rates. Gly and Ser following an Asn in protein sequences results in a higher susceptibility to deamidation.
[0220] In certain embodiments, the liquid formulation of the present disclosure may be preserved under conditions of pH and humidity to prevent deamination of the protein product. [0221] The aqueous vehicle or excipient of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation. Illustrative vehicles or excipients include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution ( e.g ., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
[0222] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.
[0223] Intravenous (IV) formulations may be the preferred administration route in particular instances, such as when a patient is in the hospital after transplantation receiving all drugs via the IV route. In certain embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.
[0224] In certain embodiments, a salt or buffer components may be added in an amount of 10 mM - 200 mM. The salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion.
[0225] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.
[0226] The aqueous vehicle excipient of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation. Illustrative vehicles or excipients include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
[0227] The single-chain polypeptide of the present disclosure could exist in a lyophilized formulation including the single-chain polypeptides and a lyoprotectant. The lyoprotectant may be sugar, e.g, disaccharides. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and/or a preservative.
[0228] The amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1 :2 protein to sucrose or maltose. In certain embodiments, the protein to sucrose or maltose weight ratio may be of from 1 :2 to 1:5.
[0229] In certain embodiments, the pH of the formulation, prior to lyophilization, may be set by addition of a pharmaceutically acceptable acid and/or base. In certain embodiments the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base may be sodium hydroxide.
[0230] Before lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted between 6 to 8. In certain embodiments, the pH range for the lyophilized drug product may be from 7 to 8.
[0231] In certain embodiments, a salt or buffer components may be added in an amount of 10 mM - 200 mM. The salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion.
[0232] In certain embodiments, a “bulking agent” may be added. A “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake ( e.g ., facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents.
[0233] A preservative may be optionally added to the formulations herein to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.
[0234] In certain embodiments, the lyophilized drug product may be constituted with an aqueous vehicle or excipient. The aqueous vehicle or excipient of interest herein is one which is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization. Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution ( e.g ., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
[0235] In certain embodiments, the lyophilized drug product of the current disclosure is reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% sodium chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into a solution.
[0236] In certain embodiments, the lyophilized protein product of the instant disclosure is constituted to about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution).
[0237] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
[0238] The specific dose can be a uniform dose for each patient, for example, 50-5000 mg of protein. Alternatively, a patient’s dose can be tailored to the approximate body weight or surface area of the patient. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially in light of the dosage information and assays disclosed herein. The dosage can also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data. An individual patient's dosage can be adjusted as the progress of the disease is monitored. Blood levels of the targetable construct or complex in a patient can be measured to see if the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics may be used to determine which targetable constructs and/or complexes, and dosages thereof, are most likely to be effective for a given individual (Schmitz et al., Clinica ChimicaActa 308: 43-53, 2001; Steimer et al ., Clinica ChimicaActa 308: 33-41, 2001).
[0239] In general, dosages based on body weight are from about 0.01 μg to about 100 mg per kg of body weight, such as about 0.01 μg to about 100 mg/kg of body weight, about 0.01 μg to about 50 mg/kg of body weight, about 0.01 μg to about 10 mg/kg of body weight, about 0.01 μg to about 1 mg/kg of body weight, about 0.01 μg to about 100 μg/kg of body weight, about 0.01 μg to about 50 μg/kg of body weight, about 0.01 μg to about 10 μg/kg of body weight, about 0.01 μg to about 1 μg/kg of body weight, about 0.01 μg to about 0.1 μg/kg of body weight, about 0.1 μg to about 100 mg/kg of body weight, about 0.1 μg to about 50 mg/kg of body weight, about 0.1 μg to about 10 mg/kg of body weight, about 0.1 μg to about 1 mg/kg of body weight, about 0.1 μg to about 100 μg/kg of body weight, about 0.1 μg to about 10 μg/kg of body weight, about 0.1 μg to about 1 μg/kg of body weight, about 1 μg to about 100 mg/kg of body weight, about 1 μg to about 50 mg/kg of body weight, about 1 μg to about 10 mg/kg of body weight, about 1 μg to about 1 mg/kg of body weight, about 1 μg to about 100 μg/kg of body weight, about 1 μg to about 50 μg/kg of body weight, about 1 μg to about 10 μg/kg of body weight, about 10 μg to about 100 mg/kg of body weight, about 10 μg to about 50 mg/kg of body weight, about 10 μg to about 10 mg/kg of body weight, about 10 μg to about 1 mg/kg of body weight, about 10 μg to about 100 μg/kg of body weight, about 10 μg to about 50 μg/kg of body weight, about 50 μg to about 100 mg/kg of body weight, about 50 μg to about 50 mg/kg of body weight, about 50 μg to about 10 mg/kg of body weight, about 50 μg to about 1 mg/kg of body weight, about 50 μg to about 100 μg/kg of body weight, about 100 μg to about 100 mg/kg of body weight, about 100 μg to about 50 mg/kg of body weight, about 100 μg to about 10 mg/kg of body weight, about 100 μg to about 1 mg/kg of body weight, about 1 mg to about 100 mg/kg of body weight, about 1 mg to about 50 mg/kg of body weight, about 1 mg to about 10 mg/kg of body weight, about 10 mg to about 100 mg/kg of body weight, about 10 mg to about 50 mg/kg of body weight, or about 50 mg to about 100 mg/kg of body weight.
[0240] Doses may be given once or more times daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the heterodimer Fc- fused protein in bodily fluids or tissues. Administration of the present invention could be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter or by direct intralesional injection. This may be administered once or more times daily, once or more times weekly, once or more times monthly, and once or more times annually.
THERAPEUTIC APPLICATIONS
[0241] The invention provides methods for treating cancer using a single-chain polypeptide described herein and/or a pharmaceutical composition described herein. The methods may be used to treat a variety of cancers by administering to a patient in need thereof a therapeutically effective amount of the single-chain polypeptide described herein. In some embodiments, the cancer that is treated by a method disclosed herein is a locally advanced malignancy. In some embodiments, the locally advanced malignancy can be fully resected. In some embodiments, the locally advanced malignancy has been fully resected, and the treatment is provided subsequent to the resection.
[0242] In some embodiments, a single-chain polypeptide of the present invention ( e.g ., a single-chain polypeptide comprising 11-15 and IL-15Rα) is used for treating an advanced malignancy as a monotherapy. In some embodiments, a single-chain polypeptide of the present invention (e.g., a single-chain polypeptide comprising IL-15 and IL-15Rα) is used as an adjuvant for active immunotherapy of severe infectious diseases. In some embodiments, a single-chain polypeptide of the present invention (e.g, a single-chain polypeptide comprising IL-15 and IL-15Rα) is used as an adjuvant for prophylactic vaccination. In some embodiments, a single-chain polypeptide of the present invention is used for treating myelosuppression following radiotherapy.
[0243] The present invention also provides a method of reducing hematopoietic toxicity. Hematopoietic toxicity can result from genetic, infective, or environmental causes, including but not limited to irradiation and chemotherapy. For example, in certain embodiments, the present invention provides a method of treating a disease or disorder associated with radiation (e.g, ionizing radiation, alpha radiation, beta radiation, gamma radiation, X radiation, or neutron radiation). For example, in certain embodiments, the present invention provides a method of treating myelosuppression following a radiation therapy, the method comprising administering to a patient in need thereof a therapeutically effective amount of a single-chain polypeptide or a formulation described herein. In certain embodiments, the dose of the radiation therapy is at least 1, 5, 10, 15, or 20 Gy. In certain embodiments, the radiation therapy causes damage in a system, organ, or tissue selected from the group consisting of bone marrow, lymphatic system, immune system, mucosal tissue, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lung, kidney, skin and brain. In certain embodiments, the method of the present invention reduces the damage.
[0244] The methods provided herein are useful in treating myelosuppression resulting from radiation. Accordingly, in certain embodiments, the present invention provides a method of treating myelosuppression occurring in the context of an accidental exposure to radiation, the method comprising administering to a patient in need thereof a therapeutically effective amount of a single-chain polypeptide or a formulation described herein. [0245] In certain embodiments, the present invention provides a method of treating acute radiation syndrome (ARS), the method comprising administering to a patient in need thereof a therapeutically effective amount of a single-chain polypeptide or a formulation described herein. ARS includes but is not limited to hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, and cutaneous radiation syndrome. For example, hematopoietic radiation syndrome results from, at least in part, depletion of hematopoietic stem cell pool and shows signs of lymphopenia and granulocytopenia. Gastrointestinal syndrome results from, at least in part, damage of stem cells and progenitor cells located in the crypts and failure to replace the cells in the surface of the villi and shows signs of watery diarrhea, dehydration, electrolyte loss, gastrointestinal bleeding, and perforation. In certain embodiments, the method of treatment provided herein is conducted at the prodromal phase of the ARS. The prodromal phase is the initial phase of acute illness, characterized by the symptoms of nausea, vomiting, anorexia, fever, headache, and/or early skin erythema, typically within 1-3 days after the exposure to radiation. In certain embodiments, the method of treatment provided herein is conducted at the latent phase of the ARS. The latent phase is a phase characterized by improvement of symptoms but exhibition of lymphopenia and granulocytopenia in lab tests, and may last hours to weeks depending on the dose of exposure. Treatment in the prodromal phase or latent phase may mitigate the development of the syndromes in the affected systems, organs, and/or tissues. In certain embodiments, the method of treatment provided herein is conducted at the manifest illness phase of the ARS. Treatment in this phase may still promote recovery from the ARS.
[0246] The present invention also provides a method of increasing the survival, proliferation, differentiation, and/or activity of an immune cell, the method comprising contacting the immune cell with a single-chain polypeptide or a formulation disclosed herein. In certain embodiments, the immune cell is a T cell ( e.g ., CD4+ T cells). In certain embodiments, the immune cell is an NK cell.
[0247] Certain of the single-chain polypeptides disclosed herein are useful in increasing or modulating the survival, proliferation, and/or activity of an immune cell. Accordingly, in another aspect, the instant disclosure provides a method of increasing or modulating the survival, proliferation, and/or activity of an immune cell, the method comprising contacting the immune cell with a single-chain polypeptide, pharmaceutical composition, or formulation disclosed herein. In certain embodiments, the immune cell is a T cell (e.g., a CD8+ T cell, a CD4+ T cell, a regulatory T cell, or an NKT cell) or an NK cell. [0248] In certain embodiments, the immune cell is contacted with the single-chain polypeptide, pharmaceutical composition, or formulation in vivo for preventing or treating a disease or disorder. The diseases and disorders suitable for such treatment include but are not limited to cancer and infection ( e.g ., viral infection, bacterial infection, or parasitic infection). Accordingly, in certain embodiments, the invention provides methods for treating cancer using a single-chain polypeptide described herein and/or a pharmaceutical composition described herein. The methods may be used to treat a variety of cancers by administering to a patient in need thereof an effective amount of a single-chain polypeptide described herein.
[0249] In some embodiments, single-chain polypeptides or compositions disclosed herein can be administered to a subject to treat a disorder associated with abnormal apoptosis or a differentiative process (e.g., cellular proliferative disorders or cellular differentiative disorders, such as cancer, by, for example, producing an active or passive immunity). In the treatment of such diseases, the disclosed Single-chain polypeptides comprising IL-15 and IL- 15Ra may possess advantageous properties, such as reduced vascular leak syndrome. Examples of cellular proliferative and/or differentiative disorders include cancer (e.g, carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g, leukemias). A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver.
[0250] The single-chain polypeptides comprising IL-15 and IL-15Rα can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma, or any viral disease. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
[0251] Additional examples of proliferative disorders include hematopoietic neoplastic disorders.
[0252] Other examples of proliferative and/or differentiative disorders include skin disorders. The skin disorder may involve the aberrant activity of a cell or a group of cells or layers in the dermal, epidermal, or hypodermal layer, or an abnormality in the dermal- epidermal junction. For example, the skin disorder may involve aberrant activity of keratinocytes (e.g, hyperproliferative basal and immediately suprabasal keratinocytes), melanocytes, Langerhans cells, Merkel cells, immune cell, and other cells found in one or more of the epidermal layers, e.g, the stratum basale (stratum germinativum), stratum spinosum, stratum granulosum, stratum lucidum or stratum comeum. In other embodiments, the disorder may involve aberrant activity of a dermal cell, for example, a dermal endothelial, fibroblast, immune cell (e.g, mast cell or macrophage) found in a dermal layer, for example, the papillary layer or the reticular layer.
[0253] In some embodiments, a single-chain polypeptide or composition disclosed herein can be administered to a patient who has a viral infection (e.g, influenza, herpesvirus such as Epstein-Barr virus or cytomegalovirus). T cells (e.g, CD8+ T cells) are typically suppressed and/or exhausted in patients with chronic viral infection, and an increase of T cell population and/or activity may augment anti-viral immunity. Therefore, in some embodiments, the viral infection that can be treated by a single-chain polypeptide or composition disclosed herein is a chronic (e.g, persistent or recurrent) viral infection. In some embodiments, the patient who has a chronic viral infection is an AIDS patient.
[0254] In some embodiments, a single-chain polypeptide of the present invention (e.g, a single-chain polypeptide comprising IL-15 and IL-15Rα) is used for treating an advanced malignancy or a chronic viral infection as a monotherapy. In some embodiments, a single- chain polypeptide of the present invention (e.g, a single-chain polypeptide comprising IL-15 and IL-15Rα) is used as an adjuvant for active immunotherapy of cancer or chronic infectious diseases. In some embodiments, a single-chain polypeptide of the present invention (e.g, a single-chain polypeptide comprising IL-15 and IL-15Rα) is used for treating is used as an adjuvant for prophylactic vaccination. The adjuvant can be administered prior to or subsequent to the administration of a vaccine. Alternatively, the adjuvant and the vaccine can be administered together in a single composition.
V. COMBINATION THERAPY
[0255] Another aspect of the invention provides for combination therapy. A single-chain polypeptide described herein can be used in combination with additional therapeutic agents to treat the cancer.
[0256] In some embodiments, a single-chain polypeptide of the present invention (e.g, a single-chain polypeptide comprising IL-15 and IL-15Rα) is used in treating an advanced malignancy in combination with another therapeutic agent selected from: cytotoxic chemotherapy; radiotherapy; an antibody that targets a molecule involved in an anti-tumor immune response, such as CTLA-4, PD-1, PD-L1, or TGF-b; an antibody that acts by ADCC on a tumor-associated antigen; a multispecific antibody binding NKG2D, CD 16, and a tumor- associated antigen, optionally administered in combination with an antibody that targets PD-1 or PD-L1; a personalized cancer vaccine; an oncolytic cancer vaccine; and a personalized vaccine administered in combination with an antibody that targets PD-1 or PD-L1.
[0257] In some embodiments, a single-chain polypeptide of the present invention ( e.g ., a single-chain polypeptide comprising IL-15 and IL-15Rα) is used in treating locally advanced malignancy that can be fully resected, in combination with a cancer vaccine or an antibody that targets PD-1 or PD-L1.
[0258] Single-chain polypeptides of the invention can also be used as an adjunct to surgical removal of the primary lesion.
[0259] The amount of single-chain polypeptide and additional therapeutic agent and the relative timing of administration may be selected in order to achieve a desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the therapeutic agents in the combination, or a pharmaceutical composition or compositions comprising the therapeutic agents, may be administered in any order such as, for example, sequentially, concurrently, together, simultaneously and the like. Further, for example, a single-chain polypeptide may be administered during a time when the additional therapeutic agent(s) exerts its prophylactic or therapeutic effect, or vice versa.
[0260] The description above describes multiple aspects and embodiments of the invention. The patent application specifically contemplates all combinations and permutations of the aspects and embodiments.
EXAMPLES
[0261] The invention now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and is not intended to limit the invention.
Example 1 - Method of Preparation
[0262] The single-chain polypeptides of the present invention were made using recombinant DNA technology.
[0263] Methods of generating recombinant DNA plasmids for protein production are well known in the art. To achieve the highest yield of the protein, different amounts of the expression vector can be explored as known in the art to determine the optimal ratio for transfection into the host cells. After transfection, single clones are isolated for cell bank generation using methods known in the art, such as limited dilution, ELISA, FACS, microscopy, or Clonepix.
[0264] For scaling up, the clones are cultured under conditions suitable for bio-reactor scale-up and maintained expression of the proteins. The proteins are isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thawing, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed-mode chromatography.
[0265] Nucleic acids comprising sequences encoding IL-15Rα sushi domain (SEQ ID NO:21); human IgGl Fc domain having heterodimerization mutations and effector silencing mutations (SEQ ID NO:25), murine IgGl having heterodimerization mutations and effector silencing mutations, or anti-human serum albumin (HSA) nanobody (VHH); IL-15 (SEQ ID NO:2) or IL-15 having a S73A mutation (SEQ ID NO:69), and linkers (SEQ ID NOs: 32 or 33) can be synthesized or PCR amplified, restriction digested and/or ligated together to form a contiguous nucleic acid encoding, in sequence from N to C-terminus, the following single- chain polypeptides (the single-chain polypeptides described below include polypeptides that are human (hu or h) or murine (mu or m)):
(i) human (hu) IgGl Fc-domain chain - Linker - huIgGl Fc-domain chain - Linker - huIL-15Rα sushi domain- Linker -huIL-15, and wherein the Fc-domain chains each comprise heterodimerization mutations and LALAPA effector function silencing mutations (Single-chain Polypeptide A; schematically represented in FIG. 2A);
(ii) huIL-15Rα sushi domain - Linker - huIgGl Fc-domain chain - Linker - huIgGl Fc-domain chain - Linker - huIL-15, and wherein the Fc-domain chains each comprise heterodimerization mutations and LALAPA effector function silencing mutations (Single- chain Polypeptide B; schematically represented in FIG. 4D and having the amino acid sequence of SEQ ID NO: 37);
(iii) anti -HSA nanobody (VHH) - Linker - huIL-15Rα sushi domain - Linker - huIL- 15 (Single-chain Polypeptide C; schematically represented in FIG. 3 A);
(iv) huIL-15 - Linker - anti- HSA nanobody (VHH) - Linker - huIL-15Rα sushi domain (Single-chain Polypeptide D; schematically represented in FIG. 5A); (v) huIL-15Rα sushi domain - Linker - anti- HSA nanobody (VHH) - Linker - huIL- 15 (Single-chain Polypeptide E; schematically represented in FIG. 5B);
(vi) huIL-15Rα sushi domain - Linker - murine (mu) IgG Fc-domain chain - Linker - muIgG Fc-domain chain - Linker - huIL-15, and wherein the Fc-domain chains each comprise heterodimerization mutations and LALAPA effector function silencing mutations (Single-chain Polypeptide F; schematically represented in FIG. 4D);
(vii) huIL-15Rα sushi domain - Linker - muIgG Fc-domain chain - Linker - muIgG Fc-domain chain - Linker - huIL-15 having a S73A mutation in the huIL-15 seuqence, and wherein the Fc-domain chains each comprise heterodimerization mutations and LALAPA effector function silencing mutations (Single-chain Polypeptide G; schematically represented in FIG. 4D);
(viii) huIL-15Rα sushi domain- Linker - muIgG Fc-domain chain - Linker - muIgG Fc-domain chain - Linker - huIL-15 having S73A and H105E mutations in the huIL-15 sequence, and wherein the Fc-domain chains each comprises heterodimerization mutations and LALAPA effector function silencing mutations (Single-chain Polypeptide H; schematically represented in FIG. 4D); and
(ix) human IL-15Rα sushi domain - Linker - human IL-15 (RLI).
[0266] Nucleic acids can be cloned into an expression vector (pET-pSURE-Puro) and stably transfected into host cells to produce the single-chain polypeptides.
Example 2 - Characterization of Single-Chain Polypeptides Comprising Fc-domain Carrier
[0267] Single-chain polypeptides were collected from host cell lysates, purified by size exclusion chromatography (SEC) (data not shown), and analyzed by SDS- polyacrylamide gel electrophoresis (PAGE) under reducing and non-reducing conditions.
[0268] When analyzed by SEC on a Superdex200 column, Single-chain Polypeptide A, and Single-chain Polypeptide B, appeared as single peaks, eluting off the column at fractions 110.29 and 112.47, respectively (data not shown) indicating that the proteins are expressed and behave as properly folded proteins.
[0269] Single-chain Polypeptide A and Single-chain Polypeptide B were further assessed by SDS-PAGE under reducing and non-reducing conditions to monitor the size and amount of intact protein as well as any degradation fragments or protein aggregates. FIG. 6 shows that despite the presence of a long linker (30 amino acids) connecting the two Fc chains, Single-chain Polypeptide A and Single-chain Polypeptide B, each resolved as a single band under reducing conditions without the presence of protein smearing or degradation products, suggesting proper protein folding (FIG. 6A). The larger molecular weight bands observed under non-reducing conditions suggests dimerization, which may be an artifact of the conditions used for analysis (FIG. 6B).
Example 3 - Characterization of a Single-Chain IL-15Ra-Linker-IL-15 Polypeptide (RLI) Comprising an Anti-Human Serum Albumin (HSA) Nanobody (VHH)
[0270] This example describes characterization of Single-chain Polypeptide C, by SEC and SDS-PAGE.
[0271] When analyzed by SEC on a Superdex 200 column, recombinantly expressed Single-chain Polypeptide C appeared as two peaks eluting at fractions 127.91 and 131.45 (data not shown). As shown in FIG. 7A, SDS PAGE of the elution fractions of each peak (“1”, “2”, as well as transition fractions between the two peaks “T”) confirmed that the proteins resolved as single bands having different molecular sizes.
[0272] Since treatment of peripheral blood mononuclear cells (PBMCs) with IL-15 results in the production of IL-2, the in vitro biological activity of Single-chain Polypeptide C was assayed using a HEK Blue IL-2 reporter assay.
[0273] IL-2R+ HEK-Blue reporter cells (InvivoGen) were harvested from culture and adjusted to lxlO6 cells/mL in culture media. Single-chain Polypeptide C, Reference Control 1, and RLI were diluted in media. 100 μL of PBMC suspension was mixed with 100 pL of diluted test polypeptide and incubated for 48 hours. The supernatant was harvested and contacted with the HEK-Blue reporter cells. Activation of signaling pathways downstream of the IL-2 receptor was detected by measurement of secreted embryonic alkaline phosphatase from the IL-2R+ HEK-Blue reporter cells following the manufacturer’s instructions. Briefly, 25 pL of supernatant was mixed with 200 pL of QUANTI-Blue reagent and incubated in the dark at RT for 10 minutes. The plate was then read with a SpectraMax i3x plate-reader at 620 nM and optical density reported to represent relative IL-2 production activity.
[0274] As shown in FIG. 7B, at the concentrations examined, the Single-chain Polypeptide C eluting in SEC peak 2 (P2) and Single-chain Polypeptide C eluting in SEC peak 1 (PI) have comparable dose responses in the IL-2 reporter assay as compared to the RLI construct without a carrier, and both have significantly increased potency in inducing IL- 2 production as compared to Reference Control 1 at the concentrations examined.
Example 4 - In vitro Characterization of Single-Chain Polypeptides
[0275] This example shows that single-chain polypeptides of the present invention are significantly more potent in inducing IL-2 production PBMCs as compared to Reference Control 1, and have comparable potencies as compared to RLI and human IL-15. This example also demonstrates that the IL-15 mutations in Single-chain Polypeptides G and H do not significantly impact potency in inducting IL-2 production as compared to Single-chain Polypeptide F ( i.e . comprising wild-type IL-15 sequence).
[0276] The in vitro biological activities of Single-chain Polypeptide A and Single-chain Polypeptide B were assayed using a HEK Blue IL-2 reporter assay as described in Example 3. As shown in FIGs. 8 A, 8B, 8C (experiments performed in triplicate) and Table 10, at the concentrations examined, the dose responses in the HEK-Blue IL-2 reporter assay were comparable between Single-chain Polypeptides A and B and RLI. All single-chain polypeptides tested (Single-chain Polypeptide A, Single-chain Polypeptide B, and RLI) showed significantly greater potency in inducing IL-2 production as compared to Reference Control 1. For example, the average EC50 for Reference Control 1 was 10.778 nM with a standard deviation of 15.289 nM, whereas the average EC50 for Single-chain Polypeptide A was 0.019 nM with a standard deviation of 0.0188 nM, the average EC50 for Single-chain Polypeptide B was 0.012 nM with a standard deviation of 0.0008 nM, and the average EC50 for RLI was 0.0182 nM with a standard deviation of 0.007 nM.
[0277] Table 10: Single-chain polypeptides show similar potency in the HEK Blue IL-2 assay
Figure imgf000083_0001
[0278] As shown in FIG. 9, Single-chain Polypeptide G and Single-chain Polypeptide H, comprising S73A and S73A H105E mutations in IL-15 respectively, exhibit comparable dose responses in inducing IL-2 production in the HEK-Blue IL-2 reporter assay as compared to Single-chain Polypeptide F (comprising wild-type IL-15). All of Single-chain Polypeptides F, G, and H have comparable potency in inducing IL-2 production in the HEK- Blue assay as compared to RLI and human IL-15, and all exhibit significantly greater potency in inducing IL-2 production in PBMCs as compared to Reference Control 1.
Example 5 - STAT5 Phosphorylation Induced by Single-chain Polypeptides
[0279] This example shows that single-chain polypeptides of the present invention have comparable potencies in inducing signaling via the IL2/IL-15 common b/g receptor and significantly greater potency as compared to Reference Control 1.
[0280] STAT5 is a signaling molecule in the IL-2 pathway. Activation of the IL-2/IL-15 common b/g receptor induces STAT5 phosphorylation. The STAT5 phosphorylation in KHYG-1 NK cells treated with IL-15Rα-IL-15-Fc Single-chain Polypeptides was measured by flow cytometry using phospho-specific STAT5 antibody.
[0281] As shown in FIGs. 10A, 10B and IOC (experiments performed in triplicate), Single-chain Polypeptide A and Single-chain Polypeptide RLI, and Reference Control 1 exhibited dose-dependent increases in STAT5 phosphorylation in KHYG-1 NK cells. As shown in Table 11, the average EC50 for Single-chain Polypeptide A was about three-fold greater than the average EC50 for Single-chain Polypeptide B. For example, the average EC50 for Single-chain Polypeptide A was 1.665 nM with a standard deviation of 2.610 nM and the average EC50 for Single-chain Polypeptide B was 0.505 nM with a standard deviation of 0.686 nM. All single-chain polypeptides tested (Single-chain Polypeptide A, Single-chain Polypeptide B, and RLI) showed significantly greater potency in inducing STAT5 phosphorylation compared to Reference Control 1 at the concentrations examined. For example, the average EC50 for Reference Control 1 was 10.524 nM with a standard deviation of 9.930 nM, whereas the average EC50 for Single-chain Polypeptide A was 1.665 nM with a standard deviation of 2.610 nM, the average EC50 for Single-chain Polypeptide B was 0.505 nM with a standard deviation of 0.686 nM, and the average EC50 for RLI was 0.040 nM with a standard deviation of 0.018 nM. [0282] Table 11: Single-chain polypeptides show similar potency in inducing STAT5 phosphorylation in KHYG-1 cells
Figure imgf000085_0001
[0283] As shown in FIG. 11, Single-chain Polypeptide F, Single-chain Polypeptide G, Single-chain Polypeptide H, human IL-15 (hu-IL-15), Reference Control 1, and Reference Control 2 all induced a dose-dependent increase in STAT5 phosphorylation in KHYG-1 NK cells. At the concentrations examined, the EC50 for Single-chain Polypeptides G and H were similar. Both, Single-chain Polypeptides G and H, comprising S73A and H105E mutations in IL-15 respectively, exhibited greater potency in inducing STAT5 phosphorylation as compared to Single-chain Polypeptide F comprising wild-type IL-15.
Example 6 - In vitro Immune Cell Proliferation by Single-chain Polypeptides
[0284] This example shows that single-chain polypeptides of the invention have potencies for inducing immune cell proliferation comparable to RLI and Reference Control 2. This example also demonstrates that single-chain polypeptides of the invention have a greater potency for inducing immune cell proliferation as compared to Reference Control 1.
[0285] The potencies of Single-chain Polypeptide A and Single-chain Polypeptide B for inducing immune cell proliferation, were measured by flow cytometry using antibodies specific for the Ki67 proliferation marker.
[0286] As shown in FIG. 12 and Table 12, Single-chain Polypeptide A and Single-chain Polypeptide B, RLI, Reference Control 1, and Reference Control 2 induced a dose-dependent increase in proliferation of CD8 T cells (FIG. 12A), NK cells (FIG. 12B), NKT cells (FIG. 12C), CD4 T cells (FIG. 12D), and Tregs (FIG. 12E). All single-chain polypeptides tested (Single-chain Polypeptide A, Single-chain Polypeptide B, and RLI) showed significantly greater potency in inducing proliferation of NK cells, NKT cells, CD8 T cells, CD4 T cells, and Tregs cells, as compared to Reference Control 1. Single-chain Polypeptide A and Single- chain Polypeptide B exhibited slightly lower potency for inducing immune cell proliferation as compared to RLI and Reference Control 2.
[0287] Table 12: Single-chain polypeptides show similar potency in the HEK Blue IL-2 assay
Figure imgf000086_0001
Example 7 - in vitro Immune Cell Activation by Single-chain Polypeptides
[0288] This example shows that single-chain polypeptides of the present invention induce dose-dependent increases in the expression of immune cell activation markers CD25, NKG2D and Granzyme B (GrzB) in CD8 T-cells, NK cells, NKT cells, and CD4 T-cells, and/or γδ T-cells. The potencies of Single-chain Polypeptides A and B are significantly greater than Reference Control 1, but slightly lower than RLI and Reference control 2.
[0289] The activities of Single-chain Polypeptide A and Single-chain Polypeptide B in inducing immune cell activation markers CD25, NKG2D and GrzB were assessed by flow cytometry using CD25, NKG2D, and GrzB-specific antibodies. [0290] As shown in FIG. 13 and Tables 13-15, Single-chain Polypeptide A and Single- chain Polypeptide B, RLI, Reference Control 1, and Reference Control 2, induced dose- dependent expression of activation markers CD25 (FIGs. 13A-D), NKG2D (FIGs. 13E-H), and GrzB (FIGs. 13I-L) in CD8 T cells (FIGs. 13A, 13E, and 131), NK cells (FIGs. 13B, 13F, and 13J), NKT cells (FIGs. 13D, 13G, and 13K), CD4 T cells (FIGs. 13C), and γδ T Cells (FIGs. 13H and 13L). All single-chain polypeptides tested (Single-chain Polypeptide A, Single-chain Polypeptide B, and RLI) showed significantly increased expression of CD25, NKG2D, and GrzB in all immune cells examined (CD8 T cells, NK cells, NKT cells and CD4 T cells) compared to Reference Control 1. Single-chain Polypeptide A and Single-chain Polypeptide B showed slightly lower activation compared to RLI and Reference Control 2.
[0291] Table 13: Activation of CD25 Expression in Immune Cell Subsets by Single- chain Polypeptides
Figure imgf000087_0001
[0292] Table 14: Activation of NKG2D Expression in Immune Cell Subsets by Single- chain Polypeptides
Figure imgf000087_0002
Figure imgf000088_0001
[0293] Table 15: Activation of GrzB Expression in Immune Cell Subsets by Single- chain Polypeptides
Figure imgf000088_0002
Example 8 - in vivo Expansion of Live Cells Induced by Single-chain Polypeptides [0294] This example shows that single-chain polypeptides of the present invention induce live cell expansion when administered to mice.
[0295] Mice were administered with treatment polypeptides according to the groupings and amounts indicated in TABLE 16 and fold-change of live cells in a peripheral blood sample was measured and compared to untreated mice. [0296] As shown in FIG. 14, mice treated with Reference Control 3, Reference Control
4, Single chain Polypeptide F, and Single-chain Polypeptide C, all exhibited an initial drop in the number of live cells in a sample on day 1 post-injection. Mice injected with human IL- 15, Reference Control 3, and Reference Control 4 demonstrated live cell expansion peaking at day 4 post-injection. An extended period of live cell expansion was observed in mice treated with Single-chain Polypeptide F and Single-chain Polypeptide C, with expansion observed even at 9 days post-injection.
[0297] Table 16 - Treatment Groups
Figure imgf000089_0001
Example 9 - in vivo Expansion of Specific Immune Cell Subsets Induced by Single- Chain Polypeptides
[0298] This example shows that single-chain polypeptides of the invention induce NK cell, NKT cell, and CD8 T cell expansion when injected into mice. [0299] Mice were administered with treatment polypeptides and measurements of the percentage of live NK cell, NKT cell and CD8 T cells at days 1, 4, and 9 post-injection were obtained.
[0300] As shown in FIG. 15 A, Single-chain Polypeptide F, Single-chain Polypeptide C, and Reference Control 4 induced a significant expansion in the percentage of live NK cells in treated mice, peaking at 4 days post-injection, whereas Reference Control 3 induced only a moderate expansion in treated mice. Elevated expansion of NK cells in mice treated with Single-chain Polypeptide F was observed even at 9 days post-injection. No significant increase in the percentage of NK cells was observed in mice treated with huIL15 or RLI.
[0301] As shown in FIG. 15B, Single-chain Polypeptide F, Single-chain Polypeptide C, Reference Control 3, and Reference Control 4 induced a significant expansion in the percentage of live NK cell in treatment mice, peaking at 4 days post injection. Elevated expansion of NKT cells in mice treated with Single-chain Polypeptide F was observed even at 9 days post-injection. No significant increase in the percentage of NKT cells was observed in mice treated with huIL15 or RLI.
[0302] As shown in FIG. 15C, Single-chain Polypeptide F, Single-chain Polypeptide C, Reference Control 3, and Reference Control 4 induced a significant expansion in the percentage of live CD8 T cells in treated mice, peaking at 4 days post-injection. Elevated expansion of CD8 T cells in mice treated with Single-chain Polypeptide F, and moderate expansion in mice treated with VHH-RLI, Reference Control 3, and Reference Control 4 was observed even at 9 days post-injection. No significant increase in the percentage of CD8 T cells was observed in mice treated with huIL15 or RLI.
[0303] As shown in FIG. 16, among the molecules that induced an expansion in live NK cells (FIG. 16A), NKT cells (FIG. 16B), and CD8 T cells (FIG. 16C) at 4 days post-injection (Single-chain Polypeptide F, Single-chain Polypeptide C, Reference Control 3, and Reference Control 4), Single-chain Polypeptide F maintained an increase in the population of NK cells and NKT cells as compared to levels in untreated mice, and exhibited even greater CD8 T cell expansion at 9 days post-injection.
Example 10 - in vivo Induction of Immune Cell Activation by Single-chain Polypeptides
[0304] This example shows that treatment of mice with Single-chain Polypeptide F, results in enhanced expression of NKGD2 on NK cells compared to Single-chain Polypeptide C, Reference Control 3, Reference Control 4, and RLI.
[0305] The in vivo activity of Single-chain Polypeptide A in inducing immune cell activation markers Ki67, GrzB, and NKG2D in NK cells and CD8 T cells was assessed by flow cytometry using Ki67, GrzB and NKG2D -specific antibodies, respectively, and compared to levels induced in mice treated with Single-chain Polypeptide C, Reference Control 3, Reference Control 4, RLI, and huIL-15.
[0306] As shown in FIG. 17, Single-chain Polypeptide F, Single-chain Polypeptide C, Reference Control 3, and Reference Control 4 all shared comparable induction of Ki67 (FIG.17 A) and GrzB (FIG. 17B) in NK cells, and Ki67 (FIG. 17D), GrzB (FIG. 17E), and NKG2D (FIG. 17F) in CD8 T cells and with similar kinetics (i.e. all peaking at day 4 post- injection). However, as shown in FIG. 17C, the number of NKG2D NK cells was significantly induced in mice treated with Single-chain Polypeptide F at day 4 post-inject compared to mice treated with Single-chain Polypeptide C, Reference Control 3, and Reference Control 4, RLI, and huIL-15, and remained elevated at day 9 post-injection.
Example 11 - HuIL-2/IL-15Rβ Binding Affinity of Single-chain Polypeptides
[0307] This example describes that Single-chain Polypeptides A, G and H have comparable binding affinities to the huIL-2R/IL- 1511b common subunit (IL-2Rβ), which are approximately two times greater than the binding affinity of wild-type IL-15, but 7X lower than Single-chain Polypeptide C.
[0308] As determined by surface plasmon resonance (SPR) and described in Table 17, Single-chain Polypeptide A, Single-chain Polypeptide G, and Single-chain Polypeptide H demonstrated similar binding kinetics (ka, kd, and KD) and steady-state affinities. For example, the steady state affinity for Single-chain Polypeptide B was 28.7 nM, the steady state affinity for Single-chain Polypeptide G was 23.6 nM, and the steady state affinity for Single-chain Polypeptide H was 24.8 nM. These values suggest that the S73A and S73A H105E mutations present in the IL-15 polypeptide of Single-chain Polypeptides G and H respectively, do not significantly impact the binding affinity of IL-15 for IL-2Rβ,
[0309] Also shown in Table 17, Single-chain Polypeptide A, Single-chain Polypeptide G, and Single-chain Polypeptide H all had about a 2-fold higher binding affinity for IL-2Rβ compared to WT IL-15, which had a stead state affinity of 53.3 nM. The higher affinity for IL-2Rβ of the single-chain polypeptides is primarily driven by faster on-rate kinetics of the 1 : 1 binding.
[0310] As also shown in Table 17, Single-chain Polypeptide A, Single-chain Polypeptide G, and Single-chain Polypeptide H all had about a 7-fold weaker binding affinity for IL-2Rβ as compared to Single-chain Polypeptide C, which had a steady state affinity of 3.84 nM. [0311] Table 17: IL-2/IL-15Rβ Binding affinities of Single Chain Polypeptides
Figure imgf000092_0001
Example 12 - Production and Characterization of Single Chain Polypeptides Comprising IL-15 Mutations
[0312] In this example, IL-15 mutant single-chain polypeptides having the structure huIL-15Rα-Linker-huFcIgGl-Linker-huFcIgGl-Linker-huIL-15, in which the huFcIgGl polypeptide chains each comprise heterodimerization mutations and effector function silencing mutations, and having a S73A point mutation, or S73A and H105E mutations in the human IL-15 (huIL-15) polypeptide unit, and produced, purified, and characterized according to methods described herein and known in the art.
[0313] Recombinant DNA techniques, standard protein expression methods known in the art, and size exclusion chromatography can be used to produce the IL-15 mutant single- chain polypeptides. in vitro assays, such as the HEC Blue IL-2 assay and flow cytometry-based analysis of STAT3 phosphorylation are used to characterize the signal transduction potential of the IL-15 mutant single-chain polypeptides. Flow cytometry analysis is also used to assess the potency of the IL-15 mutant single-chain polypeptides to induce immune cell proliferation (e.g. KI- 67) and activation (e.g. CD25, NKG2D, and GrzB) markers in in vitro cultures of total PBMCs, CD8 T cells, NK cells, NKT cells, CD4 T cells and/or γδ T cells, or in vivo in mice injected with the mutant IL-15 single-chain polypeptides. SPR is used to characterize the binding affinity of the mutant IL-15 single-chain polypeptides to measure 1 : 1 binding and steady state affinity binding kinetics. INCORPORATION BY REFERENCE
[0314] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.
EQUIVALENTS [0315] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A single-chain polypeptide comprising:
(a) an effector moiety polypeptide or a functional fragment thereof;
(b) a receptor polypeptide or a functional fragment thereof;
(c) a multi-chain or single-chain carrier; and
(d) two or more linkers, wherein a linker independently joins the multi-chain or single-chain carrier to the effector moiety polypeptide or functional fragment thereof, and/or to the receptor polypeptide or functional fragment thereof.
2. The single-chain polypeptide of claim 1, wherein when the carrier is multi-chain, two chains of the multi-chain carrier are joined by a linker.
3. A single-chain polypeptide comprising three or more units comprising an effector moiety polypeptide or a functional fragment thereof, a receptor polypeptide or a functional fragment thereof, and a multi-chain or single-chain carrier, and wherein each unit is independently joined to another unit by a linker.
4. The single-chain polypeptide of claim 3, wherein when the carrier is multi-chain, the effector moiety polypeptide or functional fragment thereof, or the receptor polypeptide or functional fragment thereof is independently joined to one chain of the multi-chain carrier.
5. The single-chain polypeptide of claim 3, wherein when the carrier is multi-chain, the effector moiety polypeptide or functional fragment thereof, or the receptor polypeptide or functional fragment thereof is joined to each chain of the multi-chain carrier by a linker.
6. The single-chain polypeptide of any one of claims 1 to 5, wherein the receptor polypeptide or functional fragment thereof binds to the effector moiety polypeptide or functional fragment thereof to form a complex.
7. The single-chain polypeptide of any one of claims 1 to 6, wherein the effector moiety polypeptide comprises a single-subunit cytokine polypeptide.
8. The single-chain polypeptide of claim 7, wherein the single- subunit cytokine polypeptide comprises an interleukin 15 (IL-15) polypeptide.
9. The single-chain polypeptide of claim 8, wherein the IL-15 polypeptide is human.
10. The single-chain polypeptide of claim 9, wherein the IL-15 polypeptide comprises one or more than one mutation.
11. The single-chain polypeptide of claim 10, wherein the one or more than one mutation removes one or more than one glycosylation site.
12. The single-chain polypeptide of claim 10 or 11, wherein the one or more than one mutation does not substantially affect binding kinetics or binding affinity to the IL-2/IL-15 receptor common b subunit (IL-15Rβ)
13. The single-chain polypeptide of any one of claims 10 to 12, wherein the one or more than one mutation comprises a mutation of the serine at position 73.
14. The single-chain polypeptide of claim 13, wherein the mutation of the serine at position 73 is to alanine.
15. The single-chain polypeptide of claim 13 or 14, wherein the one or more than one mutation further comprises a mutation of the histidine at position 105.
16. The single-chain polypeptide of claim 15, wherein the mutation of the histidine at position 105 is to glutamate.
17. The single-chain polypeptide of any one of claims 9 to 16, wherein the receptor polypeptide comprises an IL-15 receptor polypeptide or a functional fragment thereof.
18. The single-chain polypeptide of claim 17, wherein the IL-15 receptor polypeptide is an IL-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof.
19. The single-chain polypeptide of claim 18, wherein the functional fragment of IL- 15Ra comprises a sushi domain.
20. The single-chain polypeptide of any one of claims 18 or 19, wherein the IL-15Rα polypeptide is human.
21. The single-chain polypeptide of any one of claims 1 to 20, wherein the carrier comprises a unit selected from the group consisting of: a fragment crystallizable (Fc) domain, an anti-HSA nanobody (VHH), a human serum albumin (HSA) polypeptide or functional fragment thereof, an anti-HSA Fab domain, and an anti-HSA single-chain variable fragment (scFv).
22. The single-chain polypeptide of claim 21, wherein the carrier is a unit comprising a first and second immunoglobulin heavy chain polypeptide fragment, each comprising or consisting of an Fc domain polypeptide.
23. The single-chain polypeptide of claim 21, wherein the Fc domain is a human Fc domain.
24. The single-chain polypeptide of claim 23, wherein the Fc domain is an IgGl, IgG2, IgG3, or IgG4 Fc domain.
25. The single-chain polypeptide of claim 21, wherein the carrier is a unit comprising an anti-HSA Fab heavy chain polypeptide and an anti-HSA Fab light chain polypeptide.
26. The single-chain polypeptide of claim 21, wherein the carrier consists of an anti-HSA heavy chain variable domain, a linker, and an anti-HSA light chain variable domain.
27. The single-chain polypeptide of claim 1 or 3, wherein a linker joins the C-terminus of the multi-chain or single-chain carrier to the N-terminus of the effector moiety polypeptide or fragment thereof and an additional linker joins the N-terminus of the multi -chain or single- chain carrier to the C-terminus of the receptor polypeptide or fragment thereof.
28. The single-chain polypeptide of claim 1 or 3, wherein a linker joins the C-terminus of the multi-chain or single-chain carrier to the N-terminus of the receptor polypeptide or fragment thereof and an additional linker joins the N-terminus of the multi -chain or single- chain carrier to the C-terminus of the effector moiety polypeptide or fragment thereof.
29. The single-chain polypeptide of claim 1 or 3, wherein the multi-chain carrier is a unit comprising an Fc domain, wherein a linker joins the N-terminus of a first fragment of an immunoglobulin heavy chain to the C-terminus of a second fragment of an immunoglobulin heavy chain, wherein an additional linker joins the C-terminus of the first fragment of an immunoglobulin heavy chain to the N-terminus of the effector moiety polypeptide or fragment thereof, and an additional linker joins the N-terminus of the second fragment of an immunoglobulin heavy chain to the C-terminus of the receptor polypeptide or fragment thereof.
30. The single-chain polypeptide of claim 1 or 3, wherein the multi-chain carrier is a unit comprising an Fc domain, wherein a linker joins the N-terminus of a first fragment of an immunoglobulin heavy chain to the C-terminus of a second fragment of an immunoglobulin heavy chain, wherein an additional linker joins the C-terminus of the first fragment of an immunoglobulin heavy chain to the N-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the N-terminus of the second fragment of an immunoglobulin heavy chain to the C-terminus of the effector moiety polypeptide or fragment thereof.
31. The single-chain polypeptide of claim 29 or 30, wherein the first and the second fragment of an immunoglobulin heavy chain each comprises or consists of an Fc domain polypeptide.
32. The single-chain polypeptide of any one of claims 27 to 31, wherein the effector moiety polypeptide comprises a single-subunit cytokine polypeptide.
33. The single-chain polypeptide of claim 32, wherein the single- subunit cytokine polypeptide comprises an interleukin 15 (IL-15) polypeptide.
34. The single-chain polypeptide of claim 33, wherein the IL-15 polypeptide is human.
35. The single-chain polypeptide of claim 34, wherein the IL-15 polypeptide comprises one or more than one mutation.
36. The single-chain polypeptide of claim 35, wherein the one or more than one mutation removes one or more than one glycosylation site.
37. The single-chain polypeptide of claim 35 or 36, wherein the one or more than one mutation does not substantially affect binding kinetics or binding affinity to the IL-2/IL-15 receptor common b subunit (IL-15Rβ).
38. The single-chain polypeptide of any one of claims 35 to 37, wherein the one or more than one mutation comprises a mutation of the serine at position 73.
39. The single-chain polypeptide of claim 38, wherein the mutation of the serine at position 73 is to alanine.
40. The single-chain polypeptide of claim 38 or 39, wherein the one or more than one mutation further comprises a mutation of the histidine at position 105.
41. The single-chain polypeptide of claim 40, wherein the mutation of the histidine at position 105 is to glutamate.
42. The single-chain polypeptide of any one of claims 33 to 41, wherein the receptor polypeptide comprises an IL-15 receptor polypeptide or a functional fragment thereof.
43. The single-chain polypeptide of claim 42, wherein the IL-15 receptor polypeptide is an IL-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof.
44. The single-chain polypeptide of claim 43, wherein the functional fragment of IL- 15Ra comprises a sushi domain.
45. The single-chain polypeptide of any one of claims 43 or 44, wherein the IL-15Rα polypeptide is human.
46. A single-chain polypeptide comprising:
(a) a human IL-15 polypeptide or a functional fragment thereof;
(b) a human IL-15Rα polypeptide or a functional fragment thereof; and
(c) a multi-chain carrier comprising an Fc domain, wherein a linker joins the N- terminus of a first fragment of an immunoglobulin heavy chain to the C-terminus of a second fragment of an immunoglobulin heavy chain, wherein an additional linker joins the C-terminus of the first fragment of an immunoglobulin heavy chain to the N-terminus of the IL-15 polypeptide or functional fragment thereof, and an additional linker joins the N-terminus of the second fragment of an immunoglobulin heavy chain to the C-terminus of the IL-15Rα polypeptide or fragment thereof, and wherein the first and the second fragment of the immunoglobulin heavy chain each comprises or consists of an Fc domain polypeptide.
47. A single-chain polypeptide comprising the amino acid sequence of SEQ ID NO:37.
48. A single-chain polypeptide comprising the amino acid sequence of SEQ ID NO:71.
49. A single-chain polypeptide comprising the amino acid sequence of SEQ ID NO:72.
50. The single-chain polypeptide of claim 1 or 3, wherein a linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
51. The single-chain polypeptide of claim 50, wherein an additional linker joins the C- terminus of the carrier to the N-terminus of the receptor polypeptide or functional fragment thereof.
52. The single-chain polypeptide of claim 50, wherein an additional linker joins the N- terminus of the carrier to the C-terminus of the effector moiety polypeptide or functional fragment thereof.
53. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an Fc-domain, wherein an additional linker joins the C-terminus of a first fragment of an immunoglobulin heavy chain to the N-terminus of a second fragment of an immunoglobulin heavy chain, and an additional linker joins the C-terminus of the second fragment of an immunoglobulin heavy chain to the N-terminus of the receptor polypeptide or fragment thereof.
54. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an Fc-domain, wherein an additional linker joins the N-terminus of a first fragment of an immunoglobulin heavy chain to the C-terminus of a second fragment of an immunoglobulin heavy chain, and an additional linker joins the N-terminus of the second fragment of an immunoglobulin heavy chain to the C-terminus of the effector moiety polypeptide or fragment thereof.
55. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an Fc-domain, wherein an additional linker joins the C-terminus of a first fragment of an immunoglobulin heavy chain to the N-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the N-terminus of a second fragment of an immunoglobulin heavy chain to the C-terminus of the effector moiety polypeptide or fragment thereof.
56. The single-chain polypeptide of any one of claims 53 to 55, wherein the first and the second fragment of an immunoglobulin heavy chain each comprises or consists of an Fc domain polypeptide.
57. The single-chain polypeptide of claim 50, wherein the carrier comprises an HSA polypeptide or functional fragment thereof, or an anti-HSA nanobody (VHH), wherein an additional linker joins the C-terminus of the carrier to the N-terminus of the receptor polypeptide or fragment thereof.
58. The single-chain polypeptide of claim 50, wherein the carrier comprises an HSA polypeptide or functional fragment thereof, or an anti-HSA nanobody (VHH), and wherein an additional linker joins the N-terminus of the carrier to the C-terminus of the effector moiety polypeptide or fragment thereof.
59. The single-chain polypeptide of claim 1 or 3, wherein the carrier comprises an HSA polypeptide or functional fragment thereof, or an anti-HSA nanobody (VHH), and wherein a linker joins the N-terminus of the carrier to the C-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the C-terminus of the carrier to the N- terminus of the effector moiety polypeptide or fragment thereof.
60. The single-chain polypeptide of claim 1 or 3, wherein the carrier comprises an HSA polypeptide or functional fragment thereof, or an anti-HSA nanobody (VHH), and wherein a linker joins the C-terminus of the carrier to the N-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the N-terminus of the carrier to the C- terminus of the effector moiety polypeptide or fragment thereof.
61. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the C-terminus of a fragment of an immunoglobulin heavy chain to the N-terminus of an immunoglobulin light chain, and an additional linker joins the C-terminus of the immunoglobulin light chain to the N-terminus of the receptor polypeptide or fragment thereof.
62. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the C-terminus of an immunoglobulin light chain to the N-terminus of a fragment of an immunoglobulin heavy chain, and an additional linker joins the C-terminus of the fragment of an immunoglobulin heavy chain to the N- terminus of the receptor polypeptide or fragment thereof.
63. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the N-terminus of a fragment of an immunoglobulin heavy chain to the C-terminus of an immunoglobulin light chain, and an additional linker joins the N-terminus of the immunoglobulin light chain to the C-terminus of the effector moiety polypeptide or fragment thereof.
64. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the N-terminus of an immunoglobulin light chain to the C-terminus of a fragment of an immunoglobulin heavy chain, and an additional linker joins the N-terminus of the fragment of an immunoglobulin heavy chain to the C- terminus of the effector moiety polypeptide or fragment thereof.
65. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the C-terminus of a fragment of an immunoglobulin heavy chain to the N-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the N-terminus of an immunoglobulin light chain to the C -terminus of the effector moiety polypeptide or fragment thereof.
66. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the C-terminus of an immunoglobulin light chain to the N-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the N-terminus of a fragment of an immunoglobulin heavy chain to the C- terminus of the effector moiety polypeptide or fragment thereof.
67. The single-chain polypeptide of any one of claims 61 to 66, wherein the fragment of an immunoglobulin heavy chain comprises or consists of an anti-HSA Fab heavy chain polypeptide.
68. The single-chain polypeptide of any one of claims 61 to 67, wherein the immunoglobulin light chain comprises or consists of an anti-HSA Fab light chain polypeptide.
69. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein an additional linker joins the C- terminus of an immunoglobulin heavy chain variable domain to the N-terminus of an immunoglobulin light chain variable domain, and an additional linker joins the C-terminus of the an immunoglobulin light chain variable domain to the N-terminus of the receptor polypeptide or fragment thereof.
70. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein an additional linker joins the C- terminus of an immunoglobulin light chain variable domain to the N-terminus of an immunoglobulin heavy chain variable domain, and an additional linker joins the C-terminus of the immunoglobulin heavy chain variable domain to the N-terminus of the receptor polypeptide or fragment thereof.
71. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein an additional linker joins the N- terminus of an immunoglobulin heavy chain variable domain to the C-terminus of an immunoglobulin light chain variable domain, and an additional linker joins the N-terminus of the immunoglobulin light chain variable domain to the C-terminus of the effector moiety polypeptide or fragment thereof.
72. The single-chain polypeptide of claim 50, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein an additional linker joins the N- terminus of an immunoglobulin light chain variable domain to the C-terminus of an immunoglobulin heavy chain variable domain, and an additional linker joins the N-terminus of the immunoglobulin heavy chain variable domain to the C-terminus of the effector moiety polypeptide or fragment thereof.
73. The single-chain polypeptide of any one of claims 69 to 72 wherein the immunoglobulin heavy chain variable domain and light chain variable domain are joined by a linker.
74. The single-chain polypeptide of claim 1 or 3, wherein a linker joins the N-terminus of the receptor polypeptide or fragment thereof to the C-terminus of the effector moiety polypeptide or fragment thereof.
75. The single-chain polypeptide of claim 74, wherein an additional linker joins the C- terminus of the carrier to the N-terminus of the effector moiety polypeptide or fragment thereof.
76. The single-chain polypeptide of claim 74, wherein an additional linker joins the N- terminus of the carrier to the C-terminus of the receptor polypeptide or fragment thereof.
77. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an Fc-domain, wherein an additional linker joins the C-terminus of a first fragment of an immunoglobulin heavy chain to the N-terminus of a second fragment of an immunoglobulin heavy chain, and an additional linker joins the C-terminus of the second fragment of an immunoglobulin heavy chain to the N-terminus of the effector moiety polypeptide or fragment thereof.
78. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an Fc-domain, wherein an additional linker joins the N-terminus of a first fragment of an immunoglobulin heavy chain to the C-terminus of a second fragment of an immunoglobulin heavy chain, and an additional linker joins the N-terminus of the second fragment of an immunoglobulin heavy chain to the C-terminus of the receptor polypeptide or fragment thereof.
79. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an Fc-domain, wherein an additional linker joins the C-terminus of a first fragment of an immunoglobulin heavy chain to the N-terminus of the effector moiety polypeptide or fragment thereof, and wherein an additional linker joins the N-terminus of a second fragment of an immunoglobulin heavy chain to the C-terminus of the receptor polypeptide or fragment thereof.
80. The single-chain polypeptide of any one of claims 77 to 79, wherein the first and the second fragment of an immunoglobulin heavy chain each comprises or consists of an Fc domain polypeptide.
81. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the C-terminus of a fragment of an immunoglobulin heavy chain to the N-terminus of an immunoglobulin light chain, and an additional linker joins the C-terminus of the immunoglobulin light chain to the N-terminus of the effector moiety polypeptide or fragment thereof.
82. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the C-terminus of an immunoglobulin light chain to the N-terminus of a fragment of an immunoglobulin heavy chain, and an additional linker joins the C-terminus of the fragment of an immunoglobulin heavy chain to the N- terminus of the effector moiety polypeptide or fragment thereof.
83. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the N-terminus of a fragment of an immunoglobulin heavy chain to the C-terminus of an immunoglobulin light chain, and an additional linker joins the N-terminus of the immunoglobulin light chain to the C-terminus of the receptor polypeptide or fragment thereof.
84. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the N-terminus of an immunoglobulin light chain to the C-terminus of a fragment of an immunoglobulin heavy chain, and an additional linker joins the N-terminus of the fragment of an immunoglobulin heavy chain to the C- terminus of the receptor polypeptide or fragment thereof.
85. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the N-terminus of a fragment of an immunoglobulin heavy chain to the C-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the C-terminus of an immunoglobulin light chain to the N-terminus of the effector moiety polypeptide or fragment thereof.
86. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA Fab, wherein an additional linker joins the N-terminus of an immunoglobulin light chain to the C-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the C-terminus of a fragment of an immunoglobulin heavy chain to the N- terminus of the effector moiety polypeptide or fragment thereof.
87. The single-chain polypeptide of any one of claims 81 to 86, wherein the fragment of an immunoglobulin heavy chain comprises or consists of an anti-HSA Fab heavy chain polypeptide.
88. The single-chain polypeptide of any one of claims 81 to 87, wherein the immunoglobulin light chain comprises or consists of an anti-HSA Fab light chain polypeptide.
89. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein an additional linker joins the C- terminus of an immunoglobulin heavy chain variable domain to the N-terminus of an immunoglobulin light chain variable domain, and an additional linker joins the C-terminus of the immunoglobulin light chain variable domain to the N-terminus of the effector moiety polypeptide or fragment thereof.
90. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein an additional linker joins the C- terminus of an immunoglobulin light chain variable domain to the N-terminus of an immunoglobulin heavy chain variable domain, and an additional linker joins the C-terminus of the immunoglobulin heavy chain variable domain to the N-terminus of the effector moiety polypeptide or fragment thereof.
91. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein an additional linker joins the N- terminus of an immunoglobulin heavy chain variable domain to the C-terminus of an immunoglobulin light chain variable domain, and an additional linker joins the N-terminus of the immunoglobulin light chain variable domain to the C-terminus of the receptor polypeptide or fragment thereof.
92. The single-chain polypeptide of claim 74, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein an additional linker joins the N- terminus of an immunoglobulin light chain variable domain to the C-terminus of an immunoglobulin heavy chain variable domain, and an additional linker joins the N-terminus of the immunoglobulin heavy chain variable domain to the C-terminus of the receptor polypeptide or fragment thereof.
93. The single-chain polypeptide of any one of claims 89 to 92 wherein the immunoglobulin heavy chain variable domain and light chain variable domain are joined by a linker.
94. The single-chain polypeptide of claim 74, wherein the carrier comprises an HSA polypeptide or functional fragment thereof, or an anti-HSA nanobody (VHH), wherein an additional linker joins the C-terminus of the carrier to the N-terminus of the effector moiety polypeptide or fragment thereof.
95. The single-chain polypeptide of claim 74, wherein the carrier comprises an HSA polypeptide or functional fragment thereof, or an anti-HSA nanobody (VHH), and wherein an additional linker joins the N-terminus of the carrier to the C-terminus of the receptor polypeptide or fragment thereof.
96. The single-chain polypeptide of claim 1 or 3, wherein the carrier is a unit comprising an anti-HSA Fab, wherein a linker joins the N-terminus of a fragment of an immunoglobulin heavy chain to the C-terminus of a fragment of an immunoglobulin light chain, wherein an additional linker joins the C-terminus of the fragment of an immunoglobulin heavy chain to the N-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the N-terminus of the immunoglobulin light chain to the C-terminus of the effector moiety polypeptide or fragment thereof.
97. The single-chain polypeptide of claim 1 or 3, wherein the carrier is a unit comprising an anti-HSA Fab, wherein a linker joins the N-terminus of an immunoglobulin light chain to the C-terminus of a fragment of an immunoglobulin heavy chain, wherein an additional linker joins the C-terminus of the immunoglobulin light chain to the N-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the N-terminus of the fragment of an immunoglobulin heavy chain to the C-terminus of the effector moiety polypeptide or fragment thereof.
98. The single-chain polypeptide of claim 1 or 3, wherein the carrier is a unit comprising an anti-HSA Fab domain, wherein a linker joins the N-terminus of a fragment of an immunoglobulin heavy chain to the C-terminus of an immunoglobulin light chain, wherein an additional linker joins the C-terminus of the fragment of an immunoglobulin heavy chain to the N-terminus of the effector moiety polypeptide or fragment thereof, and an additional linker joins the N-terminus of the immunoglobulin light chain to the C-terminus of the receptor polypeptide or fragment thereof.
99. The single-chain polypeptide of claim 1 or 3, wherein the carrier is a unit comprising an anti-HSA Fab domain, wherein a linker joins the N-terminus of an immunoglobulin light chain to the C-terminus of a fragment of an immunoglobulin heavy chain, wherein an additional linker joins the C-terminus of the immunoglobulin light chain to the N-terminus of the effector moiety polypeptide or fragment thereof, and an additional linker joins the N- terminus of the fragment of an immunoglobulin heavy chain to the C-terminus of the receptor polypeptide or fragment thereof.
100. The single-chain polypeptide of any one of claims 96 to 99, wherein the fragment of an immunoglobulin heavy chain comprises or consists of an anti-HSA Fab heavy chain polypeptide.
101. The single-chain polypeptide of any one of claims 96 to 100, wherein the immunoglobulin light chain comprises or consists of an anti-HSA Fab light chain polypeptide.
102. The single-chain polypeptide of claim 1 or 3, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein a linker joins the N-terminus of an immunoglobulin heavy chain variable domain to the C-terminus of an immunoglobulin light chain variable domain, wherein an additional linker joins the C-terminus of the immunoglobulin heavy chain variable domain to the N-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the N-terminus of the immunoglobulin light chain variable domain to the C-terminus of the effector moiety polypeptide or fragment thereof.
103. The single-chain polypeptide of claim 1 or 3, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein a linker joins the N-terminus of an immunoglobulin light chain variable domain to the C-terminus of an immunoglobulin heavy chain variable domain, wherein an additional linker joins the C-terminus of the immunoglobulin light chain variable domain to the N-terminus of the receptor polypeptide or fragment thereof, and an additional linker joins the N-terminus of the immunoglobulin heavy chain variable domain to the C-terminus of the effector moiety polypeptide or fragment thereof.
104. The single-chain polypeptide of claim 1 or 3, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein a linker joins the N-terminus of an immunoglobulin heavy chain variable domain to the C-terminus of an immunoglobulin light chain variable domain, wherein an additional linker joins the C-terminus of the immunoglobulin heavy chain variable domain to the N-terminus of the effector moiety polypeptide or fragment thereof, and an additional linker joins the N-terminus of the immunoglobulin light chain variable domain to the C-terminus of the receptor polypeptide or fragment thereof.
105. The single-chain polypeptide of claim 1 or 3, wherein the carrier is a unit comprising an anti-HSA single-chain variable fragment (scFv), wherein a linker joins the N-terminus of an immunoglobulin light chain variable domain to the C-terminus of an immunoglobulin heavy chain variable domain, wherein an additional linker joins the C-terminus of the immunoglobulin light chain variable domain to the N-terminus of the effector moiety polypeptide or fragment thereof, and an additional linker joins the N-terminus of the immunoglobulin heavy chain variable domain to the C-terminus of the receptor polypeptide or fragment thereof.
106. The single-chain polypeptide of any one of claims 102 to 105, wherein the immunoglobulin heavy chain variable domain and light chain variable domain are joined by a linker.
107. The single-chain polypeptide of any one of claims 1-24, 27-56, and 74-80, wherein the carrier is a unit comprising an Fc domain, wherein the unit comprises a first fragment of an IgG heavy chain and a second fragment of an IgG heavy chain, and wherein the first and/or the second fragment of an IgG heavy chain comprises one or more than one mutation that reduces an effector function of the Fc domain.
108. The single-chain polypeptide of claim 107, wherein the effector function comprises the ability of an Fc domain to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC).
109. The single-chain polypeptide of claim 107 or 108, wherein the first and/or second IgG heavy chain is an IgGl heavy chain polypeptides comprising one or more than one mutation that reduces an effector function of the Fc domain at position 234, 235, 237, 329, 330, and/or 331, according to the EU numbering system.
110. The single-chain polypeptide of claim 109, wherein the first and/or second IgGl heavy chain comprises one or more than one mutation that reduces an effector function of the Fc domain selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, numbered according to the EU numbering system.
111. The single-chain polypeptide of claim 110, wherein the first and second IgGl heavy chains each comprise mutations L234A, L235A, and P329A.
112. The single-chain polypeptide of claim 110, wherein the first and second IgGl heavy chains each comprise mutations L234A and L235A.
113. The single-chain polypeptide of claim 110, wherein the first and second IgG heavy chains each comprise mutations L234A, L235A, G237A, A330S, and P331S.
114. The single-chain polypeptide of claim 107 or 108, wherein the first and second IgGl heavy chains are human IgG4 heavy chains comprising one or more than one mutation at position 235 and/or 329, according to the EU numbering system.
115. The single-chain polypeptide of claim 114, wherein the first and/or second IgG heavy chains comprise one or more than one mutation selected from L235E and P329A.
116. The single-chain polypeptide of claim 115, wherein the first and second IgG4 heavy chains each comprise mutation L235E.
117. The single-chain polypeptide of claim 115, wherein the first and second IgG4 heavy chains each comprise mutations L235E and P329A.
118. The single-chain polypeptide of any one of claims 114 to 117, wherein the first and second IgG4 heavy chains each comprise mutation S228P.
119. The single-chain polypeptide of any one of claims 50 to 118, wherein the effector moiety polypeptide comprises a single-subunit cytokine polypeptide.
120. The single-chain polypeptide of claim 119, wherein the single-subunit cytokine polypeptide comprises an interleukin 15 (IL-15) polypeptide.
121. The single-chain polypeptide of claim 120, wherein the IL-15 polypeptide is human.
122. The single-chain polypeptide of claim 121, wherein the IL-15 polypeptide comprises one or more than one mutation.
123. The single-chain polypeptide of claim 122, wherein the one or more than one mutation removes one or more than one glycosylation site.
124. The single-chain polypeptide of claim 122 or 123, wherein the one or more than one mutation does not substantially affect binding kinetics or binding affinity to the IL-2/IL-15 receptor common b subunit (IL-15IIb)
125. The single-chain polypeptide of any one of claims 122 to 124, wherein the one or more than one mutation comprises a mutation of the serine at position 73.
126. The single-chain polypeptide of claim 125, wherein the mutation of the serine at position 73 is to alanine.
127. The single-chain polypeptide of claim 125 or 126, wherein the one or more than one mutation further comprises a mutation of the histidine at position 105.
128. The single-chain polypeptide of claim 127, wherein the mutation of the histidine at position 105 is to glutamate.
129. The single-chain polypeptide of any one of claims 120 to 128, wherein the receptor polypeptide comprises an IL-15 receptor polypeptide or a functional fragment thereof.
130. The single-chain polypeptide of claim 129, wherein the IL-15 receptor polypeptide is an IL-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof.
131. The single-chain polypeptide of claim 130, wherein the functional fragment of IL- 15Ra comprises a sushi domain.
132. The single-chain polypeptide of any one of claims 130 or 131, wherein the IL-15Rα polypeptide is human.
133. The single-chain polypeptide of any one of claims 8 to 20, 33 to 49, or 120 to 134, wherein the single-chain polypeptide has higher binding affinity for IL-15Rb compared to wild-type IL-15.
134. The single-chain polypeptide of claim 133, wherein the single-chain polypeptide has binding affinity dissociation constant (KD) is 20 nM to 30 nM.
135. The single-chain polypeptide of any one of claims 8 to 20, 33 to 49, 120 to 135, wherein the single-chain polypeptide induces enhanced signal transduction as compared to wild-type IL-15 when contacted with a cell expressing the IL-15R.
136. The single-chain polypeptide of claim 135, wherein signal transduction is measured using a HEK Blue IL-2 assay.
137. The single-chain polypeptide of claim 135, wherein the single-chain polypeptide induces enhanced STAT5 phosphorylation as compared to wild-type IL-15 when contacted with the cell expressing the IL-15R.
138. The single-chain polypeptide of claim 137, wherein STAT5 phosphorylation is measured using flow cytometry or western blot analysis.
139. The single-chain polypeptide of any one of claims 135 to 137, wherein the cell is a CD8+ T-cell, a natural killer (NK) cell, a natural killer T-cell (NKT cell), a CD4+ T-cell, or a regulatory T-cell (Treg).
140. The single-chain polypeptide of any one of claims 8 to 19, 32 to 46, 117 to 128, or 129-135, wherein the single-chain polypeptide induces proliferation and/or activation of one or more than one immune cell selected from the group consisting of CD8+ T-cells, a NK cells, NKT cells, CD4+ T-cells, and Tregs, when administered to a subject.
141. The single-chain polypeptide of claim 140, wherein the single-chain polypeptide induces proliferation of the one or more than one immune cell after about 4 days.
142. The single-chain polypeptide of claim 140 or 141, wherein the polypeptide induces the expression of one or more than one protein in the one or more immune cells selected from the group consisting of CD25, Ki-67, NKG2D, and/or granzyme B (GrzB).
143. The single-chain polypeptide of claim 142, wherein the single-chain polypeptide induces the expression of the one or more than one protein after about 4 days.
144. The single-chain polypeptide of any one of claims 1 to 143, wherein the linker comprises a sequence of (GGGGS)4 (SEQ ID NO:31), (GGGGS)5 (SEQ ID NO:32), (GGGGS)6 (SEQ ID NO:33), or (SGGGG)6 (SEQ ID NO:34).
145. A single-chain polypeptide comprising:
(a) an effector moiety polypeptide or a functional fragment thereof;
(b) a receptor polypeptide or a functional fragment thereof;
(c) a carrier comprising (i) a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain, or (ii) a single-chain carrier; (d) a linker joining the carrier to the receptor polypeptide or functional fragment thereof; and
(e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the carrier.
146. The single-chain polypeptide of claim 145, wherein the additional linker joins the carrier to the effector moiety polypeptide or functional fragment thereof.
147. The single-chain polypeptide of claim 146, wherein the linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the carrier, and the additional linker joins the C-terminus of the carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
148. The single-chain polypeptide of claim 146, wherein the additional linker joins the C- terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the carrier, and the linker joins the C-terminus of the carrier to the N-terminus of the receptor polypeptide or functional fragment thereof.
149. The single-chain polypeptide of claim 145, wherein the additional linker joins the effector moiety polypeptide or functional fragment thereof to the receptor polypeptide or functional fragment thereof.
150. The single-chain polypeptide of claim 145 or 149, wherein the linker joins the C- terminus of the carrier to the N-terminus of the receptor polypeptide or functional fragment thereof.
151. The single-chain polypeptide of claim 150, the additional linker joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
152. The single-chain polypeptide of any one of claims 145 to 151, wherein the multi- chain carrier is a fragment crystallizable (Fc) domain carrier comprising two Fc-domain polypeptide chains.
153. The single-chain polypeptide of any one of claims 145 to 151, wherein the single- chain carrier is a nanobody (VHH) carrier.
154. A single-chain polypeptide comprising:
(a) an effector moiety polypeptide or a functional fragment thereof;
(b) a receptor polypeptide or a functional fragment thereof;
(c) a fragment crystallizable (Fc)-domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker;
(d) a linker joining the Fc-domain carrier to the receptor polypeptide or functional fragment thereof; and
(e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the Fc- domain carrier.
155. The single-chain polypeptide of claim 154, wherein the linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier, and the additional linker joins the C-terminus of the Fc-domain carrier to the N- terminus of the effector moiety polypeptide or functional fragment thereof.
156. The single-chain polypeptide of claim 154, wherein the linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the receptor polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
157. The single-chain polypeptide of any one of claims 154 to 156, wherein the Fc-domain carrier comprises human or murine Fc-domain polypeptide chains.
158. The single-chain polypeptide of claim 157, wherein the Fc-domain polypeptide chains are IgGl, IgG2, IgG3, or IgG4 Fc-domain polypeptide chains.
159. The single-chain polypeptide of any one of claims 154 to 158, wherein the Fc-domain polypeptide chains comprise one or more than one mutation that reduces an effector function of the Fc-domain carrier.
160. The single-chain polypeptide of claim 159, wherein the effector function comprises the ability of the Fc-domain carrier to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC).
161. The single-chain polypeptide of any one of claims 154 to 160, wherein the Fc-domain polypeptide chains are human IgGl Fc-domain polypeptide chains comprising one or more than one mutation that reduces an effector function of the Fc-domain carrier at position 234, 235, 237, 329, 330, and/or 331, according to the EU numbering system.
162. The single-chain polypeptide of claim 161, wherein one or more than one mutation that reduces an effector function of the Fc-domain carrier is selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, numbered according to the EU numbering system.
163. The single-chain polypeptide of claim 162, wherein the two Fc-domain polypeptide chains each comprise mutations L234A, L235A, and P329A.
164. The single-chain polypeptide of claim 162, wherein the two Fc-domain polypeptide chains each comprise mutations L234A, and L235A.
165. The single-chain polypeptide of claim 162, wherein the two Fc-domain polypeptide chains each comprise mutations L234A, L235A, G237A, A330S, and P331S.
166. The single-chain polypeptide of any one of claims 162 to 165, wherein the two Fc- domain polypeptide chains each further comprise mutation C220S.
167. A single-chain polypeptide comprising:
(a) an effector moiety polypeptide or a functional fragment thereof;
(b) a receptor polypeptide or a functional fragment thereof;
(c) a nanobody (VHH) carrier; (d) a linker joining the VHH carrier to the receptor polypeptide or functional fragment thereof; and
(e) an additional linker joining the effector moiety polypeptide or functional fragment thereof to (i) the receptor polypeptide or functional fragment thereof, or (ii) the VHH carrier.
168. The single-chain polypeptide of claim 167, wherein the linker joins the C-terminus of the VHH carrier to the N-terminus of the receptor polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the receptor polypeptide or functional fragment thereof, to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
169. The single-chain polypeptide of claim 167, wherein the linker joins the C-terminus of the receptor polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the additional linker joins the C-terminus of the VHH carrier to the N-terminus of the effector moiety polypeptide or functional fragment thereof.
170. The single-chain polypeptide of claim 167, wherein the additional linker joins the C- terminus of the effector moiety polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the linker joins the C-terminus of the VHH carrier to the N-terminus of the receptor polypeptide or functional fragment thereof.
171. The single-chain polypeptide of any one of claims 167 to 170, wherein the VHH carrier is an anti-human serum albumin (HSA) nanobody.
172. The single-chain polypeptide of any one of claims 145 to 171, wherein the effector moiety polypeptide or functional fragment thereof binds to the receptor polypeptide or functional fragment thereof to form a complex.
173. The single-chain polypeptide of any one of claims 145 to 172, wherein the effector moiety polypeptide or functional fragment thereof comprises a single-subunit cytokine polypeptide or a functional fragment thereof
174. The single-chain polypeptide of claim 173, wherein the single- subunit cytokine polypeptide of functional fragment thereof comprises an interleukin 15 (IL-15) polypeptide or functional fragment thereof.
175. The single-chain polypeptide of claim 174, wherein the IL-15 polypeptide or functional fragment thereof is human.
176. The single-chain polypeptide of claim 174 or 175, wherein the IL-15 polypeptide or functional fragment thereof comprises one or more than one mutation.
177. The single-chain polypeptide of claim 176, wherein the one or more than one mutation removes one or more than one glycosylation site.
178. The single-chain polypeptide of claim 176 or 177, wherein the one or more than one mutation does not substantially affect binding kinetics or binding affinity to the IL-2/IL-15 receptor common b subunit (IL-15Rβ)
179. The single-chain polypeptide of any one of claims 176 to 178, wherein the one or more than one mutation comprises a mutation of the serine at position 73.
180. The single-chain polypeptide of claim 179, wherein the mutation of the serine at position 73 is to alanine.
181. The single-chain polypeptide of claim 179 or 180, wherein the one or more than one mutation further comprises a mutation of the histidine at position 105.
182. The single-chain polypeptide of claim 181, wherein the mutation of the histidine at position 105 is to glutamate.
183. The single-chain polypeptide of any one of claims 174 to 182, wherein the receptor polypeptide or functional fragment thereof comprises an IL-15 receptor polypeptide or a functional fragment thereof.
184. The single-chain polypeptide of claim 183, wherein the IL-15 receptor polypeptide or functional fragment thereof is an IL-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof.
185. The single-chain polypeptide of claim 184, wherein the IL-15Rα polypeptide is human.
186. The single-chain polypeptide of claim 184 or 185, wherein the functional fragment of IL-15Rα comprises a sushi domain.
187. A single-chain polypeptide comprising:
(a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof;
(b) an IL-15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof;
(c) a carrier comprising (i) a multi-chain carrier comprising an inter-chain linker joining two chains of the multi-chain carrier to form a contiguous polypeptide chain, or (ii) a single-chain carrier;
(d) a linker joining the carrier to the IL-15Rα polypeptide or functional fragment thereof; and
(e) an additional linker, wherein the additional linker joining the IL-15 polypeptide or functional fragment thereof to (i) the IL-15Rα polypeptide or functional fragment thereof, or (ii) the carrier.
188. The single-chain polypeptide of claim 187, wherein the additional linker joins the carrier to the IL-15 polypeptide or functional fragment thereof.
189. The single-chain polypeptide of claim 188, wherein the linker joins the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the carrier, and the additional linker joins the C-terminus of the multi-chain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
190. The single-chain polypeptide of claim 188, wherein the additional linker joins the C- terminus of the IL-15 polypeptide or functional fragment thereof to the N-terminus of the carrier, and the linker joins the C-terminus of the carrier to the N-terminus of the IL-15Rα polypeptide or functional fragment thereof.
191. The single-chain polypeptide of claim 187, wherein the additional linker joins the IL- 15 polypeptide or functional fragment thereof to the IL-15Rα polypeptide or functional fragment thereof.
192. The single-chain polypeptide of claim 187, wherein the linker joins the C-terminus of the carrier to the N-terminus of the IL-15Rα polypeptide or functional fragment thereof.
193. The single-chain polypeptide of claim 192, wherein the additional linker joins the C- terminus of the IL-15Rα polypeptide or functional fragment thereof, to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
194. The single-chain polypeptide of any one of claims 187 to 193, wherein the multi- chain carrier is a fragment crystallizable (Fc) domain carrier comprising two Fc-domain polypeptide chains.
195. The single-chain polypeptide of any one of claims 187 to 193, wherein the single- chain carrier is a nanobody (VHH) carrier.
196. A single-chain polypeptide comprising:
(a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof;
(b) an interleukin 15 receptor alpha (IL-15Rα) polypeptide or a functional fragment thereof;
(c) a fragment crystallizable (Fc)-domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker;
(d) a linker joining the Fc-domain carrier to the receptor polypeptide or functional fragment thereof; and
(e) an additional linker joining the IL-15 polypeptide or functional fragment thereof to (i) the IL-15Rα polypeptide or functional fragment thereof, or (ii) the Fc-domain carrier.
197. The single-chain polypeptide of claim 196, wherein the linker joins the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier, and the additional linker joins the C-terminus of the Fc-domain carrier to the N- terminus of the IL-15 polypeptide or functional fragment thereof.
198. The single-chain polypeptide of claim 196, wherein the linker joins the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15Rα polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
199. The single-chain polypeptide of any one of claims 196 to 198, wherein the Fc-domain carrier comprises human or murine Fc-domain polypeptide chains.
200. The single-chain polypeptide of claim 199, wherein the Fc-domain polypeptide chains are IgGl, IgG2, IgG3, or IgG4 Fc-domain polypeptide chains.
201. The single-chain polypeptide of any one of claims 196 to 200, wherein the Fc-domain polypeptide chains comprise one or more than one mutation that reduces an effector function of the Fc-domain carrier.
202. The single-chain polypeptide of claim 201, wherein the effector function comprises the ability of an Fc-domain carrier to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement dependent cytotoxicity (CDC).
203. The single-chain polypeptide of any one of claims 196 to 202, wherein the Fc-domain polypeptide chains are human IgGl Fc-domain polypeptide chains comprising one or more than one mutation that reduces an effector function of the Fc-domain carrier at position 234, 235, 237, 329, 330, and/or 331, according to the EU numbering system.
204. The single-chain polypeptide of claim 203, wherein one or more than one mutation that reduces an effector function of the Fc-domain carrier is selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, numbered according to the EU numbering system.
205. The single-chain polypeptide of claim 204, wherein the two Fc-domain polypeptide chains each comprise mutations L234A, L235A, and P329A.
206. The single-chain polypeptide of claim 204, wherein the two Fc-domain polypeptide chains each comprise mutations L234A, and L235A.
207. The single-chain polypeptide of claim 204, wherein the two Fc-domain polypeptide chains each comprise mutations L234A, L235A, G237A, A330S, and P331S.
208. The single-chain polypeptide of any one of claims 203 to 207, wherein the two Fc- domain polypeptide chains each comprise mutation C220S.
209. A single-chain polypeptide, comprising: (a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof;
(b) an IL-15Rα polypeptide or a functional fragment thereof;
(c) a fragment crystallizable (Fc)-domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker;
(d) a linker joining the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof; and
(e) an additional linker joining the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of Fc-domain carrier.
210. A single-chain polypeptide comprising:
(a) an interleukin 15 (IL-15) polypeptide or a functional fragment thereof;
(b) an IL-15Rα polypeptide or a functional fragment thereof;
(c) a nanobody (VHH) carrier;
(d) a linker joining the VHH carrier to the IL-15Rα polypeptide or functional fragment thereof; and
(e) an additional linker joining the IL-15 polypeptide or functional fragment thereof to (i) the IL-15Rα polypeptide or functional fragment thereof, or (ii) the VHH carrier.
211. The single-chain polypeptide of claim 210, wherein the linker joins the C-terminus of the VHH carrier to the N-terminus of the IL-15Rα polypeptide or functional fragment thereof, and the additional linker joins the C-terminus of the IL-15Rα polypeptide or functional fragment thereof, to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
212. The single-chain polypeptide of claim 210, wherein the linker joins the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the additional linker joins the C-terminus of the VHH carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof.
213. The single-chain polypeptide of claim 210, wherein the additional linker joins the C- terminus of the IL-15 polypeptide or functional fragment thereof to the N-terminus of the VHH carrier, and the linker joins the C-terminus of the VHH carrier to the N-terminus of the IL-15Rα polypeptide or functional fragment thereof.
214. The single-chain polypeptide of any one of claims 210 to 213, wherein the VHH carrier is an anti-human serum albumin (HSA) nanobody.
215. The-single-chain polypeptide of any one of claims 187 to 214, wherein the IL-15 polypeptide or functional fragment thereof, and IL-15Rα polypeptide, or functional fragment thereof are human.
216. The single-chain polypeptide of any one of claims 187 to 215, wherein the IL-15 polypeptide or functional fragment thereof comprises one or more than one mutation.
217. The single-chain polypeptide of claim 216, wherein the one or more than one mutation removes one or more than one glycosylation site
218. The single-chain polypeptide of claim 216 or 217, wherein the one or more than one mutation does not substantially affect binding kinetics or binding affinity to the IL-2/IL-15 receptor common b subunit (IL-15Rβ),
219. The single-chain polypeptide of any one of claims 216 to 218, wherein the one or more than one mutation comprises a mutation of the serine at position 73.
220. The single-chain polypeptide of claim 219, wherein the mutation of the serine at position 73 is to alanine.
221. The single-chain polypeptide of claim 219 or 220, wherein the one or more than one mutation further comprises a mutation of the histidine at position 105.
222. The single-chain polypeptide of claim 221, wherein the mutation of the histidine at position 105 is to glutamate.
223. A single-chain polypeptide, comprising:
(a) a human interleukin 15 (IL-15) polypeptide or a functional fragment thereof comprising a mutation of the serine at position 73 to alanine; (b) an IL-15Rα polypeptide or a functional fragment thereof;
(c) a fragment crystallizable (Fc)-domain carrier comprising two Fc-domain polypeptide chains joined by an inter-chain linker;
(d) a linker joining the C-terminus of the Fc-domain carrier to the N-terminus of the IL-15 polypeptide or functional fragment thereof; and
(e) an additional linker joining the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier.
224. A single-chain polypeptide, comprising:
(a) a human interleukin 15 (IL-15) polypeptide or a functional fragment thereof comprising a mutation of the serine at position 73 to alanine and a mutation of the histidine at position 105 to glutamate;
(b) an IL-15Rα polypeptide or a functional fragment thereof;
(c) a fragment crystallizable (Fc)-domain carrier, comprising two Fc-domain polypeptide chains joined by an inter-chain linker; (d) a linker joining the C-terminus of the Fc-domain carrier to the N-terminus of the
IL-15 polypeptide or functional fragment thereof; and
(e) an additional linker joining the C-terminus of the IL-15Rα polypeptide or functional fragment thereof to the N-terminus of the Fc-domain carrier.
225. The single-chain polypeptide of any one of claims 187 to 224, wherein the functional fragment of IL-15Rα comprises a sushi domain.
226. The single-chain polypeptide of any one of claims 174 to 225, wherein the single- chain polypeptide has higher binding affinity for IL-15Rβ compared to wild-type IL-15.
227. The single-chain polypeptide of claim 226, wherein the single-chain polypeptide has binding affinity dissociation constant (KD) is 20 nM to 30 nM.
228. The single-chain polypeptide of any one of claims 174 to 227, wherein the single- chain polypeptide induces enhanced signal transduction as compared to wild-type IL-15 when contacted with a cell expressing the IL-15R.
229. The single-chain polypeptide of claim 228, wherein signal transduction is measured using a HEK Blue IL-2 assay.
230. The single-chain polypeptide of claim 228, wherein the single-chain polypeptide induces enhanced STAT5 phosphorylation as compared to wild-type IL-15 when contacted with the cell expressing the IL-15R.
231. The single-chain polypeptide of claim 230, wherein STAT5 phosphorylation is measured using flow cytometry or western blot analysis.
232. The single-chain polypeptide of any one of claims 228 to 231231 wherein the cell is a CD8+ T-cell, a natural killer (NK) cell, a natural killer T-cell (NKT cell), a CD4+ T-cell, or a regulatory T-cell (Treg).
233. The single-chain polypeptide of any one of claims 228 to 232, wherein the single- chain polypeptide induces proliferation and/or activation of one or more than one immune cell selected from the group consisting of CD8+ T-cells, aNK cells, NKT cells, CD4+ T-cells, and Tregs, when administered to a subject.
234. The single-chain polypeptide of claim 233, wherein the single-chain polypeptide induces proliferation of the one or more than one immune cell after about 4 days.
235. The single-chain polypeptide of claim 233 or 234, wherein the polypeptide induces the expression of one or more than one protein in the one or more immune cells selected from the group consisting of CD25, Ki-67, NKG2D, and/or granzyme B (GrzB).
236. The single-chain polypeptide of claim 235, wherein the single-chain polypeptide induces the expression of the one or more than one protein after about 4 days.
237. The single-chain polypeptide of any one of claims 145 to 236, wherein the linker comprises a sequence of (GGGGS)4 (SEQ ID NO:31), (GGGGS)5 (SEQ ID NO:32), (GGGGS)6 (SEQ ID NO:33), or (SGGGG)6 (SEQ ID NO:34).
238. A pharmaceutical composition comprising the single-chain polypeptide of any one of claims 1 to 237 and a pharmaceutically acceptable vehicle or excipient.
239. A method of treating a cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to claim 238.
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