[go: up one dir, main page]

WO2022192259A1 - PROTÉINES DE FUSION ANTI-PD-1 ET IL-15/IL-15Rα HOMODIMÉRIQUES BIFONCTIONNELLES ET LEURS UTILISATIONS - Google Patents

PROTÉINES DE FUSION ANTI-PD-1 ET IL-15/IL-15Rα HOMODIMÉRIQUES BIFONCTIONNELLES ET LEURS UTILISATIONS Download PDF

Info

Publication number
WO2022192259A1
WO2022192259A1 PCT/US2022/019344 US2022019344W WO2022192259A1 WO 2022192259 A1 WO2022192259 A1 WO 2022192259A1 US 2022019344 W US2022019344 W US 2022019344W WO 2022192259 A1 WO2022192259 A1 WO 2022192259A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
polypeptide
amino acid
region
bifunctional
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2022/019344
Other languages
English (en)
Other versions
WO2022192259A9 (fr
Inventor
Yiwen Li
Yuxiang Hu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cureimmune Therapeutics Inc
Original Assignee
Cureimmune Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cureimmune Therapeutics Inc filed Critical Cureimmune Therapeutics Inc
Publication of WO2022192259A1 publication Critical patent/WO2022192259A1/fr
Publication of WO2022192259A9 publication Critical patent/WO2022192259A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • PD-1 antibodies enhance T cell activation by blocking the co-inhibitory PD- 1/PD-Ll pathway.
  • PD-1 blockage therapy has revolutionized cancer immunotherapy.
  • Several antagonistic antibodies to the PD-1/PD-L1 pathway have been approved by the FDA for treatment of a variety of cancer indications, including nivolumab (Bristol- Myers Squibs) and pembrolizumab (Merck), cemiplimab-rwlc (Regeneron), atezolizumab (Roche), durvalumab (Astra Zeneca), and avelumab (Merck Serono).
  • nivolumab Bristol- Myers Squibs
  • pembrolizumab Merk
  • Cemiplimab-rwlc Regeneron
  • atezolizumab Roche
  • durvalumab Advanto
  • avelumab Merck Serono
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15Ra sushi domain, second linker, IL-15 polypeptide, a hinge region, and a Fc region; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment, and the Fc region does not comprise a heterodimerization variant mutation.
  • VL anti-PDl antibody light chain variable region
  • CL light chain constant region
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15 polypeptide, second linker, IL-15Ra sushi domain, a hinge region, and a Fc region; wherein the VL, CL,
  • VH, and CHI together form an anti-PD-1 Fab fragment, and the Fc region does not comprise a heterodimerization variant mutation.
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, modified IL-15Ra sushi domain comprising a S40C mutation, a hinge region, and a Fc region; and (c) a modified IL-15 polypeptide comprising a L52C mutation; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide and modified IL-15Ra sushi domain form a covalent disulfide bond; and the Fc region does not comprise a heterodimerization variant mutation.
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, modified IL-15 polypeptide comprising a L52C mutation, a hinge region, and a Fc region; and (c) a modified IL-15Ra sushi domain comprising a S40C mutation; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment; the modified IL- 15 polypeptide and modified IL-15Ra sushi domain form a covalent disulfide bond; and the Fc region does not comprise a heterodimerization variant mutation.
  • VL anti-PDl antibody light chain
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15 polypeptide, a hinge region, and a Fc region; and (c) an IL-15Ra sushi domain; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment; the IL-15 polypeptide binds to the IL15Ra sushi domain; and the Fc region does not comprise a heterodimerization variant mutation.
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15Ra sushi domain, second linker, IL-15 polypeptide, a hinge region, and a modified Fc region; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, D236, A327G, A330S, and P331S.
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15 polypeptide, second linker, IL-15Ra sushi domain, a hinge region, and a modified Fc region; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, D236, A327G, A330S, and P331S.
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15Ra sushi domain, a hinge region, and a modified Fc region; and (c) an IL-15 polypeptide; wherein the VL, CL, VH, and CHI together form an anti -PD- 1 Fab fragment; the IL-15 polypeptide binds to the IL15Ra sushi domain; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, D236, A327G, A330S, and P331S.
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15 polypeptide, a hinge region, and a modified Fc region; and (c) an IL-15Ra sushi domain; wherein the VL, CL, VH, and CHI together form an anti -PD- 1 Fab fragment; the IL-15 polypeptide binds to the IL15Ra sushi domain; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, D236, A327G, A330S, and P331S.
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, modified IL-15Ra sushi domain comprising a S40C mutation, a hinge region, and a modified Fc region; and (c) a modified IL-15 polypeptide comprising a L52C mutation; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide and modified IL-15Ra sushi domain form a covalent disulfide bond; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti -PD 1 antibody heavy chain variable region (VH), CHI domain, first linker, modified IL-15 polypeptide comprising a L52C mutation, a hinge region, and a modified Fc region; and (c) a modified IL-15Ra sushi domain comprising a S40C mutation; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide and modified IL-15Ra sushi domain form a covalent disulfide bond; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L
  • compositions comprising the bifunctional homodimeric fusion proteins of the present disclosure.
  • nucleic acid compositions encoding the bifunctional homodimeric fusion proteins of the present disclosure.
  • expression vectors comprising the nucleic acid compositions encoding the bifunctional homodimeric fusion proteins of the present disclosure.
  • host cells comprising the nucleic acid compositions or expression vectors of the present disclosure.
  • FIGS. 1A-1G show exemplary schematic representations of anti-PD-1 x IL-15 bifunctional, homodimeric fusion proteins (“BFP”): (FIG. 1 A) “BFP1”; (FIG. IB) “BFP2”; (FIG. 1C) “BFP3”; (FIG. ID) “BFP4”; (FIG. IE) “BFP5”; (FIG. IF) “BFP6”; and (FIG. 1G) “BFP7.”
  • BFP anti-PD-1 x IL-15 bifunctional, homodimeric fusion proteins
  • FIGS. 2A-2B show results from ELISA binding assay demonstrating that the anti-PD-1 binding domain in the bifunctional, homodimeric fusion protein retains the ability to bind to human (FIG. 2 A) and cynomolgus (FIG. 2B) PD-1 proteins.
  • FIG. 3 shows PD1 and IL1511b/g dual binding ELISA.
  • FIG. 4 shows flow cytometry analysis of PD-1 transfected Jurkat cells stained with anti-PD-1 antibody or various anti-PD-1 x IL-15 bifunctional, homodimeric fusion proteins.
  • FIG. 5 shows enhancement of T cell activation by various anti-PD-1 x IL-15 bifunctional, homodimeric fusion proteins in a PD-1 luciferase reporter assay.
  • FIG. 6 shows enhancement of T cell activation by various anti-PD-1 x IL-15 bifunctional, homodimeric fusion proteins in a MLR assay.
  • FIGS. 7A-7B show flow cytometry analysis of human peripheral NK cells treated with an anti -PD 1 antibody, IL-15 fusion proteins, or various anti-PD-1 x IL-15 bifunctional, homodimeric fusion proteins for CD69 expression (FIG. 7A) and CD25 expression (FIG. 7B).
  • FIG. 8 shows IFN-g secretion analysis of human peripheral NK cells co cultured with K562 cells after treatment with an anti -PD 1 antibody, IL-15 fusion proteins, or various anti-PD-1 x IL-15 bifunctional, homodimeric fusion proteins.
  • FIG. 9 shows granzyme B secretion analysis of NK cells co-cultured with K562 cells after treatment with anti-PD-1 antibody, IL-15 fusion proteins, or various anti-PD- 1 x IL-15 bifunctional, homodimeric fusion proteins.
  • FIG. 10 shows MTS assay to measure effect of anti-PD-1 antibody, IL-15 fusion proteins, or various anti-PD-1 x IL-15 bifunctional, homodimeric fusion proteins on T cell proliferation.
  • FIG. 11 shows tumor volume in MC3 mouse tumor model with human-PD-1 knock-in treated with parental PD-1, antibody, anti-PD-1 x IL-15 bifunctional, homodimeric fusion protein BFP1, BFP2, or human IgG control.
  • FIGS. 12A-12D show stability assays for exemplary anti-PD-1 x IL-15 bifunctional, homodimeric fusion proteins (“BFP”). Temperature-treated BFPs (FIG. 12A - BFP1, FIG. 12B - BFP2, FIG. 12C - BFP3, FIG. 12D - BFP4) were used in a PD-1 binding ELISA assay.
  • FIG. 13 shows results of stability test of temperature-treated BFPs in a PD-1 luciferase report assay to measure their ability to enhance T cell activation.
  • FIGS. 14A-14D show results of stability test of temperature-treated BFPs (FIG. 14A - BFP1, FIG. 14B - BFP2, FIG. 14C - BFP3, FIG. 14D - BFP4) in NK cell functional assay.
  • the present disclosure provides bifunctional, homodimeric fusion proteins comprising an anti-PD-1 binding domain component and IL-15/IL-15Ra component, which are useful in stimulating the immune response, treating cancer, optionally in combination with other anti-cancer therapeutic agents, or treating viral infections, optionally in combination with anti-viral agents.
  • the present disclosure provides a series of bifunctional, homodimeric fusion proteins comprising an anti-PD-1 antagonist antibody component and an IL-15/IL-15Ra component to overcome the shortcoming of anti-PD-1 antibody therapy.
  • the bifunctional, homodimeric fusion proteins of the present disclosure have one or more of the following features/advantages: ability to enhance T cell activation; ability to promote proliferation and expansion of T cells; ability to promote NK cell activation; enhanced stability with an Fab anti -PD 1 binding domain as compared to a scFv anti- PD1 binding domain; and relative ease in expression and/or purification due to the presence of one or more IgGl components.
  • the present disclosure hypothesizes that by increasing the number of tumor- infiltrating lymphocytes by use of IL-15 in combination with anti-PD-1 therapy, the anti-tumor efficacy could be improved in those cancer patients.
  • the bifunctionality inhibiting PD1/PDL1 signaling and enhancing IL-15 signaling
  • homodimeric fusion proteins of the present disclosure may improve anti-tumor treatment efficacy by increasing the number of tumor-infiltrating lymphocytes.
  • Anti-PD-1 component of the bifunctional, homodimeric fusion proteins of the present disclosure enhances T cell activation by removing the co-inhibitory effect of the PD-1/PD-L1 pathway.
  • the IL-15/IL-15Ra complex of the bifunctional, homodimeric fusion proteins of the present disclosure promotes T cell proliferation and expansion to increase the circulating T cells and their infiltration in the tumor microenvironment.
  • IL-15 signaling can also have a strong effect on NK cell population to attack tumor cells.
  • Embodiments of bifunctional, homodimeric fusion proteins of the present disclosure retained strong anti -PD 1 binding activity, enhanced T-cell activation, promoted proliferation and expansion of T cells, enhanced activation, proliferation, and cytotoxicity of NK cells, and/or demonstrated an anti tumor efficacy in vivo in a mouse tumor model.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer) or subranges, unless otherwise indicated.
  • antibody refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as an scFv, Fab, or Fab'2 fragment.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g ., sdAb, sdFv, nanobody).
  • Fab fragment antigen binding
  • rlgG recombinant IgG
  • scFv single chain variable fragments
  • single domain antibodies e.g ., sdAb, sdFv, nanobody.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof (IgGl, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.
  • a monoclonal antibody or antigen-binding portion thereof may be non-human, chimeric, humanized, or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al, Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).
  • variable binding regions refer to the variable binding region from an antibody light chain and an antibody heavy chain, respectively.
  • the variable binding regions comprise discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDRs complementarity determining regions
  • HVR hypervariable region
  • sequences of amino acids within antibody variable regions which, in general, together confer the antigen specificity and/or binding affinity of the antibody, wherein consecutive CDRs (i.e., CDR1 and CDR2, CDR2 and CDR3) are separated from one another in primary amino acid sequence by a framework region.
  • an antibody VH comprises four FRs and three CDRs as follows: FR1- HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and an antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4.
  • the VH and the VL together form the antigen-binding site through their respective CDRs.
  • Numbering of CDR and framework regions may be determined according to any known method or scheme, such as the Rabat, Chothia, EU, IMGT, and AHo numbering schemes (see, e.g. , Rabat etal., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.; Chothia and Lesk, J. Mol. Biol. 196:901-911 (1987)); Lefranc etal., Dev. Comp. Immunol. 27:55, 2003; Honegger and Pluckthun, J. Mol. Bio. 309:651-610 (2001)).
  • Rabat etal. "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.; Chothia and Lesk, J. Mol. Biol. 196:901-911 (1987)
  • Lefranc etal.
  • CDRs of an exemplary variable domain (VH or VL) sequence as provided herein according to one numbering scheme is not exclusive of an antibody comprising CDRs of the same variable domain as determined using a different numbering scheme.
  • CDRs of the anti -PD- 1 antibodies provided in the present disclosure are identified according to the IMGT numbering scheme unless indicated otherwise.
  • Fab or “Fab fragment” or “fragment antigen binding” refers to an antigen binding region of an antibody composed of one constant domain and one variable region of each of the heavy and light chains (VH-CHl and VL-CL).
  • a Fab fragment may be produced by enzymatic digestion of a full length antibody, chemically digestion of a full length antibody, or recombinantly synthesized.
  • Fc region refers to the heavy chain constant region segment of the Fc fragment (the “fragment crystallizable” region or Fc region) from an antibody, which can include one or more constant domains of an antibody, such as CH2, CH3, or both, and excludes the CHI domain.
  • PD-1 or “programmed cell death protein 1” or “CD279” refers to an immune checkpoint molecule expressed on the surface of T cells, B cells, and macrophages that has a role in down-regulating the immune system by promoting apoptosis of antigen-specific T cells in lymph nodes and reducing apoptosis of regulatory T cells.
  • PD-1 is a type I membrane protein and member of the CD28/CTLA- 4 family of T cell regulators.
  • the PD-1 protein includes an extracellular IgV domain, followed by a transmembrane region, and intracellular region.
  • the intracellular region contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif.
  • PD-1 binds to two ligands, PD-L1 and PD-L2.
  • PD-1 includes mammalian PD-1 proteins, e.g., human and non-human primate.
  • PD-1 is a human PD-1 (NCBI Reference Sequence NP 005009; SEQ ID NO: 15) or cynomologus monkey PD-1 (Genbank Accession No. ABR15751; SEQ ID NO:16).
  • IL-15 refers to a proinflammatory cytokine that stimulates CD8 T cell and natural killer (NK) cell activation, proliferation, and cytolytic activity.
  • IL-15 occurs in two forms: (1) soluble IL-15 (IL-15sol) and (2) complexed with its proprietary receptor IL-15Ra, forming the IL-15 receptor complex (IL-15Rc).
  • IL-15RC is trans-presented to neighboring cells expressing IL-15RP/Y, exerting enhanced bioactivity compared to IL-15 sol alone.
  • IL- 15 refers to the form of IL-15 capable of complexing with IL-15Ra.
  • IL-15 refers to human IL-15, such as a human IL-15 comprising the amino acid sequence set forth in SEQ ID NO:20.
  • IL-15 refers to a modified IL-15 containing one or more mutations.
  • a modified IL-15 comprises a L52C substitution (amino acid numbering in reference to SEQ ID NO:20), such as a modified IL-15 comprising the amino acid sequence set forth in SEQ ID NO:22.
  • IL-15Ra sushi domain or “sushi domain” or “sushi” refers to the shortest portion of the IL-15 receptor a that retains IL-15 binding activity.
  • the soluble IL-15Ra sushi domain is a 65 amino acid peptide comprising the extracellular sushi domain of IL-15Ra.
  • Sushi domains common motifs in protein-protein interactions, contain four cysteines forming two disulfide bonds in a 1-3 and 2-4 pattern.
  • IL-15Ra sushi domain refers to a human IL-15Ra sushi domain, such as a human IL-15Ra sushi domain comprising the amino acid sequence set forth in SEQ ID NO:21.
  • IL-15Ra sushi domain includes modified IL-15Ra sushi domains containing one or more mutations.
  • a modified IL-15Ra sushi domain comprises a S40C substitution (amino acid numbering in reference to SEQ ID NO:21), such as a modified IL-15Ra sushi domain comprising the amino acid sequence set forth in SEQ ID NO: 23.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g ., hydroxyproline, g-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.
  • amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
  • a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
  • modification may refer to a mutation in the nucleic acid or polypeptide sequence as compared to a reference or wild-type nucleic acid or polypeptide molecule, respectively, or an alteration to a moiety chemically linked to a protein.
  • a modification may be an altered carbohydrate or PEG structure attached to a protein.
  • protein or “polypeptide” as used herein refers to a compound made up of amino acid residues that are covalently linked by peptide bonds.
  • the term “protein” may be synonymous with the term “polypeptide” or may refer, in addition, to a complex of two or more polypeptides.
  • a polypeptide may further contain other components (e.g, covalently bound), such as a tag, a label, a bioactive molecule, or any combination thereof.
  • a polypeptide may be a fragment.
  • a "fragment” means a polypeptide that is lacking one or more amino acids that are found in a reference sequence.
  • a fragment can comprise a binding domain, antigen, or epitope found in a reference sequence.
  • a fragment of a reference polypeptide can have at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of amino acids of the amino acid sequence of the reference sequence.
  • a “variant" polypeptide species has one or more non-natural amino acids, one or more amino acid substitutions, one or more amino acid insertions, one or more amino acid deletions, or any combination thereof at one or more sites relative to a reference polypeptide as presented herein.
  • “variant” means a polypeptide having a substantially similar activity (e.g ., enzymatic function, immunogenicity, specific binding activity) or structure relative to a reference polypeptide.
  • a variant of a reference polypeptide can have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence for the reference polypeptide as determined by sequence alignment programs and parameters known in the art.
  • the variant can result from, for example, a genetic polymorphism or human manipulation.
  • Conservative substitutions of amino acids are well known and may occur naturally or may be introduced when a protein is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning:
  • Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired.
  • random or saturation mutagenesis techniques such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis may be used to prepare polypeptide variants (see, e.g, Sambrook et al., supra).
  • a “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3 : Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (lie or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or
  • amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g acidic, basic, aliphatic, aromatic, or sulfur-containing).
  • an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and He.
  • Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
  • identity in the context of two or more polypeptide or nucleic acid molecule sequences, means two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using methods known in the art, such as a sequence comparison algorithm, by manual alignment, or by visual inspection.
  • a "fusion protein” comprises a single chain polypeptide having at least two distinct domains, wherein the domains are not naturally found together in a protein.
  • a nucleic acid molecule encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be made synthetically.
  • a fusion protein may further contain other components (e.g ., covalently bound), such as a tag, linker, or bioactive molecule.
  • nucleic acid molecule or “polynucleotide” refers to a polymeric compound containing nucleotides that are covalently linked by 3’-5’ phosphodiester bonds.
  • Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes genomic DNA, mitochondrial DNA, cDNA, or vector DNA.
  • a nucleic acid molecule may be double stranded or single stranded, and if single stranded, may be the coding strand or non-coding (anti-sense strand).
  • a nucleic acid molecule may contain natural subunits or non-natural subunits.
  • a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
  • Variants of the polynucleotides of this disclosure are also contemplated. Variant polynucleotides are at least 80%, 85%, 90%, 95%, 99%, or 99.9% identical to a reference polynucleotide as described herein, or that hybridizes to a reference polynucleotide of defined sequence under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65°-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C.
  • the polynucleotide variants retain the capacity to encode an immunoglobulin-like binding protein or antigen-binding fragment thereof having the functionality described herein.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition ( e.g ., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • the term "engineered,” “recombinant,” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous or heterologous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention).
  • Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding functional RNA, proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene, or operon.
  • heterologous or exogenous nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell.
  • the source of the heterologous or exogenous nucleic acid molecule, construct or sequence may be from a different genus or species.
  • a heterologous or exogenous nucleic acid molecule is added (i.e., not endogenous or native) to a host cell or host genome by, for example, conjugation, transformation, transfection, electroporation, or the like, wherein the added molecule may integrate into the host genome or exist as extra-chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may be present in multiple copies.
  • heterologous refers to a non-native enzyme, protein, or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.
  • endogenous refers to a gene, protein, or activity that is normally present in a host cell. Moreover, a gene, protein or activity that is mutated, overexpressed, shuffled, duplicated or otherwise altered as compared to a parent gene, protein or activity is still considered to be endogenous or native to that particular host cell.
  • an endogenous control sequence from a first gene e.g ., promoter, translational attenuation sequences
  • a second native gene or nucleic acid molecule may be used to alter or regulate expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule differs from normal expression or regulation in a parent cell.
  • the term "expression” refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene.
  • the process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, posttranslational modification, or any combination thereof.
  • An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).
  • heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule (e.g., a heavy chain and a light chain of an antibody), as a single nucleic acid molecule encoding a protein (e.g, a heavy chain of an antibody), or any combination thereof.
  • a polycistronic nucleic acid molecule e.g., a heavy chain and a light chain of an antibody
  • a single nucleic acid molecule encoding a protein e.g, a heavy chain of an antibody
  • heterologous nucleic acid molecules When two or more heterologous nucleic acid molecules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof.
  • the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.
  • the term "introduced” in the context of inserting a nucleic acid sequence into a cell means “transfection", or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g ., transfected mRNA).
  • a cell e.g., chromosome, plasmid, plastid, or mitochondrial DNA
  • transiently expressed e.g ., transfected mRNA
  • Bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure comprise an anti-PD-1 binding domain.
  • the anti-PD-1 binding domain comprises a Fab fragment.
  • a Fab fragment is composed of two polypeptide chains, each comprising one constant domain and one variable region of each of the heavy and light chains.
  • a first polypeptide chain comprises from N- terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL).
  • a second polypeptide chain comprises from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH) and heavy chain constant domain 1 (CHI).
  • the VL-CL and VH-CH1 of the two polypeptide chains together form an anti-PD-1 Fab fragment.
  • the anti-PDl Fab fragment formed by the first polypeptide chain and second polypeptide chain contains one interchain disulfide bond between a cysteine residue in the CL domain and a cysteine residue in the CHI domain.
  • the anti-PDl Fab fragment formed by the first polypeptide chain and second polypeptide chain contains two or more interchain disulfide bonds between a cysteine residue in the CL domain and a cysteine residue in the CHI domain.
  • the anti-PD-1 Fab fragment is chimeric, human, or humanized.
  • the anti-PD-1 Fab fragment is of an IgGl, IgG2, IgG3, or IgG4 isotype.
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 14, and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL and CHI domains are of the IgGl, IgG2, IgG3, or IgG4 isotype.
  • the CHI domain refers to positions 118-220 according to the EU index as in Rabat.
  • the CL comprises a C kappa ("CK") constant region.
  • the CL comprises a C lambda (Ck) constant region.
  • the CL domain is an IgGl CK domain comprising the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain is an IgGl CHI domain comprising the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprising an anti -PD 1 antibody light chain variable region (VL) and light chain constant region (CL) comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the anti -PD 1 antibody light chain variable region (VL) and light chain constant region (CL) is encoded by a nucleic acid sequence set forth in SEQ ID NO:45.
  • the second polypeptide chain comprises an anti -PD 1 antibody heavy chain variable region (VH) and heavy chain constant domain 1 (CHI) comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the anti-PDl antibody heavy chain variable region (VH) and heavy chain constant domain 1 (CHI) is encoded by a nucleic acid sequence set forth in SEQ ID NO:36.
  • the first polypeptide chain comprising an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL) comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain comprises an anti-PDl antibody heavy chain variable region (VH) and heavy chain constant domain 1 (CHI) comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • Bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure comprise an IL-15 polypeptide, an IL-15Ra sushi domain, or both the IL-15 polypeptide and IL-15Ra sushi domain within the second polypeptide chain covalently linked to the C-terminus of VH-CH1 of the anti-PD-1 Fab fragment using a first linker.
  • bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure comprise an IL-15 polypeptide within the second polypeptide chain covalently linked to the C-terminus of VH-CH1 of the anti- PD-1 Fab fragment using a first linker, and an IL-15Ra sushi domain is contained in a separate, third, polypeptide chain.
  • bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure comprise an IL-15Ra sushi domain within the second polypeptide chain covalently linked to the C-terminus of VH-CH1 of the anti-PD-1 Fab fragment using a first linker, and an IL-15 polypeptide is contained in a separate, third, polypeptide chain.
  • bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure comprise an IL-15Ra sushi domain and an IL-15 polypeptide within the second polypeptide chain, with either the IL-15Ra sushi domain or the IL-15 polypeptide linked to the C-terminus of VH-CH1 of the anti-PD-1 Fab fragment using a first linker.
  • Bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure use the sushi domain of IL-15Ra to complex with an IL-15 polypeptide within the same polypeptide chain or on a separate polypeptide chain.
  • the IL-15 polypeptide is a human IL-15 polypeptide sequence. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the IL-15 polypeptide is encoded by a nucleic acid sequence set forth in SEQ ID NO:42.
  • the IL-15Ra sushi domain is a human IL-15 sushi domain.
  • the IL-15Ra sushi domain comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the IL-15Ra sushi domain is encoded by a nucleic acid sequence set forth in SEQ ID NO:40.
  • the IL-15 polypeptide is a modified IL-15 polypeptide.
  • the modified IL-15 polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:20.
  • the modified IL-15 polypeptide has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more amino acid mutations (e.g., substitutions, insertions, and/or deletions) as compared to the amino acid sequence set forth in SEQ ID NO:20.
  • the amino acid substitutions are conservative amino acid substitutions.
  • the IL-15Ra sushi domain is a modified IL-15Ra sushi domain.
  • the modified IL- 15Ra sushi domain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:21.
  • the modified IL-15Ra sushi domain has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • amino acid substitutions are conservative amino acid substitutions.
  • the modified IL-15 polypeptide comprises aN72D mutation.
  • the N72D mutation increases the binding affinity of IL-15 to the IL- 2/15R.Pyc complex and significantly enhances the IL-15 biological activities.
  • the modified IL-15 polypeptide comprises an I67E mutation (amino acid position in reference to SEQ ID NO:20). In some embodiments, the modified IL-15 I67E polypeptide comprises the amino acid sequence of SEQ ID NO:52. In some embodiments, the IL-15 I67E polypeptide is encoded by a nucleic acid sequence set forth in SEQ ID NO:53.
  • each of the IL-15 polypeptide and IL-15Ra sushi domains is engineered to contain a cysteine residue that forms a disulfide bond between the IL-15 polypeptide and IL-15Ra sushi domain.
  • the modified IL-15R polypeptide comprises a substitution selected from L45C, Q48C, V49C, L52C, E53C, E87C, E89C, or any combination thereof (amino acid position in reference to SEQ ID NO:20).
  • the modified IL-15 polypeptide comprises a L52C amino acid substitution (amino acid position in reference to SEQ ID NO:20).
  • the modified IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the modified IL-15 polypeptide is encoded by a nucleic acid sequence set forth in SEQ ID NO:43. In some embodiments, the modified IL-15Ra sushi domain comprises a substitution selected from K34C, A37C, G38C, S40C, L42C, or any combination thereof (amino acid position in reference to SEQ ID NO:21). In some embodiments, the IL-15Ra sushi domain comprises a S40C substitution (amino acid position in reference to SEQ ID NO:21). In some embodiments, the IL-15Ra sushi domain comprises the amino acid sequence set forth in SEQ ID NO:23.
  • the IL-15Ra sushi domain is encoded by a nucleic acid sequence set forth in SEQ ID NO:41.
  • the modified IL-15 polypeptide and the modified IL-15Ra sushi domain within a bifunctional, homodimeric fusion protein comprise a set of amino acid substitutions selected from L52C : S40C; V49C : S40C; E89C : K34C; Q48C : G38C; E53C : L42C; and L45C : A37C, respectively.
  • the modified IL-15 polypeptide and the modified IL-15Ra sushi domain within a bifunctional, homodimeric fusion protein comprise a set of amino acid substitutions L52C : S40C, respectively.
  • Bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure comprise an Fc region.
  • the Fc region is responsible for the effector functions of an antibody, such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors ( e.g ., CD16, CD32, FcRn), greater half-life in vivo relative to a polypeptide lacking an Fc region, protein A binding, and perhaps even placental transfer ( see Capon et al. Nature 337 : 525, 1989).
  • the Fc region comprises the CH2 and CH3 domains of an IgG antibody.
  • the Fc region is obtained from a human antibody.
  • the Fc region comprises CH2 and CH3 domains from the same antibody isotype, such as human IgGl, IgG2, IgG3, or IgG4 (e.g., CH2CH3 from human IgGl).
  • the Fc region in bifunctional, homodimeric anti-PDl/IL- 15/IL-15Ra fusion proteins of the present disclosure will be capable of mediating one or more of these effector functions.
  • the Fc region has normal effector function, meaning having less than 25%, 20%, 15%, 10%, 5%, 1% difference in effector function (e.g., ADCC, CDC, or both) as compared to a wildtype antibody of the same isotype.
  • the Fc region in bifunctional, homodimeric anti-PDl/IL- 15/IL-15Ra fusion proteins of the present disclosure has a reduction in one or more of these effector functions or lack one or more effector functions by way of, for example, one or more amino acid substitutions or deletions in the Fc region portion known in the art.
  • An antibody or antigen-binding fragment having a modified, mutated, or variant Fc region having reduced effector function means that the antibody exhibits a decrease of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
  • the Fc region in an antibody or antigen binding fragment of the present disclosure is a modified Fc region having reduced ADCC, CDC, or both reduced ADCC and CDC.
  • the Fc region is a modified Fc region comprising one, two, three, four, five, six or all of the mutations E233P, L234V, L235A, D0236, A327G, A330S, and P331 S as numbered according to the EU set forth in Rabat.
  • the Fc region is a modified IgGl Fc region comprising mutations corresponding to E233P, L234V, L235A, AG236, A327G, A330S, and P331 S, as numbered according to the EU set forth in Rabat, to attenuate Fc-mediated effector functions (ADCC and CDC).
  • the modified Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the modified Fc region is encoded by a nucleic acid sequence set forth in SEQ ID NO:44.
  • the Fc region does not comprise a heterodimerization variant mutation.
  • a “heterodimerization variant mutation” refers to a mutation in the amino acid sequence of each Fc region on two polypeptide chains, which promotes heterodimeric formation of the two polypeptide chains and/or allow for ease of purification of heterodimers over homodimers.
  • Heterodimerization variant mutations may comprise a steric mutation, knob into hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • a “steric mutation” refers to amino acid mutations in each Fc region containing polypeptide chain such that heterodimers are more likely to associate than homodimers. Steric mutations include knobs and holes mutations and charge pair mutations.
  • a “knob into hole mutation,” also known as “skew mutation,” refers to amino acid engineering in CH3 domains to create either a "knob” or a "hole” in each heavy chain to promote heterodimerization (see, e.g., Ridgway et ah, Protein Engineering (1996) 9:617; Atwell et al. J. Mol. Biol. (1997) 270:26; and U.S. Patent No. 8,216,805). Knob into hole mutations generate complementary interacting interfaces by manipulating key amino acid residues that participate in the Fc dimeric interaction.
  • a knob into hole mutation comprises a T366Y paired with Y407T mutation.
  • Additional exemplary knob into hole mutations include S364K/E357Q paired with L368D/ 370S; L368D/K370S paired with S364K; L368E/K370S paired with S364K; T411T/E360E/Q362E paired with D401K; L368D/ 370S paired with S364K/E357L, K370S paired with S364K/E357Q; and T366S/L368A/Y407V paired with T366W (optionally including a bridging disulfide, T366S/L368A/Y407V/Y349C paired with T366W/S354C).
  • Electrostatic steering mutations also referred to as “charge pairs mutations,” refer to amino acid mutations that use electrostatics to skew formation of heterodimers over homodimers (see, e.g., Gunasekaran etal., J. Biol. Chem. (2010) 285:19637). Electrostatic steering mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation.
  • Exemplary electrostatic steering mutations include D221E/P228E/L368E paired with D221R/P228R/K409R and C220E/P228E/L368E paired with C220R/E224R/P228R/K409R.
  • a “pi mutation” refers to amino acid mutations in the Fc region of one or both polypeptide chains that are directed to altering the isoelectric (pi) point of one, or both, of the Fc containing polypeptide chains such that heterodimers can be separated from homodimers.
  • the pi of one Fc region containing polypeptide chain is engineered away from the pi of a second Fc region containing polypeptide chain, or both the Fc containing polypeptide chains can be modified, with the pi of one polypeptide chain increasing and the pi of the other polypeptide chain decreasing.
  • Pi changes can be attained by removing or adding a charged residue, changing a charged residue from positive or negative to the opposite charge, or changing a charged residue to a neutral residue.
  • the number of pi variants needed on one or both polypeptide chains to get good separation of heterodimers from homodimers is dependent in part on the starting pi of the polypeptide chains. Generally, separation of heterodimers from homodimers can be accomplished if the pi values of the two polypeptide chains differ by at least 0.1 pH unit, or preferably at least 0.2, 0.3, 0.4 or 0.5 pH units.
  • Exemplary pi variants include
  • Exemplary heterodimerization variant mutations include: S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C according to the EU index.
  • the Fc region is a modified IgGl Fc region comprising mutations corresponding to E233P, L234V, L235A, AG236, A327G, A330S, and P331 S, as numbered according to the EU set forth in Rabat and does not comprise a heterodimerization variant mutation.
  • the modified Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19 and does not comprise a heterodimerization variant mutation.
  • Hinge Region Bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure comprise a hinge region.
  • Antibodies have a hinge sequence that is typically situated between the Fab and Fc region (but a lower section of the hinge may include an amino-terminal portion of the Fc region).
  • an immunoglobulin hinge acts as a flexible spacer to allow the Fab portion to move freely in space.
  • hinges are structurally diverse, varying in both sequence and length between immunoglobulin classes and even among subclasses.
  • the antibody hinge region can be divided into three regions: the upper hinge, middle (or core) hinge, and lower hinge, each having a different functional role.
  • the upper hinge allows the movement and rotation of the Fabs.
  • the central core hinge contains a variable number of cysteine residues depending on the IgG subtype that forms disulfide bonds, stabilizing the association of the antibody heavy chains.
  • On the C-terminal side is the lower hinge that allows movement of the Fc relative to the Fabs and whose amino acid residues can be involved in FcyR binding.
  • a human IgGl hinge region is composed of 23 amino acids and is freely flexible, which allows the Fab fragments to rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges.
  • a human IgG2 hinge is relatively short (19 amino acids) and contains a rigid poly-proline double helix stabilized by four inter-heavy chain disulfide bridges, which restricts the flexibility.
  • a human IgG3 hinge differs from the other subclasses by its unique extended hinge region (about four times as long as the IgGl hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix and providing greater flexibility because the Fab fragments are relatively far away from the Fc fragment.
  • human IgG4 hinge is shorter than IgGl but is similar in length as IgG2 and two inter-heavy chain disulfide bridges, and its flexibility is intermediate between that of IgGl and IgG2.
  • Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).
  • the hinge region connects the C-terminus of the IL-15 polypeptide or IL-15Ra sushi domain with the N-terminus of the Fc region within the second polypeptide chain.
  • the hinge region is about 10 to about 100 amino acids, about 10 to about 90 amino acids, about 10 to about 80 amino acids, about 10 to about 70 amino acids, about 10 to about 60 amino acids, about 10 to about 50 amino acids, about 10 to about 40 amino acids, about 10 to about 30 amino acids, about 10 to about 25 amino acids, or about 10 to about 20 amino acids in length.
  • a bifunctional, homodimeric fusion protein forms at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more disulfide bonds between the hinge regions of two homodimeric second polypeptide chains.
  • the hinge region is a modified hinge region having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a reference hinge region, e.g., an IgGl hinge - EPKSCDKTHTCPPCP (SEQ ID NO:56).
  • the hinge region is a modified hinge region having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations (substitutions, insertions, and/or deletions) as compared to a reference hinge region, e.g., an IgGl hinge - EPKSCDKTHTCPPCP (SEQ ID NO:56).
  • the hinge region comprises the amino acid sequence of SEQ ID NO: 17.
  • the modified hinge region has altered flexibility. Mutations that increase the flexibility of the region include substitution of one or more glycine residues, the substitution of a cysteine involved in the formation of a disulfide bond with an amino acid residue that cannot form a disulfide bond (e.g., serine, alanine, glycine). Mutations that decrease the flexibility of the hinge region include the substitution of one or more amino acids residues with one or more proline residues, the substitution of an amino acid residue which cannot form a disulfide bond (e.g. serine, alanine, glycine) with an amino acid residue capable of forming a disulfide bond (e.g. cysteine).
  • the hinge region is a human antibody hinge region. In some embodiments, the hinge region is an IgGl, IgG2, IgG3, or IgG4 hinge region. In some embodiments, the hinge region comprises the upper hinge or a portion thereof, middle (or core) hinge or a portion thereof, and lower hinge or a portion thereof. In some embodiments, the upper hinge or a portion thereof, middle hinge or a portion thereof, and lower hinge or a portion thereof are each obtained from the same antibody isotype ( e.g ., the upper, middle, and lower hinge are all obtained from IgGl).
  • the hinge region is a modified hinge region wherein the upper hinge or a portion thereof, middle hinge or a portion thereof, and lower hinge or a portion thereof are obtained from two or more antibody isotypes.
  • the hinge region comprises the upper hinge or a portion thereof and middle hinge or a portion thereof.
  • the upper hinge or a portion thereof and middle hinge or a portion thereof are each obtained from the same antibody isotype (e.g., the upper and middle, are both obtained from IgGl).
  • the hinge region is a modified hinge region wherein the upper hinge or a portion thereof and middle hinge or a portion thereof are obtained from two different antibody isotypes.
  • the hinge region comprises an upper hinge or a portion thereof and middle hinge obtained from the IgGl isotype.
  • the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 17.
  • Bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure comprise one or more linkers connecting various domains within a polypeptide chain.
  • the anti -PD- 1 heavy chain Fab component (VH-CH1), the IL-15 polypeptide and/or IL-15Ra sushi domain, and the hinge region-Fc region may each be separated from each other by a linker peptide.
  • a linker is an amino acid sequence of sufficient length to allow the proteins to form proper secondary and tertiary structures.
  • a linker comprises 1- 30 amino acids, 10-30 amino acids, or 15-30 amino acids.
  • the linker is a flexible linker.
  • the linker does not exhibit a propensity to forming secondary structure that could interact with the functional domains of the bifunctional, homodimeric anti-PDl/IL-15/IL-15Ra fusion protein.
  • the linker possesses minimal hydrophobic or charged residues, which could promote interaction with the fusion protein functional domains.
  • the linker comprises Gly, Asn, and/or Ser residues.
  • the linker comprises the amino acid sequence set forth in SEQ ID NO:24 or 46.
  • the linker is encoded by the nucleic acid sequence set forth in SEQ ID NOS:38, 39, or 47.
  • the linker comprises the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the linker is encoded by the nucleic acid sequence set forth in SEQ ID NO:37.
  • Bifunctional homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure may also contain junction amino acids, which refer to one or more ( e.g ., about 1-20) amino acid residues between two adjacent motifs, regions or domains of a polypeptide. Junction amino acids may result from the construct design of a chimeric protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein).
  • the present disclosure provides bifunctional homodimeric anti-PDl /IL-15/IL- 15Ra fusion proteins comprising two functional components, an IL-15/IL-15Ra sushi component (IL-15 complex), which binds with IL-1511bgo complex expressed on cell surfaces, and an anti-PD-1 component (Fab), which binds to PD-1.
  • IL-15 complex an IL-15/IL-15Ra sushi component
  • Fab anti-PD-1 component
  • the term “homodimeric,” when used in context with bifunctional homodimeric anti-PDl/IL- 15/IL-15Ra fusion proteins of the present disclosure means that the fusion protein has at least two associated Fc region containing polypeptide chains that self-assemble into a homodimeric Fc region containing fusion protein.
  • the bifunctional homodimeric anti-PDl/IL-15/IL-15Ra fusion proteins of the present disclosure may comprise more than two polypeptide chains, e.g., tetrameric (BFP1 and BFP6) or hexameric structures (BFP2-BFP5), as long as the two Fc region containing polypeptide chains form a homodimer.
  • BFP1 and BFP6 tetrameric
  • BFP2-BFP5 hexameric structures
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15Ra sushi domain, second linker, IL-15 polypeptide, a hinge region, and a Fc region; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment, and the Fc region does not comprise a heterodimerization variant mutation.
  • An exemplary bifunctional homodimeric fusion protein according this configuration is depicted in FIG. 1 A (BFP1).
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence set forth in SEQ ID NO: 14 and the VH comprises the amino acid sequence set forth in SEQ ID NO:13.
  • the CL comprises the IgGl CK or Ck domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker and/or second linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20.
  • the IL-15Ra sushi domain comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the second polypeptide chain further comprises a third linker between the IL-15 polypeptide and the hinge region.
  • the third linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:27 or amino acids 21-695 of SEQ ID NO:27.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:26.
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15Ra sushi domain, second linker, IL-15 polypeptide, a hinge region, and a modified Fc region; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, D236, A327G, A330S, and P331 S.
  • FIG. 1 A An exemplary bifunctional homodimeric fusion protein according this configuration is depicted in FIG. 1 A (BFP1)
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or Ol domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker and/or second linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the IL-15 polypeptide is a modified IL-15 having an I67E mutation and comprises the amino acid sequence set forth in SEQ ID NO:52.
  • the IL-15Ra sushi domain comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the second polypeptide chain further comprises a third linker between the IL-15 polypeptide and the hinge region.
  • the third linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the Fc region does not comprise a heterodimerization variant mutation.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:27 or amino acids 21-695 of SEQ ID NO:27.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:26.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:54 or amino acids 21-695 of SEQ ID NO:54.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:55.
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15 polypeptide, second linker, IL-15Ra sushi domain, a hinge region, and a Fc region; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment, and the Fc region does not comprise a heterodimerization variant mutation.
  • An exemplary bifunctional homodimeric fusion protein according this configuration is depicted in FIG. IF (BFP6).
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or Ck domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker and/or second linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20.
  • the IL-15Ra sushi domain comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the second polypeptide chain further comprises a third linker between the IL-15Ra sushi domain and the hinge region.
  • the third linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO: 10 or amino acids 21-695 of SEQ ID NO: 10.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:9.
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); and (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15 polypeptide, second linker, IL-15Ra sushi domain, a hinge region, and a modified Fc region; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment, and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, D236, A327G, A330S, and P331S.
  • An exemplary bifunctional homodimeric fusion protein according this configuration is depicted in FIG. IF (BFP6)
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or Ck domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker and/or second linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20.
  • the IL-15Ra sushi domain comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the second polypeptide chain further comprises a third linker between the IL-15Ra sushi domain and the hinge region.
  • the third linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the Fc region does not comprise a heterodimerization variant mutation.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO: 10 or amino acids 21-695 of SEQ ID NO: 10.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:9.
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15Ra sushi domain, a hinge region, and a modified Fc region; and (c) an IL-15 polypeptide; wherein the VL, CL, VH, and CHI together form an anti -PD- 1 Fab fragment; the IL-15 polypeptide binds to the IL15Ra sushi domain; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, D236, A327G, A330S, and P331S.
  • FIG. IB An exemplary bifunctional homodimeric fusion protein according this configuration is depicted in FIG. IB (BFP2).
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or Ol domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20.
  • the IL-15Ra sushi domain comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the second polypeptide chain further comprises a second linker between the IL-15Ra sushi domain and the hinge region.
  • the second linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the Fc region does not comprise a heterodimerization variant mutation.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:29 or amino acids 21-561 of SEQ ID NO:29.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:28.
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, modified IL-15Ra sushi domain comprising a S40C mutation, a hinge region, and a Fc region; and (c) a modified IL-15 polypeptide comprising a L52C mutation; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide and modified IL-15Ra sushi domain form a covalent disulfide bond; and the Fc region does not comprise a heterodimerization variant mutation.
  • VL anti-PDl antibody light chain variable region
  • CL light chain constant region
  • VH anti-PDl antibody heavy
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or Ol domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the modified IL-15 polypeptide comprising a L52C mutation comprises the amino acid sequence set forth in SEQ ID NO:22.
  • the modified IL-15Ra sushi domain comprising a S40C mutation comprises the amino acid sequence set forth in SEQ ID NO:23.
  • the second polypeptide chain further comprises a second linker between the modified IL-15Ra sushi domain and the hinge region.
  • the second linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:31 or amino acids 21-561 of SEQ ID NO:31.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:30.
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, modified IL-15Ra sushi domain comprising a S40C mutation, a hinge region, and a modified Fc region; and (c) a modified IL-15 polypeptide comprising a L52C mutation; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide and modified IL-15Ra sushi domain form a covalent disulfide bond; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or Ol domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the modified IL-15 polypeptide comprising a L52C mutation comprises the amino acid sequence set forth in SEQ ID NO:22.
  • the modified IL-15Ra sushi domain comprising a S40C mutation comprises the amino acid sequence set forth in SEQ ID NO:23.
  • the second polypeptide chain further comprises a second linker between the modified IL-15Ra sushi domain and the hinge region.
  • the second linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the Fc region does not comprise a heterodimerization variant mutation.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:31 or amino acids 21-561 of SEQ ID NO:31.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:30.
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, modified IL-15 polypeptide comprising a L52C mutation, a hinge region, and a Fc region; and (c) a modified IL-15Ra sushi domain comprising a S40C mutation; wherein the VL, CL,
  • VH, and CHI together form an anti-PD-1 Fab fragment; the modified IL-15 polypeptide and modified IL-15Ra sushi domain form a covalent disulfide bond; and the Fc region does not comprise a heterodimerization variant mutation.
  • An exemplary bifunctional homodimeric fusion protein according this configuration is depicted in FIG. ID (BFP4).
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or Ol domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the modified IL-15 polypeptide comprising a L52C mutation comprises the amino acid sequence set forth in SEQ ID NO:22.
  • the modified IL-15Ra sushi domain comprising a S40C mutation comprises the amino acid sequence set forth in SEQ ID NO:23.
  • the second polypeptide chain further comprises a second linker between the modified IL-15 polypeptide and the hinge region.
  • the second linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:33 or amino acids 21-610 of SEQ ID NO:33.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:32.
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, modified IL-15 polypeptide comprising a L52C mutation, a hinge region, and a modified Fc region; and (c) a modified IL-15Ra sushi domain comprising a S40C mutation; wherein the VL,
  • CL, VH, and CHI together form an anti -PD- 1 Fab fragment;
  • the modified IL-15 polypeptide and modified IL-15Ra sushi domain form a covalent disulfide bond;
  • the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, D236, A327G, A330S, and P331S.
  • An exemplary bifunctional homodimeric fusion protein according this configuration is depicted in FIG. ID (BFP4).
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or Ol domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the modified IL-15 polypeptide comprising a L52C mutation comprises the amino acid sequence set forth in SEQ ID NO:22.
  • the modified IL-15Ra sushi domain comprising a S40C mutation comprises the amino acid sequence set forth in SEQ ID NO:23.
  • the second polypeptide chain further comprises a second linker between the modified IL-15 polypeptide and the hinge region.
  • the second linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the Fc region does not comprise a heterodimerization variant mutation.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:33 or amino acids 21-610 of SEQ ID NO:33.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:32.
  • each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15 polypeptide, a hinge region, and a Fc region; and (c) an IL-15Ra sushi domain; wherein the VL, CL, VH, and CHI together form an anti-PD-1 Fab fragment; the IL-15 polypeptide binds to the IL15Ra sushi domain; and the Fc region does not comprise a heterodimerization variant mutation.
  • FIG. IE BFP5
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or Ol domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20.
  • the IL-15Ra sushi domain comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the second polypeptide chain further comprises a second linker between the IL-15 polypeptide and the hinge region.
  • the second linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S :
  • T366S/L368A/Y407V T366W
  • S267K/L368D/K370S S267K/LS364K/E357Q
  • T411T/E360E/Q362E D401K
  • L368D/K370S S364K/E357L
  • D221E/P228E/L368E D221R/P228R/K409R
  • C220E/P228E/368E C220R/E224R/K409R
  • T366S/L368A/Y407V/Y349C T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO: 8 or amino acids 21-610 of SEQ ID NO: 8.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:7.
  • the present disclosure provides a bifunctional homodimeric fusion protein, wherein each monomer of the homodimeric protein comprises: (a) a first polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody light chain variable region (VL) and light chain constant region (CL); (b) a second polypeptide chain comprising from N-terminus to C-terminus: an anti-PDl antibody heavy chain variable region (VH), CHI domain, first linker, IL-15 polypeptide, a hinge region, and a modified Fc region; and (c) an IL-15Ra sushi domain; wherein the VL, CL, VH, and CHI together form an anti -PD- 1 Fab fragment; the IL-15 polypeptide binds to the IL15Ra sushi domain; and the modified Fc region comprises one or more mutations selected from E233P, L234V, L235A, D236, A327G, A330S, and P331S.
  • the VL comprises a light-chain complementarity determining region 1 (LCDR1) of SEQ ID NO:4, a light-chain complementarity determining region 2 (LCDR2) of SEQ ID NO:5, and a light-chain complementarity determining region 3 (LCDR3) of SEQ ID NO:6, and the VH comprises a heavy-chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:l, a heavy-chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:2, and a heavy-chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:3.
  • LCDR1 light-chain complementarity determining region 1
  • HCDR2 light-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity-determining region 3
  • the VL comprises the amino acid sequence of SEQ ID NO: 14 and the VH comprises the amino acid sequence of SEQ ID NO: 13.
  • the CL comprises the IgGl CK or CX domain. In some embodiments, the CL comprises the amino acid sequence set forth in SEQ ID NO: 12.
  • the CHI domain comprises the IgGl CHI domain. In some embodiments, the CHI domain comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:34.
  • the VH and CHI of the second polypeptide chain together comprises the amino acid sequence set forth in SEQ ID NO:35 or amino acids 21-236 of SEQ ID NO:35.
  • the first linker comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20.
  • the IL-15Ra sushi domain comprises the amino acid sequence set forth in SEQ ID NO:21.
  • the second polypeptide chain further comprises a second linker between the IL-15 polypeptide and the hinge region.
  • the second linker comprises the amino acid sequence set forth in SEQ ID NO:25.
  • the hinge region is an IgGl hinge region comprising the amino acid sequence set forth in SEQ ID NO: 17.
  • the Fc region does not comprise a heterodimerization variant mutation.
  • the heterodimerization variant mutation is selected from a steric mutation, knob and hole mutation, electrostatic steering mutation, pi mutation, or any combination thereof.
  • the Fc region of each monomer does not comprise a paired set of amino acid substitutions selected from S364K/E357Q : L368D/K370S; L368D/K370S : S364K; L368E/K370S : S364K;
  • T411T/E360E/Q362E D401K; L368D/K370S : S364K/E357L; K370S : S364K/E357Q; T366S/L368A/Y407V : T366W; S267K/L368D/K370S : S267K/LS364K/E357Q; T411T/E360E/Q362E : D401K; L368D/K370S : S364K/E357L; D221E/P228E/L368E : D221R/P228R/K409R; C220E/P228E/368E : C220R/E224R/K409R; and T366S/L368A/Y407V/Y349C : T366W/S354C.
  • the Fc region is an IgGl Fc region. In some embodiments, the Fc region comprises amino acid substitutions at E233P, L234V, L235A, D236, A327G, A330S, and P331. In some embodiments, the Fc region comprises the amino acid sequence set forth in SEQ ID NO: 19.
  • the first polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO:34; and the second polypeptide chain of each monomer of the homodimeric protein comprises the amino acid sequence set forth in SEQ ID NO: 8 or amino acids 21-610 of SEQ ID NO: 8.
  • the first polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:45; and the second polypeptide chain is encoded by the nucleic acid sequence set forth in SEQ ID NO:7.
  • Polypeptide chains of the fusion proteins of the present disclosure are not covalently bound to each other and can self-assemble through ligand-ligand interactions (e.g., between IL-15 polypeptide and IL-15Ra sushi domain) and disulfide bonds (e.g., VH-VL and hinge regions of two polypeptide chains).
  • the present disclosure provides an isolated nucleic acid that encodes the bifunctional, homodimeric fusion proteins comprising an anti -PD- 1 binding domain component and IL-15/IL-15Ra component as described herein.
  • the isolated nucleic acid encodes the first polypeptide chain, the second polypeptide chain, or both the first and second polypeptide chain of BFP1 or BFP6.
  • the isolated nucleic acid encodes the first polypeptide chain, the second polypeptide chain, or both the first and second polypeptide chain of BFP2, BFP3, BFP4, or BFP5.
  • the co-expressed IL-15 polypeptide or IL-15Ra sushi domain is encoded on the same isolated nucleic acid as the first polypeptide chain and/or the second polypeptide chain or on a separate isolated nucleic acid as the first polypeptide chain and/or the second polypeptide chain of BFP2, BFP3, BFP4, or BFP5.
  • the first polypeptide chain, the second polypeptide chain, and optionally the IL-15 polypeptide or IL-15Ra sushi domain are all encoded by separate nucleic acids.
  • the present disclosure also provides isolated nucleic acid compositions comprising one or more nucleic acids encoding the bifunctional, homodimeric fusion proteins described herein.
  • the nucleic acid encoding the bifunctional, homodimeric fusion proteins comprising an anti-PD-1 binding domain component and IL-15/IL-15Ra component is codon optimized to enhance or maximize expression in certain types of cells (e.g ., Scholten et al., Clin. Immunol. 119 135-145, 2006).
  • a "codon optimized" polynucleotide is a heterologous polypeptide having codons modified with silent mutations corresponding to the abundances of host cell tRNA levels.
  • a nucleic acid molecule encoding the bifunctional, homodimeric fusion proteins comprising an anti-PD-1 binding domain component and IL-15/IL-15Ra component of the present disclosure separates different polypeptide chains contained therein (e.g., first polypeptide chain and second polypeptide chain) by a 2A self-cleaving peptide.
  • the 2A self-cleaving peptide is a porcine teschovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot-and-mouth disease virus (F2A), or any combination thereof (see, e.g, Kim et al., PLOS One 6:el8556, 2011, which 2A nucleic acid and amino acid sequences are incorporated herein by reference in their entirety).
  • P2A porcine teschovirus-1
  • E2A equine rhinitis A virus
  • T2A Thosea asigna virus
  • F2A foot-and-mouth disease virus
  • an expression construct comprising a nucleic acid encoding bifunctional, homodimeric fusion proteins as described herein.
  • a nucleic acid may be operably linked to an expression control sequence (e.g., expression construct).
  • expression construct refers to a DNA construct containing a nucleic acid molecule that is operably-linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host.
  • An expression construct may be present in a vector (e.g ., a bacterial vector, a viral vector) or may be integrated into a genome.
  • operably linked refers to the association of two or more nucleic acids on a single polynucleotide fragment so that the function of one is affected by the other.
  • a promoter is operably-linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • expression control sequence also called a regulatory sequence refers to nucleic acid sequences that effect the expression and processing of coding sequences to which they are operably linked.
  • expression control sequences may include transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
  • a nucleic acid or an expression construct encoding the bifunctional, homodimeric fusion proteins is present in a vector.
  • a "vector” is a nucleic acid molecule that is capable of transporting another nucleic acid.
  • Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi -synthetic or synthetic nucleic acids.
  • Exemplary vectors are those capable of autonomous replication (episomal vector) or expression of nucleic acids to which they are linked (expression vectors).
  • Exemplary viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).
  • ortho myxovirus e.g., influenza virus
  • rhabdovirus e.g., rabies and vesicular stomatitis virus
  • paramyxovirus
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al ., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
  • a vector is a plasmid.
  • a vector is a viral vector.
  • the viral vector is a lentiviral vector or a g-retroviral vector.
  • the present disclosure also provides an expression vector composition comprising: a first expression vector comprising a first nucleic acid encoding the first polypeptide; a second expression vector comprising a second nucleic acid encoding the second polypeptide; and optionally, a third expression vector comprising a third nucleic acid encoding an IL-15 polypeptide or IL-15Ra sushi domain.
  • the present disclosure also provides an isolated host cell comprising a nucleic acid (and nucleic acid compositions), expression construct, or vector (vector compositions) encoding the bifunctional homodimeric fusion protein as described herein.
  • the term "host” refers to a cell or microorganism targeted for genetic modification with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest (e.g ., an IL-15 polypeptide).
  • a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to biosynthesis of the heterologous or exogenous protein (e.g., inclusion of a selectable marker).
  • More than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof.
  • two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites.
  • the number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell.
  • host cells include, but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli.
  • the cells are mammalian cells.
  • the host cell is a human embryonic kidney (HEK293) cell, Y0 cell, Sp2/0 cell, NSO murine myeloma cell, PER.C6® human cell, baby hamster kidney cell (BHK), COS cell, or Chinese hamster ovary (CHO) cell.
  • Host cells are cultured using methods known in the art.
  • the present disclosure provides an isolated host cell comprising: a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:34 and a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:27 or amino acids 21-695 of SEQ ID NO:27; a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:34 and a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 10 or amino acids 21-695 of SEQ ID NO: 10; a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:34, a polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:31 or amino acids 21-561 of SEQ ID NO:31, and a polynucleotide sequence encoding
  • the present disclosure provides a process for making a bifunctional homodimeric fusion protein as described herein, comprising culturing a host cell of the present disclosure, under suitable conditions and for a sufficient time to express the bifunctional homodimeric fusion protein, and optionally isolating the bifunctional homodimeric fusion protein from the culture. Purification of the bifunctional homodimeric fusion protein may be performed according to methods known in the art.
  • the present disclosure provides a composition comprising a bifunctional homodimeric anti-PDl/IL-15/IL-15Ra fusion protein of the present disclosure as described herein and a pharmaceutically acceptable carrier, diluent, or excipient.
  • a pharmaceutically acceptable carrier for diagnostic and therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington ’s Pharmaceutical Sciences , Mack Publishing Co. (A.R. Gennaro (Ed.), 18 th Edition,
  • Exemplary pharmaceutically acceptable carriers include any adjuvant, carrier, excipient, glidant, diluent, preservative, dye/colorant, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, or any combination thereof.
  • sterile saline and phosphate buffered saline at physiological pH can be suitable pharmaceutically acceptable carriers.
  • Preservatives, stabilizers, dyes or the like may also be provided in the pharmaceutical composition.
  • antioxidants and suspending agents may also be used.
  • compositions may also contain diluents such as water, buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates (e.g ., glucose, sucrose, dextrins), chelating agents (e.g ., EDTA), glutathione, and other stabilizers and excipients.
  • diluents such as water, buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates (e.g ., glucose, sucrose, dextrins), chelating agents (e.g ., EDTA), glutathione, and other stabilizers and excipients.
  • diluents such as water, buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates (e.g ., glucose, sucrose,
  • compositions described herein can be formulated for oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration.
  • parenteral includes subcutaneous, intravenous, intramuscular, intrastemal, and intratumoral injection or infusion techniques.
  • compositions of the present invention are formulated in a single dose unit or in a form comprising a plurality of dosage units.
  • Methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000).
  • a pharmaceutical composition may be in the form of a solid, semi-solid or liquid.
  • Solid compositions may include powders and tablets.
  • the pharmaceutical compositions described here are lyophilized or in powder form for re-constitution with a suitable vehicle, e.g., sterile water, before use.
  • the pharmaceutical compositions described herein is a suspension, solution, or emulsion.
  • the present disclosure provides a method of stimulating immune response in a subject comprising administering to a patient in need thereof, an effective amount of the bifunctional homodimeric fusion protein of the present disclosure, or a pharmaceutical composition comprising the bifunctional homodimeric fusion protein of the present disclosure.
  • the present disclosure provides a method of inhibiting a tumor cell comprising contacting the tumor cell with the bifunctional homodimeric fusion protein of the present disclosure, or a pharmaceutical composition comprising the bifunctional homodimeric fusion protein of the present disclosure.
  • the bifunctional homodimeric fusion proteins of the present disclosure may be used in a method of treating cancer, comprising administering to a patient in need thereof, an effective amount of the bifunctional homodimeric fusion protein of the present disclosure, or a pharmaceutical composition comprising the bifunctional homodimeric fusion protein of the present disclosure.
  • Patients or subjects that can be treated by bifunctional homodimeric fusion proteins of the present disclosure include, but are not limited to, a mammal, such as human or non-human primates (e.g., monkeys and apes), a domesticated animal (e.g., laboratory animals, household pets, or livestock), non-domesticated animal (e.g., wildlife), dog, cat, rodent, mouse, hamster, cow, bird, chicken, fish, pig, horse, goat, sheep, rabbit, and any combination thereof.
  • the subject is human.
  • the subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric.
  • Treatment refers to medical management of a disease, disorder, or condition of a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat).
  • a subject e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat.
  • an appropriate dose or treatment regimen comprising a bifunctional homodimeric fusion protein or composition of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit.
  • Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof.
  • a “therapeutically effective amount” or “effective amount” of a bifunctional homodimeric fusion protein or composition of this disclosure refers to an amount of the molecule or composition sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner.
  • a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone.
  • a therapeutically effective amount refers to the combined amounts of active ingredients that result in a therapeutic effect, whether administered serially, sequentially, or simultaneously.
  • a combination may comprise, for example, a bifunctional homodimeric fusion protein and an anti tumor agent.
  • an appropriate dose, suitable duration, and frequency of administration of the bifunctional homodimeric fusion protein or composition thereof will be determined by such factors as the condition of the patient, size, weight, body surface area, age, sex, type and severity of the disease, particular therapy to be administered, particular form of the active ingredient, time and the method of administration, and other drugs being administered concurrently, which can readily be determined by a person skilled in the art.
  • a therapeutically effective daily dose of a bifunctional homodimeric fusion protein is (for a 70 kg mammal) from about 0.001 mg/kg ⁇ i.e., 0.07 mg) to about 100 mg/kg ( i.e ., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg ⁇ i.e., 0.7 mg) to about 50 mg/kg ⁇ i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg ⁇ i.e., 70 mg) to about 25 mg/kg ⁇ i.e., 1.75 g).
  • a bifunctional homodimeric fusion protein may be administered one or more times over a given period of time.
  • a method comprises administering the bifunctional homodimeric fusion protein to the subject at least 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more.
  • a method comprises administering the bifunctional homodimeric fusion protein to the subject a plurality of times, wherein a second or successive administration is performed at about 28 days, 21 days, 14 days, 10 days, 7 days, 3 days, 1 day, or less following a first administration.
  • Bifunctional homodimeric fusion proteins of the present disclosure may be administered to a subject by parenteral routes.
  • bifunctional homodimeric fusion proteins are administered to a subject by subcutaneous, intravenous, intraarterial, subdural, intramuscular, intracranial, intrastemal, intratumoral, intraperitoneal, or infusion techniques.
  • Cancers that may be treated by the bifunctional homodimeric fusion proteins provided in the present disclosure include hematologic malignancies and solid tumors.
  • a hematologic malignancy is a leukemia, lymphoma, or myeloma.
  • a leukemia is acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, hairy cell leukemia, B-cell prolymphocytic leukemia, T-cell prolymphocytic leukemia, or juvenile myelomonocytic leukemia.
  • a lymphoma is Hodgkin’s lymphoma; non-Hodgkin’s lymphoma; Epstein-Barr virus-associated lymphoproliferative disease; Burkitt lymphoma; large B cell lymphoma, not otherwise specified; diffuse large B cell lymphoma associated with chronic inflammation; fibrin-associated diffuse large cell lymphoma; primary effusion lymphoma; plasmablastic lymphoma; extranodal NK/T cell lymphoma, nasal type; peripheral T cell lymphoma, not otherwise specified; angioimmunoblastic T cell lymphoma; follicular T cell lymphoma; or systemic T cell lymphoma of childhood.
  • a myeloma is multiple myeloma or myelodysplastic syndrome.
  • the cancer is a Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, thymus cancer, malignant mesothelioma, pituitary tumor, thyroid tumor, melanoma, Merkel cell skin cancer, lung cancer, head and neck cancer, colorectal cancer, liver cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, esophageal cancer, gastric cancer, small intestine cancer, anal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, breast cancer, ovarian cancer, cervical cancer, vaginal cancer, vulvar cancer, endometrial cancer, eye cancer, soft tissue sarcoma, hepatocellular carcinoma, brain tumor, or spinal cord tumor.
  • Hodgkin’s lymphoma multiple myeloma
  • leukemia myelodysplastic syndrome
  • thymus cancer malignant
  • bifunctional homodimeric fusion proteins described herein may be used in combination with one or more anti-tumor agents.
  • the one or more anti-tumor agents is administered simultaneously, separately, or sequentially.
  • an anti-tumor agent is a cellular immunotherapy, antibody therapy, immune checkpoint inhibitor therapy, hormone therapy, chemotherapeutic, targeted cancer therapy, cytokine therapy or any combination thereof.
  • a cellular immunotherapy comprises a TCR-T cell therapy, dendritic cell therapy, or chimeric antigen receptor (CAR)-T cell therapy, or any combination thereof.
  • an antibody therapy comprises an agonistic, immune enhancing antibody.
  • an antibody therapy comprises an antibody-drug conjugate.
  • an antibody therapy comprises bevacizumab, nimotuzumab, lapatinib, cetuximab, panitumumab, matuzumab, trastuzumab, nimotuzumab, zalutumumab, alemtuzumab, rituxmiab, magrolimab, or any combination thereof.
  • an immune checkpoint inhibitor therapy targets PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine, GAL9, VISTA, CEACAM-1, CEACAM-3, CEACAM-5, PVRL2, PD-1, CTLA-4, BTLA, KIR, LAG3, TIM3, A2aR, CD244/2B4, CD 160, TIGIT, LAIR-1, PVRIG/CDl 12R, CD47, SIRPa, or any combination thereof.
  • an immune checkpoint inhibitor therapy comprises ipilimumab, tremelimumab, pidilizumab, nivolumab, pembrolizumab, durvalumab, atezolizumab, avelumab, urelumab, lirilumab, or any combination thereof.
  • a hormone therapy comprises abiraterone, anastrozole, exemestane, fulvestrant, letrozole, leuprolide, tamoxifen, or any combination thereof.
  • a cytokine therapy comprises IFNa, IL-2, IFNy, GM-CSF, IL-7, IL-12, IL-21, or any combination thereof.
  • a chemotherapeutic comprises an alkylating agent, a platinum based agent, a cytotoxic agent, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), a DNA repair inhibitor, or an apoptosis inducing agent.
  • an antimetabolite such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs
  • a DNA synthesis inhibitor such as an intercalating agent
  • a DNA interactive agent such as an intercalating agent
  • DNA repair inhibitor or an apoptosis inducing agent.
  • chemotherapeutic agents considered for use in combination therapies include vemurafenib, dabrafenib, trametinib, cobimetinib, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy- 5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® orNeosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytara
  • alkylating agents include nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, RevimmuneTM), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hex
  • Additional exemplary alkylating agents include, without limitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); dacarbazine (also known
  • platinum based agents include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthriplatin, and triplatin tetranitrate.
  • Exemplary apoptosis inducing agents include AMG-224, AMG-176, and AMG- 232, and venetoclax.
  • Exemplary targeted cancer therapies therapies that target specific molecules that are involved in tumor growth, progression, and metastasis (e.g., oncogenes), include angiogenesis inhibitors (e.g., a VEGF pathway inhibitors), tyrosine kinase inhibitors (e.g., an EGF pathway inhibitors), receptor tyrosine kinase inhibitors, growth factor inhibitors, GTPase inhibitors, serine/threonine kinase inhibitors, transcription factor inhibitors, B-Raf inhibitors, RAF inhibitors, MEK inhibitors, mTOR inhibitors, EGFR inhibitors, ALK inhibitors, ROS1 inhibitors, BCL-2 inhibitors, PI3K inhibitors, VEGFR inhibitors, BCR-ABL inhibitors, MET inhibitors, MYC inhibitors, ABL inhibitors, HER2 inhibitors, BTK inhibitors, H-RAS inhibitors, K-RAS inhibitors, PDGFR inhibitors, TRK inhibitors, c-KIT
  • a targeted cancer therapy comprises bevacizumab, figitumumab, ramucirumab, ranibizumab, vemurafenib, dabrafenib, encorafenib, vorinostat, binimetinib, cobimetinib, refametinib, selumetinib, trametinib, ibrutinib, tirabrutinib, acalabrutinib, spebrutinib, entrectinib, larotrectinib, lestaurtinib, imatinb, sunitinb, ponatinib, capmatinib, crizotinib, tivantinib, onartuzumab, savolitinib, tepotinib, palbociclib, ribociclib, abemaciclib, trilaciclib, defactinib, erdafit
  • bifunctional homodimeric fusion proteins of the present disclosure may be used in a method of treating a viral infection, comprising administering to a patient in need thereof, an effective amount of a bifunctional homodimeric fusion protein of the present disclosure, or a pharmaceutical composition comprising the bifunctional homodimeric fusion protein of the present disclosure.
  • the viral infection is an acute or chronic viral infection.
  • the ability of the bifunctional homodimeric fusion proteins of the present disclosure to activate T- cells would be useful to treat chronic infections.
  • Infectious viruses include eukaryotic viruses, such as adenovirus, bunyavirus, herpesvirus, papovavirus, papillomavirus (e.g., HPV), paramyxovirus, picornavirus, rhabdovirus (e.g., Rabies), orthomyxovirus (e.g., influenza), poxvirus (e.g., Vaccinia), reovirus, retrovirus, lentivirus (e.g., HIV), flavivirus (e.g., HCV, HBV), corona virus, or the like.
  • adenovirus such as adenovirus, bunyavirus, herpesvirus, papovavirus, papillomavirus (e.g., HPV), paramyxovirus, picornavirus, rhabdovirus (e.g., Rabies), orthomyxovirus (e.g., influenza), poxvirus (e.g., Vaccinia), reovirus, retrovirus
  • a viral infection to be treated with the bifunctional homodimeric fusion proteins of the present disclosure is caused by HIV, hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps rubulavirus, rotavirus, measles morbillivirus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus, or arboviral encephalitis virus.
  • herpes virus e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus
  • adenovirus e.g.,
  • a bifunctional homodimeric fusion protein described herein is used to treat a viral infection in combination with one or more anti-viral agents.
  • the one or more anti-viral agents are administered simultaneously, separately, or sequentially.
  • BFPs bifunctional, homodimeric fusion proteins
  • FIGS. 1A-1F A series of bifunctional, homodimeric fusion proteins (BFPs) were constructed comprising an anti-PD-1 antibody component and an IL-15 component plus the Sushi domain of IL-15Ra (Sushi) (see FIGS. 1A-1F).
  • Their DNA sequences were cloned into a mammalian expression vector with a human IgGl Fc containing the E233P, L234V, L235A, D236, A327G, A330S, and P331S mutations to abrogate Fc-mediated effector functions (ADCC and CDC).
  • the relevant DNA sequence of IL-15 and/or Sushi domain was inserted into the Heavy chain (H chain) of IgGl region between Fab and hinge.
  • the DNA sequence of IL-15 or Sushi domain was inserted to mammalian expression vector without IgGl backbone for the purpose of co-expression. Schematic representations of these constructs are shown in FIGS. 1 A-1F.
  • the H chain vector and the L chain vector were co-expressed in Chinese hamster ovary (CHO) cells.
  • the expression vector of H chain, L chain and IL-15 or a Sushi domain were co-expressed in CHO cells.
  • Each of these polypeptide chains of the bifunctional, homodimeric fusion proteins could also be integrated into single expression vector for fusion protein production.
  • the transfected CHO cells were cultured for 96 hours. Supernatants were then harvested and purified using Protein A affinity column.
  • ELISA enzyme-linked immunosorbent assay
  • the plate was incubated with HRP-conjugated anti human IgG Fab antibody at room temperature for 1 h.
  • the plates were washed and then incubated with chromogenic substrate 3, 3', 5, 5'-tetramethylbenzidine.
  • the absorbance at 450 nm was measured on a microplate reader. The percentage of binding over the control IgG was calculated.
  • Human PD-1 -transfected Jurkat cells were stained with anti-PDlxIL15 fusion proteins, anti-PD-1 antibody or isotype control IgGl, followed by BB700 conjugated anti-human Fab antibody. Cells were then analyzed by flow cytometry. Mean fluorescence units (MFU) were plotted against the concentrations of the protein agents.
  • a pair of engineered cell lines were used in this assay: 1) The PD- 1 + Effector Cells (GloResponse NFAT-luc2/PDl Jurkat cells), which are Jurkat T cells expressing PD-1 and a luciferase reporter driven by the NFAT response element, and 2) PD-L1 + Antigen Presenting Cells (PD-L1 aAPC/CHO-Kl Cells), which are CHO-K1 cells expressing PD-L1 and an engineered cell surface TCR-activating protein.
  • PD-1/PD-L1 interaction reduces TCR signaling and NFAT-mediated luminescence.
  • Addition of agents containing antagonist anti-PD-1 antibodies blocking the PD-1/PD-L1 interaction removes the co-inhibitory signal and leads to enhanced TCR activation and NFAT-RE-mediated luminescence.
  • PD-L1 aAPC/CHO-Kl human T-activator cells (Promega) were plated at 40,000 cells per well in a 96-well white opaque plate in 100 pi of RPMI-1640 medium with 10% FBS and incubated overnight at 37 °C at 5% CO2. Medium was removed from the assay plate the following day and various concentrations of anti-PDlxIL15 bifunctional, homodimeric fusion proteins, anti-PD-1 antibodies, control antibody or a control IL15RaSushi-IL-15-Fc fusion protein were added at 40 m ⁇ assay buffer per well.
  • GloResponse NFAT-luc2/PDl Jurkat cells (Promega) were re-suspended at 1.25 x 10 6 /ml in assay buffer and added to the plate at 40 m ⁇ per well. After 6 h of incubation, assay plates were equilibrated at room temperature for 5 min. Bio-GloTM Reagent (Promega) was added to each well at 80 m ⁇ per well. Plates were then incubated for 5 min at room temperature. Luminescence was measured in a plate reader.
  • CD4+ T cells were isolated from a healthy donor using EasySep Human CD4+
  • PBMC peripheral blood mononuclear cells
  • RPMI1640 medium 10% fetal bovine serum
  • IL-4 fetal bovine serum
  • granulocyte-macrophage colony- stimulating factor 20 ng/mL, R&D systems
  • CD4 + T cells were mixed with the allogenic DC at a ratio of 10:1 in AIM-V medium containing 0.5% BSA.
  • Bifunctional, homodimeric anti- PDlxIL15 fusion proteins, PD-1 antibodies or IgG control were then added at serially- diluted concentrations ranging from 133.25 nM to 1.33 pM. After 4 days of culture, supernatants were harvested and measured for IFN-g production by an ELISA assay.
  • Human peripheral NK cells isolated from PBMC of a healthy donor were cultured in the presence or absence of 20 nM bifunctional aPDlxIL15 fusion proteins, PD-1 antibody or IL15RSushi-IL15 fusion protein control for 24 hours. Triple staining with CD25-APC/CD56-PE/CD69-FITC were performed. After wash, cells were analyzed for NK cell activation markers by flow cytometry. CD56 + NK cells were analyzed against either CD25 or CD69.
  • Human peripheral NK cells were isolated from peripheral blood mononuclear cells (PBMC) from a healthy donor. They were maintained in culture for 48 hours in the presence of IL-2. The NK cells were co-cultured with K562 cells for 4 h in the presence or absence of bifunctional, homodimeric anti-PDlxIL15 fusion proteins, PD-1 antibody or IL15RSushi-IL15 fusion protein control. Release IFN-g or Granzyme B in the co-cultured supernatants was measure by ELISA.
  • PBMC peripheral blood mononuclear cells
  • MTS assay is a colorimetric method for the quantification of viable cells.
  • Human anti-CD3 activated T cells were maintained by 10 ng/ml IL-2. After wash, cells were treated with bifunctional, homodimeric anti-PDlxIL15 fusion proteins, control antibodies or IL-15 for 72 hours. MTS tetrazolium compound was then added to cell culture media and the plate was incubated for 2 hours. Absorbance at 490 nM was measured T cell proliferation.
  • EXAMPLE 1 CONSTRUCTION AND EXPRESSION OF ANTI-PD1XIL15 BIFUNCTIONAL,
  • BFP1 (Fab-Sushi-IL-15-Fc) was constructed by pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgGl antibody with its VH-CHl sequences linked with the Sushi sequence and IL-15 sequence followed by the Hinge-Fc sequences (SEQ ID NO:27) (FIG. 1A).
  • BFP2 (Fab-Sushi-Fc/co-IL15) was constructed by co-expressing IL-15 with a Sushi-containing IgGl -like structure.
  • This Sushi-containing IgGl -like structure was constructed by pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgGl antibody with the heavy chain VH-CHl sequence linked to the Sushi-Hinge-Fc sequences (SEQ ID NO:29) (FIG. IB).
  • BFP3 (Fab-Sushi(mu)-Fc/co-IL15 (mu)) was constructed by co-expressing IL-15 containing a point mutation (L52C) with a Sushi- containing IgGl -like structure.
  • This Sushi-containing IgGl -like structure was constructed by pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgGl antibody with the heavy chain VH-CHl sequence linked to Sushi containing a point mutation(S40C) followed by the Hinge-Fc sequences (SEQ ID NO:31) (FIG. 1C).
  • BFP4 (Fab-IL15 (mu)-Fc/co- Sushi (mu)) was constructed by co-expressing Sushi containing a point mutation (S40C) with an IL 15 -containing IgGl -like structure.
  • This IL 15 -containing IgGl -like structure was constructed by pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgGl antibody with the heavy chain VH- CHI sequence linked to IL-15 containing a point mutation (L52C) followed by the Hinge-Fc sequences (SEQ ID NO:33) (FIG. ID).
  • BFP5 (Fab-IL15-F c/co-Sushi) was constructed by co-expressing Sushi with IL- 15 containing IgGl-like structure.
  • This IL 15 -containing IgGl-like structure is constructed by pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgGl antibody with the heavy chain VH-CH1 sequence linked to IL-15 followed by the Hinge-Fc sequences (SEQ ID NO:8) (FIG. IE).
  • BFP6 (Fab-IL-15-Sushi-Fc) was constructed by pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgGl antibody with its VH-CHl sequences linked with the IL-15 sequence and Sushi sequence followed by the Hinge-Fc sequences (SEQ ID NO: 10) (FIG. IF).
  • BFP7 was constructed by pairing the VL-CL sequence (SEQ ID NO:34) of an anti-PD-1 IgGl antibody with its VH-CHl sequences linked with the Sushi sequence and IL-15 I67E sequence followed by the Hinge-Fc sequences (SEQ ID NO:54) (FIG. 16). These BFP1, BFP2, BFP3, BFP4, and BFP7 constructs were used to transfect Chinese hamster cells (CHO) for transient or stable expression. Supernatants from these expression cultures were purified using Protein A method. Endotoxin levels were determined to be below 0.2 unit per mg protein.
  • EXAMPLE 2 IN VITRO FUNCTIONAL STUDIES Anti-PDlxIL15 bifunctional, homodimeric fusion proteins retain binding activities to both recombinant human and cynomolgus PD-1
  • BFP1- BFP4 bifunctional, homodimeric anti-PDlxIL15 fusion proteins
  • PD-1 and IL-15RPY targets
  • 96-well plates were coated with 25 ng/well of recombinant human PD-l-Fc protein.
  • Series diluted antibody or fusion proteins were added. After washes, bound antibody or fusion proteins were detected using 5 ng/well of biotinylated ILl 511bg and streptavidin HRP followed by chromogenic TMB substrate.
  • Anti-PD-1 antibody and ILl 5 component in fusion proteins are capable of binding to PD-1 protein and IL15R/p simultaneously (FIG. 3).
  • Anti-PDlxIL15 bifunctional, homodimeric fusion proteins retain binding activities to cell surface PD-1
  • bifunctional, homodimeric anti-PDlxIL15 fusion proteins to bind cell line expressing human PD-1 was then tested.
  • Human PD-1 -transfected Jurkat cells were stained with anti-PDlxIL15 fusion proteins, anti-PD-1 antibody or isotype control IgGl, followed by BB700 conjugated anti-human Fab antibody. Cells were then analyzed by flow cytometry. Mean fluorescence units (MFU) were plotted against the concentrations of the protein agents. There were no significant differences in their binding activities to cell surface PD-1 among the different bifunctional fusion proteins (FIG. 4).
  • Anti-PDlxIL15 bifunctional, homodimeric fusion proteins retain the ability to enhance T cell activation in PD-1 Lucif erase reporter assay
  • bifunctional anti-PDlxIL15 fusion proteins to enhance T cell activation was compared with parental anti-PD-1 antibody in a PD-1 luciferase report assay.
  • PD-L1 aAPC/CHO-Kl human T-activator cells were co-cultured with GloResponse NFAT-luc2/PD-l Jurkat cells in the presence or absence of anti-PD-1 antibody, bifunctional homodimeric fusion proteins, control IgG, or a control IL15RaSushi-IL15 fusion protein for 6 hours.
  • Luminescence activity was measured in a plate reader (FIG. 5).
  • Anti-PDlxIL15 bifunctional, homodimeric fusion proteins retain the ability to enhance T cell activation in mixed leukocyte reaction (MLR) assay
  • bifunctional, homodimeric anti-PDlxIL15 fusion proteins to enhance T cell activation in a MLR assay was also investigated. Similar to PD-1 antibody, treatment with bifunctional anti-PDlxIL15 fusion proteins significantly enhance T cell activation as measured by IFN-g release (FIG. 6). In contrast, IL-15 itself or IL-15/IL5RaSushi complexes showed no effects on T cell activation.
  • NK cells Natural killer (NK) cells are part of the innate immune system and mediate responses against tumor cells.
  • the ability of bifunctional, homodimeric anti-PDlxIL15 fusion proteins, control IL15RaSushi-IL15-Fc fusion protein or control antibodies to activate CD56+ NK cells was analyzed.
  • CD25 and CD69 are considered to be NK cell activation markers.
  • Treatment with anti-PDlxIL15 bispecific, homodimeric fusion protein significantly increased the number of activated NK cells as analyzed by CD25 or CD69 expression, similar to IL15RaSushi-IL15-Fc fusion protein (FIGS. 7A-7B). In contrast, treatment with anti-PD-1 antibody or isotype control IgGl had no effect.
  • bifunctional, homodimeric anti-PDlxIL15 fusion proteins enhance the activity of NK cell cytotoxicity was analyzed by IFN-g and Granzyme B production.
  • Human peripheral NK cells were isolated and cultured for 48 h in the presence of IL-2.
  • K562 is a NK cell-sensitive target cell line.
  • the NK cells were co-cultured with K562 cells for 4 h in the presence or absence of bifunctional, homodimeric anti-PDlxIL15 fusion proteins, anti-PD-1 antibody or IL15RSushi-IL15-Fc fusion protein control. There were no effects in the anti-PD-1 antibody or control IgGl group.
  • NK activation can also be measured by granzyme B secretion from NK cells in a cytotoxicity assay against K562 target cells.
  • a significant increase of Granzyme B release was observed in the co-cultured supernatants of NK cells and K562 target cells treated by different types of bifunctional, homodimeric anti-PDlxIL15 fusion proteins except BFP4, IL15RSushi-IL15 fusion protein or IL15, suggesting that bifunctional aPDlxIL15 fusion proteins retained the ability of the IL15 component to enhance the activity of NK cells-mediated cytotoxicity (FIG. 9).
  • FIG. 9 In a repeated study, similar results with a dose-response effect were observed (data not shown).
  • Bifunctional anti-PDlxILl 5 bifunctional, homodimeric fusion proteins promote proliferation of T cells
  • IL-15 plays an important role in the proliferation and survival of T cells, especially memory T effector cells.
  • the ability of the IL15 component of bifunctional, homodimeric anti-PDlxIL15 fusion proteins to promote T cell proliferation was tested in a MTS assay.
  • Anti-CD3 antibody-activated T cells were cultured in the presence of bifunctional, homodimeric anti-PDlxIL15 proteins, control antibodies or IL15RaSushi- IL15 complex for 72 h. The results showed that all the bifunctional agents retained the ability of IL15 complex to promote T cell proliferation to different degrees (FIG. 10).
  • anti -PD-1 antibody had little effect on T cell proliferation.
  • mice with human PD-1 knock-in were inoculated with 1 xlO 5 MC38 tumor cells.
  • tumors reach approximately 120 mm 3 in size, mice were treated with anti-PDlxIL15 bifunctional, homodimeric fusion protein BFP1, BFP2, parental anti- PD1 antibody, or human IgG with indicated dose and intervals.
  • anti -PD 1 antibody #61 significantly slowed the growth of tumor.
  • Treatment with either BFP1 or BFP2 appeared to have better anti -tumor efficacy than anti-PDl antibody #61 treatment alone (FIG. 11).
  • Bifunctional, homodimeric fusion proteins BFP1-4 were treated at 4°C, room temperature and 37°C for 1 week (lw), 2w or 4w. Treated fusion proteins were then diluted and loaded to 96 wells plates coated with 25 ng/well of recombinant human PD- 1-Fc protein. After removing unbound fusion proteins, bound fusion proteins were detected using HRP-conjugated anti-human IgG Fab antibody and chromogenic substrate at OD 450 nM. These temperature-treated BFPs remain stable as measured by PD-1 binding activities (FIGS. 12A-12D).
  • PD-L1 aAPC/CHO-Kl human T-activator cells were co-cultured with GloResponse NFAT-luc2/PD-l Jurkat cells in the presence or absence of bifunctional, homodimeric fusion proteins BFP1-4 or control IgG for 6 hours. Luminescence activity was measured in a plate reader. These temperature- treated BFPs remain stable as measured by their ability to enhance T cell activation (FIG. 13).
  • Human peripheral NK cells were isolated and cultured for 48 hours in the presence of IL-2. The NK cells were co-cultured with K562 cells for 4 hours in the presence or absence of temperature-treated bifunctional, homodimeric fusion proteins BFP1-4. The released IFN-g in the co-cultured supernatant was measure by ELISA. These temperature-treated BFPs remained functionally stable as measured by NK cell functional assay (FIGS. 14A-14D).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Oncology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente divulgation concerne des protéines de fusion homodimériques bifonctionnelles comprenant un composant de domaine de liaison anti-PD-1 et un composant IL-15/IL-15R, des compositions comprenant les protéines de fusion homodimériques bifonctionnelles, et des méthodes de traitement du cancer ou d'une infection virale à l'aide des protéines de fusion homodimériques bifonctionnelles.
PCT/US2022/019344 2021-03-09 2022-03-08 PROTÉINES DE FUSION ANTI-PD-1 ET IL-15/IL-15Rα HOMODIMÉRIQUES BIFONCTIONNELLES ET LEURS UTILISATIONS Ceased WO2022192259A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163158801P 2021-03-09 2021-03-09
US63/158,801 2021-03-09

Publications (2)

Publication Number Publication Date
WO2022192259A1 true WO2022192259A1 (fr) 2022-09-15
WO2022192259A9 WO2022192259A9 (fr) 2023-02-09

Family

ID=80952487

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/019344 Ceased WO2022192259A1 (fr) 2021-03-09 2022-03-08 PROTÉINES DE FUSION ANTI-PD-1 ET IL-15/IL-15Rα HOMODIMÉRIQUES BIFONCTIONNELLES ET LEURS UTILISATIONS

Country Status (2)

Country Link
TW (1) TW202246357A (fr)
WO (1) WO2022192259A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116284430A (zh) * 2021-07-30 2023-06-23 广东菲鹏制药股份有限公司 靶向肿瘤的il15融合蛋白及其应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025140627A1 (fr) * 2023-12-28 2025-07-03 南通壹宸生物医药科技有限公司 Anticorps de fusion et son utilisation

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8216805B2 (en) 1995-03-01 2012-07-10 Genentech, Inc. Knobs and holes heteromeric polypeptides
WO2015109124A2 (fr) * 2014-01-15 2015-07-23 Kadmon Corporation, Llc Agents immunomodulateurs
WO2016004060A2 (fr) * 2014-06-30 2016-01-07 Altor Bioscience Corporation Molécules à base de il-15 et leurs procédés d'utilisation
WO2019191100A1 (fr) * 2018-03-26 2019-10-03 Altor Bioscience Llc Molécules d'association au récepteur du tgf-beta, il-15 et anti-pdl1
WO2019204665A1 (fr) * 2018-04-18 2019-10-24 Xencor, Inc. Protéines de fusion hétérodimères ciblant pd-1 contenant des protéines de fusion fc d'il-15/il-15ra, domaines de liaison à l'antigène pd-1 et utilisations associées
CN110448691A (zh) * 2018-05-07 2019-11-15 江苏恒瑞医药股份有限公司 抗pd-1抗体联合il-15蛋白复合物在制备治疗肿瘤的药物中的用途
WO2020077276A2 (fr) * 2018-10-12 2020-04-16 Xencor, Inc. Protéines de fusion fc d'il-15/il-15ralpha ciblant pd-1 et utilisations dans des polythérapies faisant intervenir celles-ci
WO2020165374A1 (fr) * 2019-02-14 2020-08-20 Ose Immunotherapeutics Molécule bifonctionnelle comprenant il-15ra

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8216805B2 (en) 1995-03-01 2012-07-10 Genentech, Inc. Knobs and holes heteromeric polypeptides
WO2015109124A2 (fr) * 2014-01-15 2015-07-23 Kadmon Corporation, Llc Agents immunomodulateurs
WO2016004060A2 (fr) * 2014-06-30 2016-01-07 Altor Bioscience Corporation Molécules à base de il-15 et leurs procédés d'utilisation
WO2019191100A1 (fr) * 2018-03-26 2019-10-03 Altor Bioscience Llc Molécules d'association au récepteur du tgf-beta, il-15 et anti-pdl1
WO2019204665A1 (fr) * 2018-04-18 2019-10-24 Xencor, Inc. Protéines de fusion hétérodimères ciblant pd-1 contenant des protéines de fusion fc d'il-15/il-15ra, domaines de liaison à l'antigène pd-1 et utilisations associées
CN110448691A (zh) * 2018-05-07 2019-11-15 江苏恒瑞医药股份有限公司 抗pd-1抗体联合il-15蛋白复合物在制备治疗肿瘤的药物中的用途
WO2020077276A2 (fr) * 2018-10-12 2020-04-16 Xencor, Inc. Protéines de fusion fc d'il-15/il-15ralpha ciblant pd-1 et utilisations dans des polythérapies faisant intervenir celles-ci
WO2020165374A1 (fr) * 2019-02-14 2020-08-20 Ose Immunotherapeutics Molécule bifonctionnelle comprenant il-15ra

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
"Antibodies: A Laboratory Manual", 1988, COLD SPRING HARBOR LABORATORY
"CRC Handbook of Food, Drug, and Cosmetic Excipients", 1992, CRC PRESS LLC
"Remington: The Science and Practice of Pharmacy", 2000, PHILADELPHIA COLLEGE OF PHARMACY AND SCIENCE
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING CO
ALTSCHUL ET AL.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 2007, pages 3389 - 3402, XP002905950, DOI: 10.1093/nar/25.17.3389
ATWELL ET AL., J. MOL. BIOL., vol. 270, 1997, pages 26
BIOINFORMATICS, vol. 15, 2016, pages 298 - 300
CAPON ET AL., NATURE, vol. 337, 1989, pages 525
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
COFFIN, J. M. ET AL.: "Fundamental Virology", 1996, LIPPINCOTT-RAVEN PUBLISHERS, article "Retroviridae: The viruses and their replication"
GREGORY L. MOORE ET AL: "A robust heterodimeric Fc platform engineered for efficient development of bispecific antibodies of multiple formats", METHODS, vol. 154, 1 February 2019 (2019-02-01), NL, pages 38 - 50, XP055625796, ISSN: 1046-2023, DOI: 10.1016/j.ymeth.2018.10.006 *
GUNASEKARAN ET AL., J. BIOL. CHEM., vol. 285, 2010, pages 19637
HONEGGERPLUCKTHUN, J. MOL. BIO., vol. 309, 2001, pages 657 - 670
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE NATIONAL INSTITUTES OF HEALTH
KIM ET AL., PLOS ONE, vol. 6, 2011, pages 18556
LEFRANC ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55
RIDGWAY ET AL., PROTEIN ENGINEERING, vol. 9, 1996, pages 617
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SCHOLTEN ET AL., CLIN. IMMUNOL., vol. 119, 2006, pages 135 - 145

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116284430A (zh) * 2021-07-30 2023-06-23 广东菲鹏制药股份有限公司 靶向肿瘤的il15融合蛋白及其应用

Also Published As

Publication number Publication date
TW202246357A (zh) 2022-12-01
WO2022192259A9 (fr) 2023-02-09

Similar Documents

Publication Publication Date Title
JP7184866B2 (ja) Ｐｄ１および／またはｌａｇ３結合性物質
US20250154261A1 (en) Multi-specific binding proteins that bind cd33, nkg2d, and cd16, and methods of use
ES3006183T3 (en) Antibody molecules that bind pd-l1 and cd137
JP6731403B2 (ja) 免疫応答およびがん治療の増強に使用するための組成物および方法
US20200376034A1 (en) Antibody variable domains targeting cd33, and use thereof
CN112088167A (zh) 特异性针对人类连接蛋白4的抗体
CN112300282A (zh) 靶向cd7的人源化抗体及其用途
US12258411B2 (en) Anti-CCR8 antibodies and uses thereof
KR20210010862A (ko) IL-15/IL-15Rα Fc-융합 단백질 및 PD-1 항원 결합 도메인을 함유하는 PD-1 표적화 이종이량체 융합 단백질 및 이의 용도
CN109963871A (zh) 具有激动活性的多价及多表位抗体以及使用方法
TW202039572A (zh) 雙官能抗pd-1/il-7分子
CN119661722A (zh) 结合t细胞相关癌细胞的多功能分子及其用途
JP2020522254A (ja) 骨髄増殖性白血病（ｍｐｌ）タンパク質に結合する多特異性分子およびその使用
CN113164777B (zh) Csf1r/ccr2多特异性抗体
KR20230034960A (ko) Lag3에 결합하는 항체 및 그 용도
CA3233075A1 (fr) Mutant de l'interleukine-2 et sa proteine de fusion
US20250000946A1 (en) Il-2 variants and fusion proteins thereof
WO2022192259A1 (fr) PROTÉINES DE FUSION ANTI-PD-1 ET IL-15/IL-15Rα HOMODIMÉRIQUES BIFONCTIONNELLES ET LEURS UTILISATIONS
US20240209106A1 (en) Anti-cd137 antibodies and methods of use
US20230107609A1 (en) Anti-pd-1 antibodies and methods of use
WO2024233258A1 (fr) Anticorps anti-tnfr2 et leurs utilisations
US20240209113A1 (en) Anti-gpc3 and anti-cd137 multispecific antibodies and methods of use
RU2830982C2 (ru) Улучшенные анти-flt3 антигенсвязывающие белки
HK40090273A (zh) 用於治疗癌症和感染的针对nkp46的抗体及其构建体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22713152

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22713152

Country of ref document: EP

Kind code of ref document: A1