WO2019032945A1 - Cd40-binding agents and uses thereof - Google Patents

Cd40-binding agents and uses thereof Download PDF

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Publication number
WO2019032945A1
WO2019032945A1 PCT/US2018/046186 US2018046186W WO2019032945A1 WO 2019032945 A1 WO2019032945 A1 WO 2019032945A1 US 2018046186 W US2018046186 W US 2018046186W WO 2019032945 A1 WO2019032945 A1 WO 2019032945A1
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polypeptide
agent
seq
region
fragment
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PCT/US2018/046186
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French (fr)
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WO2019032945A9 (en
Inventor
Austin L. Gurney
Minu K. SRIVASTAVA
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Oncomed Pharmaceuticals, Inc.
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Publication of WO2019032945A1 publication Critical patent/WO2019032945A1/en
Publication of WO2019032945A9 publication Critical patent/WO2019032945A9/en

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    • 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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • the present invention generally relates to agents that bind CD40, particularly agents comprising at least one copy of the extracellular domain of CD40L or a receptor binding fragment thereof, wherein the extracellular domain of CD40L or fragment thereof comprises a mutation in the integrin-binding region.
  • the invention also relates to methods of using the agents for the modulation of immune responses and/or the treatment of diseases such as cancer.
  • the basis for immunotherapy is the manipulation and/or modulation of the
  • the general aim of immunotherapy is to treat diseases by controlling the immune response to a "foreign agent," for example a pathogen or a tumor cell.
  • immunotherapy is used to treat autoimmune diseases which may arise from an abnormal immune response against proteins, molecules, and/or tissues normally present in the body. Immunotherapy may include methods to induce or enhance specific immune responses or to inhibit or reduce specific immune responses.
  • the immune system is a highly complex system made up of a great number of cell types, including but not limited to, T-cells, B-cells, natural killer cells, antigen-presenting cells, dendritic cells, monocytes, and macrophages. These cells possess complex and subtle systems for controlling their interactions and responses.
  • the cells utilize both activating and inhibitory mechanisms and feedback loops to keep responses in check and not allow negative consequences of an uncontrolled immune response (e.g., autoimmune diseases or a cytokine storm).
  • cancer immunosurveillance is based on the theory that the immune system can recognize tumor cells, mount an immune response, and suppress the development and/or progression of a tumor.
  • cancerous cells have developed mechanisms to evade the immune system which can allow for uninhibited growth of tumors.
  • Cancer/tumor immunotherapy focuses on the development of new and novel agents that can activate and/or boost the immune system to achieve a more effective attack against tumor cells resulting in increased killing of tumor cells and/or inhibition of tumor growth.
  • the present invention provides a variety of polypeptides and agents that bind
  • the term "agent” includes, but is not limited to, polypeptides, fusion proteins, homodimeric molecules, and heterodimeric molecules.
  • a polypeptide or agent binds human CD40.
  • the polypeptide or agent is a CD40 agonist.
  • the polypeptide or agent that binds CD40 is a soluble CD40 ligand (CD40L).
  • CD40L soluble CD40 ligand
  • the polypeptides and agents are used in methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response. In some embodiments, the polypeptides and agents are used in methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response to cancer, a tumor, and/or tumor cells. In some embodiments, the polypeptides and agents are used in methods of inhibiting the growth of a tumor or tumor cells. In some embodiments, the polypeptides and agents are used in methods for the treatment of cancer. In some embodiments, the methods comprise inhibiting the growth of cancer cells.
  • the invention also provides compositions comprising the agents described herein. In some embodiments, the compositions are pharmaceutical compositions comprising the polypeptides and agents described herein. Polynucleotides encoding the polypeptides and agents and methods of making the agents are also provided.
  • polypeptides comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • CD40L human CD40 ligand
  • CD40L single chain fusion human CD40 ligand (CD40L) polypeptides comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the extracellular domain of human CD40L or a fragment thereof has a lower binding affinity to integrin alpha (lib) beta 3 compared to a corresponding extracellular domain of human CD40L or a fragment thereof not comprising the mutation.
  • the integrin-binding region of CD40L comprises the KGD motif (residues 115-117 of SEQ ID NO: 12). In some embodiments, the integrin-binding region of CD40L consists of the KGD motif (residues 115-117 of SEQ ID NO: 12).
  • the mutation comprises the substitution, insertion, or
  • the mutation comprises the substitution or deletion of 1, 2, or 3 residues in the integrin- binding region.
  • the mutation in the integrin-binding region comprises a D to E substitution in the KGD motif (residues 115-117 of SEQ ID NO: 12); and/or a G to A substitution in the KGD motif (residues 115-117 of SEQ ID NO: 12).
  • the fragment of the stalk region of CD40L is selected from the group consisting of: MQKGDQ (SEQ ID NO: 16), FEMQKGDQ (SEQ ID NO: 17), EMQKGDQ (SEQ ID NO: 18), QKGDQ (SEQ ID NO: 19), and KGDQ (SEQ ID NO:20), wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the fragment of the stalk region comprises an amino acid sequence selected from the group consisting of: MQKADQ (SEQ ID NO:59),
  • FEMQKADQ (SEQ ID NO: 60), EMQKADQ (SEQ ID NO:61), QKADQ (SEQ ID NO:62), and KADQ (SEQ ID NO:63).
  • At least two of the first, second, or third copies of the extracellular domain or a fragment thereof comprise the fragment of the stalk region.
  • the second and third copies of the extracellular domain or a fragment thereof comprise the fragment of the stalk region.
  • each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the fragment of the stalk region.
  • a polypeptide described herein does not comprise an
  • exogenous peptide linker between the extracellular domains or fragments thereof of CD40L.
  • At least one of the copies of the extracellular domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments of a polypeptide described herein, at least one of the copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments of a polypeptide described herein, at least one of the copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:64.
  • each of the first, second, or third copies of the extracellular domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments of a polypeptide described herein, each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 15, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68. In some embodiments, each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:64.
  • a single chain fusion human CD40L polypeptide described herein comprises a polypeptide having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:70 or 80. In some embodiments, the single chain fusion human CD40L polypeptide comprises a polypeptide having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:70 or 80. In some embodiments, the single chain fusion human CD40L polypeptide comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • a polypeptide described herein further comprises a non-
  • the first or third copy of the extracellular domain of human CD40L or a fragment thereof is directly linked to the non-CD40L polypeptide. In some embodiments, the first or third copy of the extracellular domain of human CD40L or a fragment thereof is connected to the non-CD40L polypeptide by a linker. In some embodiments, the first copy of the extracellular domain is linked to the carboxy-terminal end of the non-CD40L polypeptide. In some embodiments, the third copy of the extracellular domain is linked to the amino-terminal end of the non-CD40L polypeptide.
  • the non-CD40L polypeptide comprises an Fc region.
  • the Fc region is a human Fc region.
  • the Fc region is from a human IgGl, IgG2, IgG3, or IgG4 immunoglobulin.
  • the Fc region is selected from the group consisting of: SEQ ID NOs:39-43. In some embodiments, the Fc region is deglycosylated. In some embodiments, the Fc region comprises a mutation in the N-linked glycosylation site of the CH2 domain. In some embodiments, the mutation is a substitution or deletion of the asparagine corresponding to residue 77 of SEQ ID NO:39. In some embodiments, the mutation is an alanine substitution of the asparagine corresponding to residue 77 of SEQ ID NO:39. In some embodiments, the mutation is a substitution or deletion of the asparagine corresponding to residue 79 of SEQ ID NO:43.
  • the mutation is an alanine substitution of the asparagine corresponding to residue 79 of SEQ ID NO:43.
  • the Fc region comprises SEQ ID NO:82.
  • the Fc region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:44 and SEQ ID NO:45.
  • a polypeptide described herein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:72. In some embodiments, a polypeptide described herein comprises a an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:72. In some embodiments, a polypeptide described herein comprises the amino acid sequence of SEQ ID NO:72.
  • a polypeptide described herein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:81. In some embodiments, a polypeptide described herein comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:81. In some embodiments, a polypeptide described herein comprises the amino acid sequence of SEQ ID NO:81.
  • the non-CD40L polypeptide comprises an immunoglobulin heavy chain.
  • the immunoglobulin heavy chain is linked to an immunoglobulin light chain.
  • the immunoglobulin heavy chain and immunoglobulin light chain form an antigen-binding site.
  • the non-CD40L polypeptide comprises a single chain antibody or a Fab.
  • described herein is an agent comprising a polypeptide described herein. In one aspect, described herein is a homodimeric agent comprising a polypeptide described herein. In one aspect, described herein is a heterodimeric agent comprising a polypeptide described herein.
  • an agent described herein activates CD40; and/or induces
  • an agent described herein has an improved therapeutic index compared to a reference agent; induces reduced systemic inflammation compared to a reference agent; induces lower serum liver enzyme level compared to a reference agent; and/or induces lower pro-inflammatory cytokine release than a reference agent, wherein the reference agent and the agent comprise the same polypeptide except that the reference agent comprises a polypeptide comprising a stalk region that does not comprise a mutation in the integrin-binding region.
  • reduced systemic inflammation is indicated by lower levels of blood neutrophils.
  • the blood neutrophil level is at least about 20%, 30%>, 40%, 50%, 60%>, or 70%) lower.
  • the blood neutrophil level is from about 20% to about 70%) lower, from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50%) to about 70%) lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower.
  • the blood neutrophil level is about 20% lower, about 30%) lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
  • the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In some embodiments, the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the liver enzyme level is from about 20% to about 70% lower, from about 20% to about 60% lower, from about 20%) to about 50%) lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower.
  • the liver enzyme level is about 20%) lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
  • the pro-inflammatory cytokine is selected from the group consisting of: IL- ⁇ , IL-6, TNF-alpha, IP-10, KC, and MTP-la. In some embodiments, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the pro-inflammatory cytokine release is from about 20% to about 70%) lower, from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower.
  • the pro-inflammatory cytokine release is about 20%) lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
  • described herein is a bispecific agent comprising: a polypeptide described herein; and an antigen-binding site from an antibody.
  • described herein is a bispecific agent comprising: a polypeptide described herein; and an immune response stimulating agent or functional fragment thereof, or an antibody or functional fragment thereof.
  • a bispecific agent described herein is a homodimer or a heterodimer.
  • a bispecific agent comprising; a) a first arm
  • a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the stalk region comprises a mutation in the integrin-binding region, and (ii) a Fc region; and b) a second arm comprising an antigen-binding site from an antibody.
  • the Fc region is deglycosylated.
  • each of the first, second, and third copies comprises the amino acid sequence of SEQ ID NO: 15, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68.
  • the first arm comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
  • the first arm comprises the amino acid sequence of SEQ ID NO:72.
  • the first arm comprises the amino acid sequence of SEQ ID NO:81.
  • the single chain fusion polypeptide is directly linked to the Fc region.
  • the single chain fusion polypeptide is connected to the Fc region by a linker.
  • the Fc region is from a human IgGl, IgG2, IgG3, or IgG4 immunoglobulin.
  • the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:39-45 and 82. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:44 or SEQ ID NO:45. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:82. [0027] In some embodiments of a bispecific agent described herein, the first arm comprises a first CH3 domain and the second arm comprises a second CH3 domain, each of which is modified to promote formation of heterodimers. In some embodiments, the first and second CH3 domains are modified based upon electrostatic effects.
  • the first arm comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:47, wherein the amino acids are replaced with glutamate or aspartate
  • the second arm comprises a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO:47, wherein the amino acids are replaced with lysine.
  • the first arm comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO:47, wherein the amino acids are replaced with lysine
  • the second arm comprises a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:47, wherein the amino acids are replaced with glutamate or aspartate.
  • the first and second CH3 domains are modified using a knobs-into-holes technique.
  • the antigen-binding site specifically binds a tumor antigen.
  • the antigen-binding site specifically binds PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3, or B7-H4.
  • a bispecific agent described herein comprises an immune response stimulating agent.
  • a bispecific agent described herein comprises an antibody.
  • the antibody specifically binds to PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, or B7-H4.
  • an agent or polypeptide described herein increases cell- mediated immunity. In some embodiments, an agent or polypeptide described herein increases antigen-presenting cell (APC) activity. In some embodiments, an agent or polypeptide described herein increases T-cell activity. In some embodiments, an agent or polypeptide described herein increases cytolytic T-cell (CTL) activity. In some embodiments, an agent or polypeptide described herein increases natural killer (NK) activity. In some embodiments, an agent or polypeptide described herein decreases or inhibits regulatory T-cell (Treg) activity. In some embodiments, an agent or polypeptide described herein decreases or inhibits myeloid-derived suppressor cell (MDSC) activity.
  • an agent or polypeptide described herein increases an effective immune response without causing substantial side effects and/or immune-based toxicities. In some embodiments, an agent or polypeptide described herein increases an effective immune response without causing cytokine release syndrome (CRS) or a cytokine storm.
  • CRM cytokine release syndrome
  • described herein are cells comprising or producing a polypeptide or agent described herein.
  • compositions or pharmaceutical compositions comprising a polypeptide or agent described herein.
  • described herein is a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide or agent described herein and a pharmaceutically acceptable carrier.
  • polynucleotides comprising a nucleotide
  • polypeptide or agent described herein encodes a polypeptide or agent described herein.
  • described herein are polynucleotides comprising a nucleotide sequence that encodes a polypeptide or agent described herein and a signal sequence.
  • vectors comprising a polynucleotide described herein.
  • isolated cells comprising a polynucleotide or a vector described herein.
  • described herein are methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of a polypeptide or agent described herein.
  • the immune response is against a tumor or cancer.
  • described herein are methods of inhibiting the growth of a tumor, comprising contacting a tumor or tumor cell with an effective amount of a polypeptide or agent described herein.
  • a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
  • the tumor is selected from the group consisting of colorectal tumor, colon tumor, ovarian tumor, pancreatic tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor.
  • the cancer is selected from the group consisting of colorectal cancer, colon cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, head and neck cancer, lymphoma and leukemia.
  • APC activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
  • described herein are methods of increasing dendritic cell activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
  • cytolytic T-cell comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
  • NK natural killer
  • T-cell (Treg) activity in a subject comprising administering to the subject a
  • described herein are methods of decreasing or inhibiting myeloid- derived suppressor cell (MDSC) activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
  • MDSC myeloid- derived suppressor cell
  • a method described herein further comprises administering at least one additional therapeutic agent.
  • the additional therapeutic agent is a chemotherapeutic agent.
  • the additional therapeutic agent is an antibody.
  • the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, or an anti-B7-H4 antibody.
  • the additional therapeutic agent is an immune response stimulating agent.
  • the immune response stimulating agent is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL- 12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-Ll antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • IL-3 interleukin 12
  • IL-1 interleukin 1
  • IL-2 interleukin 2
  • B7-1 CD80
  • the administration of a polypeptide or agent described herein (a) induces reduced systemic inflammation compared to a reference polypeptide or reference agent; (b) induces lower serum liver enzyme level compared to a reference polypeptide or reference agent; and/or (c) induces lower pro-inflammatory cytokine release than a reference polypeptide or reference agent, wherein the reference polypeptide or reference agent and the polypeptide or agent comprise the same polypeptide except that the reference polypeptide or reference agent comprise a polypeptide comprising a stalk region that does not comprise a mutation in the integrin-binding region.
  • the reduced systemic inflammation is indicated by lower levels of blood neutrophils.
  • the blood neutrophil level is at least about 20%, 30%, 40%), 50%), 60%), or 70%> lower.
  • the blood neutrophil level is from about 20%) to about 70%> lower, from about 20%> to about 60%> lower, from about 20%> to about 50%) lower, from about 20%> to about 40%> lower, from about 20%> to about 30%> lower, from about 30%> to about 70%> lower, from about 30%> to about 60%> lower, from about 30%) to about 50%> lower, from about 30%> to about 40%> lower, from about 40%> to about 70%) lower, from about 40%> to about 60%> lower, from about 40%> to about 50%> lower, from about 50%> to about 70%> lower, from about 50%> to about 60%> lower, or from about 60%) to about 70%> lower.
  • the blood neutrophil level is about 20%) lower, about 30%> lower, about 40%> lower, about 50%> lower, about 60%> lower, or about 70%) lower.
  • the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In some embodiments, the liver enzyme level is at least about 20%, 30%), 40%, 50%, 60%>, or 70% lower.
  • the liver enzyme level is from about 20% to about 70% lower, from about 20% to about 60% lower, from about 20%) to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower.
  • the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60%) lower, or about 70% lower.
  • the pro-inflammatory cytokine is selected from the group consisting of: IL- ⁇ , IL-6, TNF-alpha, IP-10, KC, and MTP-la. In some embodiments, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the pro-inflammatory cytokine release is from about 20% to about 70% lower, from about 20% to about 60% lower, from about 20%) to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower.
  • the proinflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50%) lower, about 60% lower, or about 70% lower.
  • the subject is human.
  • described herein are methods of improving the therapeutic index of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region.
  • described herein are methods of decreasing the toxicity of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region.
  • the human CD40 agonist polypeptide comprises three copies of the extracellular domain of human CD40L or a CD40-binding fragment thereof.
  • the improving the therapeutic index or decreasing the toxicity comprises
  • the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In some embodiments, the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the liver enzyme level is from about 20% to about 70% lower, from about 20% to about 60%> lower, from about 20%) to about 50%) lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower.
  • the liver enzyme level is about 20%) lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
  • the pro-inflammatory cytokine is selected from the group consisting of: IL- ⁇ , IL-6, TNF-alpha, IP-10, KC, and MTP-la. In some embodiments, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the pro-inflammatory cytokine release is from about 20% to about 70% lower, from about 20% to about 60% lower, from about 20%) to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower.
  • the proinflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50%) lower, about 60% lower, or about 70% lower.
  • described herein are methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of an improved human CD40 agonist described herein.
  • the immune response is against a tumor or cancer.
  • described herein are methods of inhibiting the growth of a tumor, comprising contacting a tumor or tumor cell with an effective amount of an improved human CD40 agonist described herein.
  • described herein are methods of inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an improved human CD40 agonist described herein.
  • the present invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members.
  • the present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
  • FIG. 1 A-1C Inhibition of tumor growth by single chain CD40L trimer-Fc
  • the murine colon tumor line CT26.WT was implanted
  • mice subcutaneously into Balb/c mice. Mice were injected on days 10, 13, and 17 with
  • Fig. 1 A The mean values ⁇ SEM for each group.
  • Fig. IB Tumor volumes of each individual mouse from the group treated with control antibody.
  • Fig. lC Tumor volumes of each individual mouse from the group treated with 340F2.
  • Figures 2A-2E Inhibition of tumor growth by single chain CD40L trimer-Fc fusion polypeptide.
  • the murine colon tumor line CT26.WT was implanted
  • Fig. 2 A Tumor volumes of each individual mouse from the group treated with control antibody.
  • Fig. 2B Tumor volumes of each individual mouse from the group treated with anti-mCD40 antibody.
  • Fig. 2C Tumor volumes of each individual mouse from the group treated with 340F2.
  • Fig. 2D The mean values ⁇ SEM for each group.
  • Fig. 2E Survival curve of the treatment groups up to Day 60.
  • FIG. 3A-3D Inhibition of tumor growth by single chain CD40L trimer-Fc fusion polypeptide.
  • the murine colon tumor line MC38 was implanted subcutaneously into C57BL/6N mice. Mice were injected on days 8, 12, and 15 with 0.25mg/mouse of single chain mCD40L trimer-Fc fusion protein 340F2, anti-mouse CD40 antibody, or a control antibody. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. Data is shown as tumor volume (mm 3 ) over days post cell injection.
  • Fig. 3A The mean values ⁇ SEM for each group.
  • Fig. 3B Tumor volumes of each individual mouse from the group treated with control antibody.
  • Fig.3C Tumor volumes of each individual mouse from the group treated with anti- mCD40 antibody.
  • Fig.3D Tumor volumes of each individual mouse from the group treated with 340F2.
  • FIG. 4A ELISpot assays for IL-2 and IFN-gamma.
  • Fig. 4A Cells were harvested from the spleens of MC38-tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti-mCD40 antibody, or a control. Cells were incubated in the absence of a peptide and then analyzed using an ELISpot kit for IL-2. Total optical density (TOD) of cells producing IL-2 is shown.
  • Fig. 4B Cells were harvested from the spleens of MC38- tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti-mCD40 antibody, or a control.
  • FIG. 4C Cells were harvested from the tumors of MC38-tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti-mCD40 antibody, or a control. Cells were incubated in the absence of a peptide and then analyzed using an ELISpot kit for IFN-gamma. TOD of cells producing IFN-gamma is shown.
  • Figures 5A-5C Cytotoxicity assays.
  • Fig. 5A Cells were harvested from the
  • YAC-1 target cells were labeled with ⁇ calcein AM and mixed with the splenocytes at an E:T ratio of 25 : 1.
  • Supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm.
  • Fig. 5B Cells were harvested from the spleens of MC38-tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti-mCD40 antibody, or a control.
  • YAC-1 target cells were labeled with ⁇ calcein AM and mixed with the splenocytes at an E:T ratio of 25: 1. Supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm.
  • Fig. 5C Cells were harvested from the spleens of MC38-tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti- mCD40 antibody, or a control.
  • MC38 target cells were labeled with ⁇ calcein AM and mixed with the splenocytes at an E:T ratio of 25 : 1. Supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm.
  • FIG. 6 Inhibition of tumor growth by single chain CD40L trimer-Fc fusion polypeptides.
  • the 30,000 cells of the murine colon tumor line CT26.WT were implanted subcutaneously into Balb/c mice.
  • mice On days 12, 15, and 18, mice were treated with lOmg/kg of single chain mCD40L trimer-Fc fusion protein 340F2, mCD40L trimer- Fc fusion protein 340F4, mCD40L trimer-Fc fusion protein 340F7, an anti-mCD40 antibody, or a control antibody.
  • Tumor growth was monitored by measuring tumor volumes at the indicated time points. Data is shown as mean tumor size ⁇ SEM (mm 3 ).
  • FIGS 7A-7C Analysis of liver enzymes in serum in single chain CD40L trimer-
  • FIG. 7A, B, and C show the serum alanine transaminase ("ALT"), serum aspartate transaminase ("ASP"), and serum alkaline phosphatase ("ALP”) activities, respectively, detected. Data points from individual animals are shown with horizontal lines representing the mean serum enzyme activity detected in each treatment group.
  • ALT serum alanine transaminase
  • ASP serum aspartate transaminase
  • ALP serum alkaline phosphatase
  • Figures 8A-8D Analysis of Complete Blood Count in single chain CD40L trimer-
  • FIG. 8A, B, C, and D show the percent of neutrophils, the neutrophil count per ⁇ , the white blood cell count per ⁇ ., and the lymphocyte count per detected, respectively. Data points from individual animals are shown with horizontal lines representing the mean values detected in each treatment group.
  • FIGS 9A-90 Analysis of Plasma Cytokines in single chain CD40L trimer-Fc fusion polypeptide treated mice. Mice were treated with lOmg/kg of single chain mCD40L trimer-Fc fusion protein 340F2, mCD40L trimer-Fc fusion protein 340F4 (only received the second dose), mCD40L trimer-Fc fusion protein 340F7, an anti-mCD40 antibody, or a control antibody as described in Example 7.
  • Plasma levels of G-CSF (A), IFN-gamma (B), IL-la (C), IL- ⁇ (D), IL-2 (E), IL-6 (F), IL-12p40 (G), IFN-gamma- induced protein 10 ("IP- 10") (H), chemokine (C-X-C motif) ligand 1 ("KC") (I), monocyte chemoattractant protein 1 ("MCP1 ”) (J), macrophage inflammatory protein 1-a (“MIP-la”) (K), MIP- ⁇ ⁇ (L), MIP2 (M), RANTES (N), and T F-alpha (O) are shown. Data points from individual animals are shown with horizontal lines representing the mean values detected in each treatment group.
  • FIGS 10 A and 10B Liver Histology of single chain CD40L trimer-Fc fusion polypeptide treated mice. Mice were treated with lOmg/kg of single chain mCD40L trimer-Fc fusion protein 340F2, mCD40L trimer-Fc fusion protein 340F4 (only received the second dose), mCD40L trimer-Fc fusion protein 340F7, an anti-mCD40 antibody, or a control antibody as described in Example 7. Liver sections stained with hematoxylin and eosin are shown at IX, 5X, or 10X magnification.
  • TNFRSF tumor necrosis factor receptor superfamily
  • TNFSF tumor necrosis factor receptor superfamily
  • TNFRSF members include, but may not be limited to, 4-1BB, BAFF, BCMA, CD27, CD30, CD40, DcR3, DcTRAIL Rl, DcTRAIL R2, DR3, DR6, EDA2R, EDAR, Fas (CD95), GITR, HVEM, lymphotoxin beta R, NGFR, osteoprotegerin, OX40, RANK, RELT, TACI, TNFRH3, TNF Rl, TNF R2, TRAIL Rl, TRAIL R2, TRAIL R3, TRAIL R4, TROY, and TWEAK R.
  • Receptors for TNF family ligands are oligomeric, type I or type III transmembrane proteins that contain multiple extracellular cysteine-rich domains. Several of these receptors also contain intracellular death domains (DDs) that recruit caspase-interacting proteins following ligand binding to initiate the extrinsic pathway of caspase activation. Other TNF superfamily receptors that lack death domains bind TNF receptor-associated factors and activate intracellular signaling pathways that can lead to proliferation or differentiation. These receptors can also initiate apoptosis, but they do so via indirect mechanisms.
  • DDs intracellular death domains
  • Other TNF superfamily receptors that lack death domains bind TNF receptor-associated factors and activate intracellular signaling pathways that can lead to proliferation or differentiation. These receptors can also initiate apoptosis, but they do so via indirect mechanisms.
  • TNF superfamily receptors are involved in regulating immune cell functions such as B-cell homeostasis and activation, natural killer cell activation, and T-cell co-stimulation.
  • TNF superfamily receptors regulate cell type-specific responses such as hair follicle development and osteoclast development.
  • TNFSF members include, but may not be limited to, 4-1BB ligand, APRIL,
  • TNFSF ligands are type II transmembrane proteins whose extracellular domains can be cleaved by specific metalloproteinases to generate soluble cytokines.
  • TNF superfamily ligands are characterized by a stalk of varying length connecting the transmembrane domain to the core region, which contains the hallmark structure of TNF family ligands, the TNF homology domain (THD) or TNF domain.
  • TNF domain is an anti-parallel beta- pleated sheet sandwich with a "jelly-roll" topology. conserveed residues within the beta- strands provide specific inter-subunit contacts, which stabilize the trimeric structure.
  • Sequences in the loops connecting adjacent beta-strands are family member-specific and are important for conferring receptor specificity.
  • agonist antibodies targeting members of the TNFR superfamily are generally dimeric molecules with each arm of the antibody binding one subunit of a TNFR, the inventors hypothesized that they may not be able to fully recapitulate the signaling impact of the native trimeric TNF family member. It was hypothesized that a therapeutic agent that presents a TNF family ligand in a stable trimeric form could be more active than an agonist TNFR antibody at eliciting signaling, and that such a trimeric TNF ligand form might therefore be a superior immunotherapeutic agent.
  • TNFSF ligand trimer One strategy to generate a TNFSF ligand trimer is to express three subunits of the trimer as a single polypeptide. It has previously been shown that TNF family members can be expressed as a single chain trimer (US Application Publication Nos. 2007/0286843 and 2011/0162095). However, a major drawback of previous single chain TNF family member trimer variants has been the introduction of exogenous linker sequences interconnecting the three subunits of the trimer. Such linkers may introduce potential instability and lability to the trimer, and/or provide a source of potential immunogenicity as the linkers are foreign sequences.
  • Another strategy is to utilize a fragment of the stalk region to bridge the distance from the C-terminus of a TNFSF ligand monomer to the N-terminus of an adjacent TNFSF ligand monomer and in this fashion construct a single chain TNFSF ligand trimer that was devoid of exogenous peptide linker sequences. See, e.g., US Patent No.
  • the present invention provides novel agents that bind CD40, including, but not limited to, polypeptides, soluble proteins, fusion proteins, homodimeric bispecific molecules, and heterodimeric bispecific molecules that modulate the immune response.
  • novel agents that bind CD40 including, but not limited to, polypeptides, soluble proteins, fusion proteins, homodimeric bispecific molecules, and heterodimeric bispecific molecules that modulate the immune response.
  • Related polypeptides and polynucleotides, compositions comprising the agents, and methods of making the agents are also provided. Methods of screening for agents that modulate the immune response are provided.
  • Methods of inhibiting an immune response, methods of suppressing an immune response, methods of decreasing activity of T-cells, and/or methods of treating autoimmune diseases are further provided.
  • CD40L integrin-binding region refers to or describes the region or domain in the CD40L stalk region that mediates the interaction between CD40L and integrin.
  • the interaction is between CD40L and integrin alpha (lib) beta 3.
  • the interaction is between human CD40L and integrin alpha (lib) beta 3.
  • the interaction between CD40L and integrin alpha (lib) beta 3 results in platelet activation as described in Prasad et al, PNAS, 100921): 12367-12371 (2003).
  • the interaction between CD40L and integrin alpha (lib) beta 3 is specific binding assayed by a competitive or non-competitive assay system using techniques such as Biacore analyses, FACS analyses,
  • assays are routine and well-known in the art.
  • the interaction between CD40L and integrin alpha (lib) beta 3 is assayed by FACS analyses determining the binding of a soluble CD40L polypeptide to platelets.
  • the interaction between CD40L and integrin alpha (lib) beta 3 is assayed by ELISA analyses determining the binding of a soluble CD40L polypeptide to an immobilized integrin alpha (lib) beta 3 polypeptide.
  • the integrin binding region of human CD40L comprises the KGD motif of the stalk region (residues 115-117 of SEQ ID NO: 12). In one embodiment, the integrin binding region of human CD40L consists of the KGD motif of the stalk region (residues 115-117 of SEQ ID NO: 12).
  • the integrin binding region of murine CD40L comprises the RGD motif of the stalk region (residues 114-116 of SEQ ID NO: 1). In one embodiment, the integrin binding region of murine CD40L consists of the RGD motif of the stalk region (residues 114-116 of SEQ ID NO: 1).
  • mutation in the CD40L integrin-binding region refers to or describes at least one substitution, insertion, or deletion in the CD40L integrin-binding region.
  • the mutation is the substitution, insertion, or deletion of at least one residue in the CD40L integrin-binding region.
  • the mutation is the substitution, insertion, or deletion of at least two residues in the CD40L integrin- binding region.
  • the mutation is the substitution, insertion, or deletion of at least three residues in the CD40L integrin-binding region.
  • the mutation is 1, 2, or 3 substitutions in the CD40L integrin-binding region.
  • the mutation comprises 1, 2, or 3 substitutions in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a substitution of the K residue in the KGD motif of the human CD40L stalk region
  • the mutation comprises a substitution of the G residue in the KGD motif of the human CD40L stalk region
  • the mutation comprises a substitution of the D residue in the KGD motif of the human CD40L stalk region
  • the mutation comprises a G to A substitution in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a D to E substitution in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation does not comprise a D to E substitution in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12).
  • the mutation comprises a substitution of the K and G residues, the K and D residues, the G and D residues, or the K, G and D residues in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises 1, 2, or 3 deletions in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a deletion of the K residue in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12).
  • the mutation comprises a deletion of the G residue in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a deletion of the D residue in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a deletion of the K and G residues, the K and D residues, the G and D residues, or the K, G and D residues in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12).
  • the mutation comprises 1, 2, or 3 substitutions in the RGD motif of the murine CD40L stalk region (residues 114-116 of SEQ ID NO: 1). In embodiment, the mutation comprises a G to A or a D to E substitution in the RGD motif of the murine CD40L stalk region (residues 114-116 of SEQ ID NO: 1). In embodiment, the mutation comprises 1, 2, or 3 deletions in the RGD motif of the murine CD40L stalk region (residues 114-1 16 of SEQ ID NO: 1). In one embodiment, the mutation in the CD40L integrin-binding region reduces or eliminates the interaction between CD40L and integrin.
  • the interaction is between CD40L and integrin alpha (lib) beta 3. In one embodiment, the interaction is between human CD40L and integrin alpha (lib) beta 3. In one embodiment, the interaction between CD40L and integrin alpha (lib) beta 3 results in platelet activation as described in Prasad et al, PNAS, 100921): 12367-12371 (2003). In one embodiment, the interaction between CD40L and integrin alpha (lib) beta 3 is assayed by FACS analyses determining the binding of a soluble CD40L polypeptide to platelets.
  • the interaction between CD40L and integrin alpha (lib) beta 3 is assayed by ELISA analyses determining the binding of a soluble CD40L polypeptide to an immobilized integrin alpha (lib) beta 3 polypeptide.
  • agonist and “agonistic” as used herein refer to or describe a
  • polypeptide or agent that is capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a target and/or a pathway.
  • agonist is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest.
  • the terms "antagonist” and “antagonistic” as used herein refer to or describe a polypeptide or agent that is capable of, directly or indirectly, partially or fully blocking, inhibiting, reducing, or neutralizing a biological activity of a target and/or pathway.
  • the term “antagonist” is used herein to include any agent that partially or fully blocks, inhibits, reduces, or neutralizes the activity of a protein or other target of interest.
  • modulation and “modulate” as used herein refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating an activity or inhibiting an activity. Modulation may be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, a pathway, a system, or other biological targets of interest.
  • soluble protein refers to a protein or a fragment thereof that can be secreted from a cell in soluble form.
  • fusion protein or "fusion polypeptide” as used herein refers to a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
  • linker or "linker region” as used herein refers to a linker inserted
  • linker is a peptide linker.
  • Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. Preferably, linkers are not antigenic and do not elicit an immune response.
  • antibody refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule.
  • the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological activity.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments
  • scFv single chain Fv
  • An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules, including but not limited to, toxins and radioisotopes.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.
  • Antibody fragment as used herein comprises an antigen-binding site or epitope-binding site.
  • variable region of an antibody refers to the variable region of an antibody
  • variable region of heavy and light chains each consist of four framework regions (FR) and three complementarity determining regions (CDRs), also known as "hypervariable regions".
  • FR framework regions
  • CDRs complementarity determining regions
  • the CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding sites of the antibody.
  • CDRs There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda MD.), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al Lazikani et al., 1997, J. Mol. Biol, 273 :927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.
  • the term "monoclonal antibody” as used herein refers to a homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include a mixture of different antibodies directed against different antigenic determinants.
  • the term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv) antibodies, fusion proteins comprising an antibody fragment, and any other modified
  • immunoglobulin molecule comprising an antigen-binding site.
  • “monoclonal antibody” refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.
  • humanized antibody refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences.
  • humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capability.
  • a non-human species e.g., mouse, rat, rabbit, or hamster
  • the Fv framework region residues of a human immunoglobulin are replaced with the
  • the humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability.
  • the humanized antibody may comprise variable domains containing all or substantially all of the CDRs that correspond to the non-human immunoglobulin whereas all or substantially all of the framework regions are those of a human immunoglobulin sequence.
  • the variable domains comprise the framework regions of a human immunoglobulin sequence.
  • the variable domains comprise the framework regions of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.
  • chimeric antibody refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding capability, while the constant regions are homologous to the sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.
  • epipe and “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody.
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein.
  • Epitopes formed from contiguous amino acids also referred to as linear epitopes
  • epitopes formed by tertiary folding also referred to as conformational epitopes
  • An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.
  • the terms “selectively binds” or “specifically binds” mean that a polypeptide or agent interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins.
  • polypeptide or agent binds a protein or target with a K D of about 0. ImM or less, but more usually less than about 1 ⁇ .
  • “specifically binds” means that a polypeptide or agent binds a target with a K D of at least about 0.1 ⁇ or less, at least about 0.01 ⁇ or less, or at least about InM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or agent that recognizes a protein or target in more than one species.
  • specific binding can include a polypeptide or agent that recognizes more than one protein or target. It is understood that, in certain embodiments, a polypeptide or agent that specifically binds a first target may or may not specifically bind a second target. As such, "specific binding" does not necessarily require (although it can include) exclusive binding, i.e. binding to a single target. Thus, a polypeptide or agent may, in certain embodiments, specifically bind more than one target. In certain embodiments, multiple targets may be bound by the same antigen-binding site on the polypeptide or agent.
  • an antibody may, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins.
  • an antibody may be bispecific and comprise at least two antigen-binding sites with differing specificities.
  • binding means "specific binding”.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids
  • the polypeptides of this invention may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the polypeptides can occur as single chains or as associated chains.
  • nucleotide and “nucleic acid” and “nucleic acid molecule” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum
  • sequence identity may be measured using sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof.
  • two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%), 96%), 97%), 98%o, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between.
  • identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between.
  • identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
  • a "conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • substitution e
  • phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides, soluble proteins, and/or antibodies of the invention do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site.
  • Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.
  • vector means a construct, which is capable of
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.
  • composition which is "isolated” is a polypeptide, soluble protein, antibody,
  • polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • substantially pure refers to material which is at least
  • immune response includes responses from both the innate immune system and the adaptive immune system. It includes both cell-mediated and/or humoral immune responses. It includes both T-cell and B-cell responses, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, etc.
  • NK natural killer
  • cancer and “cancerous” as used herein refer to or describe the
  • cancer physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and hematologic cancers such as lymphoma and leukemia.
  • tumor and "neoplasm” as used herein refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions.
  • cancer stem cell and “CSC” and “tumor stem cell” and “tumor initiating cell” are used interchangeably herein and refer to cells from a cancer or tumor that: (1) have extensive proliferative capacity; 2) are capable of asymmetric cell division to generate one or more types of differentiated cell progeny wherein the differentiated cells have reduced proliferative or developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self-maintenance. These properties confer on the cancer stem cells the ability to form or establish a tumor or cancer upon serial transplantation into an appropriate host (e.g., a mouse) compared to the majority of tumor cells that fail to form tumors. Cancer stem cells undergo self-renewal versus
  • cancer cell and “tumor cell” refer to the total population of cells derived from a cancer or tumor or pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the cancer cell population, and tumorigenic stem cells (cancer stem cells).
  • cancer stem cells tumorigenic stem cells
  • tumorigenic refers to the functional features of a cancer stem cell including the properties of self-renewal (giving rise to additional tumorigenic cancer stem cells) and proliferation to generate all other tumor cells (giving rise to differentiated and thus non-tumorigenic tumor cells).
  • tumorigenicity refers to the ability of a random sample of cells from the tumor to form palpable tumors upon serial transplantation into appropriate hosts (e.g., mice).
  • subject refers to any animal (e.g., a mammal), including, but not
  • subject and “patient” are used interchangeably herein in reference to a human subject.
  • acceptable pharmaceutical carrier refers to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one agent of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect.
  • a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.
  • an effective amount or “therapeutically effective amount” or “therapeutic effect” refer to an amount of a polypeptide or agent described herein (e.g., a fusion protein, a soluble receptor, an antibody, a polypeptide, a polynucleotide, a small organic molecule, or other drug) effective to "treat” a disease or disorder in a subject such as, a mammal.
  • a polypeptide or agent described herein e.g., a fusion protein, a soluble receptor, an antibody, a polypeptide, a polynucleotide, a small organic molecule, or other drug
  • the therapeutically effective amount of a polypeptide or agent has a therapeutic effect and as such can boost the immune response, boost the anti-tumor response, increase cytolytic activity of immune cells, increase killing of tumor cells by immune cells, reduce the number of tumor cells; decrease tumorigenicity, tumorigenic frequency or
  • tumorigenic capacity reduce the number or frequency of cancer stem cells; reduce the tumor size; reduce the cancer cell population; inhibit or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor or cancer cell metastasis; inhibit and stop tumor or cancer cell growth; relieve to some extent one or more of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects.
  • treating or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • a subject is successfully "treated” according to the methods of the present invention if the patient shows one or more of the following: an increased immune response, an increased anti-tumor response, increased cytolytic activity of immune cells, increased killing of tumor cells by immune cells, a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size;
  • the present invention provides polypeptides and agents that bind CD40 (also referred to as T FRSF5).
  • the polypeptides and agents comprise a polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof is a single chain fusion polypeptide.
  • the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12).
  • the mutation comprises at least one substitution in the KGD motif.
  • the mutation comprises at least two substitutions in the KGD motif.
  • the mutation comprises three substitutions in the KGD motif.
  • the mutation comprises a G to A or D to E substitution in the KGD motif.
  • the mutation comprises the deletion of at least one residue in the KGD motif.
  • the mutation comprises the deletion of at least two residues in the KGD motif.
  • the mutation comprises the deletion of all three residues in the KGD motif.
  • CD40 is a member of the TNF receptor superfamily (TNFRSF).
  • TNFRSF TNF receptor superfamily
  • members include, but may not be limited to, 4-1BB, BAFF, BCMA, CD27, CD30, CD40, DcR3, DcTRAIL Rl, DcTRAIL R2, DR3, DR6, EDA2R, EDAR, Fas (CD95), GITR, HVEM, lymphotoxin beta R, NGFR, osteoprotegerin, OX40, RANK, RELT, TACI, TNFRH3, TNF Rl, TNF R2, TRAIL Rl, TRAIL R2, TRAIL R3, TRAIL R4, TROY, and TWEAK R.
  • CD40 is expressed by antigen-presenting cells (APCs) including dendritic cells (DCs), B-cells, macrophages, and monocytes but is also expressed on other cell types such as epithelial cells, endothelial cells, and platelets. CD40 expression has also been demonstrated on various tumor cells, including B-cell lymphoma and renal cancer cells.
  • the natural ligand for CD40 is CD40L (CD 154) which is mainly expressed on activated CD4+ T-cells, but also on platelets, mast cells, and basophils.
  • CD40 and CD40L facilitates an improved T-cell activation including priming of naive CD4+ T-cells and CD8+ T-cells which then differentiate into CD4+ helper T-cells and CD8+ cytotoxic T-cells, respectively.
  • Cytokines secreted from CD40-expressing cells may indirectly lead to the activation of NK cells.
  • treatment with CD40 agonists may mediate a potent anti-tumor effect through boosting of APC function, T-cell activation, and NK cell activation.
  • CD40 signaling axis holds tremendous therapeutic potential (there are several agonist anti-CD40 antibodies in development and in clinical trials), at this point in time it has been difficult to develop effective therapeutic agents.
  • a principal challenge has been that CD40 is expressed by a variety of cell types as disclosed above and thus an anti-CD40 antibody can be expected to bind to each of these cell types.
  • Developing an agonist antibody against CD40 therefore has the potential drawback that there might be undesirable side effects due to the effector function activity mediated by the Fc domain of the antibody which can interact with Fc receptors on a variety of immune cells.
  • Agonist antibodies against CD40 have generally required Fc receptor binding to facilitate a clustering of antibody bound to CD40 that thereby promotes the agonist CD40 signaling activity.
  • Each of the human IgG isotypes, IgGl, IgG2, IgG3, and IgG4, are able to bind Fc receptors.
  • human IgG2 which is generally considered to have limited antibody- dependent cellular cytotoxicity (ADCC)
  • ADCC antibody- dependent cellular cytotoxicity
  • IgG2 isotype can cause an agonist anti-CD40 antibody to promote direct cell-cell contact between a CD40-expressing cell and an Fc receptor- containing immune cell and potentially elicit unintended and/or undesirable
  • the ability to create an agonist CD40 agent that does not possess the ability to engage Fc receptors can be anticipated to have the potential for greater safety and less undesired side effects. It is known in the art that the Fc region can be engineered/modified to eliminate Fc receptor binding. In some embodiments, the glycosylation site that is critical for Fc receptor binding is modified. In some
  • the glycosylation site that is critical for Fc receptor binding is eliminated.
  • a single chain CD40L trimer linked to a Fc region in which Fc receptor binding is eliminated provides the ability to achieve robust CD40 activation without the potential for undesirable toxicity due to Fc receptor-mediated events.
  • CD40-binding agents The agents described herein may be referred to herein as "CD40-binding agents”.
  • the agent is a CD40 agonist.
  • the polypeptide or agent induces, activates, enhances, increases, and/or prolongs CD40 signaling.
  • the agent is a polypeptide.
  • the agent is a soluble protein.
  • the agent is a fusion polypeptide.
  • the agent is a soluble ligand or soluble "co-receptor".
  • the ligand or "co-receptor" for CD40 is CD40L.
  • the polypeptide or agent comprises a fragment of human CD40L, wherein the fragment comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the polypeptide or agent comprises the extracellular domain of human CD40L, wherein the extracellular domain comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises a fragment of the extracellular domain of human CD40L, wherein the fragment comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a fragment of the extracellular domain of human CD40L can demonstrate altered biological activity compared to a soluble agent comprising the entire extracellular domain.
  • the polypeptide or agent binds human CD40. In some embodiments, the polypeptide or agent binds mouse CD40. In some embodiments, the agent is a polypeptide. In some embodiments, the polypeptide or agent is a soluble protein. In some embodiments, the polypeptide or agent is a soluble protein that binds human CD40. In some embodiments, the polypeptide or agent is a fusion polypeptide. In some embodiments, the agent (e.g., polypeptide) comprises at least one copy of the extracellular domain, or a CD40-binding fragment thereof, of CD40L, wherein the extracellular domain or fragment thereof comprises a mutation in the integrin-binding region.
  • the agent e.g., polypeptide
  • the agent comprises at least one copy of the extracellular domain, or a fragment thereof, of human CD40L.
  • the polypeptide or agent comprises at least one copy of the extracellular domain, or a fragment thereof, of mouse CD40L.
  • the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L.
  • the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L, wherein at least one of the extracellular domains comprises the "stalk region" of CD40L, and wherein the stalk region comprises a mutation in the integrin-binding region.
  • the stalk region of CD40L is very long in comparison to some of the other TNFSF members (i.e., GITRL and OX40L), therefore in some embodiments the extracellular domain(s) of human CD40L comprise only a short fragment of the stalk region.
  • the stalk region comprises about 4-20 amino acids. In some embodiments, the stalk region comprises about 4-10 amino acids.
  • the stalk region comprises the amino acids (e.g., 4-10 amino acids) upstream from the TNF homology domain. In some embodiments, the stalk region comprises the amino acids (e.g., 4-10 amino acids) adjacent to the TNF homology domain.
  • the fragment of the stalk region of CD40L is MQKGDQ (SEQ ID NO: 16) comprising a mutation in the integrin-binding region. In some embodiments, the fragment of the stalk region of CD40L is FEMQKGDQ (SEQ ID NO: 17), EMQKGDQ (SEQ ID NO: 18), QKGDQ (SEQ ID NO: 19), or KGDQ (SEQ ID NO:20) comprising a mutation in the integrin-binding region.
  • the mutation in the integrin-binding region can be amino acid insertions, deletions, and/or substitutions.
  • the integrin-binding region of the fragment of the stalk region of CD40L comprises at least 1, at least 2, or at least 3 insertions, deletions, and/or substitutions.
  • the stalk regions consist of amino acid sequences of CD40L, i.e., these stalk regions do not comprise any exogenous amino acids, such as an exogenous linker.
  • the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L, wherein each extracellular domain comprises a fragment of the stalk region of CD40L.
  • the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L, wherein the second and third extracellular domains include a fragment of the stalk region of CD40L.
  • the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the polypeptide does not comprise any peptide linkers.
  • the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the first copy and the second copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof.
  • the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the second copy and the third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof.
  • the agent e.g., a polypeptide
  • the agent comprises approximately amino acids 113 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the agent (e.g., a polypeptide) comprises approximately amino acids 111 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 112 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 114 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the agent e.g., a polypeptide
  • the agent comprises approximately amino acids 115 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the polypeptide or agent comprises approximately amino acids 113 to 261, 111 to 261, 112 to 261, 114 to 261, or 115 to 261 of human CD40L, wherein the sequence of human CD40L is UniProtKB No. P29965, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the polypeptide or agent comprises at least one copy of SEQ ID NO: 22 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:22 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the polypeptide or agent comprises at least one copy of SEQ ID NO: 23 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:23 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:23, wherein the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:24 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the polypeptide or agent comprises at least two copies of SEQ ID NO:24 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:24, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:25 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the polypeptide or agent comprises at least two copies of SEQ ID NO:25 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:25, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:26 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the polypeptide or agent comprises at least two copies of SEQ ID NO:26 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:26, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. [Add mutant SEQ IDNO:] In some embodiments, the
  • polypeptide or agent comprises SEQ ID NO:28.
  • polypeptide or agent comprises SEQ ID NO:36, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the integrin-binding region of the fragment of the stalk region of CD40L comprises at least 1, at least 2, or at least 3 insertions, deletions, and/or substitutions.
  • the polypeptide or agent is a fusion polypeptide or fusion protein.
  • the fusion protein comprises a non-CD40L polypeptide (i.e., a heterologous protein).
  • the fusion polypeptide comprises a Fc region.
  • the non-CD40L polypeptide comprises a Fc region. In some embodiments, the non-CD40L polypeptide comprises a human Fc region. In some embodiments, the Fc region is from an IgGl, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region is modified. In some embodiments, the Fc region is deglycosylated. In some embodiments, the Fc region is selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45. In some embodiments, the Fc region comprises SEQ ID NO:44 or SEQ ID NO:45. In some embodiments, the non-CD40L polypeptide comprises an immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is associated with an immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is associated with
  • the immunoglobulin light chain In some embodiments, the immunoglobulin heavy chain and light chain form an antigen-binding site. In some embodiments, the non-CD40L polypeptide comprises a single chain antibody or a Fab.
  • a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12).
  • the mutation is a substitution in the KGD motif.
  • the mutation is two substitutions in the KGD motif.
  • the mutation is three substitutions in the KGD motif. In one embodiment, the mutation is a G to A or D to E substitution in the KGD motif. In one embodiment, the mutation is a deletion in the KGD motif. In one embodiment, the mutation is two deletions in the KGD motif. In one embodiment, the mutation is three deletions in the KGD motif.
  • the polypeptide or agent comprises at least one copy of the extracellular domain of CD40L or a fragment thereof, wherein the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the "extracellular domain" of CD40L is approximately amino acids 47-261 of SEQ ID NO: 12. Those of skill in the art may differ in their understanding of the exact amino acids corresponding to the extracellular domain of CD40L. Thus, the N-terminus and/or C-terminus of the extracellular domain described herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. As used herein, the extracellular domain of CD40L generally comprises the "stalk region" or a fragment of the stalk region and the "TNF family domain". The stalk region of CD40L is
  • the stalk region comprises a fragment of the CD40L stalk region more equivalent in length to the stalk regions of, for example, GITRL or OX40L.
  • a copy of the extracellular domain of CD40L in the polypeptides or agents described herein comprises a fragment of the stalk region of CD40L.
  • the stalk region comprises about 4-20 amino acids. In some embodiments, the stalk region is about 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the stalk region comprises about 4-10 amino acids.
  • the stalk region comprises the amino acids (e.g., 4-10 amino acids) upstream from the TNF homology domain.
  • the stalk region of CD40L is approximately amino acids 47-112 of SEQ ID NO: 12.
  • the stalk region comprises a fragment of the CD40L stalk region.
  • fragments of the CD40L stalk region comprise MQKGDQ (SEQ ID NO: 16; fragment 1); FEMQKGDQ (SEQ ID NO: 17; fragment 2); EMQKGDQ (SEQ ID NO: 18; fragment 3); QKGDQ (SEQ ID NO: 19; fragment 4); or KGDQ (SEQ ID NO:20; fragment 5), wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • fragments of the CD40L stalk region comprise an amino acid sequence selected from the group consisting of: MQKADQ (SEQ ID NO:59), FEMQKADQ (SEQ ID NO:60), EMQKADQ (SEQ ID NO:61), QKADQ (SEQ ID NO:62), and KADQ (SEQ ID NO:63).
  • the "TNF homology domain" or "TNF family domain" of CD40L is approximately amino acids 122-261 of SEQ ID NO: 12.
  • the TNF homology domain comprises SEQ ID NO:21.
  • a polypeptide or agent comprises at least one copy of SEQ ID NO: 15.
  • a polypeptide or agent comprises at least two copies of SEQ ID NO: 15.
  • a polypeptide or agent comprises three copies of SEQ ID NO: 15. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:22, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:22, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:22, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region.
  • a polypeptide or agent comprises at least one copy of SEQ ID NO:23, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:23, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:23, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region.
  • a polypeptide or agent comprises at least one copy of SEQ ID NO:24, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:24, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:24, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region.
  • the polypeptide or agent comprises at least one copy of SEQ ID NO:25, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:25, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:25, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region.
  • the polypeptide or agent comprises at least one copy of SEQ ID NO:26, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:26, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:26, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:59.
  • a polypeptide or agent comprises at least two copies of SEQ ID NO:59. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:59. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:60. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:60. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:60. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:61. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:61.
  • a polypeptide or agent comprises three copies of SEQ ID NO:61. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:62. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:62. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:62. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:63. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:63. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:63.
  • a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of CD40L or a CD40-binding fragment thereof. In certain embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of CD40L or a fragment thereof as a single chain polypeptide. In certain embodiments, a polypeptide or agent comprises SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region.
  • the polypeptide or agent comprises SEQ ID NO:36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO: 36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region.
  • a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO: 36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%), 98%), or 99% sequence identity to SEQ ID NO: 36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent comprises SEQ ID NO:70.
  • a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:70.
  • a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:70.
  • a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:70.
  • a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:70. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:70. In certain embodiments, the polypeptide or agent comprises SEQ ID NO:80. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:80. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:80. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:80. In some
  • a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:80. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:80. In one embodiment, the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12). In one embodiment, the mutation is a substitution in the KGD motif. In one embodiment, the mutation is a G to A or D to E substitution in the KGD motif. In certain embodiments, a polypeptide or agent comprises at least a first, second, and third copy of a fragment of the extracellular domain of CD40L.
  • the copies of the extracellular domain of CD40L consist of the same amino acid sequence. In some embodiments, the copies of the extracellular domain of CD40L are not identical. In some embodiments, the copies of the extracellular domain of CD40L comprise substitutions, deletions, and/or additions to the amino acid sequence of human CD40L as compared to the wild-type or parental sequence.
  • a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof, wherein each extracellular domain comprises the stalk region or a stalk region fragment, wherein the stalk region or fragment of the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L, wherein each extracellular domain comprises the stalk region or a stalk region fragment, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region, and the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers).
  • the extracellular domain of CD40L comprises amino acids 113-261 of SEQ ID NO: 12, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the extracellular domain of CD40L comprises SEQ ID NO: 15.
  • the extracellular domain of CD40L comprises SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some
  • the extracellular domain of CD40L comprises SEQ ID NO:23, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:23, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:24, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:24, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the extracellular domain of CD40L comprises SEQ ID NO:25, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:25, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:26, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:26, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the single chain fusion polypeptide comprises SEQ ID NO:28, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:28, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the single chain fusion polypeptide comprises SEQ ID NO:36, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:36, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12).
  • the mutation is a substitution in the KGD motif.
  • the mutation is a G to A or D to E substitution in the KGD motif.
  • the extracellular domain of CD40L comprises SEQ ID NO:59.
  • the extracellular domain of CD40L consists of SEQ ID NO :59.
  • the extracellular domain of CD40L comprises
  • the extracellular domain of CD40L consists of
  • the extracellular domain of CD40L comprises
  • the extracellular domain of CD40L consists of
  • the extracellular domain of CD40L comprises
  • the extracellular domain of CD40L consists of
  • the extracellular domain of CD40L comprises
  • the extracellular domain of CD40L consists of
  • the single chain fusion polypeptide comprises
  • the single chain fusion polypeptide consists of
  • the single chain fusion polypeptide comprises
  • the single chain fusion polypeptide consists of
  • a polypeptide or agent comprises a variant of the
  • the agent is a polypeptide. In some embodiments, the
  • polypeptide is a fusion protein.
  • the fusion protein comprises at least one copy of the extracellular domain of human CD40L or a fragment thereof, and further comprises a non-CD40L polypeptide.
  • the fusion protein may include an extracellular domain or fragment thereof linked to a heterologous functional and structural polypeptide including, but not limited to, a human Fc region, one or more protein tags (e.g., myc, FLAG, GST), other endogenous proteins or protein fragments, or any other useful protein sequence including any peptide sequence between the extracellular domain and the non-CD40L polypeptide.
  • the non-CD40L polypeptide comprises a human Fc region.
  • the Fc region can be obtained from any of the classes of immunoglobulin, IgG, IgA, IgM, IgD and IgE.
  • the Fc region is a human IgGl Fc region.
  • the Fc region is a human IgG2 Fc region.
  • the Fc region is a wild-type Fc region.
  • the Fc region is a natural variant of a wild-type Fc region.
  • the Fc region is a mutated Fc region.
  • the Fc region is truncated at the N-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, (e.g., in the hinge domain).
  • the Fc region is truncated at the C- terminal end (e.g., lysine is absent).
  • an amino acid in the hinge domain is changed to hinder undesirable disulfide bond formation.
  • a cysteine is replaced with a different amino acid to hinder undesirable disulfide bond formation.
  • a cysteine is replaced with a serine to hinder undesirable disulfide bond formation.
  • Fc receptors e.g., FcyRI, Fcyll, FcyRIII, FcyRIIB
  • FcyRIIB FcyRI receptors
  • Clq component of complement these biological activities include, but are not limited to, ADCC and CDC.
  • Modification of the glycosylation site or sites in a Fc region can inhibit and/or eliminate binding of Fc receptors to a Fc region. For example, substitution of the asparagine at a glycosylation site with a different amino acid such as an alanine residue results in deglycosylation of the Fc region and generally results in reduced ADCC activity.
  • a Fc region is modified to destroy a glycosylation site or sites.
  • a Fc region is modified to reduce binding of a Fc receptor to a Fc region.
  • a Fc region is modified to reduce biological activities mediated by binding of a Fc receptor to a Fc region.
  • the Fc region comprises SEQ ID NO:39, SEQ ID NO:40,
  • SEQ ID NO:41 SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82.
  • the polypeptide or agent comprises (i) SEQ ID NO:69,
  • a polypeptide or agent comprises SEQ ID NO:70 and SEQ ID NO:44. In some embodiments, a polypeptide or agent comprises SEQ ID NO:70 and SEQ ID NO:45. In some embodiments, a polypeptide or agent comprises SEQ ID NO:70 and SEQ ID NO:82. In some embodiments, a polypeptide or agent comprises SEQ ID NO:80 and SEQ ID NO:45. In some embodiments, a polypeptide or agent comprises SEQ ID NO:80 and SEQ ID NO:44. In some embodiments, a polypeptide or agent comprises SEQ ID NO:80 and SEQ ID NO:82.
  • the polypeptide or agent is a single chain CD40L trimer-Fc protein. In some embodiments, the polypeptide or agent is a single chain CD40L trimer- IgGl Fc protein. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgGl Fc protein, wherein the single chain CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or 80, and the IgGl Fc comprises the amino acid sequence of SEQ ID NO:39.
  • the polypeptide or agent is a single chain CD40L trimer-IgGl Fc protein, wherein the single chain CD40L trimer consists of the amino acid sequence of SEQ ID NO:70 or 80, and the IgGl Fc consists of the amino acid sequence of SEQ ID NO:39.
  • the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein.
  • the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein, wherein the single chain CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or 80, and the IgG2 Fc comprises the amino acid sequence of SEQ ID NO:42 or 43.
  • the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein, wherein the single chain CD40L trimer consists of the amino acid sequence of SEQ ID NO:70 or 80, and the IgG2 Fc consists of the amino acid sequence of SEQ ID NO:42 or 43.
  • the polypeptide or agent is a single chain CD40L trimer-IgGl Fc protein, wherein the Fc region is modified.
  • the polypeptide or agent is a single chain CD40L trimer- IgGl Fc protein, wherein the Fc region is deglycosylated.
  • the term "deglycosylation of the Fc region” and “deglycosylated Fc region” as used herein refer to or describe an Fc region comprising a mutation in the N-linked glycosylation site of the CH2 domain.
  • the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein, wherein the Fc region is modified.
  • the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein, wherein the Fc region is deglycosylated.
  • the polypeptide or agent comprises SEQ ID NO:72.
  • the polypeptide or agent comprises SEQ ID NO:81.
  • the polypeptide or agent consists essentially of SEQ ID NO:72 or SEQ ID NO:81. In some embodiments, the polypeptide or agent consists of SEQ ID NO:72. In some embodiments, the polypeptide or agent consists of SEQ ID NO:81.
  • the non-CD40L polypeptide comprises SEQ ID NO:39,
  • the non-CD40L polypeptide consists essentially of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82.
  • the non-CD40L polypeptide consists of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82.
  • the non-CD40L polypeptide comprises an
  • the immunoglobulin heavy chain is associated with an immunoglobulin light chain. In some embodiments, the immunoglobulin heavy chain is associated with an immunoglobulin light chain.
  • the non-CD40L polypeptide comprises an antibody. In certain embodiments, the non-CD40L polypeptide comprises a single chain antibody or Fab.
  • a fusion protein comprises at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof and a non- CD40L polypeptide, wherein the C-terminal end of the non-CD40L polypeptide is linked to the extracellular domain(s) of CD40L, wherein at least one of the first, second, or third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • a fusion protein comprises at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof and a non-CD40L polypeptide, wherein the N- terminal end of the non-CD40L polypeptide is linked to the extracellular domain(s) of CD40L, wherein at least one of the first, second, or third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the first copy of the extracellular domain of CD40L is linked to the C-terminal end of the non-CD40L polypeptide.
  • the third copy of the extracellular domain of CD40L is linked to the N- terminal end of the non-CD40L polypeptide. In some embodiments, the extracellular domain(s) of CD40L is linked to the C-terminal end of a Fc region. In some
  • the extracellular domain(s) of CD40L is linked to the N-terminal end of a Fc region. In some embodiments, the extracellular domain(s) of CD40L is directly linked to the Fc region (i.e. without an intervening peptide linker). In some embodiments, the extracellular domain(s) of CD40L is linked to the Fc region via a peptide linker.
  • linker refers to a linker inserted between a first polypeptide (e.g., a extracellular domain of CD40L or a fragment thereof comprising a fragment of the stalk region of CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region) and a second polypeptide (e.g., a Fc region).
  • the linker is a peptide linker.
  • Linkers should not adversely affect the expression, secretion, or bioactivity of the fusion protein. Linkers should not be antigenic and should not elicit an immune response.
  • Suitable linkers are known to those of skill in the art and often include mixtures of glycine and serine residues and often include amino acids that are sterically unhindered. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. Linkers can range in length, for example from 1-50 amino acids in length, 1-22 amino acids in length, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3 amino acids in length.
  • Linkers may include, but are not limited to, SerGly, GGSG, GSGS, GGGS, S(GGS)n where n is 1-7, GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO:50), ESGGGGVT (SEQ ID NO:51), LESGGGGVT (SEQ ID NO:52), GRAQVT (SEQ ID NO:53), WRAQVT (SEQ ID NO:54), and ARGRAQVT (SEQ ID NO:55).
  • the linker may comprise a cleavage site.
  • the linker may comprise an enzyme cleavage site, so that the second polypeptide may be separated from the first polypeptide.
  • a linker is an intervening peptide sequence that does not include amino acid residues from either the C-terminus or N-terminus of the first polypeptide (e.g., an extracellular domain of CD40L or a fragment thereof comprising a fragment of the stalk region of CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region) or C-terminus or the N-terminus of the second polypeptide (e.g., the Fc region).
  • a polypeptide e.g., a fusion polypeptide described herein comprises: (a) a first polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region; and (b) a second polypeptide comprising a Fc region, wherein the Fc region is deglycosylated.
  • CD40L human CD40 ligand
  • the fragment of the stalk region of CD40L is selected from the group consisting of: MQKGDQ (SEQ ID NO: 16), FEMQKGDQ (SEQ ID NO: 17), EMQKGDQ (SEQ ID NO: 18), QKGDQ (SEQ ID NO: 19), and KGDQ (SEQ ID NO:20), wherein the fragment of the stalk region comprises a mutation in the integrin- binding region.
  • the fragment of the stalk region of CD40L is MQKGDQ (SEQ ID NO: 16), wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the fragment of the stalk region of CD40L comprises an amino acid sequence selected from the group consisting of: MQKADQ (SEQ ID NO:59), FEMQKADQ (SEQ ID NO:60), EMQKADQ (SEQ ID NO:61), QKADQ (SEQ ID NO:62), and KADQ (SEQ ID NO:63).
  • the fragment of the stalk region of CD40L comprises the amino acid sequence of
  • the polypeptide does not comprise an exogenous peptide linker between any of the copies of the extracellular domain or a fragment thereof of CD40L. In some embodiments, at least one of the copies of the extracellular domain or a fragment thereof comprises SEQ ID NO: 15.
  • At least one of the copies of the extracellular domain or a fragment thereof comprises SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • extracellular domain or a fragment thereof comprises SEQ ID NO: 15.
  • each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide comprises SEQ ID NO:70.
  • a polypeptide comprises SEQ ID NO:80.
  • a polypeptide comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:70.
  • a polypeptide comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:70.
  • a polypeptide comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:80.
  • a polypeptide comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:80.
  • the first polypeptide is directly linked to the second polypeptide.
  • the first polypeptide is connected to the second polypeptide by a linker.
  • the first copy of the extracellular domain of the first polypeptide is linked to the C-terminal end of the second polypeptide.
  • the third copy of the extracellular domain of the first polypeptide is linked to the N-terminal end of the second polypeptide.
  • the second polypeptide comprises a human Fc region.
  • a human Fc region is from an IgGl, IgG2, IgG3, or IgG4 immunoglobulin.
  • the Fc region is selected from the group consisting of: SEQ ID NO:44 and SEQ ID NO:45.
  • the Fc region comprises the amino acid sequence of SEQ ID NO: 82.
  • the polypeptide comprises SEQ ID NO:72 or SEQ ID NO:81.
  • an additional polypeptide is linked to the first polypeptide.
  • an additional polypeptide is linked to the second polypeptide.
  • the second polypeptide comprises an immunoglobulin heavy chain.
  • the immunoglobulin heavy chain is associated with an immunoglobulin light chain. In some embodiments, the immunoglobulin heavy chain and immunoglobulin light chain form an antigen-binding site. In some embodiments, the second polypeptide comprises a single chain antibody or a Fab. In some embodiments, the polypeptide comprises a third polypeptide.
  • a polypeptide or agent described herein specifically binds
  • CD40 and acts as a CD40 agonist.
  • a polypeptide or agent described herein specifically binds CD40 and activates CD40 signaling.
  • a polypeptide or agent described herein specifically binds CD40 and induces, activates, promotes, increases, enhances, or prolongs CD40 activity.
  • a polypeptide or agent described herein specifically binds
  • CD40 and modulates an immune response.
  • a polypeptide or agent described herein specifically binds CD40 and induces, augments, increases, and/or prolongs an immune response.
  • a polypeptide or agent described herein specifically binds
  • the polypeptide or agent binds CD40 with a K D of from about 1 ⁇ to about O. lnM, or any range of values thereof (e.g., from about ⁇ ⁇ to about InM, from about ⁇ ⁇ to about lOnM, from about ⁇ ⁇ to about 20nM, from about ⁇ ⁇ to about 40nM, from about ⁇ ⁇ to about ⁇ , from about ⁇ to about O.
  • lnM from about ⁇ to about InM, from about ⁇ to about lOnM, from about ⁇ to about 20nM, from about ⁇ to about 40nM, from about 40nM to about 0. InM, from about 40nM to about InM, from about 40nM to about lOnM, from about 40nM to about 20nM, from about 20nM to about O. lnM, from about 20nM to about InM, from about 20nM to about lOnM, from about lOnM to about O. lnM, from about lOnM to about InM, or from about InM to about O. lnM).
  • the polypeptide or agent binds CD40 with an K D of about ⁇ ⁇ , about ⁇ , about 40nM, about 20nM, about lOnM, about InM, or about O. lnM. In some embodiments, the polypeptide or agent binds CD40 with a K D of about InM or less. In some embodiments, the polypeptide or agent binds CD40 with a K D of about 0. InM or less. In some embodiments, the polypeptide or agent binds human CD40 and/or mouse CD40 with a K D of about lOnM or less. In some embodiments, the polypeptide or agent binds human CD40 with a K D of about lOnM or less.
  • a polypeptide or agent described herein binds human CD40 and/or mouse CD40 with a K D of about lOnM or less. In some embodiments, a polypeptide or agent described herein binds human CD40 and/or mouse CD40 with a K D of about InM or less. In some embodiments, a polypeptide or agent described herein binds human CD40 and/or mouse CD40 with a K D of about 0. InM or less. In some embodiments, a polypeptide or agent described herein binds human CD40 and does not bind mouse CD40. In some embodiments, a polypeptide or agent described herein binds human CD40 with a K D of about lOnM or less.
  • a polypeptide or agent described herein binds human CD40 with a K D of about InM or less. In some embodiments, a polypeptide or agent described herein binds human CD40 with a K D of about O. lnM or less.
  • CD40 is the dissociation constant determined using a CD40 fusion protein comprising at least a portion of a CD40 extracellular domain immobilized on a Biacore chip.
  • a polypeptide or agent binds CD40 with a half maximal effective concentration (EC 50 ) of about ⁇ ⁇ or less, about ⁇ or less, about 40nM or less, about 20nM or less, about lOnM or less, about InM or less, or about O. lnM or less. In one embodiment, a polypeptide or agent binds CD40 with an EC 50 of from about ⁇ ⁇ to about O.
  • lnM or any range of values thereof (e.g., from about ⁇ ⁇ to about InM, from about ⁇ ⁇ to about lOnM, from about ⁇ ⁇ to about 20nM, from about ⁇ ⁇ to about 40nM, from about ⁇ ⁇ to about ⁇ , from about ⁇ to about O. lnM, from about ⁇ to about InM, from about ⁇ to about lOnM, from about ⁇ to about 20nM, from about ⁇ to about 40nM, from about 40nM to about O. lnM, from about 40nM to about InM, from about 40nM to about lOnM, from about 40nM to about 20nM, from about 20nM to about 0.
  • a polypeptide or agent binds CD40 with an EC 50 of about ⁇ ⁇ , about ⁇ , about 40nM, about 20nM, about lOnM, about InM, or about O. lnM.
  • a polypeptide or agent described herein has an improved therapeutic index compared to a reference polypeptide or reference agent. In some embodiments, a polypeptide or agent described herein induces reduced systemic inflammation compared to a reference polypeptide or reference agent. In some embodiments, a polypeptide or agent described herein induces lower serum liver enzyme level compared to a reference polypeptide or reference agent. In some embodiments, a polypeptide or agent described herein induces lower pro-inflammatory cytokine release than a reference polypeptide or reference agent.
  • the reference polypeptide or reference agent comprise the same polypeptide as the polypeptide or agent described herein, except that reference polypeptide or reference agent comprise a polypeptide comprising a CD40L stalk region that does not comprise a mutation in the integrin-binding region.
  • the reduced systemic inflammation is indicated by lower levels of blood neutrophils. In one embodiment, the blood neutrophil level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the blood neutrophil level is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60%> lower, from about 40% to about 50% lower, from about 50%) to about 70%) lower, from about 50% to about 60%> lower, or from about 60%> to about 70%) lower).
  • the blood neutrophil level is about 20% lower, about 30%) lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
  • the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In one embodiment, the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the liver enzyme level is from about 20%) to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60%) lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30%) to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40%) lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50%) to about 60% lower, or from about 60% to about 70% lower).
  • the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
  • the pro-inflammatory cytokine is selected from the group consisting of: IL- ⁇ , IL-6, TNF- alpha, IP- 10, KC, and ⁇ - ⁇ . In one embodiment, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the pro-inflammatory cytokine release is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50%) lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30%) to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60%) to about 70% lower).
  • the pro-inflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60%) lower, or about 70% lower.
  • provided herein is a method of improving the therapeutic index of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region.
  • a method of decreasing the toxicity of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region.
  • the human CD40 agonist polypeptide comprises three copies of the extracellular domain of human CD40L or a CD40-binding fragment thereof.
  • the improving the therapeutic index or decreasing the toxicity comprises induction of reduced systemic inflammation compared to the human CD40 agonist.
  • the improving the therapeutic index or decreasing the toxicity comprises induction of lower serum liver enzyme level compared to the human CD40 agonist.
  • the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP).
  • the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the liver enzyme level is from about 20%) to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60%) lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30%) to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40%) lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50%) to about 60% lower, or from about 60% to about 70% lower).
  • the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
  • the improving the therapeutic index or decreasing the toxicity comprises induction of lower pro-inflammatory cytokine compared to the human CD40 agonist.
  • the pro-inflammatory cytokine is selected from the group consisting of: IL- ⁇ , IL-6, T F- alpha, IP- 10, KC, and ⁇ - ⁇ .
  • the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the pro-inflammatory cytokine release is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30%) to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60%) to about 70% lower).
  • the pro-inflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60%) lower, or about 70% lower.
  • an improved human CD40 agonist produced by a method described herein.
  • a method of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject comprising administering a therapeutically effective amount of the improved human CD40 agonist described herein.
  • the immune response is against a tumor or cancer.
  • a method of inhibiting the growth of a tumor comprising contacting a tumor or tumor cell with an effective amount of the improved human CD40 agonist described herein.
  • provided herein is a method of inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the improved human CD40 agonist described herein.
  • a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the improved human CD40 agonist described herein.
  • fusion polypeptides are made using recombinant DNA techniques as known to one skilled in the art.
  • polynucleotides encoding a specific protein or a fragment thereof are isolated from mammalian cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the gene encoding the protein, and the nucleotide sequence is determined using conventional techniques.
  • the isolated polynucleotides encoding the protein may be cloned into suitable expression vectors which produce the polypeptide when transfected into host cells such as E. coli, simian COS cells, or Chinese hamster ovary (CHO) cells.
  • recombinant proteins, or fragments thereof can be isolated from phage display libraries or using other cell surface display techniques.
  • the polynucleotide(s) encoding a protein can be modified in a number of different manners using recombinant DNA technology to generate alternative or variant proteins. Site-directed or high-density mutagenesis of a protein can be used to optimize specificity, affinity, stability, etc. of a recombinant protein.
  • Proteins generally contain a signal sequence that directs the transport of the
  • Signal sequences are located at the N-terminus of nascent polypeptides. They target the polypeptide to the endoplasmic reticulum and the proteins are sorted to their destinations, for example, to the inner space of an organelle, to an interior membrane, to the cell outer membrane, or to the cell exterior via secretion. Most signal sequences are cleaved from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum. The cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent upon amino acid residues within the signal sequence.
  • the polypeptides as described herein may comprise a mixture of polypeptides with different N-termini.
  • the N-termini differ in length by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.
  • the N-termini differ in length by 1, 2, 3, 4, or 5 amino acids.
  • the polypeptide is substantially homogeneous, i.e., the polypeptides have the same N-terminus.
  • the signal sequence of the polypeptide comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) amino acid substitutions and/or deletions as compared to the native sequence of the protein.
  • the signal sequence of the polypeptide comprises amino acid substitutions and/or deletions that allow one cleavage site to be dominant, thereby resulting in a substantially homogeneous polypeptide with one N-terminus.
  • the signal sequence of a fusion polypeptide is not the native signal sequence of the protein(s) contained within the fusion polypeptide.
  • a polypeptide, agent, or fusion polypeptide described herein comprises the Fc region of an immunoglobulin.
  • the polypeptides or agents of this invention will comprise fusion proteins in which at least a portion of the Fc region has been deleted or otherwise altered so as to provide desired biochemical characteristics, such as increased cancer cell localization, increased tumor penetration, reduced serum half-life, or increased serum half-life, when compared with a fusion protein of approximately the same
  • immunogenicity comprising a native or unaltered Fc region.
  • Modifications to the Fc region may include additions, deletions, or substitutions of one or more amino acids in one or more domains.
  • the modified fusion proteins disclosed herein may comprise alterations or modifications to one or more of the two heavy chain constant domains (CH2 or CH3) or to the hinge region. In other embodiments, the entire CH2 domain may be removed (ACH2 constructs).
  • the omitted constant region domain is replaced by a short amino acid spacer (e.g., 10 aa residues) that provides some of the molecular flexibility typically imparted by the absent constant region domain.
  • the modified fusion proteins are engineered to link the
  • CH3 domain directly to the hinge region or to the first polypeptide.
  • a peptide spacer or linker is inserted between the hinge region or the first polypeptide and the modified CH2 and/or CH3 domains.
  • constructs may be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region or first polypeptide with a 5-20 amino acid spacer.
  • a spacer may be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible.
  • amino acid spacers may, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct.
  • any spacer added to the construct will be relatively non-immunogenic so as to maintain the desired biological qualities of the fusion protein.
  • the modified fusion proteins may have only a partial
  • deletion of a constant domain or substitution of a few or even a single amino acid may be enough to substantially reduce Fc binding and thereby increase cancer cell localization and/or tumor penetration.
  • Such partial deletions of the constant regions may improve selected characteristics of the polypeptide or agent (e.g., serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
  • the constant regions of the disclosed fusion proteins may be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct.
  • the modified fusion proteins comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function, or provide for more cytotoxin or carbohydrate attachment sites.
  • binding of the CI component of complement to the Fc region of IgG or IgM antibodies activates the complement system. Activation of
  • complement is important in the opsonization and lysis of cell pathogens.
  • the activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity.
  • the Fc region can bind to a cell expressing a Fc receptor (FcR).
  • Fc receptors There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
  • the modified fusion proteins provide for altered effector functions that, in turn, affect the biological profile of the polypeptide or agent.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified agent, thereby increasing cancer cell localization and/or tumor penetration.
  • the constant region modifications increase or reduce the serum half-life of the polypeptide or agent.
  • the constant region is modified to eliminate disulfide linkages or oligosaccharide moiety attachment sites.
  • a modified fusion protein does not have one or more effector functions normally associated with an Fc region.
  • the polypeptide or agent has no antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or no complement-dependent cytotoxicity (CDC) activity.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the polypeptide or agent does not bind to the Fc receptor and/or complement factors.
  • the polypeptide or agent has no effector function normally associated with an Fc region.
  • polypeptides and agents of the present invention can be assayed for specific binding to a target by any method known in the art.
  • the immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore analyses, FACS analyses, immunofluorescence,
  • immunocytochemistry Western blot analyses, radioimmunoassays, ELISAs, "sandwich” immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A
  • immunoassays Such assays are routine and well-known in the art.
  • test agent e.g., a polypeptide
  • CD40 may be determined using ELISA.
  • An ELISA assay comprises preparing CD40 protein, coating wells of a 96-well microtiter plate with the CD40, adding the test agent conjugated to a detectable compound such as an enzymatic substrate (e.g. horseradish peroxidase or alkaline phosphatase) to the well, incubating for a period of time and detecting the presence of the agent bound to CD40.
  • a detectable compound such as an enzymatic substrate (e.g. horseradish peroxidase or alkaline phosphatase)
  • the test agent is not conjugated to a detectable compound, but instead a labeled secondary antibody that recognizes the agent is added to the well.
  • the test agent instead of coating the well with CD40, the test agent can be coated to the well, soluble CD40 is added, and a second antibody conjugated to a detectable compound that recognizes CD40 can be used to detect binding.
  • a detectable compound that recognizes CD40 can be used to detect binding.
  • the specific binding of a test agent e.g., a polypeptide) to human CD40 may be determined using FACS.
  • a FACS screening assay may comprise generating a cDNA construct that expresses CD40, transfecting the construct into cells, expressing CD40 on the surface of the cells, mixing the test agent with the transfected cells, and incubating for a period of time.
  • the cells bound by the test agent may be identified by using a secondary antibody conjugated to a detectable compound (e.g., PE- conjugated anti-Fc antibody) and a flow cytometer.
  • a detectable compound e.g., PE- conjugated anti-Fc antibody
  • the binding affinity of a test agent to a target (e.g., human CD40) and the off-rate of an agent-target interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled target (e.g., 3 H or 125 I-labeled CD40), or fragment or variant thereof, with the agent of interest in the presence of increasing amounts of unlabeled target followed by the detection of the agent bound to the labeled target.
  • labeled target e.g., 3 H or 125 I-labeled CD40
  • the affinity of the agent for a target (e.g., human CD40) and the binding off-rates can be determined from the data by
  • Biacore kinetic analysis is used to determine the binding on and off rates of agents that bind a target (e.g., human CD40).
  • Biacore kinetic analysis comprises analyzing the binding and dissociation of agents from chips with immobilized target (e.g., human CD40) on the chip surface.
  • This invention also encompasses homodimenc agents and heterodimeric
  • the homodimeric agents are polypeptides.
  • the heterodimeric molecules are polypeptides. Generally the homodimeric molecule comprises two identical polypeptides. Generally the heterodimeric molecule comprises two non-identical polypeptides. In some embodiments, a
  • heterodimeric molecule is capable of binding at least two targets, e.g., a bispecific agent.
  • the targets may be, for example, two different proteins on a single cell or two different proteins on two separate cells.
  • the bispecific agents are polypeptides.
  • one polypeptide of the heterodimeric molecule comprises a polypeptide described herein (e.g., a single chain trimer-Fc protein that binds CD40) and one polypeptide of the heterodimeric molecule is an antibody.
  • the term "arm” may be used herein to describe the structure of a homodimeric agent, a heterodimeric agent, and/or a bispecific agent. As used herein, each "arm" is directed against a target.
  • one "arm” may comprise an antigen-binding site from an antibody. In some embodiments, one “arm” may comprise a binding portion of a receptor. In some embodiments, a homodimeric agent comprises two identical am s. In some embodiments, a heterodimeric agent comprises two different arms. In some embodiments, a bispecific agent comprises two different arms. [0161] In some embodiments, a bispecific agent comprises the polypeptides or agents described herein. In some embodiments, the bispecific agent is a homodimeric protein. In some embodiments, the homodimer bispecific agent comprises a polypeptide comprising a heavy chain immunoglobulin and a CD40L trimer.
  • the heavy chain immunoglobulin is associated with a light chain to form an antigen-binding site.
  • the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain CD40L trimer. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising a single-chain antibody and a single chain CD40L trimer. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds a tumor antigen. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds an antigen on an immune cell.
  • the homodimeric bispecific agent comprises an antibody that specifically binds PD-1, PD-L1, CTLA-4, LAG-3, GITR, OX-40, TIGIT, TEVI3, or B7-H4.
  • the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
  • the bispecific agent is a heterodimeric protein.
  • the heterodimeric bispecific agent comprises an antigen-binding site from an antibody (e.g., an antigen-binding site formed by an immunoglobulin heavy chain and an immunoglobulin light chain) and a CD40L trimer.
  • a bispecific agent comprises an immune response stimulating agent or functional fragment thereof and a CD40L trimer.
  • the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
  • a heterodimeric bispecific agent is capable of binding one target and also comprises a "non-binding" function.
  • one polypeptide of the heterodimeric bispecific agent comprises a polypeptide described herein (e.g., binds CD40) and one polypeptide of the heterodimeric agent is an additional immune response stimulating agent.
  • immune response stimulating agent is used in the broadest sense and refers to a substance that directly or indirectly stimulates the immune system by inducing activation or increasing activity of any of the immune system's components.
  • immune response stimulating agents may include cytokines, as well as various antigens including tumor antigens, and antigens derived from pathogens.
  • the immune response stimulating agent includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), an antibody that blocks immunosuppressive functions (e.g., an anti-CTLA-4 antibody, anti-CD28 antibody, anti-PD-1 antibody, anti-PD-Ll antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86).
  • GM-CSF granulocyte
  • a heterodimeric bispecific agent comprises a first
  • a bispecific agent with a binding specificity for a tumor antigen can be used to direct the CD40L trimer polypeptide to a tumor.
  • the bispecific agent may be used to direct the CD40L trimer polypeptide to a tumor that expresses the tumor antigen or overexpresses the tumor antigen. This may be useful to induce and/or enhance an immune response near or within the tumor microenvironment.
  • a bispecific agent may be used to induce or enhance the activity of tumor infiltrating immune cells.
  • the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
  • a heterodimeric bispecific agent comprises a first
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds an immune response molecule.
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds an immune checkpoint protein.
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds PD-1.
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds PD-L1.
  • a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds B7-H4.
  • the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
  • a bispecific agent either heterodimeric or homodimeric, has enhanced potency as compared to an individual agent. It is known to those of skill in the art that any agent (e.g., a soluble protein or a cytokine) may have unique
  • a bispecific agent has the ability to synchronize the PK of two active agents and/or polypeptides wherein the two individual agents and/or polypeptides have different PK profiles.
  • a bispecific molecule has the ability to concentrate the actions of two agents and/or polypeptides in a common area (e.g., a tumor and/or tumor
  • a bispecific molecule has the ability to concentrate the actions of two agents and/or polypeptides to a common target (e.g., a tumor or a tumor cell).
  • a bispecific agent has the ability to target the actions of two agents and/or polypeptides to more than one biological pathway or more than one aspect of the immune response.
  • the bispecific agent has decreased toxicity and/or side effects than either of the polypeptides and/or agents alone.
  • the bispecific agent has decreased toxicity and/or side effects as compared to a mixture of the two individual polypeptides and/or agents.
  • the bispecific agent has an increased therapeutic index.
  • the bispecific agent has an increased therapeutic index as compared to a mixture of the two individual polypeptides and/or agents or the polypeptides and/or agents as single agents.
  • a single chain CD40L trimer molecule may be more active than an anti-CD40 agonist antibody, because a single chain CD40L trimer would function, i.e., bind to three CD40 molecules, in a very similar manner as three native CD40L molecules.
  • an anti-CD40 antibody is able to engage only two CD40 molecules, thereby reducing any potential effect.
  • To make a bispecific molecule comprising an antibody generally involves the antibody being monovalent (i.e., one-armed antibody). This reduces, if not completely eliminates, the effect of an agonist antibody, especially if activation depends upon clustering of the target molecules.
  • a single chain CD40L trimer is able to bind three CD40 molecules, thus as part of a heterodimeric or homodimeric bispecific molecule, it does not lose any functionality or potency.
  • the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
  • a heterodimeric bispecific molecule comprises a first
  • polypeptide comprising a single chain CD40L trimer and a second polypeptide comprising an antagonist antibody
  • the single chain CD40L trimer comprises a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
  • a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the stalk region comprises a mutation in the integrin-binding region, and (b) a second arm comprising an antigen-binding site from an antibody.
  • a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the stalk region comprises a mutation in the integrin
  • a heterodimeric bispecific agent comprises: (a) a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the stalk region comprises a mutation in the integrin-binding region, and (b) a second arm comprising an immune response stimulating agent.
  • CD40L human CD40 ligand
  • At least one copy of the extracellular domain of CD40L of the first arm comprises SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • at least one copy of the extracellular domain of CD40L of the first arm comprises the amino acid sequence of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68.
  • the bispecific agent comprises a first arm comprising SEQ ID NO:28 or SEQ ID NO:36, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
  • the bispecific agent comprises a first arm comprising the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
  • the heterodimeric bispecific agent comprises a first arm which further comprises a non-CD40L polypeptide.
  • the heterodimeric bispecific agent comprises a single chain fusion CD40L polypeptide described herein which is directly linked to a non-CD40L polypeptide.
  • the single chain fusion polypeptide is connected to the non-CD40L polypeptide by a linker.
  • the non-CD40L polypeptide comprises a human Fc region.
  • the non-CD40L polypeptide comprises SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82.
  • the non-CD40L polypeptide comprises SEQ ID NO:44.
  • the non-CD40L polypeptide comprises SEQ ID NO:45.
  • the non-CD40L polypeptide comprises SEQ ID NO:82.
  • a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain CD40 trimer and a second polypeptide comprising an immune response stimulating agent.
  • the multimeric molecule e.g., a bispecific agent
  • first CH3 domain comprises a first CH3 domain and a second CH3 domain, each of which is modified to promote formation of heteromultimers or heterodimers.
  • first and second CH3 domains are modified using a knobs-into-holes technique.
  • the first and second CH3 domains comprise changes in amino acids that result in altered electrostatic interactions.
  • the first and second CH3 domains comprise changes in amino acids that result in altered hydrophobic/hydrophilic interactions (see, for example, U.S. Patent App. Publication No. 2011/0123532).
  • the heterodimeric molecule e.g., a bispecific agent
  • a human IgGl constant region wherein the amino acids at positions corresponding to positions 253 and 292 of SEQ ID NO:46 are replaced with glutamate or aspartate, and a second human IgGl constant region, wherein the amino acids at positions corresponding to 240 and 282 of SEQ ID NO:46 are replaced with lysine
  • a first human IgG2 constant region wherein the amino acids at positions corresponding to positions 249 and 288 of SEQ ID NO:47 are replaced with glutamate or aspartate, and a second human IgG2 constant region wherein the amino acids at positions corresponding to positions 236 and 278 of SEQ ID NO:47 are replaced with lysine
  • a first human IgG3 constant region wherein the amino acids at positions corresponding to positions 300 and 339 of SEQ ID NO:48 are replaced with glutamate or aspartate, and a second human IgG3 constant region wherein the amino acids at positions corresponding to positions 300 and 339 of SEQ ID NO:48 are replaced with gluta
  • the heterodimeric molecule e.g., a bispecific agent
  • CH2 and CH3 domains selected from the group consisting of: (a) a first human IgGl CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 253 and 292 of SEQ ID NO:46 are replaced with glutamate or aspartate, and a second human IgGl CH2 and CH3 domain, wherein the amino acids at positions corresponding to 240 and 282 of SEQ ID NO:46 are replaced with lysine; (b) a first human IgG2 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 249 and 288 of SEQ ID NO:47 are replaced with glutamate or aspartate, and a second human IgG2 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 236 and 278 of SEQ ID NO:47 are replaced with lysine; (c) a first human IgG3 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 300 and 339 of SEQ ID NO:48
  • the polypeptides or agents are monovalent. In some embodiments, the polypeptides or agents are monovalent. In some embodiments, the polypeptides or agents are monovalent.
  • the polypeptide or agent is a soluble protein that is monovalent. In some embodiments, the polypeptides or agents described herein are bivalent. In some embodiments, the polypeptides or agents described herein are trivalent. In some embodiments, the polypeptides or agents described herein are monospecific. In some embodiments, the polypeptides or agents described herein are bispecific. In some embodiments, the polypeptides or agents described herein are multispecific. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is monovalent. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is bivalent. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is trivalent (i.e., binds three target molecules).
  • the polypeptides or agents comprise polypeptides that are substantially homologous to the fusion proteins and/or polypeptides described herein. These agents can contain, for example, conservative substitution mutations, i.e. the substitution of one or more amino acids by similar amino acids. For example,
  • conservative substitution refers to the substitution of an amino acid with another within the same general class such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art and described herein.
  • a polypeptide or agent described herein binds CD40 and modulates an immune response. In some embodiments, a polypeptide or agent described herein activates and/or increases an immune response. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances cell-mediated immunity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances innate cell-mediated immunity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances adaptive cell- mediated immunity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances APC activity.
  • a polypeptide or agent described herein increases, promotes, or enhances adaptive dendritic cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances T-cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances CD4+ T-cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances CD8+ T-cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances CTL activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances K cell activity. In some embodiments, or enhances
  • a polypeptide or agent described herein increases, promotes, or enhances lymphokine-activated killer cell (LAK) activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances tumor-infiltrating lymphocyte (TIL) activity. In some embodiments, a polypeptide or agent described herein inhibits or decreases Treg cell activity. In some embodiments, a polypeptide or agent described herein inhibits or decreases MDSC cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances tumor cell killing. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances the inhibition of tumor growth.
  • LAK lymphokine-activated killer cell
  • TIL tumor-infiltrating lymphocyte
  • a polypeptide or agent described herein inhibits or decreases Treg cell activity. In some embodiments, a polypeptide or agent described herein inhibits or decreases MDSC cell activity. In some embodiments, a poly
  • a polypeptide or agent described herein increases or enhances an effective immune response without causing substantial side effects and/or immune-based toxicities. In some embodiments, a polypeptide or agent described herein increases or enhances an effective immune response without causing cytokine release syndrome (CRS) or a cytokine storm.
  • CRS cytokine release syndrome
  • a polypeptide or agent described herein binds CD40 and induces, enhances, increases, and/or prolongs CD40 signaling.
  • a polypeptide or agent described herein is an agonist
  • a polypeptide or agent is an agonist of CD40 and activates and/or increases an immune response.
  • a polypeptide or agent is an agonist of CD40 and activates and/or increases activity of APCs (e.g., dendritic cells, B-cells).
  • a polypeptide or agent is an agonist of CD40 and activates and/or increases activity of K cells and/or T- cells (e.g., cytolytic activity or cytokine production).
  • a polypeptide or agent increases the activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%). In certain embodiments, a polypeptide or agent increases the activity by from about 10%) to about 100%, or any range of values thereof (e.g., from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, from about 10% to about 30%), from about 10% to about 20%, from about 20% to about 100%, from about 20% to about 90%, from about 20% to about 75%, from about 20% to about 50%, from about 20% to about 30%, from about 30% to about 100%, from about 30% to about 90%, from about 30% to about 75%, from about 30% to about 50%, from about 50% to about 100%, from about 50% to about 90%, from about 50% to about 75%, from about 75% to about 100%, from about 75% to about 90%, or from about 90% to about 100%).
  • a polypeptide or agent increases the activity by about 10%, about 20%, about 30%, about 50%, about 75%, about 90%, or about 100%.
  • a polypeptide or agent described herein increases activation of a NK cell.
  • a polypeptide or agent increases activation of a T-cell.
  • the activation of a K cell and/or a T-cell by a polypeptide or agent results in an increase in the level of activation of a NK cell and/or a T-cell of at least about 10%, at least about 25%, at least about 50%, at least about 75%), at least about 90%, or at least about 95%.
  • the activation of a NK cell and/or a T-cell by a polypeptide or agent results in an increase in the level of activation of a NK cell and/or a T-cell of from about 10% to about 95%, or any range of values thereof (e.g., from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, from about 10% to about 25%, from about 25% to about 95%, from about 25% to about 90%, from about 25% to about 75%, from about 25% to about 50%, from about 50% to about 95%, from about 50% to about 90%, from about 50% to about 75%), from about 75% to about 95%, from about 75% to about 90%, or from about 90%) to about 95%).
  • values thereof e.g., from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, from about 10% to about 25%, from about 25% to about 95%, from about 25% to about 90%, from about 25% to about 75%, from about 25% to about 50%, from about 50% to about 9
  • the activation of a NK cell and/or a T-cell by a polypeptide or agent results in an increase in the level of activation of a NK cell and/or a T-cell of about 10%, about 25%, about 50%, about 75%, about 90%, or about 95%.
  • a polypeptide or agent described herein inhibits or
  • a polypeptide or agent inhibits activity of a Treg cell.
  • the inhibition of suppressive activity of a Treg cell by a polypeptide or agent results in an inhibition of suppressive activity of a Treg cell of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.
  • the inhibition of suppressive activity of a Treg cell by a polypeptide or agent results in an inhibition of suppressive activity of a Treg cell of from about 10% to about 95%, or any range of values thereof (e.g., from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, from about 10% to about 25%, from about 25% to about 95%), from about 25% to about 90%, from about 25% to about 75%, from about 25% to about 50%, from about 50% to about 95%, from about 50% to about 90%, from about 50%) to about 75%, from about 75% to about 95%, from about 75% to about 90%, or from about 90% to about 95%).
  • the inhibition of suppressive activity of a Treg cell by a polypeptide or agent results in an inhibition of suppressive activity of a Treg cell of about 10%, about 25%, about 50%, about 75%, about 90%, or about 95%.
  • a polypeptide or agent described herein inhibits or
  • a polypeptide or agent inhibits activity of a MDSC.
  • the inhibition of suppressive activity of a MDSC by a polypeptide or agent results in an inhibition of suppressive activity of a MDSC of at least about 10%, at least about 25%, at least about 50%, at least about 75%), at least about 90%, or at least about 95%.
  • the inhibition of suppressive activity of a MDSC by a polypeptide or agent results in an inhibition of suppressive activity of a MDSC of from about 10% to about 95%, or any range of values thereof (e.g., from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, from about 10% to about 25%, from about 25% to about 95%), from about 25% to about 90%, from about 25% to about 75%, from about 25% to about 50%, from about 50% to about 95%, from about 50% to about 90%, from about 50%) to about 75%, from about 75% to about 95%, from about 75% to about 90%, or from about 90% to about 95%).
  • the inhibition of suppressive activity of a MDSC by a polypeptide or agent results in an inhibition of suppressive activity of a MDSC of about 10%, about 25%, about 50%, about 75%, about 90%, or about 95%.
  • a functional assay that detects T-cell activation can be used.
  • a functional assay that detects Treg activity can be used.
  • a functional assay that detects MDSC activity can be used.
  • a functional assay that detects K cell activity can be used.
  • a functional assay that detects cytolytic T-cell activity can be used.
  • an assay that detects cytokine production can be used.
  • an assay that detects cytokine-producing cells can be used.
  • a polypeptide or agent described herein is capable of inhibiting tumor growth. In certain embodiments, the polypeptide or agent is capable of inhibiting tumor growth in vivo (e.g., in a mouse model and/or in a human having cancer). [0184] In certain embodiments, a polypeptide or agent described herein is capable of reducing the tumorigenicity of a tumor. In certain embodiments, the polypeptide or agent is capable of reducing the tumorigenicity of a tumor in an animal model, such as a mouse model. In certain embodiments, the polypeptide or agent is capable of reducing the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a mouse model.
  • the number or frequency of cancer stem cells in a tumor is reduced by at least about two-fold, about three-fold, about five-fold, about tenfold, about 50-fold, about 100-fold, or about 1000-fold.
  • the reduction in the number or frequency of cancer stem cells is determined by limiting dilution assay using an animal model. Additional examples and guidance regarding the use of limiting dilution assays to determine a reduction in the number or frequency of cancer stem cells in a tumor can be found, e.g., in International Publication Number WO 2008/042236; U.S. Patent Publication No. 2008/0064049; and U.S. Patent Publication No. 2008/0178305.
  • a polypeptide or agent described herein has one or more of the following effects: inhibits proliferation of tumor cells, inhibits tumor growth, reduces the tumorigenicity of a tumor, reduces the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, triggers cell death of tumor cells, increases cell contact-dependent growth inhibition, increases tumor cell apoptosis, reduces epithelial mesenchymal transition (EMT), or decreases survival of tumor cells.
  • the polypeptide or agent has one or more of the following effects: inhibits viral infection, inhibits chronic viral infection, reduces viral load, triggers cell death of virus-infected cells, or reduces the number or percentage of virus-infected cells.
  • a polypeptide or agent described herein has a circulating half-life in mice, rats, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, or at least 3 weeks.
  • the polypeptide or agent is an IgG (e.g., IgGl or IgG2) fusion protein that has a circulating half-life in mice, rats, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, at least about 2 weeks, or at least 3 weeks.
  • IgG e.g., IgGl or IgG2
  • Methods of increasing (or decreasing) the half- life of agents such as polypeptides and soluble receptors are known in the art.
  • known methods of increasing the circulating half-life of IgG fusion proteins include the introduction of mutations in the Fc region which increase the pH-dependent binding of the antibody to the neonatal Fc receptor (FcRn) at pH 6.0.
  • Known methods of increasing the circulating half-life of soluble receptors lacking a Fc region include such techniques as PEGylation.
  • the agent is a polypeptide.
  • the polypeptide can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide that binds CD40. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the structure or function of the protein. Thus, the invention also includes variations of the polypeptides which show substantial binding activity to CD40. In some embodiments, amino acid sequence variations of the polypeptides include deletions, insertions, inversions, repeats, and/or other types of substitutions.
  • polypeptides, analogs and variants thereof can be further modified to contain additional chemical moieties not normally part of the polypeptide.
  • the derivatized moieties can improve the solubility, the biological half-life, and/or absorption of the polypeptide.
  • the moieties can also reduce or eliminate undesirable side effects of the polypeptides and variants.
  • polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host.
  • a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest.
  • the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof.
  • a DNA sequence encoding a polypeptide of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer.
  • Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
  • a recombinant expression vector is used to amplify and express DNA encoding a polypeptide or agent described herein.
  • a recombinant expression vector can be a replicable DNA construct which has synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an agent operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes.
  • a transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences.
  • Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated.
  • DNA regions are
  • DNA for a signal peptide is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein when expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as Ml 3 and other filamentous single-stranded DNA phages.
  • Suitable host cells for expression of a polypeptide include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters.
  • Prokaryotes include gram-negative or gram-positive organisms, for example E. coli or Bacillus.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known by those skilled in the art.
  • polypeptides expression of recombinant proteins in mammalian cells can be preferred because such proteins are generally correctly folded, appropriately modified, and biologically functional.
  • suitable mammalian host cell lines include COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), CI 27 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), and HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.
  • Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • Baculovirus also offers a robust method for producing correctly folded and biologically functional proteins.
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • the present invention provides cells comprising the polypeptides and agents described herein.
  • the cells produce the polypeptides and agents described herein.
  • the cells produce a fusion protein.
  • the cells produce a soluble receptor/ligand.
  • the cells produce an antibody.
  • the cells produce a bispecific agent.
  • the cells produce a bispecific antibody.
  • the cells produce a homodimeric bispecific agent.
  • the cells produce a heterodimeric bispecific agent.
  • the proteins produced by a transformed host can be purified according to any suitable method.
  • Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
  • Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (MR), high performance liquid chromatography (HPLC), and x-ray crystallography.
  • recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
  • an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
  • a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • a commercially available protein concentration filter for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • the concentrate can be applied to a suitable purification matrix.
  • an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups
  • hydroxyapatite media can be employed, including but not limited to, ceramic
  • one or more reverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a polypeptide or agent. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.
  • recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. HPLC can be employed for final purification steps.
  • Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • a polypeptide or agent described herein is a polypeptide that does not comprise an immunoglobulin Fc region.
  • the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, protein G, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.
  • phage display technology may be used to produce and/or identify a binding polypeptide.
  • mammalian cell display technology may be used to produce and/or identify a binding polypeptide.
  • Heteroconjugate molecules are also within the scope of the present invention.
  • Heteroconjugate molecules are composed of two covalently joined polypeptides. Such molecules have, for example, been proposed to target immune cells to unwanted cells, such as tumor cells. It is also contemplated that the heteroconjugate molecules can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
  • a polypeptide or agent described herein can be used in any one of a number of conjugated (i.e. an immunoconjugate or radioconjugate) or non- conjugated forms.
  • the polypeptides or agents can be used in a non-conjugated form to harness the subject's natural defense mechanisms including CDC and ADCC to eliminate malignant or cancer cells.
  • an agent described herein is a small molecule.
  • small molecule generally refers to a low molecular weight organic compound which is by definition not a peptide/protein.
  • a polypeptide or agent described herein is conjugated to a cytotoxic agent.
  • the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents.
  • the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain ricin A chain
  • abrin A chain abrin A chain
  • modeccin A chain alpha-s
  • the cytotoxic agent is a radioisotope to produce a radioconjugate or a radioconjugated agent.
  • a radionuclides are available for the production of radioconjugated agents including, but not limited to, 90 Y, 125 I, 131 I, 123 I, U1 ln, 131 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re,
  • Conjugates of a polypeptide or agent and one or more small molecule toxins such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, can also be used.
  • Conjugates of a polypeptide or agent and cytotoxic agent are made using a variety of bifunctional protein- coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as
  • glutaraldehyde bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
  • diisocyanates such as toluene 2,6-diisocyanate
  • bis-active fluorine compounds such as l,5-difluoro-2,4-dinitrobenzene
  • the invention encompasses polynucleotides comprising polynucleotides that encode a polypeptide or agent described herein.
  • polynucleotides that encode a polypeptide encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences.
  • the polynucleotides of the invention can be in the form of RNA or in the form of DNA.
  • DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.
  • a polypeptide or agent described herein comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the CD40L stalk region, and wherein the stalk region comprises a mutation in the integrin-binding region.
  • each of the first, second, and third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, wherein the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • a polypeptide or agent described herein further comprises an Fc Region.
  • the Fc Region is a human Fc region.
  • the Fc region is deglycosylated.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the agent is a homodimer. In one embodiment, the agent is a heterodimer.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:78, and SEQ ID NO:79, wherein the integrin binding region comprises at least one mutation.
  • the integrin binding region comprises or consists of the RGD motif (residues 114-116 of SEQ ID NO: 1).
  • the at least one mutation is a substitution of a residue in the RGD motif.
  • the at least one mutation is a G to A or a D to E substitution in the RGD motif.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:57, and SEQ ID NO:58, wherein the integrin binding region comprises at least one mutation.
  • the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12).
  • the at least one mutation is a substitution in the KGD motif. In one embodiment, the at least one mutation is a G to A or D to E substitution in the KGD motif.
  • the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO: 80, and SEQ ID NO:81.
  • a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the CD40-binding agents described herein.
  • a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the CD40-binding agents described herein and a signal sequence.
  • a vector comprises the polynucleotide.
  • a cell comprises the polynucleotide.
  • a cell comprises the vector.
  • the cell is isolated.
  • a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77.
  • a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:80, and SEQ ID NO:81.
  • polynucleotide that comprises a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77. Also provided is a
  • polynucleotide that comprises a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:80, and SEQ ID NO:81.
  • the hybridization is under conditions of high stringency as known to those of skill in the art.
  • a polynucleotide comprises the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell).
  • the polypeptide having a leader sequence is a pre-protein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides can also encode for a pro-protein which is the mature protein plus additional 5' amino acid residues.
  • a mature protein having a pro-sequence is a pro-protein and is an inactive form of the protein. Once the pro-sequence is cleaved an active mature protein remains.
  • a polynucleotide comprises the coding sequence for the mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, for purification of the encoded polypeptide.
  • the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used.
  • the marker sequence is a FLAG-tag, a peptide of sequence DYKDDDDK (SEQ ID NO:56) which can be used in conjunction with other affinity tags.
  • the present invention further relates to variants of the hereinabove described polynucleotides encoding, for example, fragments, analogs, and/or derivatives.
  • the present invention provides a polynucleotide
  • polynucleotide having a nucleotide sequence at least about 80% identical, at least about 85%> identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding a polypeptide comprising a polypeptide or agent described herein.
  • a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence.
  • These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the polynucleotide variants can contain alterations in the coding regions, non- coding regions, or both.
  • a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide.
  • a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code).
  • Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.
  • a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
  • At least one polynucleotide variant is produced (without changing the amino acid sequence of the encoded polypeptide) to increase production of a heterodimeric molecule. In some embodiments, at least one polynucleotide variant is produced (without changing the amino acid sequence of the encoded polypeptide) to increase production of a bispecific agent, a bispecific antibody, or a heterodimeric agent.
  • the polynucleotides are isolated. In certain embodiments, the polynucleotides are substantially pure.
  • an expression vector comprises a polynucleotide molecule.
  • a host cell comprises an expression vector comprising the polynucleotide molecule.
  • a host cell comprises a
  • polypeptides or agents of the invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as immunotherapy for cancer.
  • a polypeptide or agent described herein is useful for activating, promoting, increasing, and/or enhancing an immune response, inhibiting tumor growth, reducing tumor volume, inducing tumor regression, increasing tumor cell apoptosis, and/or reducing the tumorigenicity of a tumor.
  • the polypeptides or agents of the invention are also useful for immunotherapy against pathogens, such as viruses.
  • a polypeptide or agent described herein is useful for inhibiting viral infection, reducing viral infection, increasing virally- infected cell apoptosis, and/or increasing killing of virus-infected cells.
  • the methods of use may be in vitro, ex vivo, or in vivo methods.
  • a polypeptide or agent described herein comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the CD40L stalk region, and wherein the stalk region comprises a mutation in the integrin-binding region.
  • each of the first, second, and third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, wherein the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • a polypeptide or agent described herein further comprises an Fc Region.
  • the Fc Region is a human Fc region.
  • the Fc region is deglycosylated.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the agent is a homodimer.
  • the agent is a heterodimer.
  • the present invention provides methods for activating an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the invention provides methods for promoting an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the invention provides methods for increasing an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the invention provides methods for enhancing an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing cell-mediated immunity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing antigen-presenting cell (APC) activity.
  • APC antigen-presenting cell
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing Thl-type responses. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD4+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD8+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing NK cell activity.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of Treg cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of MDSCs. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing the number of the percentage of memory T-cells.
  • the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term immune memory function. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term memory. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of substantial side effects and/or immune-based toxicities. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of cytokine release syndrome (CRS) or a cytokine storm. In some embodiments, the immune response is a result of antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor cell. In some embodiments, the antigenic stimulation is cancer. In some
  • the antigenic stimulation is a pathogen. In some embodiments, the antigenic stimulation is a virally-infected cell.
  • a polypeptide or agent described herein comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the CD40L stalk region, and wherein the stalk region comprises a mutation in the integrin-binding region.
  • each of the first, second, and third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, wherein the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • a polypeptide or agent described herein further comprises an Fc Region.
  • the Fc Region is a human Fc region.
  • the Fc region is deglycosylated.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the agent is a homodimer.
  • the agent is a heterodimer.
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent binds human CD40. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40.
  • a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain integrin binding-region mutant CD40L trimer.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region.
  • the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region, wherein the Fc region is deglycosylated.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • a method of activating or enhancing a persistent or long-term immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human CD40.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds CD40.
  • a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain integrin binding-region mutant CD40L trimer.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region.
  • the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region, wherein the Fc region is deglycosylated.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO: 72 or 81.
  • a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human CD40.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40.
  • a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain integrin binding-region mutant CD40L trimer.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region.
  • the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region, wherein the Fc region is deglycosylated.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human CD40.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40.
  • a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain integrin binding-region mutant CD40L trimer.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region.
  • the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region, wherein the Fc region is deglycosylated.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the present invention also provides methods for inhibiting growth of a tumor using a polypeptide or agent described herein.
  • the method of inhibiting growth of a tumor comprises contacting a cell mixture with a polypeptide or agent in vitro.
  • a cell mixture with a polypeptide or agent in vitro.
  • an immortalized cell line or a cancer cell line mixed with immune cells e.g., APCs, T-cells, cytolytic T-cells, or K cells
  • tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample, mixed with immune cells (e.g., APCs, T-cells, cytolytic T-cell, and/or NK cells), and cultured in medium to which is added a test agent.
  • the polypeptide or agent increases, promotes, and/or enhances the activity of the immune cells.
  • the polypeptide or agent inhibits tumor cell growth.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the method of inhibiting growth of a tumor comprises contacting the tumor or tumor cells with a polypeptide or agent described herein in vivo.
  • contacting a tumor or tumor cell with a polypeptide or agent is undertaken in an animal model.
  • a test agent may be administered to mice which have tumors.
  • the polypeptide or agent increases, promotes, and/or enhances the activity of immune cells in the mice.
  • the polypeptide or agent inhibits tumor growth.
  • the polypeptide or agent is administered at the same time or shortly after introduction of tumor cells into the animal to prevent tumor growth ("preventative model").
  • the polypeptide or agent is administered as a therapeutic after tumors have grown to a specified size ("therapeutic model").
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein.
  • the subject is a human.
  • the subject has a tumor or the subject had a tumor which was removed.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO: 72 or 81.
  • the invention provides a method of inhibiting growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
  • the tumor comprises cancer stem cells.
  • the frequency of cancer stem cells in the tumor is reduced by administration of the polypeptide or agent.
  • a method of reducing the frequency of cancer stem cells in a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent is provided.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the invention provides a method of reducing the tumorigenicity of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
  • the tumor comprises cancer stem cells.
  • the tumorigenicity of a tumor is reduced by reducing the frequency of cancer stem cells in the tumor.
  • the methods comprise using the polypeptides or agents described herein.
  • the frequency of cancer stem cells in the tumor is reduced by administration of a polypeptide or agent.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the administration of the polypeptide or agent described herein induces reduced systemic inflammation compared to a reference polypeptide or reference agent.
  • the reduced systemic inflammation is indicated by lower levels of blood neutrophils.
  • the blood neutrophil level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the blood neutrophil level is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60%> lower, from about 20% to about 50% lower, from about 20%) to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40%) to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70%) lower, from about 50% to about 60% lower, or from about 60% to about 70% lower).
  • the blood neutrophil level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
  • the administration of the polypeptide or agent described herein induces lower serum liver enzyme level compared to a reference polypeptide or reference agent.
  • the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP).
  • ALT alanine aminotransferase
  • ASP aspartate aminotransferase
  • ALP alkaline phosphatase
  • the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the liver enzyme level is from about 20% to about 70%) lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20%) to about 30% lower, from about 30% to about 70% lower, from about 30% to about 60%) lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60%> lower, from about 40%) to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60%) lower, or from about 60% to about 70% lower).
  • the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60%) lower, or about 70% lower.
  • the administration of the polypeptide or agent described herein induces lower pro-inflammatory cytokine release than a reference polypeptide or reference agent.
  • the proinflammatory cytokine is selected from the group consisting of: IL- ⁇ , IL-6, TNF-alpha, IP- 10, KC, and MUM a.
  • the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
  • the proinflammatory cytokine release is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50%) to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower).
  • the pro-inflammatory cytokine release is about 20%) lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70%) lower.
  • the reference polypeptide or reference agent comprise the same polypeptide as the polypeptide or agent except that the reference polypeptide or reference agent comprise a polypeptide comprising a CD40L stalk region that does not comprise a mutation in the integrin-binding region.
  • the tumor is a solid tumor.
  • the tumor is a tumor selected from the group consisting of: colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, neuroendocrine tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor.
  • the tumor is a colorectal tumor.
  • the tumor is an ovarian tumor.
  • the tumor is a lung tumor.
  • the tumor is a pancreatic tumor.
  • the tumor is a melanoma tumor.
  • the tumor is a bladder tumor.
  • the tumor expresses or overexpresses a tumor antigen targeted by the polypeptide or agent, such as a bispecific agent which comprises an antigen-binding site that specifically binds the tumor antigen.
  • the present invention further provides methods for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a
  • polypeptide or agent described herein binds CD40 and inhibits or reduces growth of the cancer.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the present invention provides for methods of treating cancer comprising
  • a subject e.g., a subject in need of treatment
  • a therapeutically effective amount of a polypeptide or agent described herein is administered to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of a polypeptide or agent described herein.
  • the subject is a human.
  • the subject has a cancerous tumor.
  • the subject has had a tumor removed.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, neuroendocrine cancer, bladder cancer, brain cancer, glioblastoma, and head and neck cancer.
  • the cancer is pancreatic cancer.
  • the cancer is ovarian cancer.
  • the cancer is colorectal cancer.
  • the cancer is breast cancer.
  • the cancer is prostate cancer.
  • the cancer is lung cancer. In certain
  • the cancer is melanoma. In some embodiments, the cancer is bladder cancer.
  • the cancer is a hematologic cancer.
  • the cancer is selected from the group consisting of: acute myelogenous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T-cell lymphoma (CTCL).
  • AML acute myelogenous leukemia
  • T-ALL T-cell acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • non-Hodgkin lymphoma diffuse large B-cell lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lympho
  • the invention also provides a method of activating or enhancing CD40 signaling in a cell comprises contacting the cell with an effective amount of a CD40-binding polypeptide or agent described herein.
  • the cell is a T-cell.
  • the cell is a cytolytic cell.
  • the cell is a CTL.
  • the cell is a NK cell.
  • the method is an in vivo method wherein the step of contacting the cell with the polypeptide or agent comprises administering a therapeutically effective amount of the polypeptide or agent to the subject.
  • the method is an in vitro or ex vivo method.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO: 72 or 81.
  • the present invention provides methods of determining the level of expression of
  • CD40 and/or CD40L are CD40 and/or CD40L.
  • the level of expression of CD40 is determined.
  • the level of expression of CD40L is determined.
  • Methods for determining the level of nucleic acid expression in a cell, tumor, or cancer are known by those of skill in the art. These methods include, but are not limited to, PCR- based assays, microarray analyses, and nucleotide sequencing (e.g., NextGen
  • Methods for determining the level of protein expression in a cell, tumor, or cancer include, but are not limited to, Western blot analyses, protein arrays, ELISAs, immunohistochemistry (IHC), and FACS.
  • expression of a nucleic acid or protein can use a variety of samples. In some
  • the sample is taken from a subject having a tumor or cancer.
  • the sample is a fresh tumor/cancer sample.
  • the sample is a frozen tumor/cancer sample.
  • the sample is a formalin- fixed paraffin-embedded sample.
  • the sample is a blood sample.
  • the sample is a plasma sample.
  • the sample is processed to a cell lysate.
  • the sample is processed to DNA or RNA.
  • the pharmaceutical compositions find use in immuno-oncology. In some embodiments, the compositions find use in inhibiting tumor growth. In some embodiments, the pharmaceutical compositions find use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the
  • compositions find use in treating cancer.
  • the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient).
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO: 72 or 81.
  • Formulations are prepared for storage and use by combining a purified agent of the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient).
  • a pharmaceutically acceptable vehicle e.g., a carrier or excipient.
  • pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.
  • the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01% polysorbate 20. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01%) polysorbate 20 at pH 5.5. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01%> polysorbate 20 at pH 6.0.
  • the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01%> polysorbate 20 at pH 6.5. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, lOOmM NaCl, 150mM sucrose, and 0.01% polysorbate 20 at pH 6.0. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of lOmM potassium phosphate and 0.04% polysorbate 20 at pH 7.5.
  • compositions or pharmaceutical compositions comprising a polypeptide or agent described herein and further comprising about 20mM histidine, about 40mM NaCl, about 5% sucrose, and about 0.01% polysorbate 20.
  • pH of the composition is about pH 5.5, about pH 6.0, or about pH 6.5.
  • a polypeptide or agent described herein is lyophilized
  • a formulation comprising a polypeptide or agent described herein is lyophilized.
  • Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, hist
  • compositions of the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops,
  • suppositories sprays, liquids and powders
  • pulmonary by inhalation or insufflation of powders or aerosols including by nebulizer, intratracheal, and intranasal
  • oral or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or
  • the therapeutic formulation can be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
  • the solid preformulation composition is then subdivided into unit dosage forms of a type described above.
  • the tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner composition covered by an outer component.
  • the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release.
  • enteric layers or coatings such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • polypeptides or agents described herein can also be entrapped in
  • microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22 nd Edition, 2012, Pharmaceutical Press, London.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • pharmaceutical formulations include a polypeptide or agent of the present invention complexed with liposomes.
  • Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter.
  • sustained-release preparations comprising the
  • sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing a polypeptide or agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules).
  • sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
  • polyesters such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)
  • polylactides copolymers of L-glutamic acid and 7 ethyl-L-glutamate
  • non-degradable ethylene-vinyl acetate non-degradable ethylene-vinyl a
  • the method or treatment further comprises administering at least one additional immune response stimulating agent.
  • the additional immune response stimulating agent includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), a checkpoint inhibitor, an antibody that blocks immunosuppressive functions (e.g., an anti- CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86).
  • a colony stimulating factor e.g., granulocyte-macrophage colony stimulating factor (GM
  • an additional immune response stimulating agent can be administered prior to, concurrently with, and/or subsequently to, administration of the polypeptide or agent.
  • Pharmaceutical compositions comprising a polypeptide or agent and the immune response stimulating agent(s) are also provided.
  • the immune response stimulating agent comprises 1, 2, 3, or more immune response stimulating agents.
  • the method or treatment further comprises administering at least one additional therapeutic agent.
  • An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the polypeptide or agent.
  • Pharmaceutical compositions comprising a polypeptide or agent and the additional therapeutic agent(s) are also provided.
  • the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.
  • Combination therapy with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the polypeptide or agent(s).
  • Combination therapy may decrease the likelihood that resistant cancer cells will develop.
  • combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells.
  • the combination therapy results in an increase in the therapeutic index of the polypeptide or agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the polypeptide or agent. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s).
  • Useful classes of therapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri- nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, anti-metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.
  • the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor.
  • Therapeutic agents that may be administered in combination with the polypeptides or agents described herein include chemotherapeutic agents.
  • the method or treatment involves the administration of a polypeptide or agent of the present invention in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents.
  • Treatment with a polypeptide or agent can occur prior to, concurrently with, or subsequent to administration of chemotherapies.
  • Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4 th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.
  • Chemotherapeutic agents useful in the present invention include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide,
  • triethylenethiophosphoramide and trimethylolomelamime nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
  • streptonigrin streptozocin, tubercidin, ubenimex, zinostatin, zorubicin
  • anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as
  • folic acid replenishers such as folinic acid
  • aceglatone aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium;
  • arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide;
  • mitomycin C mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;
  • DMFO difluoromethylomithine
  • XELODA capecitabine
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide, nilutamide, b
  • the additional therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin.
  • the chemotherapeutic agent is a topoisomerase inhibitor.
  • Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II).
  • Topoisomerase inhibitors include, but are not limited to, doxorubicin hydrochloride (HCl), daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the additional therapeutic agent is irinotecan.
  • the chemotherapeutic agent is an anti-metabolite.
  • An anti-metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division.
  • Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as
  • the additional therapeutic agent is gemcitabine.
  • the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin.
  • the agent is a taxane.
  • the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel.
  • the agent is paclitaxel (TAXOL), docetaxel
  • the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof.
  • the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plkl .
  • the additional therapeutic agent is paclitaxel. In certain embodiments, the additional therapeutic agent is nab-paclitaxel.
  • an additional therapeutic agent comprises an agent such as a small molecule.
  • treatment can involve the combined administration of a polypeptide or agent of the present invention with a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.
  • a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.
  • a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.
  • a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF.
  • a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (Erb
  • polypeptide or agent of the present invention is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTEVIA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib ( EXAVAR), and pazopanib (GW786034B).
  • a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTEVIA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib ( EXAVAR), and pazopanib (GW786034B).
  • a protein kinase inhibitor selected from the group consisting of: ge
  • the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Hippo pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the mTOR/AKR pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the RSPO/LGR pathway.
  • an additional therapeutic agent comprises a biological molecule, such as an antibody.
  • treatment can involve the combined administration of a polypeptide or agent of the present invention with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF.
  • the additional therapeutic agent is an antibody specific for a cancer stem cell marker.
  • the additional therapeutic agent is an antibody that binds a component of the Notch pathway.
  • the additional therapeutic agent is an antibody that binds a component of the Wnt pathway.
  • the additional therapeutic agent is an antibody that inhibits a cancer stem cell pathway.
  • the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits ⁇ -catenin signaling. In certain embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody).
  • angiogenesis inhibitor e.g., an anti-VEGF or VEGF receptor antibody.
  • the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).
  • the additional therapeutic agent is an antibody that modulates the immune response.
  • the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti- CTLA-4 antibody, or an anti-TIGIT antibody.
  • treatment with a polypeptide or agent described herein can include combination treatment with other biologic molecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by a treating physician.
  • the additional therapeutic agent is an immune response stimulating agent.
  • the polypeptide or agent can be combined with a growth factor selected from the group consisting of:
  • AM adrenomedullin
  • Ang angiopoietin
  • BMPs BDNF
  • EGF erythropoietin
  • FGF FGF
  • GDNF G-CSF
  • GM-CSF GDF9
  • HGF HGF
  • HDGF HDGF
  • IGF migration-stimulating factor
  • myostatin (GDF-8) NGF, neurotrophins, PDGF, thrombopoietin, TGF-a, TGF- ⁇ , TNF-a, VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.
  • the additional therapeutic agent is an immune response stimulating agent.
  • the immune response stimulating agent is selected from the group consisting of granulocyte- macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-OX40 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-Ll antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.
  • GM-CSF granulocyte- macrophage colony stimulating factor
  • M-CSF macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • IL-3 interleukin 12
  • IL-12 interleukin
  • an immune response in some embodiments of the methods described herein, an immune response
  • a modulator of PD-1 activity is selected from the group consisting of: a modulator of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4- IBB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDOl activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an immunostimul
  • an immune response stimulating agent is selected from the group consisting of: a PD-1 antagonist, a PD-Ll antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, and/or an IDOl antagonist.
  • the PD-1 antagonist is an antibody that specifically binds PD-1.
  • the antibody that binds PD- 1 is KEYTRUDA (MK-3475), pidilizumab (CT-011), nivolumab (OPDIVO, BMS- 936558, MDX-1106), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI-A1110.
  • the antibody that binds PD-1 is described in PCT Publication WO 2014/179664, for example, an antibody identified as APE2058,
  • the PD-1 antagonist is a fusion protein that includes PD-L2, for example, AMP-224.
  • the PD-1 antagonist is a peptide inhibitor, for example, AU P-12.
  • the PD-Ll antagonist is an antibody that specifically binds
  • the antibody that binds PD-Ll is atezolizumab (RG7446, MPDL3280A), MEDI4736, BMS-936559 (MDX-1105), avelumab (MSB0010718C), KD033, the antibody portion of KD033, or STI-A1014.
  • the antibody that binds PD-Ll is described in PCT Publication WO 2014/055897, for example, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52, or Ab-55, or an antibody that contains the CDR regions of any of these antibodies.
  • the CTLA-4 antagonist is an antibody that specifically binds CTLA-4.
  • the antibody that binds CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206).
  • the CTLA-4 antagonist a CTLA-4 fusion protein, for example, KAHR-102.
  • the LAG3 antagonist is an antibody that specifically binds
  • the antibody that binds LAG3 is IMP701, IMP731, BMS- 986016, LAG525, and GSK2831781.
  • the LAG3 antagonist includes a soluble LAG3 receptor, for example, IMP321.
  • the KIR antagonist is an antibody that specifically binds
  • an immune response stimulating agent is selected from the group consisting of: a CD28 agonist, a 4- IBB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist.
  • the OX40 agonist includes OX40 ligand, or an OX40- binding portion thereof.
  • the OX40 agonist may be MEDI6383.
  • the OX40 agonist is an antibody that specifically binds OX40.
  • the antibody that binds OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888).
  • the OX40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing OX40 ligand.
  • a vector e.g., an expression vector or virus, such as an adenovirus
  • the OX40-expressing vector is Delta-24-RGDOX or DNX2401.
  • the 4-1BB (CD137) agonist is a binding molecule, such as an anticalin.
  • the anticalin is PRS-343.
  • the 4- IBB agonist is an antibody that specifically binds 4- IBB.
  • antibody that binds 4- IBB is PF-2566 (PF-05082566) or urelumab (BMS-663513).
  • the CD27 agonist is an antibody that specifically binds
  • CD27 In some embodiments, the antibody that binds CD27 is varlilumab (CDX-1127).
  • the GITR agonist comprises GITR ligand or a GITR- binding portion thereof.
  • the GITR agonist is an antibody that specifically binds GITR.
  • the antibody that binds GITR is TRX518, MK-4166, or INBRX-110.
  • immune response stimulating agents include, but are not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family.
  • immune response stimulating agents include immunostimulatory oligonucleotides, such as CpG dinucleotides.
  • an immune response stimulating agent includes, but is not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti- CTLA-4 antibodies, anti-CD28 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-4-lBB antibodies, anti-OX40 antibodies, anti -KIR antibodies, anti-Tim-3 antibodies, anti-LAG3 antibodies, anti-CD27 antibodies, anti-CD40 antibodies, anti-GITR
  • a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a CD40-binding polypeptide or agent described herein in combination with a checkpoint inhibitor.
  • the checkpoint inhibitor is an anti-PD-1 antibody.
  • the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is melanoma.
  • the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is lung cancer.
  • the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is bladder cancer.
  • the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is a hematologic cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is melanoma. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is lung cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is bladder cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is breast cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is a hematologic cancer.
  • the treatment involves the administration of a polypeptide or agent of the present invention in combination with radiation therapy.
  • Treatment with a polypeptide or agent can occur prior to, concurrently with, or subsequent to administration of radiation therapy. Dosing schedules for such radiation therapy can be determined by the skilled medical practitioner.
  • the treatment involves the administration of a polypeptide or agent of the present invention in combination with anti-viral therapy.
  • Treatment with a polypeptide or agent can occur prior to, concurrently with, or subsequent to administration of antiviral therapy.
  • the anti-viral drug used in combination therapy will depend upon the virus the subject is infected with.
  • Combined administration can include co-administration, either in a single
  • the combination of a polypeptide or agent described herein and at least one additional therapeutic agent may be administered in any order or concurrently.
  • the polypeptide or agent will be administered to patients that have previously undergone treatment with a second therapeutic agent.
  • the polypeptide or agent and a second therapeutic agent will be administered substantially simultaneously or concurrently.
  • a subject may be given a polypeptide or agent while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy).
  • a polypeptide or agent will be administered within 1 year of the treatment with a second therapeutic agent.
  • a polypeptide or agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, a polypeptide or agent will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, a polypeptide or agent will be administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).
  • the appropriate dosage of a polypeptide or agent of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the polypeptide or agent is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on, all at the discretion of the treating physician.
  • the polypeptide or agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size).
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual agent.
  • dosage is from 0.01 ⁇ g to lOOmg/kg of body weight, from O. ⁇ g to lOOmg/kg of body weight, from ⁇ g to lOOmg/kg of body weight, from lmg to lOOmg/kg of body weight, lmg to 80mg/kg of body weight from lOmg to lOOmg/kg of body weight, from lOmg to 75mg/kg of body weight, or from lOmg to 50mg/kg of body weight.
  • dosage is from 0.01 ⁇ g to lOOmg/kg of body weight, from O. ⁇ g to lOOmg/kg of body weight, from ⁇ g to lOOmg/kg of body weight, from lmg to lOOmg/kg of body weight, from lmg to 80mg/kg of body weight from lOmg to lOOmg/kg of body weight, from lOmg to 75mg/kg of body
  • the dosage of the polypeptide or agent is from about 0. lmg to about 20mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about O. lmg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 0.25mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 0.5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about lmg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 1.5mg/kg of body weight. In some
  • the dosage of the polypeptide or agent is about 2mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 2.5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 7.5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about lOmg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 12.5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 15mg/kg of body weight. In certain embodiments, the dosage can be given once or more daily, weekly, monthly, or yearly. In certain embodiments, the polypeptide or agent is given once every week, once every two weeks, once every three weeks, or once every four weeks.
  • a polypeptide or agent may be administered at an initial higher "loading" dose, followed by one or more lower doses.
  • the frequency of administration may also change.
  • a dosing regimen may comprise administering an initial dose, followed by additional doses (or
  • a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose.
  • a dosing regimen may comprise administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week.
  • a dosing regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.
  • any therapeutic agent may lead to side effects and/or toxicities.
  • the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose.
  • drug therapy must be discontinued, and other agents may be tried.
  • many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent.
  • the dosing schedule may be limited to a specific number of administrations or "cycles".
  • the polypeptide or agent is
  • the polypeptide or agent is administered for 3, 4, 5, 6, 7, 8, or more cycles.
  • the polypeptide or agent is administered every 2 weeks for 6 cycles, the polypeptide or agent is administered every 3 weeks for 6 cycles, the polypeptide or agent is administered every 2 weeks for 4 cycles, the polypeptide or agent is administered every 3 weeks for 4 cycles, etc.
  • Dosing schedules can be decided upon and subsequently modified by those skilled in the art.
  • the present invention provides methods of administering to a subject the polypeptides or agents described herein comprising using an intermittent dosing strategy for administering one or more agents, which may reduce side effects and/or toxicities associated with administration of a polypeptide or agent, chemotherapeutic agent, etc.
  • a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of a polypeptide or agent in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy.
  • the intermittent dosing strategy comprises administering an initial dose of a polypeptide or agent to the subject, and administering subsequent doses of the polypeptide or agent about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a polypeptide or agent to the subject, and administering subsequent doses of the polypeptide or agent about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a polypeptide or agent to the subject, and administering subsequent doses of the polypeptide or agent about once every 4 weeks. In some embodiments, the polypeptide or agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.
  • the present invention provides screening methods to identify agents that modulate the immune response.
  • the present invention provides methods for screening candidate agents, including but not limited to, proteins, antibodies, peptides, peptidomimetics, small molecules, compounds, or other drugs, which modulate the immune response.
  • a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent has an effect on immune response cells. In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent is capable of increasing the activity of immune cells. In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent is capable of increasing the activity of cytolytic cells, such as CTLs and/or K cells. In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent is capable of inhibiting the activity of suppressor cells, such as Tregs and/or MDSCs.
  • kits that comprise the polypeptides or agents
  • kits comprises at least one purified agent in one or more containers.
  • the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.
  • the disclosed agents of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.
  • a polypeptide or agent described herein comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the CD40L stalk region, and wherein the stalk region comprises a mutation in the integrin-binding region.
  • each of the first, second, and third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, wherein the stalk region comprises a mutation in the integrin-binding region.
  • a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80.
  • a polypeptide or agent described herein further comprises an Fc Region.
  • the Fc Region is a human Fc region.
  • the Fc region is deglycosylated.
  • the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
  • the agent is a homodimer. In one embodiment, the agent is a heterodimer.
  • kits that comprise a polypeptide or agent as well as at least one additional therapeutic agent.
  • the second (or more) therapeutic agent is a chemotherapeutic agent.
  • the second (or more) therapeutic agent is an angiogenesis inhibitor.
  • Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the present disclosure.
  • CD40L proteins organize into a homotrimer at the surface of cells and interact and/or engage with three CD40 molecules.
  • a soluble single chain mouse CD40L trimer-construct was generated comprising three copies of a fragment of the stalk region and TNF family domain of mCD40L (amino acids 112-260 of SEQ ID NO: l; also see SEQ ID NO:6 and SEQ ID NO:7).
  • the N-terminal end of this construct was attached to the C-terminal end of a mouse IgG2a Fc region (340F1; SEQ ID NO:8 and SEQ ID NO:9).
  • a deglycosylated variant of 340F1 antibody was generated that comprised an alanine residue at position 74 of SEQ ID NO: 11 and is referred to herein as 340F2 (SEQ ID NO: 10 and SEQ ID NO: 11).
  • the alanine residue replaces the asparagine residue in the native Fc region and results in reduced effector function of the molecule.
  • 340F4 was generated that comprised three copies of a fragment of the stalk region and TNF family domain of mCD40L (amino acids 112-260 of SEQ ID NO: l; also see SEQ ID NO: 6). The N-terminal end of this construct was attached to the C-terminal end of a mouse IgG2a Fc region (340F4; SEQ ID NO:78 and SEQ ID NO:79).
  • 340F7 a single chain integrin binding mutant mouse CD40L trimer was generated comprising three copies of a fragment of the stalk region and TNF family domain of mCD40L (amino acids 112-260 of SEQ ID NO: 1; also see SEQ ID NO:6).
  • the C- terminal end of this construct was attached to the N-terminal end of a deglycosylated variant of mouse IgG2a Fc region (340F7; SEQ ID NO:76 and SEQ ID NO:77).
  • An additional construct was generated that comprised three copies of a fragment of the stalk region and TNF family domain of human CD40L (amino acids 113-261 of SEQ ID NO: 12; also see SEQ ID NO:22 and SEQ ID NO:28).
  • the C-terminal end of the hCD40L trimer construct was attached to the N-terminal end of a human IgGl Fc region (SEQ ID NO:29 with signal sequence and SEQ ID NO:30 without signal sequence).
  • a deglycosylated variant of the hCD40L trimer-Fc IgGl construct was generated (SEQ ID NO: 57 with signal sequence and SEQ ID NO: 58 without signal sequence) that comprises an alanine residue at position 524 of SEQ ID NO: 58.
  • the alanine residue replaces the asparagine residue in the native Fc region and results in deglycosylation of the Fc region and reduced effector function of the molecule.
  • the C-terminal end of the hCD40L trimer construct was attached to the N-terminal end of a human IgG2 Fc region (SEQ ID NO:31 with signal sequence and SEQ ID NO:32 without signal sequence).
  • a deglycosylated variant of the hCD40L trimer-Fc IgG2 construct was generated that comprises an alanine residue at position 526 of SEQ ID NO:34.
  • the alanine residue replaces the asparagine residue in the native Fc region and results in deglycosylation of the Fc region and reduced effector function of the molecule.
  • the murine colon tumor line CT26 The murine colon tumor line CT26.
  • WT was implanted subcutaneously (30,000 cells/mouse) in Balb/c mice and allowed to grow to an average size of approximately 159mm 3 .
  • Figure 2E is a survival curve at Day 60 of each group and shows that treatment with 340F2 had a significantly higher survival rate than the anti-mCD40 antibody.
  • the murine colon tumor line MC38 was implanted subcutaneously (400,000
  • mice C57BL/6N mice and allowed to grow to an average size of approximately 108mm 3 .
  • ELISpot is a highly sensitive immunoassay for the detection of cytokine-secreting cells.
  • an ELISpot assay employs a capture antibody specific for a desired cytokine, pre-coated onto the wells of a microplate. Stimulated cells are dispersed into the wells and the immobilized antibody in the immediate vicinity of any cytokine-secreting cell binds the secreted cytokine. Standard wash steps and incubation with appropriate detection reagents follow. For example, a combination of a biotinylated detection antibody followed by streptavidin conjugated to alkaline-phosphatase and a colored substrate solution is commonly used.
  • a colored precipitate forms at the sites of cytokine localization and appears as a spot, with each individual spot representing an individual cytokine-secreting cell.
  • the spots may be counted with an automated reader system or manually using a microscope.
  • an image of each well is captured using an automated reader system, and total spots, spot area, or total optical density (TOD) is used for quantification.
  • TOD total optical density
  • IL-2 secreting cells were detected using a mouse IL-2 ELISpot kit (MabTech).
  • Splenocytes (5 x 10 5 /well) from each mouse in each treatment group were dispensed into a 96-well plate coated with an antibody specific for mouse IL-2 and incubated without peptide for 48 hours. Cells secreting IL-2 were detected following the manufacturer's instructions. Images were captured using a Bioreader 6000-F-z instrument (BioSys) and spot number, spot area, and total optical density were determined. Results are shown as total optical density (TOD) and data are expressed as mean ⁇ S.E.M.
  • mice treated with mCD40L trimer-Fc 340F2 significantly increased in mice treated with an anti-mCD40 antibody or a control antibody.
  • IFN-gamma secreting cells were detected using a mouse IFN-gamma ELISpot kit
  • IFN-gamma secreting cells isolated from the spleen were significantly increased in mice treated with mCD40L trimer-Fc 340F2 as compared to mice treated with an anti-mCD40 antibody or a control antibody.
  • Tumor cells including tumor-associated immune cells (5 x 10 5 /well) from each mouse in each treatment group were dispensed into a 96-well plate coated with an antibody specific for mouse IFN-gamma. The cells were incubated for 48 hours. Cells secreting IFN-gamma were detected following the manufacturer's instructions. Images were captured using a Bioreader 6000-F-z instrument (BioSys) and spot number, spot area, and total optical density were determined. Results are shown as TOD and data are expressed as mean ⁇ S.E.M.
  • IFN-gamma secreting cells isolated from tumors were significantly increased in mice treated with mCD40L trimer-Fc 340F2 as compared to mice treated with an anti-mCD40 antibody or a control antibody.
  • IL-2 is a multifunctional protein generally produced by T-cells and is a potent growth factor for T-cells, B-cells, and NK cells. IL-2 also stimulates IFN-gamma production by T-cells. IFN-gamma is generally produced by NK cells, CD4+ T-cells, CD8+ T-cells, APCs, and B-cells. IFN-gamma is a known regulator of anti-tumor activity. Cells from mice treated with a CD40L trimer-Fc molecule produced more IL-2 and IFN-gamma than cells from mice treated with an anti-CD40 antibody or a control. These results support the idea that a CD40L trimer-Fc may be more effective therapeutic agent than an anti-CD40 antibody.
  • NK cells are a type of cytotoxic lymphocyte critical to the innate immune system. NK cell activity in mice treated with mCD40L trimer-Fc 340F2 was assessed by measuring the cytotoxic activity of cells on tumor targets. For these NK cytotoxicity assays, the mouse lymphoblast cell line YAC-1 was used as target cells. Cells were harvested from the spleens of the CT26.WT tumor-bearing mice described above in Example 2.
  • YAC-1 target cells were labeled with 10 ⁇ calcein AM (Life Technologies) for 1 hour at 37°C and then combined with the splenocytes at an effector :target (E:T) ratio of 25: 1.
  • % lysis 100 x (ER-SR)/(MR-SR), where ER, SR, and MR represent experimental, spontaneous, and maximum calcein release, respectively.
  • Spontaneous release is the fluorescence emitted by target cells incubated in media alone (i.e., in the absence of effector cells), while maximum release is determined by lysing target cells with an equal volume of 10% SDS.
  • YAC-1 target cells were labeled with 10 ⁇ calcein AM (Life Technologies) for 1 hour at 37°C and then combined with the splenocytes at an effector :target (E:T) ratio of 25 : 1. Following a 4 hour incubation at 37°C, cell-free supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm.
  • FBS fetal bovine serum
  • E:T effector :target
  • % lysis 100 x (ER-SR)/(MR-SR), where ER, SR, and MR represent experimental, spontaneous, and maximum calcein release, respectively.
  • Spontaneous release is the fluorescence emitted by target cells incubated in media alone (i.e., in the absence of effector cells), while maximum release is determined by lysing target cells with an equal volume of 10% SDS.
  • NK cells from MC38 tumor-bearing mice treated with mCD40L trimer-Fc 340F2 demonstrated an increased ability to kill YAC-1 target cells as compared to cells from mice treated with control.
  • NK cells from MC38 tumor-bearing mice treated with an anti-mCD40 antibody also had an increased ability to kill target cells but to a smaller extent than 340F2.
  • Antigen-specific cytotoxic T-cells are critical to the adaptive immune system.
  • CD8+ cytotoxic cell activity in mice treated with mCD40L trimer-Fc 340F2 was assessed by measuring the cytotoxic activity of CD8+ T-cells on tumor targets.
  • Cells were harvested from the spleens of the MC38 tumor-bearing mice described above in Example 4.
  • a CD8 + T-cell specific MHC class I tumor peptide sequence is not known for the MC38 cell line, therefore the MC38 cells were used as stimulators.
  • MC38 cells were treated with 25 ⁇ g/ml mitomycin C (Sigma-Aldrich) for 30 minutes at 37°C, washed, and resuspended at 10 7 cells/ml in RPMI-1640 media containing 10% FCS, 2mM L- glutamine, and antibiotics.
  • Splenocytes were co-cultured with the mitomycin-treated MC38 cells in the presence of IL-2 (2ng/ml), incubated for 5 days at 37°C, harvested, counted, and used in cytotoxicity assays as described above.
  • Calcein AM-labeled MC38 cells were used as targets at an effectontarget ratio of 25 : 1. Calcein release was determined after 4 hours and specific lysis was calculated as described above.
  • CD8 + cytotoxic cells from MC38 tumor-bearing mice demonstrated an increased ability to kill MC38 target cells when the mice had been treated with mCD40L trimer-Fc 340F2 as compared to cells from mice treated with anti- CD40 antibody or control.
  • CD40L trimer-Fc molecule can increase both NK cell and antigen-specific cytotoxic T-cell activity and therefore enhance anti-tumor immune responses, consistent with increased IL-2 and IFN-gamma production.
  • This increased cytotoxic activity may be due to a direct or indirect effect of CD40L-Fc, i.e., increasing APC activity, B-cell maturation, and/or inhibiting the effect of suppressor cells.
  • the murine colon tumor line CT26 The murine colon tumor line CT26.
  • WT was implanted subcutaneously (30,000 cells/mouse) in Balb/c mice and allowed to grow to an average size of 113 mm 3 .
  • mice All groups of mice were euthanized at day 19, except mice treated with mCD40L trimer-Fc 340F4, which were euthanized on day 15 after the second dose due to low body condition score ("BCS").
  • BCS low body condition score
  • the low BCS of 340F4 treated mice was likely due to the high endotoxin levels (6.4 EU/ml) in the 340F4 preparation. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.
  • mice treated with lOmg/kg of single chain mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F7, anti-mCD40 antibody, or a control antibody from Example 7 were euthanized at day 19 and serum was isolated from each animal. From the serum, alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP) levels were determined for each mouse. As shown in Figures 7A-7C, mice treated with mCD40L trimer-Fc 340F7 had lower levels of ALT and ALP than mice treated with mCD40L trimer-Fc 340F2. This result is indicative of less liver damage in mice treated with the integrin binding mutant mCD40L trimer-Fc 340F7 than mice treated with mCD40L trimer-Fc 340F2.
  • ALT alanine aminotransferase
  • ASP aspartate aminotransferase
  • mice treated with lOmg/kg of single chain mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F7, anti-mCD40 antibody, or a control antibody from Example 7 were euthanized at day 19 and a complete blood count was performed.
  • mice treated with mCD40L trimer-Fc 340F7 had lower levels of neutrophils than mice treated with mCD40L trimer-Fc 340F2.
  • Figure 8D shows that mice treated with mCD40L trimer-Fc 340F7 had higher levels of lymphocytes than mice treated with mCD40L trimer-Fc 340F2.
  • Millipore panel by following Luminex assay protocol with adaption of the Drop Array system (Curiox Biosystem). Briefly, the plate was blocked with lOuL 1%BSA/PBS for 30 minutes at room temperature. STD and control were prepared as Luminex assay, 5uL (1/5) bead mix was added to each DropArray wall-less plate, 5uL buffer/well was added to the plasma sample well, and matrix buffer was added to the STDs and control wells. 5uL STD or control samples per well was added with tips changed after each addition. The plate was carefully inserted into shaker and was shaken for 10 seconds @1000RPM. The assay plate was placed on top of the Magnetic stand into a Humidified box and shaken overnight at 4°C.
  • the Wash Plate was washed 3X with DropArray LT washing station MX96 (Curiox Biosystems), the detection ab was added, 5uL/well and incubated on shaker for 60 minutes. 5uL/well of the Streptavidin /PE substrate was added, incubated for 30 minutes with shaking. Plates were washed 3X with DropArray LT washing station MX96, 75uL Sheath fluid was added and the plate was read with the adaptor. Luminex data was analyzed using the EMD Millipore's Milliplex Analyst software. Statistical analysis was performed using the One-Way ANOVA Bonferroni multiple comparison test.
  • mice treated with lOmg/kg of single chain mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F4, mCD40L trimer-Fc 340F7, anti-mCD40 antibody, or a control antibody from Example 7 were euthanized and serum levels of G-CSF, IFN-gamma, IL- la, IL- ⁇ , IL-2, IL-6, IL-12p40, IFN-gamma-induced protein 10 ("IP-10"), chemokine (C-X-C motif) ligand 1 ("KC"), monocyte chemoattractant protein 1 ("MCP1 "), macrophage inflammatory protein 1-a ("MIP-la”), ⁇ , MIP2, Regulated on G-CSF, IFN-gamma, IL- la, IL- ⁇ , IL-2, IL-6, IL-12p40, IFN-gamma-induced protein 10 ("IP-10"), chemokine (C-X-C
  • mice treated with mCD40L trimer-Fc 340F7 had lower levels of pro-inflammatory cytokines including IL- ⁇ , IL-6, TNF-alpha, IP-10, KC, MIP-la than mice treated with mCD40L trimer-Fc 340F2.
  • pro-inflammatory cytokines including IL- ⁇ , IL-6, TNF-alpha, IP-10, KC, MIP-la
  • mice treated with mCD40L trimer-Fc 340F2 had lower cytokine production and reduced inflammation than mCD40L trimer-Fc 340F2-treated mice.
  • mice treated with lOmg/kg of single chain mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F4, mCD40L trimer-Fc 340F7, anti-mCD40 antibody, or a control antibody from Example 7 were euthanized and livers were isolated. Liver sections were stained with hematoxylin and eosin following standard protocols.
  • mice treated with mCD40L trimer-Fc 340F7 had large thrombi and multifocally extensive coagulative necrosis.
  • Mice treated with mCD40L trimer-Fc 340F2 had multifocal infiltrates, with and without associated hepatocyte necrosis, and occasional thrombi.
  • Mice treated with anti-mCD40 antibody had multifocally extensive perivascular infiltrates.
  • mice treated with mCD40L trimer-Fc 340F4 had rare infiltrates.
  • liver histology of 340F4 treated mice which were euthanized earlier than the other groups due to low BCS, is consistent with these animals getting sick from the high endotoxin levels in the 340F4 preparation.
  • the liver histology indicates that 340F7 is capable of inducing liver damage at the very robust dose tested.
  • mice treated with single chain integrin binding mutant mCD40L trimer-Fc 340F7 had a lower cytokine response, reduced systemic inflammation, and decreased serum liver enzymes (indicating less liver damage) compared to mice treated with mCD40L trimer-Fc 340F2. As indicated by the liver histology, 340F7 is still capable of inducing liver damage at the very robust dose tested. These results in total indicate that with mCD40L trimer-Fc 340F7 has an improved safety profile, which could enable an improved therapeutic index.
  • a soluble single chain integrin binding mutant human CD40L trimer-construct will be generated comprising three copies of a fragment of the stalk region and TNF family domain of human CD40L (amino acids 113-261 of SEQ ID NO: 12; also see SEQ ID NO:69 and SEQ ID NO:70).
  • the N-terminal end of this construct will be attached to the C-terminal end of a deglycosylated variant of a human IgGl Fc region (SEQ ID NO:71 with signal sequence and SEQ ID NO: 72 without signal sequence).
  • the deglycosylated variant of the human IgGl Fc region (SEQ ID NO:82) comprises an alanine residue at position 526 of SEQ ID NO:34.
  • the alanine residue replaces the asparagine residue in the native Fc region and results in deglycosylation of the Fc region and reduced effector function of the molecule.
  • a humanized mouse model will be used to study the efficacy of treatment with a single chain integrin binding mutant human CD40L trimer-Fc protein on a human tumor.
  • the humanized mice can be obtained from Jackson Laboratories. These mice can be created by injecting human hematopoietic stem cells (CD34+ cells) into irradiated NSG mice. After 15 weeks, the presence of mature human lymphocytes can be confirmed by flow cytometry. Each mouse will be injected subcutaneously with patient-derived human tumor cells, for example, with melanoma tumor cells (OMP-M9, 75,000 cells/mouse). Tumors will be allowed to grow, for example for 16 days until they reach an average volume of approximately 60mm 3 .

Abstract

Polypeptides and agents that bind human CD40 are disclosed. The polypeptides or agents may include fusion polypeptides, particularly polypeptides comprising the extracellular domain of CD40L or a fragment thereof, wherein the extracellular domain of CD40L or fragment thereof comprises a mutation in the integrin-binding region. The agents may include bispecific and multispecific agents. Also disclosed are methods of using the polypeptides or agents for inducing and/or enhancing the immune response, as well as methods for the treatment of diseases such as cancer.

Description

CD40-BINDING AGENTS AND USES THEREOF
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No.
62/543,734, filed August 10, 2017, which is incorporated herein by reference in its entirety.
REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB
[0002] The content of the electronically submitted sequence listing (Name:
2293.170PC01_sequencelisting.ST25.txt, Size: 220,291 bytes; and Date of Creation: July 30, 2018) is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention generally relates to agents that bind CD40, particularly agents comprising at least one copy of the extracellular domain of CD40L or a receptor binding fragment thereof, wherein the extracellular domain of CD40L or fragment thereof comprises a mutation in the integrin-binding region. The invention also relates to methods of using the agents for the modulation of immune responses and/or the treatment of diseases such as cancer.
BACKGROUND OF THE INVENTION
[0004] The basis for immunotherapy is the manipulation and/or modulation of the
immune system, including both innate immune responses and adaptive immune responses. The general aim of immunotherapy is to treat diseases by controlling the immune response to a "foreign agent," for example a pathogen or a tumor cell. However, in some instances immunotherapy is used to treat autoimmune diseases which may arise from an abnormal immune response against proteins, molecules, and/or tissues normally present in the body. Immunotherapy may include methods to induce or enhance specific immune responses or to inhibit or reduce specific immune responses. [0005] The immune system is a highly complex system made up of a great number of cell types, including but not limited to, T-cells, B-cells, natural killer cells, antigen-presenting cells, dendritic cells, monocytes, and macrophages. These cells possess complex and subtle systems for controlling their interactions and responses. The cells utilize both activating and inhibitory mechanisms and feedback loops to keep responses in check and not allow negative consequences of an uncontrolled immune response (e.g., autoimmune diseases or a cytokine storm).
[0006] The concept of cancer immunosurveillance is based on the theory that the immune system can recognize tumor cells, mount an immune response, and suppress the development and/or progression of a tumor. However, it is clear that many cancerous cells have developed mechanisms to evade the immune system which can allow for uninhibited growth of tumors. Cancer/tumor immunotherapy (immuno-oncology) focuses on the development of new and novel agents that can activate and/or boost the immune system to achieve a more effective attack against tumor cells resulting in increased killing of tumor cells and/or inhibition of tumor growth.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention provides a variety of polypeptides and agents that bind
CD40. As used herein, the term "agent" includes, but is not limited to, polypeptides, fusion proteins, homodimeric molecules, and heterodimeric molecules. In some embodiments, a polypeptide or agent binds human CD40. In certain embodiments, the polypeptide or agent is a CD40 agonist. In some embodiments, the polypeptide or agent that binds CD40 is a soluble CD40 ligand (CD40L). The invention provides methods of using the polypeptides and agents described herein. In some embodiments, the invention provides methods of using the polypeptides and agents for cancer immunotherapy or immuno-oncology. In some embodiments, the polypeptides and agents are used in methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response. In some embodiments, the polypeptides and agents are used in methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response to cancer, a tumor, and/or tumor cells. In some embodiments, the polypeptides and agents are used in methods of inhibiting the growth of a tumor or tumor cells. In some embodiments, the polypeptides and agents are used in methods for the treatment of cancer. In some embodiments, the methods comprise inhibiting the growth of cancer cells. The invention also provides compositions comprising the agents described herein. In some embodiments, the compositions are pharmaceutical compositions comprising the polypeptides and agents described herein. Polynucleotides encoding the polypeptides and agents and methods of making the agents are also provided.
[0008] In one aspect, described herein are polypeptides comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In one aspect, described herein are single chain fusion human CD40 ligand (CD40L) polypeptides comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of human CD40L or a fragment thereof has a lower binding affinity to integrin alpha (lib) beta 3 compared to a corresponding extracellular domain of human CD40L or a fragment thereof not comprising the mutation.
[0009] In some embodiments, the integrin-binding region of CD40L comprises the KGD motif (residues 115-117 of SEQ ID NO: 12). In some embodiments, the integrin-binding region of CD40L consists of the KGD motif (residues 115-117 of SEQ ID NO: 12).
[0010] In some embodiments, the mutation comprises the substitution, insertion, or
deletion of at least one residue in the integrin-binding region. In some embodiments, the mutation comprises the substitution or deletion of 1, 2, or 3 residues in the integrin- binding region. In some embodiments, the mutation in the integrin-binding region comprises a D to E substitution in the KGD motif (residues 115-117 of SEQ ID NO: 12); and/or a G to A substitution in the KGD motif (residues 115-117 of SEQ ID NO: 12).
[0011] In some embodiments, the fragment of the stalk region of CD40L is selected from the group consisting of: MQKGDQ (SEQ ID NO: 16), FEMQKGDQ (SEQ ID NO: 17), EMQKGDQ (SEQ ID NO: 18), QKGDQ (SEQ ID NO: 19), and KGDQ (SEQ ID NO:20), wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the fragment of the stalk region comprises an amino acid sequence selected from the group consisting of: MQKADQ (SEQ ID NO:59),
FEMQKADQ (SEQ ID NO: 60), EMQKADQ (SEQ ID NO:61), QKADQ (SEQ ID NO:62), and KADQ (SEQ ID NO:63).
[0012] In some embodiments of a polypeptide described herein, at least two of the first, second, or third copies of the extracellular domain or a fragment thereof comprise the fragment of the stalk region. In some embodiments of a polypeptide described herein, the second and third copies of the extracellular domain or a fragment thereof comprise the fragment of the stalk region. In some embodiments of a polypeptide described herein, each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the fragment of the stalk region.
[0013] In some embodiments, a polypeptide described herein does not comprise an
exogenous peptide linker between the extracellular domains or fragments thereof of CD40L.
[0014] In some embodiments of a polypeptide described herein, at least one of the copies of the extracellular domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments of a polypeptide described herein, at least one of the copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments of a polypeptide described herein, at least one of the copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:64. In some embodiments of a polypeptide described herein, each of the first, second, or third copies of the extracellular domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments of a polypeptide described herein, each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments of a polypeptide described herein, each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 15, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68. In some embodiments, each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:64.
[0015] In some embodiments, a single chain fusion human CD40L polypeptide described herein comprises a polypeptide having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:70 or 80. In some embodiments, the single chain fusion human CD40L polypeptide comprises a polypeptide having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:70 or 80. In some embodiments, the single chain fusion human CD40L polypeptide comprises the amino acid sequence of SEQ ID NO:70 or 80.
[0016] In some embodiments, a polypeptide described herein further comprises a non-
CD40L polypeptide. In some embodiments, the first or third copy of the extracellular domain of human CD40L or a fragment thereof is directly linked to the non-CD40L polypeptide. In some embodiments, the first or third copy of the extracellular domain of human CD40L or a fragment thereof is connected to the non-CD40L polypeptide by a linker. In some embodiments, the first copy of the extracellular domain is linked to the carboxy-terminal end of the non-CD40L polypeptide. In some embodiments, the third copy of the extracellular domain is linked to the amino-terminal end of the non-CD40L polypeptide.
[0017] In some embodiments, the non-CD40L polypeptide comprises an Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, the Fc region is from a human IgGl, IgG2, IgG3, or IgG4 immunoglobulin. In some
embodiments, the Fc region is selected from the group consisting of: SEQ ID NOs:39-43. In some embodiments, the Fc region is deglycosylated. In some embodiments, the Fc region comprises a mutation in the N-linked glycosylation site of the CH2 domain. In some embodiments, the mutation is a substitution or deletion of the asparagine corresponding to residue 77 of SEQ ID NO:39. In some embodiments, the mutation is an alanine substitution of the asparagine corresponding to residue 77 of SEQ ID NO:39. In some embodiments, the mutation is a substitution or deletion of the asparagine corresponding to residue 79 of SEQ ID NO:43. In some embodiments, the mutation is an alanine substitution of the asparagine corresponding to residue 79 of SEQ ID NO:43. In some embodiments, the Fc region comprises SEQ ID NO:82. In some embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:44 and SEQ ID NO:45.
[0018] In some embodiments, a polypeptide described herein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:72. In some embodiments, a polypeptide described herein comprises a an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:72. In some embodiments, a polypeptide described herein comprises the amino acid sequence of SEQ ID NO:72.
[0019] In some embodiments, a polypeptide described herein comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:81. In some embodiments, a polypeptide described herein comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:81. In some embodiments, a polypeptide described herein comprises the amino acid sequence of SEQ ID NO:81.
[0020] In some embodiments, the non-CD40L polypeptide comprises an immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is linked to an immunoglobulin light chain. In some embodiments, the immunoglobulin heavy chain and immunoglobulin light chain form an antigen-binding site.
[0021] In some embodiments, the non-CD40L polypeptide comprises a single chain antibody or a Fab.
[0022] In one aspect, described herein is an agent comprising a polypeptide described herein. In one aspect, described herein is a homodimeric agent comprising a polypeptide described herein. In one aspect, described herein is a heterodimeric agent comprising a polypeptide described herein.
[0023] In some embodiments, an agent described herein activates CD40; and/or induces
CD40 activity. In some embodiments, an agent described herein has an improved therapeutic index compared to a reference agent; induces reduced systemic inflammation compared to a reference agent; induces lower serum liver enzyme level compared to a reference agent; and/or induces lower pro-inflammatory cytokine release than a reference agent, wherein the reference agent and the agent comprise the same polypeptide except that the reference agent comprises a polypeptide comprising a stalk region that does not comprise a mutation in the integrin-binding region. In some embodiments, reduced systemic inflammation is indicated by lower levels of blood neutrophils. In some embodiments, the blood neutrophil level is at least about 20%, 30%>, 40%, 50%, 60%>, or 70%) lower. In some embodiments, the blood neutrophil level is from about 20% to about 70%) lower, from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50%) to about 70%) lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower. In some embodiments, the blood neutrophil level is about 20% lower, about 30%) lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower. In some embodiments, the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In some embodiments, the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In some embodiments, the liver enzyme level is from about 20% to about 70% lower, from about 20% to about 60% lower, from about 20%) to about 50%) lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower. In some embodiments, the liver enzyme level is about 20%) lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-Ιβ, IL-6, TNF-alpha, IP-10, KC, and MTP-la. In some embodiments, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In some embodiments, the pro-inflammatory cytokine release is from about 20% to about 70%) lower, from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower. In some embodiments, the pro-inflammatory cytokine release is about 20%) lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
[0025] In one aspect, described herein is a bispecific agent comprising: a polypeptide described herein; and an antigen-binding site from an antibody. In one aspect, described herein is a bispecific agent comprising: a polypeptide described herein; and an immune response stimulating agent or functional fragment thereof, or an antibody or functional fragment thereof. In some embodiment, a bispecific agent described herein is a homodimer or a heterodimer.
[0026] In one aspect, described herein is a bispecific agent comprising; a) a first arm
comprising (i) a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the stalk region comprises a mutation in the integrin-binding region, and (ii) a Fc region; and b) a second arm comprising an antigen-binding site from an antibody. In some embodiments, the Fc region is deglycosylated. In some embodiments, each of the first, second, and third copies comprises the amino acid sequence of SEQ ID NO: 15, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO:72. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO:81. In some embodiments, the single chain fusion polypeptide is directly linked to the Fc region. In some embodiments, the single chain fusion polypeptide is connected to the Fc region by a linker. In some embodiments, the Fc region is from a human IgGl, IgG2, IgG3, or IgG4 immunoglobulin. In some
embodiments, the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:39-45 and 82. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:44 or SEQ ID NO:45. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO:82. [0027] In some embodiments of a bispecific agent described herein, the first arm comprises a first CH3 domain and the second arm comprises a second CH3 domain, each of which is modified to promote formation of heterodimers. In some embodiments, the first and second CH3 domains are modified based upon electrostatic effects. In some embodiments, the first arm comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:47, wherein the amino acids are replaced with glutamate or aspartate, and the second arm comprises a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO:47, wherein the amino acids are replaced with lysine. In some embodiments, the first arm comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO:47, wherein the amino acids are replaced with lysine, and the second arm comprises a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:47, wherein the amino acids are replaced with glutamate or aspartate. In some embodiments, the first and second CH3 domains are modified using a knobs-into-holes technique.
[0028] In some embodiments of a bispecific agent described herein, the antigen-binding site specifically binds a tumor antigen.
[0029] In some embodiments of a bispecific agent described herein, the antigen-binding site specifically binds PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3, or B7-H4.
[0030] In some embodiments, a bispecific agent described herein comprises an immune response stimulating agent.
[0031] In some embodiments, a bispecific agent described herein comprises an antibody.
In some embodiments, the antibody specifically binds to PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, or B7-H4.
[0032] In some embodiments, an agent or polypeptide described herein increases cell- mediated immunity. In some embodiments, an agent or polypeptide described herein increases antigen-presenting cell (APC) activity. In some embodiments, an agent or polypeptide described herein increases T-cell activity. In some embodiments, an agent or polypeptide described herein increases cytolytic T-cell (CTL) activity. In some embodiments, an agent or polypeptide described herein increases natural killer (NK) activity. In some embodiments, an agent or polypeptide described herein decreases or inhibits regulatory T-cell (Treg) activity. In some embodiments, an agent or polypeptide described herein decreases or inhibits myeloid-derived suppressor cell (MDSC) activity. In some embodiments, an agent or polypeptide described herein increases an effective immune response without causing substantial side effects and/or immune-based toxicities. In some embodiments, an agent or polypeptide described herein increases an effective immune response without causing cytokine release syndrome (CRS) or a cytokine storm.
[0033] In one aspect, described herein are cells comprising or producing a polypeptide or agent described herein.
[0034] In one aspect, described herein are compositions or pharmaceutical compositions comprising a polypeptide or agent described herein.
[0035] In one aspect, described herein is a pharmaceutical composition comprising a polypeptide or agent described herein and a pharmaceutically acceptable carrier.
[0036] In one aspect, described herein are polynucleotides comprising a nucleotide
sequence that encodes a polypeptide or agent described herein. In one aspect, described herein are polynucleotides comprising a nucleotide sequence that encodes a polypeptide or agent described herein and a signal sequence.
[0037] In one aspect, described herein are vectors comprising a polynucleotide described herein.
[0038] In one aspect, described herein are isolated cells comprising a polynucleotide or a vector described herein.
[0039] In one aspect, described herein are methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the immune response is against a tumor or cancer.
[0040] In one aspect, described herein are methods of inhibiting the growth of a tumor, comprising contacting a tumor or tumor cell with an effective amount of a polypeptide or agent described herein.
[0041] In one aspect, described herein are methods of inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the tumor is selected from the group consisting of colorectal tumor, colon tumor, ovarian tumor, pancreatic tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor.
[0042] In one aspect, described herein are methods of treating cancer in a subject,
comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, colon cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, head and neck cancer, lymphoma and leukemia.
[0043] In one aspect, described herein are methods of increasing T-cell activity in a
subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
[0044] In one aspect, described herein are methods of increasing antigen-presenting cell
(APC) activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
[0045] In one aspect, described herein are methods of increasing dendritic cell activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
[0046] In one aspect, described herein are methods of increasing cytolytic T-cell (CTL) activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
[0047] In one aspect, described herein are methods of increasing natural killer (NK)
activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
[0048] In one aspect, described herein are methods of decreasing or inhibiting regulatory
T-cell (Treg) activity in a subject, comprising administering to the subject a
therapeutically effective amount of a polypeptide or agent described herein.
[0049] In one aspect, described herein are methods of decreasing or inhibiting myeloid- derived suppressor cell (MDSC) activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.
[0050] In some embodiments, a method described herein further comprises administering at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the additional therapeutic agent is an antibody. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, or an anti-B7-H4 antibody. In some embodiments, the additional therapeutic agent is an immune response stimulating agent. In some embodiments, the immune response stimulating agent is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL- 12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-Ll antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.
In some embodiments, the administration of a polypeptide or agent described herein (a) induces reduced systemic inflammation compared to a reference polypeptide or reference agent; (b) induces lower serum liver enzyme level compared to a reference polypeptide or reference agent; and/or (c) induces lower pro-inflammatory cytokine release than a reference polypeptide or reference agent, wherein the reference polypeptide or reference agent and the polypeptide or agent comprise the same polypeptide except that the reference polypeptide or reference agent comprise a polypeptide comprising a stalk region that does not comprise a mutation in the integrin-binding region. In some embodiments, the reduced systemic inflammation is indicated by lower levels of blood neutrophils. In some embodiments, the blood neutrophil level is at least about 20%, 30%, 40%), 50%), 60%), or 70%> lower. In some embodiments, the blood neutrophil level is from about 20%) to about 70%> lower, from about 20%> to about 60%> lower, from about 20%> to about 50%) lower, from about 20%> to about 40%> lower, from about 20%> to about 30%> lower, from about 30%> to about 70%> lower, from about 30%> to about 60%> lower, from about 30%) to about 50%> lower, from about 30%> to about 40%> lower, from about 40%> to about 70%) lower, from about 40%> to about 60%> lower, from about 40%> to about 50%> lower, from about 50%> to about 70%> lower, from about 50%> to about 60%> lower, or from about 60%) to about 70%> lower. In some embodiments, the blood neutrophil level is about 20%) lower, about 30%> lower, about 40%> lower, about 50%> lower, about 60%> lower, or about 70%) lower. In some embodiments, the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In some embodiments, the liver enzyme level is at least about 20%, 30%), 40%, 50%, 60%>, or 70% lower. In some embodiments, the liver enzyme level is from about 20% to about 70% lower, from about 20% to about 60% lower, from about 20%) to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower. In some embodiments, the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60%) lower, or about 70% lower. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-Ιβ, IL-6, TNF-alpha, IP-10, KC, and MTP-la. In some embodiments, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In some embodiments, the pro-inflammatory cytokine release is from about 20% to about 70% lower, from about 20% to about 60% lower, from about 20%) to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower. In some embodiments, the proinflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50%) lower, about 60% lower, or about 70% lower. In some embodiments, the subject is human.
In one aspect, described herein are methods of improving the therapeutic index of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region. In one aspect, described herein are methods of decreasing the toxicity of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region. In some embodiments, the human CD40 agonist polypeptide comprises three copies of the extracellular domain of human CD40L or a CD40-binding fragment thereof. In some embodiments, the improving the therapeutic index or decreasing the toxicity comprises
(a) induction of reduced systemic inflammation compared to the human CD40 agonist;
(b) induction of lower serum liver enzyme level compared to the human CD40 agonist; and/or (c) induction of lower pro-inflammatory cytokine compared to the human CD40 agonist. In some embodiments, the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In some embodiments, the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In some embodiments, the liver enzyme level is from about 20% to about 70% lower, from about 20% to about 60%> lower, from about 20%) to about 50%) lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower. In some embodiments, the liver enzyme level is about 20%) lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-Ιβ, IL-6, TNF-alpha, IP-10, KC, and MTP-la. In some embodiments, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In some embodiments, the pro-inflammatory cytokine release is from about 20% to about 70% lower, from about 20% to about 60% lower, from about 20%) to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30%) lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40%) to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50%) lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower. In some embodiments, the proinflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50%) lower, about 60% lower, or about 70% lower. [0053] In one aspect, described herein are improved human CD40 agonists produced by a method described herein.
[0054] In one aspect, described herein are methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of an improved human CD40 agonist described herein. In some embodiments, the immune response is against a tumor or cancer.
[0055] In one aspect, described herein are methods of inhibiting the growth of a tumor, comprising contacting a tumor or tumor cell with an effective amount of an improved human CD40 agonist described herein.
[0056] In one aspect, described herein are methods of inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an improved human CD40 agonist described herein.
[0057] In one aspect, described herein are methods of treating cancer in a subject,
comprising administering to the subject a therapeutically effective amount of an improved human CD40 agonist described herein.
[0058] Where aspects or embodiments of the invention are described in terms of a
Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
BRIEF DESCRIPTION OF THE FIGURES
[0059] Figures 1 A-1C. Inhibition of tumor growth by single chain CD40L trimer-Fc
fusion polypeptide. The murine colon tumor line CT26.WT was implanted
subcutaneously into Balb/c mice. Mice were injected on days 10, 13, and 17 with
12.5mg/kg of single chain mCD40L trimer-Fc fusion protein 340F2 or a control antibody. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. Data is shown as tumor volume (mm3) over days post cell injection. Fig. 1 A. The mean values ± SEM for each group. Fig. IB. Tumor volumes of each individual mouse from the group treated with control antibody. Fig. lC. Tumor volumes of each individual mouse from the group treated with 340F2.
[0060] Figures 2A-2E. Inhibition of tumor growth by single chain CD40L trimer-Fc fusion polypeptide. The murine colon tumor line CT26.WT was implanted
subcutaneously into Balb/c mice. Mice were injected on days 12, 15, and 19 with 0.25mg/mouse of single chain mCD40L trimer-Fc fusion protein 340F2, an anti-mouse CD40 antibody, or a control antibody. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. Data is shown as tumor volume (mm3) over days post cell injection. Fig. 2 A. Tumor volumes of each individual mouse from the group treated with control antibody. Fig. 2B. Tumor volumes of each individual mouse from the group treated with anti-mCD40 antibody. Fig. 2C. Tumor volumes of each individual mouse from the group treated with 340F2. Fig. 2D. The mean values ± SEM for each group. Fig. 2E. Survival curve of the treatment groups up to Day 60.
[0061] Figures 3 A-3D. Inhibition of tumor growth by single chain CD40L trimer-Fc fusion polypeptide. The murine colon tumor line MC38 was implanted subcutaneously into C57BL/6N mice. Mice were injected on days 8, 12, and 15 with 0.25mg/mouse of single chain mCD40L trimer-Fc fusion protein 340F2, anti-mouse CD40 antibody, or a control antibody. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. Data is shown as tumor volume (mm3) over days post cell injection. Fig. 3A. The mean values ± SEM for each group. Fig. 3B. Tumor volumes of each individual mouse from the group treated with control antibody. Fig.3C. Tumor volumes of each individual mouse from the group treated with anti- mCD40 antibody. Fig.3D. Tumor volumes of each individual mouse from the group treated with 340F2.
[0062] Figure 4A-4C. ELISpot assays for IL-2 and IFN-gamma. Fig. 4A. Cells were harvested from the spleens of MC38-tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti-mCD40 antibody, or a control. Cells were incubated in the absence of a peptide and then analyzed using an ELISpot kit for IL-2. Total optical density (TOD) of cells producing IL-2 is shown. Fig. 4B. Cells were harvested from the spleens of MC38- tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti-mCD40 antibody, or a control. Cells were incubated in the absence of a peptide and then analyzed using an ELISpot kit for IFN-gamma. TOD of cells producing IFN-gamma is shown. Fig. 4C. Cells were harvested from the tumors of MC38-tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti-mCD40 antibody, or a control. Cells were incubated in the absence of a peptide and then analyzed using an ELISpot kit for IFN-gamma. TOD of cells producing IFN-gamma is shown.
[0063] Figures 5A-5C. Cytotoxicity assays. Fig. 5A. Cells were harvested from the
spleens of CT26.WT -tumor bearing mice treated with mCD40L trimer-Fc 340F2 or a control. YAC-1 target cells were labeled with ΙΟμΜ calcein AM and mixed with the splenocytes at an E:T ratio of 25 : 1. Supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm. Fig. 5B. Cells were harvested from the spleens of MC38-tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti-mCD40 antibody, or a control. YAC-1 target cells were labeled with ΙΟμΜ calcein AM and mixed with the splenocytes at an E:T ratio of 25: 1. Supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm. Fig. 5C. Cells were harvested from the spleens of MC38-tumor bearing mice treated with mCD40L trimer-Fc 340F2, anti- mCD40 antibody, or a control. MC38 target cells were labeled with ΙΟμΜ calcein AM and mixed with the splenocytes at an E:T ratio of 25 : 1. Supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm.
[0064] Figure 6. Inhibition of tumor growth by single chain CD40L trimer-Fc fusion polypeptides. On day 0, the 30,000 cells of the murine colon tumor line CT26.WT were implanted subcutaneously into Balb/c mice. On days 12, 15, and 18, mice were treated with lOmg/kg of single chain mCD40L trimer-Fc fusion protein 340F2, mCD40L trimer- Fc fusion protein 340F4, mCD40L trimer-Fc fusion protein 340F7, an anti-mCD40 antibody, or a control antibody. Tumor growth was monitored by measuring tumor volumes at the indicated time points. Data is shown as mean tumor size ± SEM (mm3).
[0065] Figures 7A-7C. Analysis of liver enzymes in serum in single chain CD40L trimer-
Fc fusion polypeptide treated mice. Mice were treated with lOmg/kg of single chain mCD40L trimer-Fc fusion protein 340F2, mCD40L trimer-Fc fusion protein 340F7, an anti-mCD40 antibody, or a control antibody as described in Example 7. Fig. 7A, B, and C show the serum alanine transaminase ("ALT"), serum aspartate transaminase ("ASP"), and serum alkaline phosphatase ("ALP") activities, respectively, detected. Data points from individual animals are shown with horizontal lines representing the mean serum enzyme activity detected in each treatment group.
[0066] Figures 8A-8D. Analysis of Complete Blood Count in single chain CD40L trimer-
Fc fusion polypeptide treated mice. Mice were treated with lOmg/kg of single chain mCD40L trimer-Fc fusion protein 340F2, mCD40L trimer-Fc fusion protein 340F7, an anti-mCD40 antibody, or a control antibody as described in Example 7. Fig. 8A, B, C, and D show the percent of neutrophils, the neutrophil count per μΕ, the white blood cell count per μΐ., and the lymphocyte count per detected, respectively. Data points from individual animals are shown with horizontal lines representing the mean values detected in each treatment group.
[0067] Figures 9A-90. Analysis of Plasma Cytokines in single chain CD40L trimer-Fc fusion polypeptide treated mice. Mice were treated with lOmg/kg of single chain mCD40L trimer-Fc fusion protein 340F2, mCD40L trimer-Fc fusion protein 340F4 (only received the second dose), mCD40L trimer-Fc fusion protein 340F7, an anti-mCD40 antibody, or a control antibody as described in Example 7. Plasma levels of G-CSF (A), IFN-gamma (B), IL-la (C), IL-Ιβ (D), IL-2 (E), IL-6 (F), IL-12p40 (G), IFN-gamma- induced protein 10 ("IP- 10") (H), chemokine (C-X-C motif) ligand 1 ("KC") (I), monocyte chemoattractant protein 1 ("MCP1 ") (J), macrophage inflammatory protein 1-a ("MIP-la") (K), MIP-Ι β (L), MIP2 (M), RANTES (N), and T F-alpha (O) are shown. Data points from individual animals are shown with horizontal lines representing the mean values detected in each treatment group.
[0068] Figures 10 A and 10B. Liver Histology of single chain CD40L trimer-Fc fusion polypeptide treated mice. Mice were treated with lOmg/kg of single chain mCD40L trimer-Fc fusion protein 340F2, mCD40L trimer-Fc fusion protein 340F4 (only received the second dose), mCD40L trimer-Fc fusion protein 340F7, an anti-mCD40 antibody, or a control antibody as described in Example 7. Liver sections stained with hematoxylin and eosin are shown at IX, 5X, or 10X magnification.
DETAILED DESCRIPTION OF THE INVENTION
[0069] Proteins belonging to the tumor necrosis factor receptor superfamily (TNFRSF) and their ligands (TNFSF) are intimately involved in the activation, differentiation, and survival of cells of the immune system. TNFRSF members include, but may not be limited to, 4-1BB, BAFF, BCMA, CD27, CD30, CD40, DcR3, DcTRAIL Rl, DcTRAIL R2, DR3, DR6, EDA2R, EDAR, Fas (CD95), GITR, HVEM, lymphotoxin beta R, NGFR, osteoprotegerin, OX40, RANK, RELT, TACI, TNFRH3, TNF Rl, TNF R2, TRAIL Rl, TRAIL R2, TRAIL R3, TRAIL R4, TROY, and TWEAK R. Receptors for TNF family ligands are oligomeric, type I or type III transmembrane proteins that contain multiple extracellular cysteine-rich domains. Several of these receptors also contain intracellular death domains (DDs) that recruit caspase-interacting proteins following ligand binding to initiate the extrinsic pathway of caspase activation. Other TNF superfamily receptors that lack death domains bind TNF receptor-associated factors and activate intracellular signaling pathways that can lead to proliferation or differentiation. These receptors can also initiate apoptosis, but they do so via indirect mechanisms. In addition to regulating apoptosis, several TNF superfamily receptors are involved in regulating immune cell functions such as B-cell homeostasis and activation, natural killer cell activation, and T-cell co-stimulation. Several others regulate cell type-specific responses such as hair follicle development and osteoclast development.
TNFSF members include, but may not be limited to, 4-1BB ligand, APRIL,
BAFF, CD27 ligand (CD27L), CD30 ligand (CD30L), CD40 ligand (CD40L), EDA, EDA-A1, EDA-A2, Fas ligand (CD95L), GITR ligand (GITRL), LIGHT, lymphotoxin, lymphotoxin beta, lymphotoxin-alpha, OX40 ligand (OX40L), TL1A, TNF-alpha, TRAIL, TRANCE, and TWEAK. Most TNFSF ligands are type II transmembrane proteins whose extracellular domains can be cleaved by specific metalloproteinases to generate soluble cytokines. Cleaved and non-cleaved ligands are active as non-covalent homotnmers except for lymphotoxin beta (which forms heterotrimers with TNF-beta) and BAFF (which forms heterotrimers with APRIL). TNF superfamily ligands are characterized by a stalk of varying length connecting the transmembrane domain to the core region, which contains the hallmark structure of TNF family ligands, the TNF homology domain (THD) or TNF domain. The TNF domain is an anti-parallel beta- pleated sheet sandwich with a "jelly-roll" topology. Conserved residues within the beta- strands provide specific inter-subunit contacts, which stabilize the trimeric structure. Sequences in the loops connecting adjacent beta-strands are family member-specific and are important for conferring receptor specificity. [0071] Because agonist antibodies targeting members of the TNFR superfamily are generally dimeric molecules with each arm of the antibody binding one subunit of a TNFR, the inventors hypothesized that they may not be able to fully recapitulate the signaling impact of the native trimeric TNF family member. It was hypothesized that a therapeutic agent that presents a TNF family ligand in a stable trimeric form could be more active than an agonist TNFR antibody at eliciting signaling, and that such a trimeric TNF ligand form might therefore be a superior immunotherapeutic agent.
[0072] One strategy to generate a TNFSF ligand trimer is to express three subunits of the trimer as a single polypeptide. It has previously been shown that TNF family members can be expressed as a single chain trimer (US Application Publication Nos. 2007/0286843 and 2011/0162095). However, a major drawback of previous single chain TNF family member trimer variants has been the introduction of exogenous linker sequences interconnecting the three subunits of the trimer. Such linkers may introduce potential instability and lability to the trimer, and/or provide a source of potential immunogenicity as the linkers are foreign sequences.
[0073] Another strategy is to utilize a fragment of the stalk region to bridge the distance from the C-terminus of a TNFSF ligand monomer to the N-terminus of an adjacent TNFSF ligand monomer and in this fashion construct a single chain TNFSF ligand trimer that was devoid of exogenous peptide linker sequences. See, e.g., US Patent No.
9,724,390 and International Patent Application No. PCT/US 17/45281 filed on August 3, 2017, each of which is incorporated by reference herein in its entirety.
[0074] The present invention provides novel agents that bind CD40, including, but not limited to, polypeptides, soluble proteins, fusion proteins, homodimeric bispecific molecules, and heterodimeric bispecific molecules that modulate the immune response. Related polypeptides and polynucleotides, compositions comprising the agents, and methods of making the agents are also provided. Methods of screening for agents that modulate the immune response are provided. Methods of using the novel agents, such as methods of activating an immune response, methods of stimulating an immune response, methods of promoting an immune response, methods of increasing an immune response, methods of activating NK cells, methods of activating T-cells, including CTLs, methods of activating APCs, methods of increasing the activity of NK cells, methods of increasing the activity of T-cells, including CTLs, methods of increasing the activity of APCs, methods of promoting the activity of NK cells, methods of promoting the activity of T- cells, including CTLs, methods of inhibiting the activity of Tregs, methods of inhibiting the activity of MDSCs, methods of inhibiting tumor growth, methods of treating cancer, and/or methods of treating viral diseases are provided. Methods of inhibiting an immune response, methods of suppressing an immune response, methods of decreasing activity of T-cells, and/or methods of treating autoimmune diseases are further provided.
I. Definitions
[0075] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.
[0076] The term "CD40L integrin-binding region" as used herein refers to or describes the region or domain in the CD40L stalk region that mediates the interaction between CD40L and integrin. In one embodiment, the interaction is between CD40L and integrin alpha (lib) beta 3. In one embodiment, the interaction is between human CD40L and integrin alpha (lib) beta 3. In one embodiment, the interaction between CD40L and integrin alpha (lib) beta 3 results in platelet activation as described in Prasad et al, PNAS, 100921): 12367-12371 (2003). In one embodiment, the interaction between CD40L and integrin alpha (lib) beta 3 is specific binding assayed by a competitive or non-competitive assay system using techniques such as Biacore analyses, FACS analyses,
immunofluorescence, immunocytochemistry, Western blot analyses, radioimmunoassays, ELISAs, "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well-known in the art. In one embodiment, the interaction between CD40L and integrin alpha (lib) beta 3 is assayed by FACS analyses determining the binding of a soluble CD40L polypeptide to platelets. In one embodiment, the interaction between CD40L and integrin alpha (lib) beta 3 is assayed by ELISA analyses determining the binding of a soluble CD40L polypeptide to an immobilized integrin alpha (lib) beta 3 polypeptide. In one embodiment, the integrin binding region of human CD40L comprises the KGD motif of the stalk region (residues 115-117 of SEQ ID NO: 12). In one embodiment, the integrin binding region of human CD40L consists of the KGD motif of the stalk region (residues 115-117 of SEQ ID NO: 12). In one embodiment, the integrin binding region of murine CD40L comprises the RGD motif of the stalk region (residues 114-116 of SEQ ID NO: 1). In one embodiment, the integrin binding region of murine CD40L consists of the RGD motif of the stalk region (residues 114-116 of SEQ ID NO: 1).
The term "mutation in the CD40L integrin-binding region" as used herein refers to or describes at least one substitution, insertion, or deletion in the CD40L integrin-binding region. In one embodiment, the mutation is the substitution, insertion, or deletion of at least one residue in the CD40L integrin-binding region. In embodiment, the mutation is the substitution, insertion, or deletion of at least two residues in the CD40L integrin- binding region. In embodiment, the mutation is the substitution, insertion, or deletion of at least three residues in the CD40L integrin-binding region. In embodiment, the mutation is 1, 2, or 3 substitutions in the CD40L integrin-binding region. In embodiment, the mutation comprises 1, 2, or 3 substitutions in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a substitution of the K residue in the KGD motif of the human CD40L stalk region
(residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a substitution of the G residue in the KGD motif of the human CD40L stalk region
(residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a substitution of the D residue in the KGD motif of the human CD40L stalk region
(residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a G to A substitution in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a D to E substitution in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation does not comprise a D to E substitution in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a substitution of the K and G residues, the K and D residues, the G and D residues, or the K, G and D residues in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises 1, 2, or 3 deletions in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a deletion of the K residue in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a deletion of the G residue in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a deletion of the D residue in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises a deletion of the K and G residues, the K and D residues, the G and D residues, or the K, G and D residues in the KGD motif of the human CD40L stalk region (residues 115-117 of SEQ ID NO: 12). In embodiment, the mutation comprises 1, 2, or 3 substitutions in the RGD motif of the murine CD40L stalk region (residues 114-116 of SEQ ID NO: 1). In embodiment, the mutation comprises a G to A or a D to E substitution in the RGD motif of the murine CD40L stalk region (residues 114-116 of SEQ ID NO: 1). In embodiment, the mutation comprises 1, 2, or 3 deletions in the RGD motif of the murine CD40L stalk region (residues 114-1 16 of SEQ ID NO: 1). In one embodiment, the mutation in the CD40L integrin-binding region reduces or eliminates the interaction between CD40L and integrin. In one embodiment, the interaction is between CD40L and integrin alpha (lib) beta 3. In one embodiment, the interaction is between human CD40L and integrin alpha (lib) beta 3. In one embodiment, the interaction between CD40L and integrin alpha (lib) beta 3 results in platelet activation as described in Prasad et al, PNAS, 100921): 12367-12371 (2003). In one embodiment, the interaction between CD40L and integrin alpha (lib) beta 3 is assayed by FACS analyses determining the binding of a soluble CD40L polypeptide to platelets. In one embodiment, the interaction between CD40L and integrin alpha (lib) beta 3 is assayed by ELISA analyses determining the binding of a soluble CD40L polypeptide to an immobilized integrin alpha (lib) beta 3 polypeptide.
[0078] The terms "agonist" and "agonistic" as used herein refer to or describe a
polypeptide or agent that is capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a target and/or a pathway. The term "agonist" is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest.
[0079] The terms "antagonist" and "antagonistic" as used herein refer to or describe a polypeptide or agent that is capable of, directly or indirectly, partially or fully blocking, inhibiting, reducing, or neutralizing a biological activity of a target and/or pathway. The term "antagonist" is used herein to include any agent that partially or fully blocks, inhibits, reduces, or neutralizes the activity of a protein or other target of interest. [0080] The terms "modulation" and "modulate" as used herein refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating an activity or inhibiting an activity. Modulation may be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, a pathway, a system, or other biological targets of interest.
[0081] The term "soluble protein" as used herein refers to a protein or a fragment thereof that can be secreted from a cell in soluble form.
[0082] The term "fusion protein" or "fusion polypeptide" as used herein refers to a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
[0083] The term "linker" or "linker region" as used herein refers to a linker inserted
between a first polypeptide (e.g., copies of a CD40L extracellular domain or fragments thereof) and a second polypeptide (e.g., a Fc region). In some embodiments, the linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. Preferably, linkers are not antigenic and do not elicit an immune response.
[0084] The term "antibody" as used herein refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological activity. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, including but not limited to, toxins and radioisotopes.
[0085] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. "Antibody fragment" as used herein comprises an antigen-binding site or epitope-binding site.
[0086] The term "variable region" of an antibody refers to the variable region of an
antibody light chain, or the variable region of an antibody heavy chain, either alone or in combination. Generally, the variable region of heavy and light chains each consist of four framework regions (FR) and three complementarity determining regions (CDRs), also known as "hypervariable regions". The CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding sites of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda MD.), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al Lazikani et al., 1997, J. Mol. Biol, 273 :927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.
[0087] The term "monoclonal antibody" as used herein refers to a homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include a mixture of different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv) antibodies, fusion proteins comprising an antibody fragment, and any other modified
immunoglobulin molecule comprising an antigen-binding site. Furthermore, "monoclonal antibody" refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.
[0088] The term "humanized antibody" as used herein refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capability. In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the
corresponding residues in an antibody from a non-human species. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability. The humanized antibody may comprise variable domains containing all or substantially all of the CDRs that correspond to the non-human immunoglobulin whereas all or substantially all of the framework regions are those of a human immunoglobulin sequence. In some embodiments, the variable domains comprise the framework regions of a human immunoglobulin sequence. In some embodiments, the variable domains comprise the framework regions of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. A humanized antibody is usually considered distinct from a chimeric antibody.
[0089] The term "human antibody" as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.
[0090] The term "chimeric antibody" as used herein refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding capability, while the constant regions are homologous to the sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species. [0091] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.
[0092] The terms "selectively binds" or "specifically binds" mean that a polypeptide or agent interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. In certain
embodiments "specifically binds" means, for instance, that a polypeptide or agent binds a protein or target with a KD of about 0. ImM or less, but more usually less than about 1 μΜ. In certain embodiments, "specifically binds" means that a polypeptide or agent binds a target with a KD of at least about 0.1 μΜ or less, at least about 0.01 μΜ or less, or at least about InM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or agent that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or agent that recognizes more than one protein or target. It is understood that, in certain embodiments, a polypeptide or agent that specifically binds a first target may or may not specifically bind a second target. As such, "specific binding" does not necessarily require (although it can include) exclusive binding, i.e. binding to a single target. Thus, a polypeptide or agent may, in certain embodiments, specifically bind more than one target. In certain embodiments, multiple targets may be bound by the same antigen-binding site on the polypeptide or agent. For example, an antibody may, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In certain alternative embodiments, an antibody may be bispecific and comprise at least two antigen-binding sites with differing specificities. Generally, but not necessarily, reference to "binding" means "specific binding". [0093] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the polypeptides can occur as single chains or as associated chains.
[0094] The terms "polynucleotide" and "nucleic acid" and "nucleic acid molecule" are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
[0095] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum
correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%), 96%), 97%), 98%o, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.
[0096] A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a
phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and/or antibodies of the invention do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site. Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.
[0097] The term "vector" as used herein means a construct, which is capable of
delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.
[0098] A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or
composition which is "isolated" is a polypeptide, soluble protein, antibody,
polynucleotide, vector, cell, or composition which is in a form not found in nature.
Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
[0099] The term "substantially pure" as used herein refers to material which is at least
50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
[0100] The term "immune response" as used herein includes responses from both the innate immune system and the adaptive immune system. It includes both cell-mediated and/or humoral immune responses. It includes both T-cell and B-cell responses, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, etc.
[0101] The terms "cancer" and "cancerous" as used herein refer to or describe the
physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and hematologic cancers such as lymphoma and leukemia.
[0102] The terms "tumor" and "neoplasm" as used herein refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions.
[0103] The term "metastasis" as used herein refers to the process by which a cancer
spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A "metastatic" or
"metastasizing" cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures. [0104] The terms "cancer stem cell" and "CSC" and "tumor stem cell" and "tumor initiating cell" are used interchangeably herein and refer to cells from a cancer or tumor that: (1) have extensive proliferative capacity; 2) are capable of asymmetric cell division to generate one or more types of differentiated cell progeny wherein the differentiated cells have reduced proliferative or developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self-maintenance. These properties confer on the cancer stem cells the ability to form or establish a tumor or cancer upon serial transplantation into an appropriate host (e.g., a mouse) compared to the majority of tumor cells that fail to form tumors. Cancer stem cells undergo self-renewal versus
differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur.
[0105] The terms "cancer cell" and "tumor cell" refer to the total population of cells derived from a cancer or tumor or pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the cancer cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the terms "cancer cell" or "tumor cell" will be modified by the term "non-tumorigenic" when referring solely to those cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.
[0106] The term "tumorigenic" as used herein refers to the functional features of a cancer stem cell including the properties of self-renewal (giving rise to additional tumorigenic cancer stem cells) and proliferation to generate all other tumor cells (giving rise to differentiated and thus non-tumorigenic tumor cells).
[0107] The term "tumorigenicity" as used herein refers to the ability of a random sample of cells from the tumor to form palpable tumors upon serial transplantation into appropriate hosts (e.g., mice).
[0108] The term "subject" refers to any animal (e.g., a mammal), including, but not
limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.
[0109] The term "pharmaceutically acceptable" refers to a substance approved or
approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
[0110] The terms "pharmaceutically acceptable excipient, carrier or adjuvant" or
"acceptable pharmaceutical carrier" refer to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one agent of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect. In general, those of skill in the art and the U.S. FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.
[0111] The terms "effective amount" or "therapeutically effective amount" or "therapeutic effect" refer to an amount of a polypeptide or agent described herein (e.g., a fusion protein, a soluble receptor, an antibody, a polypeptide, a polynucleotide, a small organic molecule, or other drug) effective to "treat" a disease or disorder in a subject such as, a mammal. In the case of cancer or a tumor, the therapeutically effective amount of a polypeptide or agent (e.g., polypeptide, soluble protein, or antibody) has a therapeutic effect and as such can boost the immune response, boost the anti-tumor response, increase cytolytic activity of immune cells, increase killing of tumor cells by immune cells, reduce the number of tumor cells; decrease tumorigenicity, tumorigenic frequency or
tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce the tumor size; reduce the cancer cell population; inhibit or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor or cancer cell metastasis; inhibit and stop tumor or cancer cell growth; relieve to some extent one or more of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects.
[0112] The terms "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In the case of cancer or a tumor, a subject is successfully "treated" according to the methods of the present invention if the patient shows one or more of the following: an increased immune response, an increased anti-tumor response, increased cytolytic activity of immune cells, increased killing of tumor cells by immune cells, a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size;
inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality;
improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of effects.
[0113] As used in the present disclosure and claims, the singular forms "a", "an" and
"the" include plural forms unless the context clearly dictates otherwise.
[0114] It is understood that wherever embodiments are described herein with the
language "comprising" otherwise analogous embodiments described in terms of
"consisting of and/or "consisting essentially of are also provided. It is also understood that wherever embodiments are described herein with the language "consisting essentially of otherwise analogous embodiments described in terms of "consisting of are also provided.
[0115] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".
[0116] The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
II. CD40-binding agents
[0117] The present invention provides polypeptides and agents that bind CD40 (also referred to as T FRSF5). The polypeptides and agents comprise a polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In one embodiment, the polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof is a single chain fusion polypeptide. In one embodiment, the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12). In one embodiment, the mutation comprises at least one substitution in the KGD motif. In one embodiment, the mutation comprises at least two substitutions in the KGD motif. In one embodiment, the mutation comprises three substitutions in the KGD motif. In one embodiment, the mutation comprises a G to A or D to E substitution in the KGD motif. In one embodiment, the mutation comprises the deletion of at least one residue in the KGD motif. In one embodiment, the mutation comprises the deletion of at least two residues in the KGD motif. In one embodiment, the mutation comprises the deletion of all three residues in the KGD motif.
[0118] CD40 is a member of the TNF receptor superfamily (TNFRSF). TNFRSF
members include, but may not be limited to, 4-1BB, BAFF, BCMA, CD27, CD30, CD40, DcR3, DcTRAIL Rl, DcTRAIL R2, DR3, DR6, EDA2R, EDAR, Fas (CD95), GITR, HVEM, lymphotoxin beta R, NGFR, osteoprotegerin, OX40, RANK, RELT, TACI, TNFRH3, TNF Rl, TNF R2, TRAIL Rl, TRAIL R2, TRAIL R3, TRAIL R4, TROY, and TWEAK R. CD40 is expressed by antigen-presenting cells (APCs) including dendritic cells (DCs), B-cells, macrophages, and monocytes but is also expressed on other cell types such as epithelial cells, endothelial cells, and platelets. CD40 expression has also been demonstrated on various tumor cells, including B-cell lymphoma and renal cancer cells. The natural ligand for CD40 is CD40L (CD 154) which is mainly expressed on activated CD4+ T-cells, but also on platelets, mast cells, and basophils. Interaction between CD40 and CD40L facilitates an improved T-cell activation including priming of naive CD4+ T-cells and CD8+ T-cells which then differentiate into CD4+ helper T-cells and CD8+ cytotoxic T-cells, respectively. Cytokines secreted from CD40-expressing cells may indirectly lead to the activation of NK cells. Thus, treatment with CD40 agonists may mediate a potent anti-tumor effect through boosting of APC function, T-cell activation, and NK cell activation.
[0119] While the CD40 signaling axis holds tremendous therapeutic potential (there are several agonist anti-CD40 antibodies in development and in clinical trials), at this point in time it has been difficult to develop effective therapeutic agents. A principal challenge has been that CD40 is expressed by a variety of cell types as disclosed above and thus an anti-CD40 antibody can be expected to bind to each of these cell types. Developing an agonist antibody against CD40 therefore has the potential drawback that there might be undesirable side effects due to the effector function activity mediated by the Fc domain of the antibody which can interact with Fc receptors on a variety of immune cells. Agonist antibodies against CD40 have generally required Fc receptor binding to facilitate a clustering of antibody bound to CD40 that thereby promotes the agonist CD40 signaling activity. Each of the human IgG isotypes, IgGl, IgG2, IgG3, and IgG4, are able to bind Fc receptors. Even human IgG2, which is generally considered to have limited antibody- dependent cellular cytotoxicity (ADCC), is still able to engage the inhibitory Fc receptor FcyRIIB. As a result, even the IgG2 isotype can cause an agonist anti-CD40 antibody to promote direct cell-cell contact between a CD40-expressing cell and an Fc receptor- containing immune cell and potentially elicit unintended and/or undesirable
consequences. Therefore, the ability to create an agonist CD40 agent that does not possess the ability to engage Fc receptors can be anticipated to have the potential for greater safety and less undesired side effects. It is known in the art that the Fc region can be engineered/modified to eliminate Fc receptor binding. In some embodiments, the glycosylation site that is critical for Fc receptor binding is modified. In some
embodiments, the glycosylation site that is critical for Fc receptor binding is eliminated. In some embodiments, a single chain CD40L trimer linked to a Fc region in which Fc receptor binding is eliminated provides the ability to achieve robust CD40 activation without the potential for undesirable toxicity due to Fc receptor-mediated events.
[0120] The agents described herein may be referred to herein as "CD40-binding agents".
In certain embodiments, the agent is a CD40 agonist. In certain embodiments, the polypeptide or agent induces, activates, enhances, increases, and/or prolongs CD40 signaling.
[0121] In certain embodiments, the agent is a polypeptide. In certain embodiments, the agent is a soluble protein. In some embodiments, the agent is a fusion polypeptide. In some embodiments, the agent is a soluble ligand or soluble "co-receptor". The ligand or "co-receptor" for CD40 is CD40L. Thus, in some embodiments, the polypeptide or agent comprises a fragment of human CD40L, wherein the fragment comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises the extracellular domain of human CD40L, wherein the extracellular domain comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises a fragment of the extracellular domain of human CD40L, wherein the fragment comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a fragment of the extracellular domain of human CD40L can demonstrate altered biological activity compared to a soluble agent comprising the entire extracellular domain.
In some embodiments, the polypeptide or agent binds human CD40. In some embodiments, the polypeptide or agent binds mouse CD40. In some embodiments, the agent is a polypeptide. In some embodiments, the polypeptide or agent is a soluble protein. In some embodiments, the polypeptide or agent is a soluble protein that binds human CD40. In some embodiments, the polypeptide or agent is a fusion polypeptide. In some embodiments, the agent (e.g., polypeptide) comprises at least one copy of the extracellular domain, or a CD40-binding fragment thereof, of CD40L, wherein the extracellular domain or fragment thereof comprises a mutation in the integrin-binding region. In some embodiments, the agent (e.g., polypeptide) comprises at least one copy of the extracellular domain, or a fragment thereof, of human CD40L. In some embodiments, the polypeptide or agent comprises at least one copy of the extracellular domain, or a fragment thereof, of mouse CD40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L, wherein at least one of the extracellular domains comprises the "stalk region" of CD40L, and wherein the stalk region comprises a mutation in the integrin-binding region. The stalk region of CD40L is very long in comparison to some of the other TNFSF members (i.e., GITRL and OX40L), therefore in some embodiments the extracellular domain(s) of human CD40L comprise only a short fragment of the stalk region. In some embodiments, the stalk region comprises about 4-20 amino acids. In some embodiments, the stalk region comprises about 4-10 amino acids. In some embodiments, the stalk region comprises the amino acids (e.g., 4-10 amino acids) upstream from the TNF homology domain. In some embodiments, the stalk region comprises the amino acids (e.g., 4-10 amino acids) adjacent to the TNF homology domain. In some embodiments, the fragment of the stalk region of CD40L is MQKGDQ (SEQ ID NO: 16) comprising a mutation in the integrin-binding region. In some embodiments, the fragment of the stalk region of CD40L is FEMQKGDQ (SEQ ID NO: 17), EMQKGDQ (SEQ ID NO: 18), QKGDQ (SEQ ID NO: 19), or KGDQ (SEQ ID NO:20) comprising a mutation in the integrin-binding region. The mutation in the integrin-binding region can be amino acid insertions, deletions, and/or substitutions. In some embodiments, the integrin-binding region of the fragment of the stalk region of CD40L comprises at least 1, at least 2, or at least 3 insertions, deletions, and/or substitutions. As used herein, the stalk regions consist of amino acid sequences of CD40L, i.e., these stalk regions do not comprise any exogenous amino acids, such as an exogenous linker. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L, wherein each extracellular domain comprises a fragment of the stalk region of CD40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L, wherein the second and third extracellular domains include a fragment of the stalk region of CD40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the polypeptide does not comprise any peptide linkers. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the first copy and the second copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the second copy and the third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 113 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 111 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 112 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 114 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 115 to 261 of human CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises approximately amino acids 113 to 261, 111 to 261, 112 to 261, 114 to 261, or 115 to 261 of human CD40L, wherein the sequence of human CD40L is UniProtKB No. P29965, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO: 22 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:22 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO: 23 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:23 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:23, wherein the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:24 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:24 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:24, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:25 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:25 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:25, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:26 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:26 or a fragment thereof, wherein the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:26, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. [Add mutant SEQ IDNO:] In some embodiments, the
polypeptide or agent comprises SEQ ID NO:28. In some embodiments, the polypeptide or agent comprises SEQ ID NO:36, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the integrin-binding region of the fragment of the stalk region of CD40L comprises at least 1, at least 2, or at least 3 insertions, deletions, and/or substitutions. In some embodiments, the polypeptide or agent is a fusion polypeptide or fusion protein. In some embodiments, the fusion protein comprises a non-CD40L polypeptide (i.e., a heterologous protein). In some embodiments, the fusion polypeptide comprises a Fc region. In some embodiments, the non-CD40L polypeptide comprises a Fc region. In some embodiments, the non-CD40L polypeptide comprises a human Fc region. In some embodiments, the Fc region is from an IgGl, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region is modified. In some embodiments, the Fc region is deglycosylated. In some embodiments, the Fc region is selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45. In some embodiments, the Fc region comprises SEQ ID NO:44 or SEQ ID NO:45. In some embodiments, the non-CD40L polypeptide comprises an immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is associated with an
immunoglobulin light chain. In some embodiments, the immunoglobulin heavy chain and light chain form an antigen-binding site. In some embodiments, the non-CD40L polypeptide comprises a single chain antibody or a Fab.
[0124] In some embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In one embodiment, the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12). In one embodiment, the mutation is a substitution in the KGD motif. In one embodiment, the mutation is two substitutions in the KGD motif. In one embodiment, the mutation is three substitutions in the KGD motif. In one embodiment, the mutation is a G to A or D to E substitution in the KGD motif. In one embodiment, the mutation is a deletion in the KGD motif. In one embodiment, the mutation is two deletions in the KGD motif. In one embodiment, the mutation is three deletions in the KGD motif.
[0125] The full-length amino acid (aa) sequence of human CD40L is known in the art
(UniProtKB No. P29965) and is provided herein as SEQ ID NO: 12. In some
embodiments, the polypeptide or agent comprises at least one copy of the extracellular domain of CD40L or a fragment thereof, wherein the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In certain
embodiments, the "extracellular domain" of CD40L is approximately amino acids 47-261 of SEQ ID NO: 12. Those of skill in the art may differ in their understanding of the exact amino acids corresponding to the extracellular domain of CD40L. Thus, the N-terminus and/or C-terminus of the extracellular domain described herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. As used herein, the extracellular domain of CD40L generally comprises the "stalk region" or a fragment of the stalk region and the "TNF family domain". The stalk region of CD40L is
approximately 72 amino acids, much longer than the stalk regions of other proteins in the TNFSF family, e.g., GITRL and OX40L. In some embodiments, to allow for proper folding and conformation of a single chain CD40L trimer, the stalk region comprises a fragment of the CD40L stalk region more equivalent in length to the stalk regions of, for example, GITRL or OX40L. Thus, in some embodiments, a copy of the extracellular domain of CD40L in the polypeptides or agents described herein comprises a fragment of the stalk region of CD40L. In some embodiments, the stalk region comprises about 4-20 amino acids. In some embodiments, the stalk region is about 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the stalk region comprises about 4-10 amino acids. In some embodiments, the stalk region comprises the amino acids (e.g., 4-10 amino acids) upstream from the TNF homology domain. The stalk region of CD40L is approximately amino acids 47-112 of SEQ ID NO: 12. In some embodiments, the stalk region comprises a fragment of the CD40L stalk region. In some embodiments, fragments of the CD40L stalk region comprise MQKGDQ (SEQ ID NO: 16; fragment 1); FEMQKGDQ (SEQ ID NO: 17; fragment 2); EMQKGDQ (SEQ ID NO: 18; fragment 3); QKGDQ (SEQ ID NO: 19; fragment 4); or KGDQ (SEQ ID NO:20; fragment 5), wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, fragments of the CD40L stalk region comprise an amino acid sequence selected from the group consisting of: MQKADQ (SEQ ID NO:59), FEMQKADQ (SEQ ID NO:60), EMQKADQ (SEQ ID NO:61), QKADQ (SEQ ID NO:62), and KADQ (SEQ ID NO:63). The "TNF homology domain" or "TNF family domain" of CD40L is approximately amino acids 122-261 of SEQ ID NO: 12. The TNF homology domain comprises SEQ ID NO:21. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO: 15. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO: 15. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO: 15. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:22, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:22, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:22, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:23, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:23, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:23, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:24, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:24, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:24, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:25, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:25, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:25, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:26, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:26, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:26, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:59. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:59. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:59. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:60. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:60. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:60. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:61. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:61. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:61. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:62. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:62. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:62. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:63. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:63. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:63. In certain embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of CD40L or a CD40-binding fragment thereof. In certain embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of CD40L or a fragment thereof as a single chain polypeptide. In certain embodiments, a polypeptide or agent comprises SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:28, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In certain embodiments, the polypeptide or agent comprises SEQ ID NO:36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO: 36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO: 36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%), 98%), or 99% sequence identity to SEQ ID NO: 36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:36, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin-binding region. In certain embodiments, a polypeptide or agent comprises SEQ ID NO:70. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:70. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:70. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:70. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:70. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:70. In certain embodiments, the polypeptide or agent comprises SEQ ID NO:80. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:80. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:80. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:80. In some
embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:80. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:80. In one embodiment, the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12). In one embodiment, the mutation is a substitution in the KGD motif. In one embodiment, the mutation is a G to A or D to E substitution in the KGD motif. In certain embodiments, a polypeptide or agent comprises at least a first, second, and third copy of a fragment of the extracellular domain of CD40L. In some embodiments, the copies of the extracellular domain of CD40L consist of the same amino acid sequence. In some embodiments, the copies of the extracellular domain of CD40L are not identical. In some embodiments, the copies of the extracellular domain of CD40L comprise substitutions, deletions, and/or additions to the amino acid sequence of human CD40L as compared to the wild-type or parental sequence.
In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof, wherein each extracellular domain comprises the stalk region or a stalk region fragment, wherein the stalk region or fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L, wherein each extracellular domain comprises the stalk region or a stalk region fragment, wherein the stalk region or the fragment of the stalk region comprises a mutation in the integrin- binding region, and the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers). In some embodiments, the extracellular domain of CD40L comprises amino acids 113-261 of SEQ ID NO: 12, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO: 15. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some
embodiments, the extracellular domain of CD40L comprises SEQ ID NO:23, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:23, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:24, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:24, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:25, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:25, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:26, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO:26, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the single chain fusion polypeptide comprises SEQ ID NO:28, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:28, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the single chain fusion polypeptide comprises SEQ ID NO:36, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:36, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In one embodiment, the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12). In one embodiment, the mutation is a substitution in the KGD motif. In one embodiment, the mutation is a G to A or D to E substitution in the KGD motif. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:59. In some embodiments, the extracellular domain of CD40L consists of SEQ ID NO :59. In some embodiments, the extracellular domain of CD40L comprises
SEQ ID NO :60. In some embodiments, the extracellular domain of CD40L consists of
SEQ ID NO :60. In some embodiments, the extracellular domain of CD40L comprises
SEQ ID NO :61. In some embodiments, the extracellular domain of CD40L consists of
SEQ ID NO :61. In some embodiments, the extracellular domain of CD40L comprises
SEQ ID NO :62. In some embodiments, the extracellular domain of CD40L consists of
SEQ ID NO :62. In some embodiments, the extracellular domain of CD40L comprises
SEQ ID NO :63. In some embodiments, the extracellular domain of CD40L consists of
SEQ ID NO :63. In some embodiments, the single chain fusion polypeptide comprises
SEQ ID NO :70. In some embodiments, the single chain fusion polypeptide consists of
SEQ ID NO :70. In some embodiments, the single chain fusion polypeptide comprises
SEQ ID NO :80. In some embodiments, the single chain fusion polypeptide consists of
SEQ ID NO :80.
[0127] In certain embodiments, a polypeptide or agent comprises a variant of the
extracellular domain CD40L amino acid sequence or a fragment thereof that comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) conservative substitutions and is capable of binding CD40.
[0128] In some embodiments, the agent is a polypeptide. In some embodiments, the
polypeptide is a fusion protein. In certain embodiments, the fusion protein comprises at least one copy of the extracellular domain of human CD40L or a fragment thereof, and further comprises a non-CD40L polypeptide. In some embodiments, the fusion protein may include an extracellular domain or fragment thereof linked to a heterologous functional and structural polypeptide including, but not limited to, a human Fc region, one or more protein tags (e.g., myc, FLAG, GST), other endogenous proteins or protein fragments, or any other useful protein sequence including any peptide sequence between the extracellular domain and the non-CD40L polypeptide. In certain embodiments, the non-CD40L polypeptide comprises a human Fc region. The Fc region can be obtained from any of the classes of immunoglobulin, IgG, IgA, IgM, IgD and IgE. In some embodiments, the Fc region is a human IgGl Fc region. In some embodiments, the Fc region is a human IgG2 Fc region. In some embodiments, the Fc region is a wild-type Fc region. In some embodiments, the Fc region is a natural variant of a wild-type Fc region. In some embodiments, the Fc region is a mutated Fc region. In some embodiments, the Fc region is truncated at the N-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, (e.g., in the hinge domain). In some embodiments, the Fc region is truncated at the C- terminal end (e.g., lysine is absent). In some embodiments, an amino acid in the hinge domain is changed to hinder undesirable disulfide bond formation. In some embodiments, a cysteine is replaced with a different amino acid to hinder undesirable disulfide bond formation. In some embodiments, a cysteine is replaced with a serine to hinder undesirable disulfide bond formation.
[0129] It has been shown that glycosylation of a Fc region is essential for optimal
expression of biological activities mediated through Fc receptors (e.g., FcyRI, Fcyll, FcyRIII, FcyRIIB) and the Clq component of complement. These biological activities include, but are not limited to, ADCC and CDC. Modification of the glycosylation site or sites in a Fc region can inhibit and/or eliminate binding of Fc receptors to a Fc region. For example, substitution of the asparagine at a glycosylation site with a different amino acid such as an alanine residue results in deglycosylation of the Fc region and generally results in reduced ADCC activity. In some embodiments, a Fc region is modified to destroy a glycosylation site or sites. In some embodiments, a Fc region is modified to reduce binding of a Fc receptor to a Fc region. In some embodiments, a Fc region is modified to reduce biological activities mediated by binding of a Fc receptor to a Fc region.
[0130] In some embodiments, the Fc region comprises SEQ ID NO:39, SEQ ID NO:40,
SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82.
[0131] In some embodiments, the polypeptide or agent comprises (i) SEQ ID NO:69,
SEQ ID NO:70, or SEQ ID NO:80; and (ii) SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82. In some embodiments, a polypeptide or agent comprises SEQ ID NO:70 and SEQ ID NO:44. In some embodiments, a polypeptide or agent comprises SEQ ID NO:70 and SEQ ID NO:45. In some embodiments, a polypeptide or agent comprises SEQ ID NO:70 and SEQ ID NO:82. In some embodiments, a polypeptide or agent comprises SEQ ID NO:80 and SEQ ID NO:45. In some embodiments, a polypeptide or agent comprises SEQ ID NO:80 and SEQ ID NO:44. In some embodiments, a polypeptide or agent comprises SEQ ID NO:80 and SEQ ID NO:82.
[0132] In some embodiments, the polypeptide or agent is a single chain CD40L trimer-Fc protein. In some embodiments, the polypeptide or agent is a single chain CD40L trimer- IgGl Fc protein. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgGl Fc protein, wherein the single chain CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or 80, and the IgGl Fc comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgGl Fc protein, wherein the single chain CD40L trimer consists of the amino acid sequence of SEQ ID NO:70 or 80, and the IgGl Fc consists of the amino acid sequence of SEQ ID NO:39. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein, wherein the single chain CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or 80, and the IgG2 Fc comprises the amino acid sequence of SEQ ID NO:42 or 43. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein, wherein the single chain CD40L trimer consists of the amino acid sequence of SEQ ID NO:70 or 80, and the IgG2 Fc consists of the amino acid sequence of SEQ ID NO:42 or 43. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgGl Fc protein, wherein the Fc region is modified. In some embodiments, the polypeptide or agent is a single chain CD40L trimer- IgGl Fc protein, wherein the Fc region is deglycosylated. The term "deglycosylation of the Fc region" and "deglycosylated Fc region" as used herein refer to or describe an Fc region comprising a mutation in the N-linked glycosylation site of the CH2 domain. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein, wherein the Fc region is modified. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein, wherein the Fc region is deglycosylated. In some embodiments, the polypeptide or agent comprises SEQ ID NO:72. In some embodiments, the polypeptide or agent comprises SEQ ID NO:81. In some embodiments, the polypeptide or agent consists essentially of SEQ ID NO:72 or SEQ ID NO:81. In some embodiments, the polypeptide or agent consists of SEQ ID NO:72. In some embodiments, the polypeptide or agent consists of SEQ ID NO:81.
In certain embodiments, the non-CD40L polypeptide comprises SEQ ID NO:39,
SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82. In certain embodiments, the non-CD40L polypeptide consists essentially of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82. In certain embodiments, the non-CD40L polypeptide consists of SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82.
[0134] In certain embodiments, the non-CD40L polypeptide comprises an
immunoglobulin heavy chain. In certain embodiments, the immunoglobulin heavy chain is associated with an immunoglobulin light chain. In some embodiments, the
immunoglobulin heavy chain and the immunoglobulin light chain form an antigen- binding site. In certain embodiments, the non-CD40L polypeptide comprises an antibody. In certain embodiments, the non-CD40L polypeptide comprises a single chain antibody or Fab.
[0135] In certain embodiments, a fusion protein comprises at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof and a non- CD40L polypeptide, wherein the C-terminal end of the non-CD40L polypeptide is linked to the extracellular domain(s) of CD40L, wherein at least one of the first, second, or third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In certain embodiments, a fusion protein comprises at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof and a non-CD40L polypeptide, wherein the N- terminal end of the non-CD40L polypeptide is linked to the extracellular domain(s) of CD40L, wherein at least one of the first, second, or third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the first copy of the extracellular domain of CD40L is linked to the C-terminal end of the non-CD40L polypeptide. In some embodiments, the third copy of the extracellular domain of CD40L is linked to the N- terminal end of the non-CD40L polypeptide. In some embodiments, the extracellular domain(s) of CD40L is linked to the C-terminal end of a Fc region. In some
embodiments, the extracellular domain(s) of CD40L is linked to the N-terminal end of a Fc region. In some embodiments, the extracellular domain(s) of CD40L is directly linked to the Fc region (i.e. without an intervening peptide linker). In some embodiments, the extracellular domain(s) of CD40L is linked to the Fc region via a peptide linker. [0136] As used herein, the term "linker" refers to a linker inserted between a first polypeptide (e.g., a extracellular domain of CD40L or a fragment thereof comprising a fragment of the stalk region of CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region) and a second polypeptide (e.g., a Fc region). In some embodiments, the linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the fusion protein. Linkers should not be antigenic and should not elicit an immune response. Suitable linkers are known to those of skill in the art and often include mixtures of glycine and serine residues and often include amino acids that are sterically unhindered. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. Linkers can range in length, for example from 1-50 amino acids in length, 1-22 amino acids in length, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3 amino acids in length. Linkers may include, but are not limited to, SerGly, GGSG, GSGS, GGGS, S(GGS)n where n is 1-7, GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO:50), ESGGGGVT (SEQ ID NO:51), LESGGGGVT (SEQ ID NO:52), GRAQVT (SEQ ID NO:53), WRAQVT (SEQ ID NO:54), and ARGRAQVT (SEQ ID NO:55). In some embodiments, the linker may comprise a cleavage site. In some embodiments, the linker may comprise an enzyme cleavage site, so that the second polypeptide may be separated from the first polypeptide. As used herein, a linker is an intervening peptide sequence that does not include amino acid residues from either the C-terminus or N-terminus of the first polypeptide (e.g., an extracellular domain of CD40L or a fragment thereof comprising a fragment of the stalk region of CD40L, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region) or C-terminus or the N-terminus of the second polypeptide (e.g., the Fc region).
[0137] In some embodiments, a polypeptide (e.g., a fusion polypeptide) described herein comprises: (a) a first polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region; and (b) a second polypeptide comprising a Fc region, wherein the Fc region is deglycosylated. In some embodiments, the fragment of the stalk region of CD40L is selected from the group consisting of: MQKGDQ (SEQ ID NO: 16), FEMQKGDQ (SEQ ID NO: 17), EMQKGDQ (SEQ ID NO: 18), QKGDQ (SEQ ID NO: 19), and KGDQ (SEQ ID NO:20), wherein the fragment of the stalk region comprises a mutation in the integrin- binding region. In some embodiments, the fragment of the stalk region of CD40L is MQKGDQ (SEQ ID NO: 16), wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the fragment of the stalk region of CD40L comprises an amino acid sequence selected from the group consisting of: MQKADQ (SEQ ID NO:59), FEMQKADQ (SEQ ID NO:60), EMQKADQ (SEQ ID NO:61), QKADQ (SEQ ID NO:62), and KADQ (SEQ ID NO:63). In some embodiments, the fragment of the stalk region of CD40L comprises the amino acid sequence of
MQKADQ (SEQ ID NO:59). In some embodiments, at least two of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L. In some embodiments, each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L. In some embodiments, the polypeptide does not comprise an exogenous peptide linker between any of the copies of the extracellular domain or a fragment thereof of CD40L. In some embodiments, at least one of the copies of the extracellular domain or a fragment thereof comprises SEQ ID NO: 15. In some embodiments, at least one of the copies of the extracellular domain or a fragment thereof comprises SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, each of the first, second, and third copies of the
extracellular domain or a fragment thereof comprises SEQ ID NO: 15. In some
embodiments, each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, a polypeptide comprises SEQ ID NO:70. In some embodiments, a polypeptide comprises SEQ ID NO:80. In some embodiments, a polypeptide comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:70. In some embodiments, a polypeptide comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:70. In some embodiments, a polypeptide comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:80. In some embodiments, a polypeptide comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:80. In some embodiments, the first polypeptide is directly linked to the second polypeptide. In some embodiments, the first polypeptide is connected to the second polypeptide by a linker. In some embodiments, the first copy of the extracellular domain of the first polypeptide is linked to the C-terminal end of the second polypeptide. In some embodiments, the third copy of the extracellular domain of the first polypeptide is linked to the N-terminal end of the second polypeptide. In some embodiments, the second polypeptide comprises a human Fc region. In some embodiments, a human Fc region is from an IgGl, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region is selected from the group consisting of: SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the Fc region comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the polypeptide comprises SEQ ID NO:72 or SEQ ID NO:81. In some embodiments, an additional polypeptide is linked to the first polypeptide. In some embodiments, an additional polypeptide is linked to the second polypeptide. In some embodiments, the second polypeptide comprises an immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is associated with an immunoglobulin light chain. In some embodiments, the immunoglobulin heavy chain and immunoglobulin light chain form an antigen-binding site. In some embodiments, the second polypeptide comprises a single chain antibody or a Fab. In some embodiments, the polypeptide comprises a third polypeptide.
[0138] In some embodiments, a polypeptide or agent described herein specifically binds
CD40 and acts as a CD40 agonist. In some embodiments, a polypeptide or agent described herein specifically binds CD40 and activates CD40 signaling. In some embodiments, a polypeptide or agent described herein specifically binds CD40 and induces, activates, promotes, increases, enhances, or prolongs CD40 activity.
[0139] In some embodiments, a polypeptide or agent described herein specifically binds
CD40 and modulates an immune response. In some embodiments, a polypeptide or agent described herein specifically binds CD40 and induces, augments, increases, and/or prolongs an immune response.
[0140] In some embodiments, a polypeptide or agent described herein specifically binds
CD40 with a dissociation constant (KD) of about ΙμΜ or less, about lOOnM or less, about 40nM or less, about 20nM or less, about lOnM or less, about InM or less, or about O. lnM or less. In some embodiments, the polypeptide or agent binds CD40 with a KD of from about 1 μΜ to about O. lnM, or any range of values thereof (e.g., from about Ι μΜ to about InM, from about Ι μΜ to about lOnM, from about Ι μΜ to about 20nM, from about Ι μΜ to about 40nM, from about Ι μΜ to about ΙΟΟηΜ, from about ΙΟΟηΜ to about O. lnM, from about ΙΟΟηΜ to about InM, from about ΙΟΟηΜ to about lOnM, from about ΙΟΟηΜ to about 20nM, from about ΙΟΟηΜ to about 40nM, from about 40nM to about 0. InM, from about 40nM to about InM, from about 40nM to about lOnM, from about 40nM to about 20nM, from about 20nM to about O. lnM, from about 20nM to about InM, from about 20nM to about lOnM, from about lOnM to about O. lnM, from about lOnM to about InM, or from about InM to about O. lnM). In some embodiments, the polypeptide or agent binds CD40 with an KD of about Ι μΜ, about ΙΟΟηΜ, about 40nM, about 20nM, about lOnM, about InM, or about O. lnM. In some embodiments, the polypeptide or agent binds CD40 with a KD of about InM or less. In some embodiments, the polypeptide or agent binds CD40 with a KD of about 0. InM or less. In some embodiments, the polypeptide or agent binds human CD40 and/or mouse CD40 with a KD of about lOnM or less. In some embodiments, the polypeptide or agent binds human CD40 with a KD of about lOnM or less.
[0141] In some embodiments, a polypeptide or agent described herein binds human CD40 and/or mouse CD40 with a KD of about lOnM or less. In some embodiments, a polypeptide or agent described herein binds human CD40 and/or mouse CD40 with a KD of about InM or less. In some embodiments, a polypeptide or agent described herein binds human CD40 and/or mouse CD40 with a KD of about 0. InM or less. In some embodiments, a polypeptide or agent described herein binds human CD40 and does not bind mouse CD40. In some embodiments, a polypeptide or agent described herein binds human CD40 with a KD of about lOnM or less. In some embodiments, a polypeptide or agent described herein binds human CD40 with a KD of about InM or less. In some embodiments, a polypeptide or agent described herein binds human CD40 with a KD of about O. lnM or less.
[0142] In some embodiments, the dissociation constant of the polypeptide or agent to
CD40 is the dissociation constant determined using a CD40 fusion protein comprising at least a portion of a CD40 extracellular domain immobilized on a Biacore chip.
[0143] In some embodiments, a polypeptide or agent binds CD40 with a half maximal effective concentration (EC50) of about Ι μΜ or less, about ΙΟΟηΜ or less, about 40nM or less, about 20nM or less, about lOnM or less, about InM or less, or about O. lnM or less. In one embodiment, a polypeptide or agent binds CD40 with an EC50 of from about Ι μΜ to about O. lnM, or any range of values thereof (e.g., from about Ι μΜ to about InM, from about Ι μΜ to about lOnM, from about Ι μΜ to about 20nM, from about Ι μΜ to about 40nM, from about Ι μΜ to about ΙΟΟηΜ, from about ΙΟΟηΜ to about O. lnM, from about ΙΟΟηΜ to about InM, from about ΙΟΟηΜ to about lOnM, from about ΙΟΟηΜ to about 20nM, from about ΙΟΟηΜ to about 40nM, from about 40nM to about O. lnM, from about 40nM to about InM, from about 40nM to about lOnM, from about 40nM to about 20nM, from about 20nM to about 0. InM, from about 20nM to about InM, from about 20nM to about lOnM, from about lOnM to about O. lnM, from about lOnM to about InM, or from about InM to about O. lnM). In one embodiment, a polypeptide or agent binds CD40 with an EC50 of about Ι μΜ, about ΙΟΟηΜ, about 40nM, about 20nM, about lOnM, about InM, or about O. lnM.
In some embodiments, a polypeptide or agent described herein has an improved therapeutic index compared to a reference polypeptide or reference agent. In some embodiments, a polypeptide or agent described herein induces reduced systemic inflammation compared to a reference polypeptide or reference agent. In some embodiments, a polypeptide or agent described herein induces lower serum liver enzyme level compared to a reference polypeptide or reference agent. In some embodiments, a polypeptide or agent described herein induces lower pro-inflammatory cytokine release than a reference polypeptide or reference agent. In one embodiment, the reference polypeptide or reference agent comprise the same polypeptide as the polypeptide or agent described herein, except that reference polypeptide or reference agent comprise a polypeptide comprising a CD40L stalk region that does not comprise a mutation in the integrin-binding region. In one embodiment, the reduced systemic inflammation is indicated by lower levels of blood neutrophils. In one embodiment, the blood neutrophil level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In one embodiment, the blood neutrophil level is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60%> lower, from about 40% to about 50% lower, from about 50%) to about 70%) lower, from about 50% to about 60%> lower, or from about 60%> to about 70%) lower). In one embodiment, the blood neutrophil level is about 20% lower, about 30%) lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower. In one embodiment, the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In one embodiment, the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In one embodiment, the liver enzyme level is from about 20%) to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60%) lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30%) to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40%) lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50%) to about 60% lower, or from about 60% to about 70% lower). In one embodiment, the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower. In one embodiment, the pro-inflammatory cytokine is selected from the group consisting of: IL-Ιβ, IL-6, TNF- alpha, IP- 10, KC, and ΜΙΡ-Ια. In one embodiment, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In one embodiment, the pro-inflammatory cytokine release is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50%) lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30%) to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60%) to about 70% lower). In one embodiment, the pro-inflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60%) lower, or about 70% lower.
In one aspect, provided herein is a method of improving the therapeutic index of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region. In another aspect, provided herein is a method of decreasing the toxicity of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region. In one embodiment, the human CD40 agonist polypeptide comprises three copies of the extracellular domain of human CD40L or a CD40-binding fragment thereof. In one embodiment, the improving the therapeutic index or decreasing the toxicity comprises induction of reduced systemic inflammation compared to the human CD40 agonist. In one embodiment, the improving the therapeutic index or decreasing the toxicity comprises induction of lower serum liver enzyme level compared to the human CD40 agonist. In one embodiment, the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In one embodiment, the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In one embodiment, the liver enzyme level is from about 20%) to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60%) lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30%) to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40%) lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50%) to about 60% lower, or from about 60% to about 70% lower). In one embodiment, the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower. In one embodiment, the improving the therapeutic index or decreasing the toxicity comprises induction of lower pro-inflammatory cytokine compared to the human CD40 agonist. In one embodiment, the pro-inflammatory cytokine is selected from the group consisting of: IL-Ιβ, IL-6, T F- alpha, IP- 10, KC, and ΜΙΡ-Ια. In one embodiment, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In one embodiment, the pro-inflammatory cytokine release is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30%) to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60%) to about 70% lower). In one embodiment, the pro-inflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60%) lower, or about 70% lower.
[0146] In another aspect, provided herein is an improved human CD40 agonist produced by a method described herein. In another aspect, provided herein is a method of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of the improved human CD40 agonist described herein. In one embodiment, the immune response is against a tumor or cancer. In another aspect, provided herein is a method of inhibiting the growth of a tumor, comprising contacting a tumor or tumor cell with an effective amount of the improved human CD40 agonist described herein. In another aspect, provided herein is a method of inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the improved human CD40 agonist described herein. In another aspect, provided herein is a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of the improved human CD40 agonist described herein.
[0147] In certain embodiments, fusion polypeptides are made using recombinant DNA techniques as known to one skilled in the art. In some embodiments, polynucleotides encoding a specific protein or a fragment thereof are isolated from mammalian cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the gene encoding the protein, and the nucleotide sequence is determined using conventional techniques. The isolated polynucleotides encoding the protein may be cloned into suitable expression vectors which produce the polypeptide when transfected into host cells such as E. coli, simian COS cells, or Chinese hamster ovary (CHO) cells. In other embodiments, recombinant proteins, or fragments thereof, can be isolated from phage display libraries or using other cell surface display techniques. [0148] The polynucleotide(s) encoding a protein can be modified in a number of different manners using recombinant DNA technology to generate alternative or variant proteins. Site-directed or high-density mutagenesis of a protein can be used to optimize specificity, affinity, stability, etc. of a recombinant protein.
[0149] Proteins generally contain a signal sequence that directs the transport of the
proteins. Signal sequences (also referred to as signal peptides or leader sequences) are located at the N-terminus of nascent polypeptides. They target the polypeptide to the endoplasmic reticulum and the proteins are sorted to their destinations, for example, to the inner space of an organelle, to an interior membrane, to the cell outer membrane, or to the cell exterior via secretion. Most signal sequences are cleaved from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum. The cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent upon amino acid residues within the signal sequence. Although there is usually one specific cleavage site, more than one cleavage site may be recognized and/or used by a signal peptidase resulting in a non-homogenous N-terminus of the polypeptide. For example, the use of different cleavage sites within a signal sequence can result in a polypeptide expressed with different N-terminal amino acids. Accordingly, in some embodiments, the polypeptides as described herein may comprise a mixture of polypeptides with different N-termini. In some embodiments, the N-termini differ in length by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, the N-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In some embodiments, the polypeptide is substantially homogeneous, i.e., the polypeptides have the same N-terminus. In some embodiments, the signal sequence of the polypeptide comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) amino acid substitutions and/or deletions as compared to the native sequence of the protein. In some embodiments, the signal sequence of the polypeptide comprises amino acid substitutions and/or deletions that allow one cleavage site to be dominant, thereby resulting in a substantially homogeneous polypeptide with one N-terminus. In some embodiments, the signal sequence of a fusion polypeptide is not the native signal sequence of the protein(s) contained within the fusion polypeptide.
[0150] In certain embodiments, a polypeptide, agent, or fusion polypeptide described herein comprises the Fc region of an immunoglobulin. Those skilled in the art will appreciate that some of the polypeptides or agents of this invention will comprise fusion proteins in which at least a portion of the Fc region has been deleted or otherwise altered so as to provide desired biochemical characteristics, such as increased cancer cell localization, increased tumor penetration, reduced serum half-life, or increased serum half-life, when compared with a fusion protein of approximately the same
immunogenicity comprising a native or unaltered Fc region. Modifications to the Fc region may include additions, deletions, or substitutions of one or more amino acids in one or more domains. The modified fusion proteins disclosed herein may comprise alterations or modifications to one or more of the two heavy chain constant domains (CH2 or CH3) or to the hinge region. In other embodiments, the entire CH2 domain may be removed (ACH2 constructs). In some embodiments, the omitted constant region domain is replaced by a short amino acid spacer (e.g., 10 aa residues) that provides some of the molecular flexibility typically imparted by the absent constant region domain.
[0151] In some embodiments, the modified fusion proteins are engineered to link the
CH3 domain directly to the hinge region or to the first polypeptide. In other
embodiments, a peptide spacer or linker is inserted between the hinge region or the first polypeptide and the modified CH2 and/or CH3 domains. For example, constructs may be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region or first polypeptide with a 5-20 amino acid spacer. Such a spacer may be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers may, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct.
Accordingly, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic so as to maintain the desired biological qualities of the fusion protein.
[0152] In some embodiments, the modified fusion proteins may have only a partial
deletion of a constant domain or substitution of a few or even a single amino acid. For example, the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase cancer cell localization and/or tumor penetration. Similarly, it may be desirable to simply delete that part of one or more constant region domains that control a specific effector function (e.g., complement Clq binding). Such partial deletions of the constant regions may improve selected characteristics of the polypeptide or agent (e.g., serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
Moreover, as alluded to above, the constant regions of the disclosed fusion proteins may be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it may be possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified fusion protein. In certain embodiments, the modified fusion proteins comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function, or provide for more cytotoxin or carbohydrate attachment sites.
[0153] It is known in the art that the constant region mediates several effector functions.
For example, binding of the CI component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of
complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity. In addition, the Fc region can bind to a cell expressing a Fc receptor (FcR). There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
[0154] In some embodiments, the modified fusion proteins provide for altered effector functions that, in turn, affect the biological profile of the polypeptide or agent. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified agent, thereby increasing cancer cell localization and/or tumor penetration. In other embodiments, the constant region modifications increase or reduce the serum half-life of the polypeptide or agent. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moiety attachment sites.
[0155] In certain embodiments, a modified fusion protein does not have one or more effector functions normally associated with an Fc region. In some embodiments, the polypeptide or agent has no antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or no complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the polypeptide or agent does not bind to the Fc receptor and/or complement factors. In certain embodiments, the polypeptide or agent has no effector function normally associated with an Fc region.
[0156] The polypeptides and agents of the present invention can be assayed for specific binding to a target by any method known in the art. The immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore analyses, FACS analyses, immunofluorescence,
immunocytochemistry, Western blot analyses, radioimmunoassays, ELISAs, "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays. Such assays are routine and well-known in the art.
[0157] For example, the specific binding of a test agent (e.g., a polypeptide) to human
CD40 may be determined using ELISA. An ELISA assay comprises preparing CD40 protein, coating wells of a 96-well microtiter plate with the CD40, adding the test agent conjugated to a detectable compound such as an enzymatic substrate (e.g. horseradish peroxidase or alkaline phosphatase) to the well, incubating for a period of time and detecting the presence of the agent bound to CD40. In some embodiments, the test agent is not conjugated to a detectable compound, but instead a labeled secondary antibody that recognizes the agent is added to the well. In some embodiments, instead of coating the well with CD40, the test agent can be coated to the well, soluble CD40 is added, and a second antibody conjugated to a detectable compound that recognizes CD40 can be used to detect binding. One of skill in the art would know which parameters can be modified to increase the signal detected and/or increase the overall efficiency of the assay.
[0158] In another example, the specific binding of a test agent e.g., a polypeptide) to human CD40 may be determined using FACS. A FACS screening assay may comprise generating a cDNA construct that expresses CD40, transfecting the construct into cells, expressing CD40 on the surface of the cells, mixing the test agent with the transfected cells, and incubating for a period of time. The cells bound by the test agent may be identified by using a secondary antibody conjugated to a detectable compound (e.g., PE- conjugated anti-Fc antibody) and a flow cytometer. One of skill in the art would know which parameters can be modified to increase the signal detected and/or increase the overall efficiency of the assay.
[0159] The binding affinity of a test agent to a target (e.g., human CD40) and the off-rate of an agent-target interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled target (e.g., 3H or 125I-labeled CD40), or fragment or variant thereof, with the agent of interest in the presence of increasing amounts of unlabeled target followed by the detection of the agent bound to the labeled target. The affinity of the agent for a target (e.g., human CD40) and the binding off-rates can be determined from the data by
Scatchard plot analysis. In some embodiments, Biacore kinetic analysis is used to determine the binding on and off rates of agents that bind a target (e.g., human CD40). Biacore kinetic analysis comprises analyzing the binding and dissociation of agents from chips with immobilized target (e.g., human CD40) on the chip surface.
[0160] This invention also encompasses homodimenc agents and heterodimeric
agents/molecules. In some embodiments, the homodimeric agents are polypeptides. In some embodiments, the heterodimeric molecules are polypeptides. Generally the homodimeric molecule comprises two identical polypeptides. Generally the heterodimeric molecule comprises two non-identical polypeptides. In some embodiments, a
heterodimeric molecule is capable of binding at least two targets, e.g., a bispecific agent. The targets may be, for example, two different proteins on a single cell or two different proteins on two separate cells. In some embodiments, the bispecific agents are polypeptides. Thus, in some embodiments, one polypeptide of the heterodimeric molecule comprises a polypeptide described herein (e.g., a single chain trimer-Fc protein that binds CD40) and one polypeptide of the heterodimeric molecule is an antibody. The term "arm" may be used herein to describe the structure of a homodimeric agent, a heterodimeric agent, and/or a bispecific agent. As used herein, each "arm" is directed against a target. In some embodiments, one "arm" may comprise an antigen-binding site from an antibody. In some embodiments, one "arm" may comprise a binding portion of a receptor. In some embodiments, a homodimeric agent comprises two identical am s. In some embodiments, a heterodimeric agent comprises two different arms. In some embodiments, a bispecific agent comprises two different arms. [0161] In some embodiments, a bispecific agent comprises the polypeptides or agents described herein. In some embodiments, the bispecific agent is a homodimeric protein. In some embodiments, the homodimer bispecific agent comprises a polypeptide comprising a heavy chain immunoglobulin and a CD40L trimer. In some embodiments, the heavy chain immunoglobulin is associated with a light chain to form an antigen-binding site. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain CD40L trimer. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising a single-chain antibody and a single chain CD40L trimer. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds a tumor antigen. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds an antigen on an immune cell. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds PD-1, PD-L1, CTLA-4, LAG-3, GITR, OX-40, TIGIT, TEVI3, or B7-H4. In some embodiments, the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
[0162] In some embodiments, the bispecific agent is a heterodimeric protein. In some embodiments, the heterodimeric bispecific agent comprises an antigen-binding site from an antibody (e.g., an antigen-binding site formed by an immunoglobulin heavy chain and an immunoglobulin light chain) and a CD40L trimer. In certain embodiments, a bispecific agent comprises an immune response stimulating agent or functional fragment thereof and a CD40L trimer. In some embodiments, the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
[0163] In some embodiments, a heterodimeric bispecific agent is capable of binding one target and also comprises a "non-binding" function. Thus in some embodiments, one polypeptide of the heterodimeric bispecific agent comprises a polypeptide described herein (e.g., binds CD40) and one polypeptide of the heterodimeric agent is an additional immune response stimulating agent. As used herein, the phrase "immune response stimulating agent" is used in the broadest sense and refers to a substance that directly or indirectly stimulates the immune system by inducing activation or increasing activity of any of the immune system's components. For example, immune response stimulating agents may include cytokines, as well as various antigens including tumor antigens, and antigens derived from pathogens. In some embodiments, the immune response stimulating agent includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), an antibody that blocks immunosuppressive functions (e.g., an anti-CTLA-4 antibody, anti-CD28 antibody, anti-PD-1 antibody, anti-PD-Ll antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86).
[0164] In some embodiments, a heterodimeric bispecific agent comprises a first
polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds a tumor antigen. A bispecific agent with a binding specificity for a tumor antigen can be used to direct the CD40L trimer polypeptide to a tumor. For example the bispecific agent may be used to direct the CD40L trimer polypeptide to a tumor that expresses the tumor antigen or overexpresses the tumor antigen. This may be useful to induce and/or enhance an immune response near or within the tumor microenvironment. In some embodiments, a bispecific agent may be used to induce or enhance the activity of tumor infiltrating immune cells. In some embodiments, the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
[0165] In some embodiments, a heterodimeric bispecific agent comprises a first
polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds an immune response molecule. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds an immune checkpoint protein. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds PD-1. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds PD-L1. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds B7-H4. In some embodiments, the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
[0166] In some embodiments, a bispecific agent, either heterodimeric or homodimeric, has enhanced potency as compared to an individual agent. It is known to those of skill in the art that any agent (e.g., a soluble protein or a cytokine) may have unique
pharmacokinetics (PK) (e.g., circulating half-life). In some embodiments, a bispecific agent has the ability to synchronize the PK of two active agents and/or polypeptides wherein the two individual agents and/or polypeptides have different PK profiles. In some embodiments, a bispecific molecule has the ability to concentrate the actions of two agents and/or polypeptides in a common area (e.g., a tumor and/or tumor
microenvironment). In some embodiments, a bispecific molecule has the ability to concentrate the actions of two agents and/or polypeptides to a common target (e.g., a tumor or a tumor cell). In some embodiments, a bispecific agent has the ability to target the actions of two agents and/or polypeptides to more than one biological pathway or more than one aspect of the immune response. In some embodiments, the bispecific agent has decreased toxicity and/or side effects than either of the polypeptides and/or agents alone. In some embodiments, the bispecific agent has decreased toxicity and/or side effects as compared to a mixture of the two individual polypeptides and/or agents. In some embodiments, the bispecific agent has an increased therapeutic index. In some embodiments, the bispecific agent has an increased therapeutic index as compared to a mixture of the two individual polypeptides and/or agents or the polypeptides and/or agents as single agents.
[0167] It is believed that a single chain CD40L trimer molecule may be more active than an anti-CD40 agonist antibody, because a single chain CD40L trimer would function, i.e., bind to three CD40 molecules, in a very similar manner as three native CD40L molecules. In contrast, an anti-CD40 antibody is able to engage only two CD40 molecules, thereby reducing any potential effect. To make a bispecific molecule comprising an antibody, generally involves the antibody being monovalent (i.e., one-armed antibody). This reduces, if not completely eliminates, the effect of an agonist antibody, especially if activation depends upon clustering of the target molecules. A single chain CD40L trimer is able to bind three CD40 molecules, thus as part of a heterodimeric or homodimeric bispecific molecule, it does not lose any functionality or potency. In some embodiments, the CD40L trimer comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
[0168] In some embodiments, a heterodimeric bispecific molecule comprises a first
polypeptide comprising a single chain CD40L trimer and a second polypeptide comprising an antagonist antibody, wherein the single chain CD40L trimer comprises a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the CD40L trimer comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
In some embodiments, a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the stalk region comprises a mutation in the integrin-binding region, and (b) a second arm comprising an antigen-binding site from an antibody. In some embodiments, a heterodimeric bispecific agent comprises: (a) a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the stalk region comprises a mutation in the integrin-binding region, and (b) a second arm comprising an immune response stimulating agent. In some embodiments, at least one copy of the extracellular domain of CD40L of the first arm comprises SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, at least one copy of the extracellular domain of CD40L of the first arm comprises the amino acid sequence of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68. In some embodiments, the bispecific agent comprises a first arm comprising SEQ ID NO:28 or SEQ ID NO:36, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region. In some embodiments, the bispecific agent comprises a first arm comprising the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80. In some embodiments, the heterodimeric bispecific agent comprises a first arm which further comprises a non-CD40L polypeptide. In some embodiments, the heterodimeric bispecific agent comprises a single chain fusion CD40L polypeptide described herein which is directly linked to a non-CD40L polypeptide. In some embodiments, the single chain fusion polypeptide is connected to the non-CD40L polypeptide by a linker. In some embodiments, the non-CD40L polypeptide comprises a human Fc region. In some embodiments, the non-CD40L polypeptide comprises SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:82. In some embodiments, the non-CD40L polypeptide comprises SEQ ID NO:44. In some embodiments, the non-CD40L polypeptide comprises SEQ ID NO:45. In some embodiments, the non-CD40L polypeptide comprises SEQ ID NO:82.
[0170] In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain CD40 trimer and a second polypeptide comprising an immune response stimulating agent.
[0171] In some embodiments, the multimeric molecule (e.g., a bispecific agent)
comprises a first CH3 domain and a second CH3 domain, each of which is modified to promote formation of heteromultimers or heterodimers. In some embodiments, the first and second CH3 domains are modified using a knobs-into-holes technique. In some embodiments, the first and second CH3 domains comprise changes in amino acids that result in altered electrostatic interactions. In some embodiments, the first and second CH3 domains comprise changes in amino acids that result in altered hydrophobic/hydrophilic interactions (see, for example, U.S. Patent App. Publication No. 2011/0123532).
[0172] In some embodiments, the heterodimeric molecule (e.g., a bispecific agent)
comprises heavy chain constant regions selected from the group consisting of: (a) a first human IgGl constant region, wherein the amino acids at positions corresponding to positions 253 and 292 of SEQ ID NO:46 are replaced with glutamate or aspartate, and a second human IgGl constant region, wherein the amino acids at positions corresponding to 240 and 282 of SEQ ID NO:46 are replaced with lysine; (b) a first human IgG2 constant region, wherein the amino acids at positions corresponding to positions 249 and 288 of SEQ ID NO:47 are replaced with glutamate or aspartate, and a second human IgG2 constant region wherein the amino acids at positions corresponding to positions 236 and 278 of SEQ ID NO:47 are replaced with lysine; (c) a first human IgG3 constant region, wherein the amino acids at positions corresponding to positions 300 and 339 of SEQ ID NO:48 are replaced with glutamate or aspartate, and a second human IgG3 constant region wherein the amino acids at positions corresponding to positions 287 and 329 of SEQ ID NO:48 are replaced with lysine; and (d) a first human IgG4 constant region, wherein the amino acids at positions corresponding to positions 250 and 289 of SEQ ID NO:49 are replaced with glutamate or aspartate, and a second IgG4 constant region wherein the amino acids at positions corresponding to positions 237 and 279 of SEQ ID NO:49 are replaced with lysine.
[0173] In some embodiments, the heterodimeric molecule (e.g., a bispecific agent)
comprises heavy chain CH2 and CH3 domains selected from the group consisting of: (a) a first human IgGl CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 253 and 292 of SEQ ID NO:46 are replaced with glutamate or aspartate, and a second human IgGl CH2 and CH3 domain, wherein the amino acids at positions corresponding to 240 and 282 of SEQ ID NO:46 are replaced with lysine; (b) a first human IgG2 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 249 and 288 of SEQ ID NO:47 are replaced with glutamate or aspartate, and a second human IgG2 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 236 and 278 of SEQ ID NO:47 are replaced with lysine; (c) a first human IgG3 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 300 and 339 of SEQ ID NO:48 are replaced with glutamate or aspartate, and a second human IgG3 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 287 and 329 of SEQ ID NO:48 are replaced with lysine; and (d) a first human IgG4 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 250 and 289 of SEQ ID NO:49 are replaced with glutamate or aspartate, and a second IgG4 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 237 and 279 of SEQ ID NO:49 are replaced with lysine.
[0174] In some embodiments, the polypeptides or agents are monovalent. In some
embodiments, the polypeptide or agent is a soluble protein that is monovalent. In some embodiments, the polypeptides or agents described herein are bivalent. In some embodiments, the polypeptides or agents described herein are trivalent. In some embodiments, the polypeptides or agents described herein are monospecific. In some embodiments, the polypeptides or agents described herein are bispecific. In some embodiments, the polypeptides or agents described herein are multispecific. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is monovalent. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is bivalent. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is trivalent (i.e., binds three target molecules).
[0175] In some embodiments, the polypeptides or agents comprise polypeptides that are substantially homologous to the fusion proteins and/or polypeptides described herein. These agents can contain, for example, conservative substitution mutations, i.e. the substitution of one or more amino acids by similar amino acids. For example,
conservative substitution refers to the substitution of an amino acid with another within the same general class such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art and described herein.
[0176] In certain embodiments, a polypeptide or agent described herein binds CD40 and modulates an immune response. In some embodiments, a polypeptide or agent described herein activates and/or increases an immune response. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances cell-mediated immunity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances innate cell-mediated immunity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances adaptive cell- mediated immunity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances APC activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances adaptive dendritic cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances T-cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances CD4+ T-cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances CD8+ T-cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances CTL activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances K cell activity. In some
embodiments, a polypeptide or agent described herein increases, promotes, or enhances lymphokine-activated killer cell (LAK) activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances tumor-infiltrating lymphocyte (TIL) activity. In some embodiments, a polypeptide or agent described herein inhibits or decreases Treg cell activity. In some embodiments, a polypeptide or agent described herein inhibits or decreases MDSC cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances tumor cell killing. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances the inhibition of tumor growth. In some embodiments, a polypeptide or agent described herein increases or enhances an effective immune response without causing substantial side effects and/or immune-based toxicities. In some embodiments, a polypeptide or agent described herein increases or enhances an effective immune response without causing cytokine release syndrome (CRS) or a cytokine storm.
[0177] In some embodiments, a polypeptide or agent described herein binds CD40 and induces, enhances, increases, and/or prolongs CD40 signaling.
[0178] In certain embodiments, a polypeptide or agent described herein is an agonist
(either directly or indirectly) of human CD40. In some embodiments, a polypeptide or agent is an agonist of CD40 and activates and/or increases an immune response. In some embodiments, a polypeptide or agent is an agonist of CD40 and activates and/or increases activity of APCs (e.g., dendritic cells, B-cells). In some embodiments, a polypeptide or agent is an agonist of CD40 and activates and/or increases activity of K cells and/or T- cells (e.g., cytolytic activity or cytokine production). In certain embodiments, a polypeptide or agent increases the activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%). In certain embodiments, a polypeptide or agent increases the activity by from about 10%) to about 100%, or any range of values thereof (e.g., from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, from about 10% to about 30%), from about 10% to about 20%, from about 20% to about 100%, from about 20% to about 90%, from about 20% to about 75%, from about 20% to about 50%, from about 20% to about 30%, from about 30% to about 100%, from about 30% to about 90%, from about 30% to about 75%, from about 30% to about 50%, from about 50% to about 100%, from about 50% to about 90%, from about 50% to about 75%, from about 75% to about 100%, from about 75% to about 90%, or from about 90% to about 100%). In certain embodiments, a polypeptide or agent increases the activity by about 10%, about 20%, about 30%, about 50%, about 75%, about 90%, or about 100%. [0179] In certain embodiments, a polypeptide or agent described herein increases activation of a NK cell. In certain embodiments, a polypeptide or agent increases activation of a T-cell. In certain embodiments, the activation of a K cell and/or a T-cell by a polypeptide or agent results in an increase in the level of activation of a NK cell and/or a T-cell of at least about 10%, at least about 25%, at least about 50%, at least about 75%), at least about 90%, or at least about 95%. In certain embodiments, the activation of a NK cell and/or a T-cell by a polypeptide or agent results in an increase in the level of activation of a NK cell and/or a T-cell of from about 10% to about 95%, or any range of values thereof (e.g., from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, from about 10% to about 25%, from about 25% to about 95%, from about 25% to about 90%, from about 25% to about 75%, from about 25% to about 50%, from about 50% to about 95%, from about 50% to about 90%, from about 50% to about 75%), from about 75% to about 95%, from about 75% to about 90%, or from about 90%) to about 95%). In certain embodiments, the activation of a NK cell and/or a T-cell by a polypeptide or agent results in an increase in the level of activation of a NK cell and/or a T-cell of about 10%, about 25%, about 50%, about 75%, about 90%, or about 95%.
[0180] In certain embodiments, a polypeptide or agent described herein inhibits or
decreases the suppressive activity of a Treg cell. In certain embodiments, a polypeptide or agent inhibits activity of a Treg cell. In certain embodiments, the inhibition of suppressive activity of a Treg cell by a polypeptide or agent results in an inhibition of suppressive activity of a Treg cell of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In certain embodiments, the inhibition of suppressive activity of a Treg cell by a polypeptide or agent results in an inhibition of suppressive activity of a Treg cell of from about 10% to about 95%, or any range of values thereof (e.g., from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, from about 10% to about 25%, from about 25% to about 95%), from about 25% to about 90%, from about 25% to about 75%, from about 25% to about 50%, from about 50% to about 95%, from about 50% to about 90%, from about 50%) to about 75%, from about 75% to about 95%, from about 75% to about 90%, or from about 90% to about 95%). In certain embodiments, the inhibition of suppressive activity of a Treg cell by a polypeptide or agent results in an inhibition of suppressive activity of a Treg cell of about 10%, about 25%, about 50%, about 75%, about 90%, or about 95%.
[0181] In certain embodiments, a polypeptide or agent described herein inhibits or
decreases the suppressive activity of a MDSC. In certain embodiments, a polypeptide or agent inhibits activity of a MDSC. In certain embodiments, the inhibition of suppressive activity of a MDSC by a polypeptide or agent results in an inhibition of suppressive activity of a MDSC of at least about 10%, at least about 25%, at least about 50%, at least about 75%), at least about 90%, or at least about 95%. In certain embodiments, the inhibition of suppressive activity of a MDSC by a polypeptide or agent results in an inhibition of suppressive activity of a MDSC of from about 10% to about 95%, or any range of values thereof (e.g., from about 10% to about 90%, from about 10% to about 75%, from about 10% to about 50%, from about 10% to about 25%, from about 25% to about 95%), from about 25% to about 90%, from about 25% to about 75%, from about 25% to about 50%, from about 50% to about 95%, from about 50% to about 90%, from about 50%) to about 75%, from about 75% to about 95%, from about 75% to about 90%, or from about 90% to about 95%). In certain embodiments, the inhibition of suppressive activity of a MDSC by a polypeptide or agent results in an inhibition of suppressive activity of a MDSC of about 10%, about 25%, about 50%, about 75%, about 90%, or about 95%.
[0182] In vivo and in vitro assays for determining whether a polypeptide or agent (or candidate binding agent) modulates an immune response are known in the art or are being developed. In some embodiments, a functional assay that detects T-cell activation can be used. In some embodiments, a functional assay that detects Treg activity can be used. In some embodiments, a functional assay that detects MDSC activity can be used. In some embodiments, a functional assay that detects K cell activity can be used. In some embodiments, a functional assay that detects cytolytic T-cell activity can be used. In some embodiments, an assay that detects cytokine production can be used. In some
embodiments, an assay that detects cytokine-producing cells can be used.
[0183] In certain embodiments, a polypeptide or agent described herein is capable of inhibiting tumor growth. In certain embodiments, the polypeptide or agent is capable of inhibiting tumor growth in vivo (e.g., in a mouse model and/or in a human having cancer). [0184] In certain embodiments, a polypeptide or agent described herein is capable of reducing the tumorigenicity of a tumor. In certain embodiments, the polypeptide or agent is capable of reducing the tumorigenicity of a tumor in an animal model, such as a mouse model. In certain embodiments, the polypeptide or agent is capable of reducing the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a mouse model. In certain embodiments, the number or frequency of cancer stem cells in a tumor is reduced by at least about two-fold, about three-fold, about five-fold, about tenfold, about 50-fold, about 100-fold, or about 1000-fold. In certain embodiments, the reduction in the number or frequency of cancer stem cells is determined by limiting dilution assay using an animal model. Additional examples and guidance regarding the use of limiting dilution assays to determine a reduction in the number or frequency of cancer stem cells in a tumor can be found, e.g., in International Publication Number WO 2008/042236; U.S. Patent Publication No. 2008/0064049; and U.S. Patent Publication No. 2008/0178305.
[0185] In certain embodiments, a polypeptide or agent described herein has one or more of the following effects: inhibits proliferation of tumor cells, inhibits tumor growth, reduces the tumorigenicity of a tumor, reduces the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, triggers cell death of tumor cells, increases cell contact-dependent growth inhibition, increases tumor cell apoptosis, reduces epithelial mesenchymal transition (EMT), or decreases survival of tumor cells. In some embodiments, the polypeptide or agent has one or more of the following effects: inhibits viral infection, inhibits chronic viral infection, reduces viral load, triggers cell death of virus-infected cells, or reduces the number or percentage of virus-infected cells.
[0186] In certain embodiments, a polypeptide or agent described herein has a circulating half-life in mice, rats, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, or at least 3 weeks. In certain embodiments, the polypeptide or agent is an IgG (e.g., IgGl or IgG2) fusion protein that has a circulating half-life in mice, rats, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, at least about 2 weeks, or at least 3 weeks. Methods of increasing (or decreasing) the half- life of agents such as polypeptides and soluble receptors are known in the art. For example, known methods of increasing the circulating half-life of IgG fusion proteins include the introduction of mutations in the Fc region which increase the pH-dependent binding of the antibody to the neonatal Fc receptor (FcRn) at pH 6.0. Known methods of increasing the circulating half-life of soluble receptors lacking a Fc region include such techniques as PEGylation.
[0187] In some embodiments of the present invention, the agent is a polypeptide. The polypeptide can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide that binds CD40. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the structure or function of the protein. Thus, the invention also includes variations of the polypeptides which show substantial binding activity to CD40. In some embodiments, amino acid sequence variations of the polypeptides include deletions, insertions, inversions, repeats, and/or other types of substitutions.
[0188] The polypeptides, analogs and variants thereof, can be further modified to contain additional chemical moieties not normally part of the polypeptide. The derivatized moieties can improve the solubility, the biological half-life, and/or absorption of the polypeptide. The moieties can also reduce or eliminate undesirable side effects of the polypeptides and variants. An overview for chemical moieties can be found in
Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.
[0189] The polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof.
[0190] In some embodiments, a DNA sequence encoding a polypeptide of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer.
Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
[0191] Once assembled (by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
[0192] In certain embodiments, a recombinant expression vector is used to amplify and express DNA encoding a polypeptide or agent described herein. For example, a recombinant expression vector can be a replicable DNA construct which has synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an agent operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are
"operatively linked" when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In other embodiments, where recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
[0193] The choice of an expression control sequence and an expression vector depends upon the choice of host. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as Ml 3 and other filamentous single-stranded DNA phages.
[0194] Suitable host cells for expression of a polypeptide (or a protein to use as a target) include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, for example E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known by those skilled in the art.
[0195] Various mammalian cell culture systems are used to express recombinant
polypeptides. Expression of recombinant proteins in mammalian cells can be preferred because such proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), CI 27 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), and HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
[0196] Expression of recombinant proteins in insect cell culture systems (e.g.,
baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
[0197] Thus, the present invention provides cells comprising the polypeptides and agents described herein. In some embodiments, the cells produce the polypeptides and agents described herein. In certain embodiments, the cells produce a fusion protein. In some embodiments, the cells produce a soluble receptor/ligand. In some embodiments, the cells produce an antibody. In some embodiments, the cells produce a bispecific agent. In some embodiments, the cells produce a bispecific antibody. In some embodiments, the cells produce a homodimeric bispecific agent. In some embodiments, the cells produce a heterodimeric bispecific agent.
[0198] The proteins produced by a transformed host can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance ( MR), high performance liquid chromatography (HPLC), and x-ray crystallography.
[0199] In some embodiments, supernatants from expression systems which secrete
recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification. In some embodiments, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In some embodiments, a
hydroxyapatite media can be employed, including but not limited to, ceramic
hydroxyapatite (CHT). In certain embodiments, one or more reverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a polypeptide or agent. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.
[0200] In some embodiments, recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. HPLC can be employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
[0201] In certain embodiments, a polypeptide or agent described herein is a polypeptide that does not comprise an immunoglobulin Fc region. In certain embodiments, the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, protein G, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin. A variety of methods for identifying and producing non- antibody polypeptides that bind with high affinity to a protein target are known in the art. In certain embodiments, phage display technology may be used to produce and/or identify a binding polypeptide. In certain embodiments, mammalian cell display technology may be used to produce and/or identify a binding polypeptide.
[0202] It can further be desirable to modify a polypeptide in order to increase (or
decrease) its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the polypeptide by mutation of the appropriate region in the polypeptide or by incorporating the epitope into a peptide tag that is then fused to the polypeptide at either end or in the middle (e.g., by DNA or peptide synthesis). [0203] Heteroconjugate molecules are also within the scope of the present invention.
Heteroconjugate molecules are composed of two covalently joined polypeptides. Such molecules have, for example, been proposed to target immune cells to unwanted cells, such as tumor cells. It is also contemplated that the heteroconjugate molecules can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
[0204] In certain embodiments, a polypeptide or agent described herein can be used in any one of a number of conjugated (i.e. an immunoconjugate or radioconjugate) or non- conjugated forms. In certain embodiments, the polypeptides or agents can be used in a non-conjugated form to harness the subject's natural defense mechanisms including CDC and ADCC to eliminate malignant or cancer cells.
[0205] In certain embodiments, an agent described herein is a small molecule. The term
"small molecule" generally refers to a low molecular weight organic compound which is by definition not a peptide/protein.
[0206] In some embodiments, a polypeptide or agent described herein is conjugated to a cytotoxic agent. In some embodiments, the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents. In some embodiments, the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In some embodiments, the cytotoxic agent is a radioisotope to produce a radioconjugate or a radioconjugated agent. A variety of radionuclides are available for the production of radioconjugated agents including, but not limited to, 90Y, 125I, 131I, 123I, U1ln, 131In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re,
188 212
Re, and Bi. Conjugates of a polypeptide or agent and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, can also be used. Conjugates of a polypeptide or agent and cytotoxic agent are made using a variety of bifunctional protein- coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as
glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
III. Polynucleotides
In certain embodiments, the invention encompasses polynucleotides comprising polynucleotides that encode a polypeptide or agent described herein. The term
"polynucleotides that encode a polypeptide" encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences. The polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand. In one embodiment, a polypeptide or agent described herein comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the CD40L stalk region, and wherein the stalk region comprises a mutation in the integrin-binding region. In one embodiment, each of the first, second, and third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, wherein the stalk region comprises a mutation in the integrin-binding region. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, a polypeptide or agent described herein further comprises an Fc Region. In one embodiment, the Fc Region is a human Fc region. In one embodiment, the Fc region is deglycosylated. In one
embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81. In one embodiment, the agent is a homodimer. In one embodiment, the agent is a heterodimer.
In certain embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:78, and SEQ ID NO:79, wherein the integrin binding region comprises at least one mutation. In one embodiment, the integrin binding region comprises or consists of the RGD motif (residues 114-116 of SEQ ID NO: 1). In one embodiment, the at least one mutation is a substitution of a residue in the RGD motif. In one embodiment, the at least one mutation is a G to A or a D to E substitution in the RGD motif. In certain
embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77. In certain embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:57, and SEQ ID NO:58, wherein the integrin binding region comprises at least one mutation. In one embodiment, the integrin binding region comprises or consists of the KGD motif (residues 115-117 of SEQ ID NO: 12). In one embodiment, the at least one mutation is a substitution in the KGD motif. In one embodiment, the at least one mutation is a G to A or D to E substitution in the KGD motif. In certain embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72, SEQ ID NO: 80, and SEQ ID NO:81. In some embodiments, a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the CD40-binding agents described herein. In some embodiments, a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the CD40-binding agents described herein and a signal sequence. In some embodiments, a vector comprises the polynucleotide. In some embodiments, a cell comprises the polynucleotide. In some embodiments, a cell comprises the vector. In some embodiments, the cell is isolated. [0209] In certain embodiments, a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77. In certain
embodiments, a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:80, and SEQ ID NO:81.
[0210] Also provided is a polynucleotide that comprises a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77. Also provided is a
polynucleotide that comprises a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:80, and SEQ ID NO:81. In certain embodiments, the hybridization is under conditions of high stringency as known to those of skill in the art.
[0211] In certain embodiments, a polynucleotide comprises the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a pre-protein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also encode for a pro-protein which is the mature protein plus additional 5' amino acid residues. A mature protein having a pro-sequence is a pro-protein and is an inactive form of the protein. Once the pro-sequence is cleaved an active mature protein remains.
[0212] In certain embodiments, a polynucleotide comprises the coding sequence for the mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, for purification of the encoded polypeptide. For example, the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. In some embodiments, the marker sequence is a FLAG-tag, a peptide of sequence DYKDDDDK (SEQ ID NO:56) which can be used in conjunction with other affinity tags.
[0213] The present invention further relates to variants of the hereinabove described polynucleotides encoding, for example, fragments, analogs, and/or derivatives.
[0214] In certain embodiments, the present invention provides a polynucleotide
comprising a polynucleotide having a nucleotide sequence at least about 80% identical, at least about 85%> identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding a polypeptide comprising a polypeptide or agent described herein.
[0215] As used herein, the phrase a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
[0216] The polynucleotide variants can contain alterations in the coding regions, non- coding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli). In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.
[0217] In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.
[0218] In some embodiments, at least one polynucleotide variant is produced (without changing the amino acid sequence of the encoded polypeptide) to increase production of a heterodimeric molecule. In some embodiments, at least one polynucleotide variant is produced (without changing the amino acid sequence of the encoded polypeptide) to increase production of a bispecific agent, a bispecific antibody, or a heterodimeric agent.
[0219] In certain embodiments, the polynucleotides are isolated. In certain embodiments, the polynucleotides are substantially pure.
[0220] Vectors and cells comprising the polynucleotides described herein are also
provided. In some embodiments, an expression vector comprises a polynucleotide molecule. In some embodiments, a host cell comprises an expression vector comprising the polynucleotide molecule. In some embodiments, a host cell comprises a
polynucleotide molecule.
IV. Methods of use and pharmaceutical compositions
[0221] The polypeptides or agents of the invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as immunotherapy for cancer. In certain embodiments, a polypeptide or agent described herein is useful for activating, promoting, increasing, and/or enhancing an immune response, inhibiting tumor growth, reducing tumor volume, inducing tumor regression, increasing tumor cell apoptosis, and/or reducing the tumorigenicity of a tumor. In certain embodiments, the polypeptides or agents of the invention are also useful for immunotherapy against pathogens, such as viruses. In certain embodiments, a polypeptide or agent described herein is useful for inhibiting viral infection, reducing viral infection, increasing virally- infected cell apoptosis, and/or increasing killing of virus-infected cells. The methods of use may be in vitro, ex vivo, or in vivo methods. In one embodiment, a polypeptide or agent described herein comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the CD40L stalk region, and wherein the stalk region comprises a mutation in the integrin-binding region. In one embodiment, each of the first, second, and third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, wherein the stalk region comprises a mutation in the integrin-binding region. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, a polypeptide or agent described herein further comprises an Fc Region. In one embodiment, the Fc Region is a human Fc region. In one embodiment, the Fc region is deglycosylated. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81. In one embodiment, the agent is a homodimer. In one embodiment, the agent is a heterodimer.
The present invention provides methods for activating an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the invention provides methods for promoting an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the invention provides methods for increasing an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the invention provides methods for enhancing an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing cell-mediated immunity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing antigen-presenting cell (APC) activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing Thl-type responses. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD4+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD8+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of Treg cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of MDSCs. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing the number of the percentage of memory T-cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term immune memory function. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term memory. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of substantial side effects and/or immune-based toxicities. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of cytokine release syndrome (CRS) or a cytokine storm. In some embodiments, the immune response is a result of antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor cell. In some embodiments, the antigenic stimulation is cancer. In some
embodiments, the antigenic stimulation is a pathogen. In some embodiments, the antigenic stimulation is a virally-infected cell. In one embodiment, a polypeptide or agent described herein comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the CD40L stalk region, and wherein the stalk region comprises a mutation in the integrin-binding region. In one embodiment, each of the first, second, and third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, wherein the stalk region comprises a mutation in the integrin-binding region. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, a polypeptide or agent described herein further comprises an Fc Region. In one embodiment, the Fc Region is a human Fc region. In one embodiment, the Fc region is deglycosylated. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81. In one embodiment, the agent is a homodimer. In one embodiment, the agent is a heterodimer.
[0223] In vivo and in vitro assays for determining whether an agent or polypeptide
modulates, activates, or inhibits an immune response are known in the art and are continually being revised and/or developed.
[0224] In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent binds human CD40. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain integrin binding-region mutant CD40L trimer. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In some embodiments, the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region. In some embodiments, the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region, wherein the Fc region is deglycosylated. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
[0225] In certain embodiments of the methods described herein, a method of activating or enhancing a persistent or long-term immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human CD40. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds CD40. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain integrin binding-region mutant CD40L trimer. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In some embodiments, the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region. In some embodiments, the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region, wherein the Fc region is deglycosylated. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO: 72 or 81.
In certain embodiments of the methods described herein, a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human CD40. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain integrin binding-region mutant CD40L trimer. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In some embodiments, the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region. In some embodiments, the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region, wherein the Fc region is deglycosylated. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
[0227] In certain embodiments of the methods described herein, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human CD40. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain integrin binding-region mutant CD40L trimer. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In some embodiments, the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region. In some embodiments, the polypeptide comprises a single chain integrin binding-region mutant CD40L trimer and a Fc region, wherein the Fc region is deglycosylated. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
[0228] The present invention also provides methods for inhibiting growth of a tumor using a polypeptide or agent described herein. In certain embodiments, the method of inhibiting growth of a tumor comprises contacting a cell mixture with a polypeptide or agent in vitro. For example, an immortalized cell line or a cancer cell line mixed with immune cells (e.g., APCs, T-cells, cytolytic T-cells, or K cells) is cultured in medium to which is added a test agent. In some embodiments, tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample, mixed with immune cells (e.g., APCs, T-cells, cytolytic T-cell, and/or NK cells), and cultured in medium to which is added a test agent. In some embodiments, the polypeptide or agent increases, promotes, and/or enhances the activity of the immune cells. In some embodiments, the polypeptide or agent inhibits tumor cell growth. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81. [0229] In some embodiments, the method of inhibiting growth of a tumor comprises contacting the tumor or tumor cells with a polypeptide or agent described herein in vivo. In certain embodiments, contacting a tumor or tumor cell with a polypeptide or agent is undertaken in an animal model. For example, a test agent may be administered to mice which have tumors. In some embodiments, the polypeptide or agent increases, promotes, and/or enhances the activity of immune cells in the mice. In some embodiments, the polypeptide or agent inhibits tumor growth. In some embodiments, the polypeptide or agent is administered at the same time or shortly after introduction of tumor cells into the animal to prevent tumor growth ("preventative model"). In some embodiments, the polypeptide or agent is administered as a therapeutic after tumors have grown to a specified size ("therapeutic model"). In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
[0230] In certain embodiments, the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or the subject had a tumor which was removed. In one
embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO: 72 or 81.
[0231] In addition, the invention provides a method of inhibiting growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of the polypeptide or agent. In some embodiments, a method of reducing the frequency of cancer stem cells in a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent is provided. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
[0232] In addition, the invention provides a method of reducing the tumorigenicity of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In certain embodiments, the tumor comprises cancer stem cells. In some embodiments, the tumorigenicity of a tumor is reduced by reducing the frequency of cancer stem cells in the tumor. In some
embodiments, the methods comprise using the polypeptides or agents described herein. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of a polypeptide or agent. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
In one embodiment, the administration of the polypeptide or agent described herein induces reduced systemic inflammation compared to a reference polypeptide or reference agent. In one embodiment, the reduced systemic inflammation is indicated by lower levels of blood neutrophils. In one embodiment, the blood neutrophil level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In one embodiment, the blood neutrophil level is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60%> lower, from about 20% to about 50% lower, from about 20%) to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40%) to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70%) lower, from about 50% to about 60% lower, or from about 60% to about 70% lower). In one embodiment, the blood neutrophil level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower. In one embodiment, the administration of the polypeptide or agent described herein induces lower serum liver enzyme level compared to a reference polypeptide or reference agent. In one embodiment, the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP). In one embodiment, the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In one embodiment, the liver enzyme level is from about 20% to about 70%) lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20%) to about 30% lower, from about 30% to about 70% lower, from about 30% to about 60%) lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60%> lower, from about 40%) to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60%) lower, or from about 60% to about 70% lower). In one embodiment, the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60%) lower, or about 70% lower. In one embodiment, the administration of the polypeptide or agent described herein induces lower pro-inflammatory cytokine release than a reference polypeptide or reference agent. In one embodiment, the proinflammatory cytokine is selected from the group consisting of: IL-Ιβ, IL-6, TNF-alpha, IP- 10, KC, and MUM a. In one embodiment, the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower. In one embodiment, the proinflammatory cytokine release is from about 20% to about 70% lower, or any range of values thereof (e.g., from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70% lower, from about 30% to about 60% lower, from about 30% to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50%) to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower). In one embodiment, the pro-inflammatory cytokine release is about 20%) lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70%) lower. In one embodiment, the reference polypeptide or reference agent comprise the same polypeptide as the polypeptide or agent except that the reference polypeptide or reference agent comprise a polypeptide comprising a CD40L stalk region that does not comprise a mutation in the integrin-binding region.
In some embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a tumor selected from the group consisting of: colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, neuroendocrine tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In certain embodiments, the tumor is a colorectal tumor. In certain embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a lung tumor. In certain embodiments, the tumor is a pancreatic tumor. In certain embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a bladder tumor. [0235] In some embodiments, the tumor expresses or overexpresses a tumor antigen targeted by the polypeptide or agent, such as a bispecific agent which comprises an antigen-binding site that specifically binds the tumor antigen.
[0236] The present invention further provides methods for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a
polypeptide or agent described herein. In some embodiments, the polypeptide or agent binds CD40 and inhibits or reduces growth of the cancer. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
[0237] The present invention provides for methods of treating cancer comprising
administering to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of a polypeptide or agent described herein. In certain embodiments, the subject is a human. In certain embodiments, the subject has a cancerous tumor. In certain embodiments, the subject has had a tumor removed. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81.
[0238] In certain embodiments, the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, neuroendocrine cancer, bladder cancer, brain cancer, glioblastoma, and head and neck cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is lung cancer. In certain
embodiments, the cancer is melanoma. In some embodiments, the cancer is bladder cancer.
[0239] In some embodiments, the cancer is a hematologic cancer. In some embodiment, the cancer is selected from the group consisting of: acute myelogenous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T-cell lymphoma (CTCL).
[0240] The invention also provides a method of activating or enhancing CD40 signaling in a cell comprises contacting the cell with an effective amount of a CD40-binding polypeptide or agent described herein. In certain embodiments, the cell is a T-cell. In some embodiments, the cell is a cytolytic cell. In some embodiments, the cell is a CTL. In some embodiments, the cell is a NK cell. In certain embodiments, the method is an in vivo method wherein the step of contacting the cell with the polypeptide or agent comprises administering a therapeutically effective amount of the polypeptide or agent to the subject. In some embodiments, the method is an in vitro or ex vivo method. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO: 72 or 81.
[0241] The present invention provides methods of determining the level of expression of
CD40 and/or CD40L. In some embodiments, the level of expression of CD40 is determined. In some embodiments, the level of expression of CD40L is determined. Methods for determining the level of nucleic acid expression in a cell, tumor, or cancer are known by those of skill in the art. These methods include, but are not limited to, PCR- based assays, microarray analyses, and nucleotide sequencing (e.g., NextGen
sequencing). Methods for determining the level of protein expression in a cell, tumor, or cancer include, but are not limited to, Western blot analyses, protein arrays, ELISAs, immunohistochemistry (IHC), and FACS.
[0242] Methods for determining whether a tumor or cancer has an elevated level of
expression of a nucleic acid or protein can use a variety of samples. In some
embodiments, the sample is taken from a subject having a tumor or cancer. In some embodiments, the sample is a fresh tumor/cancer sample. In some embodiments, the sample is a frozen tumor/cancer sample. In some embodiments, the sample is a formalin- fixed paraffin-embedded sample. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is processed to a cell lysate. In some embodiments, the sample is processed to DNA or RNA. [0243] The present invention provides compositions comprising a polypeptide or agent described herein. The present invention also provides pharmaceutical compositions comprising a polypeptide or agent described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in
immunotherapy. In some embodiments, the pharmaceutical compositions find use in immuno-oncology. In some embodiments, the compositions find use in inhibiting tumor growth. In some embodiments, the pharmaceutical compositions find use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the
compositions find use in treating cancer. In some embodiments, the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient). In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO: 72 or 81.
[0244] Formulations are prepared for storage and use by combining a purified agent of the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those of skill in the art generally consider pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.
[0245] In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01% polysorbate 20. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01%) polysorbate 20 at pH 5.5. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01%> polysorbate 20 at pH 6.0. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, 40mM NaCl, 5% sucrose, and 0.01%> polysorbate 20 at pH 6.5. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20mM histidine, lOOmM NaCl, 150mM sucrose, and 0.01% polysorbate 20 at pH 6.0. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of lOmM potassium phosphate and 0.04% polysorbate 20 at pH 7.5. [0246] Thus, in some embodiments the invention provides compositions or pharmaceutical compositions comprising a polypeptide or agent described herein and further comprising about 20mM histidine, about 40mM NaCl, about 5% sucrose, and about 0.01% polysorbate 20. In some embodiments the pH of the composition is about pH 5.5, about pH 6.0, or about pH 6.5.
[0247] In some embodiments, a polypeptide or agent described herein is lyophilized
and/or stored in a lyophilized form. In some embodiments, a formulation comprising a polypeptide or agent described herein is lyophilized.
[0248] Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG). {Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012,
Pharmaceutical Press, London.).
[0249] The pharmaceutical compositions of the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or
intraventricular). [0250] The therapeutic formulation can be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. The solid preformulation composition is then subdivided into unit dosage forms of a type described above. The tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner composition covered by an outer component. Furthermore, the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
[0251] The polypeptides or agents described herein can also be entrapped in
microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press, London.
[0252] In certain embodiments, pharmaceutical formulations include a polypeptide or agent of the present invention complexed with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter. [0253] In certain embodiments, sustained-release preparations comprising the
polypeptides or agents described herein can be produced. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing a polypeptide or agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0254] In certain embodiments, in addition to administering a polypeptide or agent
described herein, the method or treatment further comprises administering at least one additional immune response stimulating agent. In some embodiments, the additional immune response stimulating agent includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), a checkpoint inhibitor, an antibody that blocks immunosuppressive functions (e.g., an anti- CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86). An additional immune response stimulating agent can be administered prior to, concurrently with, and/or subsequently to, administration of the polypeptide or agent. Pharmaceutical compositions comprising a polypeptide or agent and the immune response stimulating agent(s) are also provided. In some embodiments, the immune response stimulating agent comprises 1, 2, 3, or more immune response stimulating agents.
[0255] In certain embodiments, in addition to administering a polypeptide or agent
described herein, the method or treatment further comprises administering at least one additional therapeutic agent. An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the polypeptide or agent. Pharmaceutical compositions comprising a polypeptide or agent and the additional therapeutic agent(s) are also provided. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents. [0256] Combination therapy with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the polypeptide or agent(s). Combination therapy may decrease the likelihood that resistant cancer cells will develop. In some embodiments, combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells.
[0257] In some embodiments of the methods described herein, the combination of a
polypeptide or agent described herein and at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the polypeptide or agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the polypeptide or agent. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s).
[0258] Useful classes of therapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri- nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, anti-metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor.
[0259] Therapeutic agents that may be administered in combination with the polypeptides or agents described herein include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves the administration of a polypeptide or agent of the present invention in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents. Treatment with a polypeptide or agent can occur prior to, concurrently with, or subsequent to administration of chemotherapies. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.
Chemotherapeutic agents useful in the present invention include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide,
triethylenethiophosphoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin,
streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as
aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium;
tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide;
mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;
difluoromethylomithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and
pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the additional therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin.
In certain embodiments of the methods described herein, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin hydrochloride (HCl), daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In some embodiments, the additional therapeutic agent is irinotecan. [0262] In certain embodiments, the chemotherapeutic agent is an anti-metabolite. An anti-metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division. Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as
pharmaceutically acceptable salts, acids, or derivatives of any of these. In certain embodiments, the additional therapeutic agent is gemcitabine.
[0263] In certain embodiments of the methods described herein, the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin. In some embodiments, the agent is a taxane. In certain embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel
(TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXA E), DHA- paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plkl . In certain embodiments, the additional therapeutic agent is paclitaxel. In certain embodiments, the additional therapeutic agent is nab-paclitaxel.
[0264] In some embodiments of the methods described herein, an additional therapeutic agent comprises an agent such as a small molecule. For example, treatment can involve the combined administration of a polypeptide or agent of the present invention with a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF. In some embodiments, a
polypeptide or agent of the present invention is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTEVIA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib ( EXAVAR), and pazopanib (GW786034B). In some embodiments, an additional therapeutic agent comprises an mTOR inhibitor. [0265] In certain embodiments of the methods described herein, the additional therapeutic agent is a small molecule that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Hippo pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the mTOR/AKR pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the RSPO/LGR pathway.
[0266] In some embodiments of the methods described herein, an additional therapeutic agent comprises a biological molecule, such as an antibody. For example, treatment can involve the combined administration of a polypeptide or agent of the present invention with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF. In certain embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In some embodiments, the additional therapeutic agent is an antibody that binds a component of the Notch pathway. In some embodiments, the additional therapeutic agent is an antibody that binds a component of the Wnt pathway. In certain embodiments, the additional therapeutic agent is an antibody that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits β-catenin signaling. In certain embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).
[0267] In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates the immune response. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti- CTLA-4 antibody, or an anti-TIGIT antibody. [0268] Furthermore, treatment with a polypeptide or agent described herein can include combination treatment with other biologic molecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by a treating physician. In some embodiments, the additional therapeutic agent is an immune response stimulating agent.
[0269] In some embodiments of the methods described herein, the polypeptide or agent can be combined with a growth factor selected from the group consisting of:
adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-a, TGF-β, TNF-a, VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.
[0270] In some embodiments of the methods described herein, the additional therapeutic agent is an immune response stimulating agent. In some embodiments, the immune response stimulating agent is selected from the group consisting of granulocyte- macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-OX40 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-Ll antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.
[0271] In some embodiments of the methods described herein, an immune response
stimulating agent is selected from the group consisting of: a modulator of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4- IBB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDOl activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an immunostimulatory oligonucleotide. [0272] In some embodiments of the methods described herein, an immune response stimulating agent is selected from the group consisting of: a PD-1 antagonist, a PD-Ll antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, and/or an IDOl antagonist.
[0273] In some embodiments of the methods described herein, the PD-1 antagonist is an antibody that specifically binds PD-1. In some embodiments, the antibody that binds PD- 1 is KEYTRUDA (MK-3475), pidilizumab (CT-011), nivolumab (OPDIVO, BMS- 936558, MDX-1106), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI-A1110. In some embodiments, the antibody that binds PD-1 is described in PCT Publication WO 2014/179664, for example, an antibody identified as APE2058,
APE1922, APE1923, APE1924, APE 1950, or APE1963, or an antibody containing the CDR regions of any of these antibodies. In other embodiments, the PD-1 antagonist is a fusion protein that includes PD-L2, for example, AMP-224. In other embodiments, the PD-1 antagonist is a peptide inhibitor, for example, AU P-12.
[0274] In some embodiments, the PD-Ll antagonist is an antibody that specifically binds
PD-Ll . In some embodiments, the antibody that binds PD-Ll is atezolizumab (RG7446, MPDL3280A), MEDI4736, BMS-936559 (MDX-1105), avelumab (MSB0010718C), KD033, the antibody portion of KD033, or STI-A1014. In some embodiments, the antibody that binds PD-Ll is described in PCT Publication WO 2014/055897, for example, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52, or Ab-55, or an antibody that contains the CDR regions of any of these antibodies.
[0275] In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds CTLA-4. In some embodiments, the antibody that binds CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206). In some embodiments, the CTLA-4 antagonist a CTLA-4 fusion protein, for example, KAHR-102.
[0276] In some embodiments, the LAG3 antagonist is an antibody that specifically binds
LAG3. In some embodiments, the antibody that binds LAG3 is IMP701, IMP731, BMS- 986016, LAG525, and GSK2831781. In some embodiments, the LAG3 antagonist includes a soluble LAG3 receptor, for example, IMP321.
[0277] In some embodiments, the KIR antagonist is an antibody that specifically binds
KIR. In some embodiments, the antibody that binds KIR is lirilumab. [0278] In some embodiments, an immune response stimulating agent is selected from the group consisting of: a CD28 agonist, a 4- IBB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist.
[0279] In some embodiments, the OX40 agonist includes OX40 ligand, or an OX40- binding portion thereof. For example, the OX40 agonist may be MEDI6383. In some embodiments, the OX40 agonist is an antibody that specifically binds OX40. In some embodiments, the antibody that binds OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888). In some embodiments, the OX40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing OX40 ligand. In some
embodiments the OX40-expressing vector is Delta-24-RGDOX or DNX2401.
[0280] In some embodiments, the 4-1BB (CD137) agonist is a binding molecule, such as an anticalin. In some embodiments, the anticalin is PRS-343. In some embodiments, the 4- IBB agonist is an antibody that specifically binds 4- IBB. In some embodiments, antibody that binds 4- IBB is PF-2566 (PF-05082566) or urelumab (BMS-663513).
[0281] In some embodiments, the CD27 agonist is an antibody that specifically binds
CD27. In some embodiments, the antibody that binds CD27 is varlilumab (CDX-1127).
[0282] In some embodiments, the GITR agonist comprises GITR ligand or a GITR- binding portion thereof. In some embodiments, the GITR agonist is an antibody that specifically binds GITR. In some embodiments, the antibody that binds GITR is TRX518, MK-4166, or INBRX-110.
[0283] In some embodiments, immune response stimulating agents include, but are not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family. In some embodiments, immune response stimulating agents include immunostimulatory oligonucleotides, such as CpG dinucleotides.
[0284] In some embodiments, an immune response stimulating agent includes, but is not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti- CTLA-4 antibodies, anti-CD28 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-4-lBB antibodies, anti-OX40 antibodies, anti -KIR antibodies, anti-Tim-3 antibodies, anti-LAG3 antibodies, anti-CD27 antibodies, anti-CD40 antibodies, anti-GITR
antibodies, anti-TIGIT antibodies, anti-CD20 antibodies, anti-CD96 antibodies, or anti- IDOl antibodies. [0285] In some embodiments, a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a CD40-binding polypeptide or agent described herein in combination with a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is melanoma. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is lung cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is bladder cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is a hematologic cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is melanoma. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is lung cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is bladder cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is breast cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-Ll antibody and the cancer is a hematologic cancer.
[0286] In certain embodiments of the methods described herein, the treatment involves the administration of a polypeptide or agent of the present invention in combination with radiation therapy. Treatment with a polypeptide or agent can occur prior to, concurrently with, or subsequent to administration of radiation therapy. Dosing schedules for such radiation therapy can be determined by the skilled medical practitioner.
[0287] In certain embodiments of the methods described herein, the treatment involves the administration of a polypeptide or agent of the present invention in combination with anti-viral therapy. Treatment with a polypeptide or agent can occur prior to, concurrently with, or subsequent to administration of antiviral therapy. The anti-viral drug used in combination therapy will depend upon the virus the subject is infected with.
[0288] Combined administration can include co-administration, either in a single
pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
[0289] It will be appreciated that the combination of a polypeptide or agent described herein and at least one additional therapeutic agent may be administered in any order or concurrently. In some embodiments, the polypeptide or agent will be administered to patients that have previously undergone treatment with a second therapeutic agent. In certain other embodiments, the polypeptide or agent and a second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject may be given a polypeptide or agent while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, a polypeptide or agent will be administered within 1 year of the treatment with a second therapeutic agent. In certain alternative embodiments, a polypeptide or agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, a polypeptide or agent will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, a polypeptide or agent will be administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).
For the treatment of a disease, the appropriate dosage of a polypeptide or agent of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the polypeptide or agent is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on, all at the discretion of the treating physician. The polypeptide or agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size). Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual agent. The administering physician can determine optimum dosages, dosing methodologies, and repetition rates. In certain embodiments, dosage is from 0.01 μg to lOOmg/kg of body weight, from O. ^g to lOOmg/kg of body weight, from ^g to lOOmg/kg of body weight, from lmg to lOOmg/kg of body weight, lmg to 80mg/kg of body weight from lOmg to lOOmg/kg of body weight, from lOmg to 75mg/kg of body weight, or from lOmg to 50mg/kg of body weight. In certain
embodiments, the dosage of the polypeptide or agent is from about 0. lmg to about 20mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about O. lmg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 0.25mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 0.5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about lmg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 1.5mg/kg of body weight. In some
embodiments, the dosage of the polypeptide or agent is about 2mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 2.5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 7.5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about lOmg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 12.5mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 15mg/kg of body weight. In certain embodiments, the dosage can be given once or more daily, weekly, monthly, or yearly. In certain embodiments, the polypeptide or agent is given once every week, once every two weeks, once every three weeks, or once every four weeks.
[0291] In some embodiments, a polypeptide or agent may be administered at an initial higher "loading" dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also change. In some embodiments, a dosing regimen may comprise administering an initial dose, followed by additional doses (or
"maintenance" doses) once a week, once every two weeks, once every three weeks, or once every month. For example, a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose. Or a dosing regimen may comprise administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week. Or a dosing regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.
[0292] As is known to those of skill in the art, administration of any therapeutic agent may lead to side effects and/or toxicities. In some cases, the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose. In some cases, drug therapy must be discontinued, and other agents may be tried. However, many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent.
[0293] In some embodiments, the dosing schedule may be limited to a specific number of administrations or "cycles". In some embodiments, the polypeptide or agent is
administered for 3, 4, 5, 6, 7, 8, or more cycles. For example, the polypeptide or agent is administered every 2 weeks for 6 cycles, the polypeptide or agent is administered every 3 weeks for 6 cycles, the polypeptide or agent is administered every 2 weeks for 4 cycles, the polypeptide or agent is administered every 3 weeks for 4 cycles, etc. Dosing schedules can be decided upon and subsequently modified by those skilled in the art.
[0294] Thus, the present invention provides methods of administering to a subject the polypeptides or agents described herein comprising using an intermittent dosing strategy for administering one or more agents, which may reduce side effects and/or toxicities associated with administration of a polypeptide or agent, chemotherapeutic agent, etc. In some embodiments, a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of a polypeptide or agent in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a polypeptide or agent to the subject, and administering subsequent doses of the polypeptide or agent about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a polypeptide or agent to the subject, and administering subsequent doses of the polypeptide or agent about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a polypeptide or agent to the subject, and administering subsequent doses of the polypeptide or agent about once every 4 weeks. In some embodiments, the polypeptide or agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.
V. Screening
[0295] The present invention provides screening methods to identify agents that modulate the immune response. In some embodiments, the present invention provides methods for screening candidate agents, including but not limited to, proteins, antibodies, peptides, peptidomimetics, small molecules, compounds, or other drugs, which modulate the immune response.
[0296] In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent has an effect on immune response cells. In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent is capable of increasing the activity of immune cells. In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent is capable of increasing the activity of cytolytic cells, such as CTLs and/or K cells. In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent is capable of inhibiting the activity of suppressor cells, such as Tregs and/or MDSCs.
VI. Kits comprising agents described herein
[0297] The present invention provides kits that comprise the polypeptides or agents
described herein and that can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one purified agent in one or more containers. In some embodiments, the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results. One skilled in the art will readily recognize that the disclosed agents of the present invention can be readily incorporated into one of the established kit formats which are well known in the art. In one embodiment, a polypeptide or agent described herein comprises a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the CD40L stalk region, and wherein the stalk region comprises a mutation in the integrin-binding region. In one embodiment, each of the first, second, and third copies of the extracellular domain of human CD40L or a fragment thereof comprises a fragment of the stalk region of CD40L, wherein the stalk region comprises a mutation in the integrin-binding region. In one embodiment, a polypeptide or agent comprises the amino acid sequence of SEQ ID NO:70 or 80. In one embodiment, a polypeptide or agent described herein further comprises an Fc Region. In one embodiment, the Fc Region is a human Fc region. In one embodiment, the Fc region is deglycosylated. In some embodiments, the polypeptide or agent comprises the amino acid sequence of SEQ ID NO:72 or 81. In one embodiment, the agent is a homodimer. In one embodiment, the agent is a heterodimer.
[0298] Further provided are kits that comprise a polypeptide or agent as well as at least one additional therapeutic agent. In certain embodiments, the second (or more) therapeutic agent is a chemotherapeutic agent. In certain embodiments, the second (or more) therapeutic agent is an angiogenesis inhibitor.
[0299] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the present disclosure.
EXAMPLES
Example 1
Generation of single chain CD40L trimer constructs
[0300] As discussed herein CD40L proteins organize into a homotrimer at the surface of cells and interact and/or engage with three CD40 molecules. A soluble single chain mouse CD40L trimer-construct was generated comprising three copies of a fragment of the stalk region and TNF family domain of mCD40L (amino acids 112-260 of SEQ ID NO: l; also see SEQ ID NO:6 and SEQ ID NO:7). The N-terminal end of this construct was attached to the C-terminal end of a mouse IgG2a Fc region (340F1; SEQ ID NO:8 and SEQ ID NO:9). A deglycosylated variant of 340F1 antibody was generated that comprised an alanine residue at position 74 of SEQ ID NO: 11 and is referred to herein as 340F2 (SEQ ID NO: 10 and SEQ ID NO: 11). The alanine residue replaces the asparagine residue in the native Fc region and results in reduced effector function of the molecule.
[0301] An additional construct, 340F4 was generated that comprised three copies of a fragment of the stalk region and TNF family domain of mCD40L (amino acids 112-260 of SEQ ID NO: l; also see SEQ ID NO: 6). The N-terminal end of this construct was attached to the C-terminal end of a mouse IgG2a Fc region (340F4; SEQ ID NO:78 and SEQ ID NO:79).
[0302] 340F7, a single chain integrin binding mutant mouse CD40L trimer was generated comprising three copies of a fragment of the stalk region and TNF family domain of mCD40L (amino acids 112-260 of SEQ ID NO: 1; also see SEQ ID NO:6). The C- terminal end of this construct was attached to the N-terminal end of a deglycosylated variant of mouse IgG2a Fc region (340F7; SEQ ID NO:76 and SEQ ID NO:77).
[0303] An additional construct was generated that comprised three copies of a fragment of the stalk region and TNF family domain of human CD40L (amino acids 113-261 of SEQ ID NO: 12; also see SEQ ID NO:22 and SEQ ID NO:28). The C-terminal end of the hCD40L trimer construct was attached to the N-terminal end of a human IgGl Fc region (SEQ ID NO:29 with signal sequence and SEQ ID NO:30 without signal sequence). A deglycosylated variant of the hCD40L trimer-Fc IgGl construct was generated (SEQ ID NO: 57 with signal sequence and SEQ ID NO: 58 without signal sequence) that comprises an alanine residue at position 524 of SEQ ID NO: 58. The alanine residue replaces the asparagine residue in the native Fc region and results in deglycosylation of the Fc region and reduced effector function of the molecule. In addition, the C-terminal end of the hCD40L trimer construct was attached to the N-terminal end of a human IgG2 Fc region (SEQ ID NO:31 with signal sequence and SEQ ID NO:32 without signal sequence). A deglycosylated variant of the hCD40L trimer-Fc IgG2 construct was generated that comprises an alanine residue at position 526 of SEQ ID NO:34. The alanine residue replaces the asparagine residue in the native Fc region and results in deglycosylation of the Fc region and reduced effector function of the molecule.
Example 2
In vivo tumor growth inhibition by single chain CD40L trimer-Fc protein
[0304] The murine colon tumor line CT26.WT was implanted subcutaneously (30,000 cells/mouse) in Balb/c mice and allowed to grow to an average size of 73mm3. Mice were treated with 12.5mg/kg of single chain mCD40L trimer-Fc 340F2 or a control antibody (n = 15 per group). Mice were dosed by intraperitoneal injection on days 10, 13, and 17. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.
[0305] As is shown in Figure 1 A, treatment with mCD40L trimer-Fc 340F2 strongly inhibited and/or prevented growth of the CT26.WT tumors. A more nuanced picture of the difference between animals treated with 340F2 and control antibody can be seen by looking at the results from the individual mice within each group. As shown in Figure 1C, at Day 28 treatment with 340F2 inhibited tumor growth to a size smaller than 100mm3 in 9 of 13 surviving mice as compared to control antibody treatment where tumor inhibition was observed in only one of 11 surviving mouse.
[0306] These results indicate that a single chain CD40L trimer-Fc molecule is active as an immunotherapeutic agent.
Example 3
In vivo tumor growth inhibition by single chain CD40L trimer-Fc protein
[0307] The murine colon tumor line CT26. WT was implanted subcutaneously (30,000 cells/mouse) in Balb/c mice and allowed to grow to an average size of approximately 159mm3. Mice were treated with 0.25mg/mouse of single chain mCD40L trimer-Fc 340F2, an anti-mCD40 antibody, or a control antibody (n = 19 per group). Mice were dosed by intraperitoneal injection on days 12, 15, and 19. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.
[0308] As is shown in Figure 2D, treatment with mCD40L trimer-Fc 340F2 strongly inhibited and/or prevented growth of the CT26.WT tumors. The tumor growth inhibition was greater than inhibition observed with an anti-mCD40 antibody. A more nuanced picture of the difference between 340F2, anti-mCD40 antibody, and control can be seen by looking at the results from the individual mice within each group (Fig. 2A-C). Nine of the 340F2-treated mice had tumors that regressed to a size that was smaller on Day 26 than when treatment started on Day 12. Importantly, two of the 13 responding mice had undetectable tumors on Day 26 and five mice had undetectable tumors on Day 32. Figure 2E is a survival curve at Day 60 of each group and shows that treatment with 340F2 had a significantly higher survival rate than the anti-mCD40 antibody. [0309] These results indicate that a single chain CD40L trimer is active as an immunotherapeutic agent and suggest that the CD40L trimer-Fc may be more effective than an anti-CD40 antibody.
Example 4
In vivo tumor growth inhibition by single chain CD40L trimer-Fc protein
[0310] The murine colon tumor line MC38 was implanted subcutaneously (400,000
cells/mouse) in C57BL/6N mice and allowed to grow to an average size of approximately 108mm3. Mice were treated with 0.25mg/mouse of single chain mCD40L trimer-Fc 340F2, anti-mouse CD40 antibody (IgG2; Bioxcell), or a control antibody (n = 10 per group). Mice were dosed by intraperitoneal injection on days 8, 12, and 15. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.
[0311] As is shown in Figure 3 A, treatment with mCD40L trimer-Fc 340F2 strongly inhibited growth of the MC38 tumors. The tumor growth inhibition with 340F2 was greater than inhibition observed with the anti-mCD40 antibody. A more nuanced picture of the difference between 340F2, anti-mCD40 antibody, and control can be seen by looking at the results from the individual mice within each group (Fig. 3B-D).
[0312] These results serve as support that the single chain CD40L trimer is active as an immunotherapeutic agent in different syngeneic mouse models, i.e., Balb/c and C57BL.
Example 5
Cytokine production
[0313] ELISpot is a highly sensitive immunoassay for the detection of cytokine-secreting cells. Briefly, an ELISpot assay employs a capture antibody specific for a desired cytokine, pre-coated onto the wells of a microplate. Stimulated cells are dispersed into the wells and the immobilized antibody in the immediate vicinity of any cytokine-secreting cell binds the secreted cytokine. Standard wash steps and incubation with appropriate detection reagents follow. For example, a combination of a biotinylated detection antibody followed by streptavidin conjugated to alkaline-phosphatase and a colored substrate solution is commonly used. A colored precipitate forms at the sites of cytokine localization and appears as a spot, with each individual spot representing an individual cytokine-secreting cell. The spots may be counted with an automated reader system or manually using a microscope. In some embodiments, an image of each well is captured using an automated reader system, and total spots, spot area, or total optical density (TOD) is used for quantification.
[0314] IL-2 secreting cells were detected using a mouse IL-2 ELISpot kit (MabTech).
Cells were isolated from the spleens of MC38 tumor-bearing mice treated with mCD40L- Fc 340F2, an anti-mCD40 antibody, or an isotype-matched control antibody (n = 5) (see Example 4). Splenocytes (5 x 105/well) from each mouse in each treatment group were dispensed into a 96-well plate coated with an antibody specific for mouse IL-2 and incubated without peptide for 48 hours. Cells secreting IL-2 were detected following the manufacturer's instructions. Images were captured using a Bioreader 6000-F-z instrument (BioSys) and spot number, spot area, and total optical density were determined. Results are shown as total optical density (TOD) and data are expressed as mean ± S.E.M.
[0315] As shown in Figure 4A, IL-2 secreting cells isolated from the spleen were
significantly increased in mice treated with mCD40L trimer-Fc 340F2 as compared to mice treated with an anti-mCD40 antibody or a control antibody.
[0316] IFN-gamma secreting cells were detected using a mouse IFN-gamma ELISpot kit
(MabTech). As described above cells were isolated from the spleens of MC38 tumor- bearing mice treated with mCD40L-Fc 340F2, an anti-mCD40 antibody, or an isotype- matched control antibody (n = 5) (see Example 4). Splenocytes (5 x 105/well) from each mouse in each treatment group were dispensed into a 96-well plate coated with an antibody specific for mouse IFN-gamma. The cells were incubated for 48 hours. Cells secreting IFN-gamma were detected following the manufacturer's instructions. Images were captured using a Bioreader 6000-F-z instrument (BioSys) and spot number, spot area, and total optical density were determined. Results are shown as TOD and data are expressed as mean ± S.E.M.
[0317] As shown in Figure 4B, IFN-gamma secreting cells isolated from the spleen were significantly increased in mice treated with mCD40L trimer-Fc 340F2 as compared to mice treated with an anti-mCD40 antibody or a control antibody.
[0318] Cells were also isolated from the tumors of MC38 tumor-bearing mice treated with mCD40L-Fc 340F2, an anti-mCD40 antibody, or an isotype-matched control antibody (n = 5) (see Example 4). Tumor cells including tumor-associated immune cells (5 x 105/well) from each mouse in each treatment group were dispensed into a 96-well plate coated with an antibody specific for mouse IFN-gamma. The cells were incubated for 48 hours. Cells secreting IFN-gamma were detected following the manufacturer's instructions. Images were captured using a Bioreader 6000-F-z instrument (BioSys) and spot number, spot area, and total optical density were determined. Results are shown as TOD and data are expressed as mean ± S.E.M.
[0319] As shown in Figure 4C, IFN-gamma secreting cells isolated from tumors were significantly increased in mice treated with mCD40L trimer-Fc 340F2 as compared to mice treated with an anti-mCD40 antibody or a control antibody.
[0320] IL-2 is a multifunctional protein generally produced by T-cells and is a potent growth factor for T-cells, B-cells, and NK cells. IL-2 also stimulates IFN-gamma production by T-cells. IFN-gamma is generally produced by NK cells, CD4+ T-cells, CD8+ T-cells, APCs, and B-cells. IFN-gamma is a known regulator of anti-tumor activity. Cells from mice treated with a CD40L trimer-Fc molecule produced more IL-2 and IFN-gamma than cells from mice treated with an anti-CD40 antibody or a control. These results support the idea that a CD40L trimer-Fc may be more effective therapeutic agent than an anti-CD40 antibody.
Example 6
Cell cytotoxicity assays
[0321] Natural killer (NK) cells are a type of cytotoxic lymphocyte critical to the innate immune system. NK cell activity in mice treated with mCD40L trimer-Fc 340F2 was assessed by measuring the cytotoxic activity of cells on tumor targets. For these NK cytotoxicity assays, the mouse lymphoblast cell line YAC-1 was used as target cells. Cells were harvested from the spleens of the CT26.WT tumor-bearing mice described above in Example 2. Cells were plated in 96-well V-bottom plates in RPMI 1640 culture medium (Gibco/Life Technologies, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS), 2mM L-glutamine, lOOU/ml penicillin, and 100μg/ml streptomycin (Gibco). YAC-1 target cells were labeled with 10μΜ calcein AM (Life Technologies) for 1 hour at 37°C and then combined with the splenocytes at an effector :target (E:T) ratio of 25: 1. Following a 4 hour incubation at 37°C, cell-free supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm. The percentage of specific cell lysis was determined as: % lysis = 100 x (ER-SR)/(MR-SR), where ER, SR, and MR represent experimental, spontaneous, and maximum calcein release, respectively. Spontaneous release is the fluorescence emitted by target cells incubated in media alone (i.e., in the absence of effector cells), while maximum release is determined by lysing target cells with an equal volume of 10% SDS.
[0322] As shown in Figure 5A, NK cells from CT26.WT tumor-bearing mice
demonstrated an increased ability to kill YAC-1 target cells when the mice had been treated with mCD40L trimer-Fc 340F2 as compared to cells from mice treated with a control antibody.
[0323] In another experiment, cells were harvested from the spleens of the MC38 tumor- bearing mice described above in Example 4. Cells were plated in 96-well V-bottom plates in RPMI 1640 culture medium (Gibco/Life Technologies, Grand Island, NY)
supplemented with 10% (v/v) fetal bovine serum (FBS), 2mM L-glutamine, lOOU/ml penicillin, and 100μg/ml streptomycin (Gibco). YAC-1 target cells were labeled with 10μΜ calcein AM (Life Technologies) for 1 hour at 37°C and then combined with the splenocytes at an effector :target (E:T) ratio of 25 : 1. Following a 4 hour incubation at 37°C, cell-free supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm. The percentage of specific cell lysis was determined as: % lysis = 100 x (ER-SR)/(MR-SR), where ER, SR, and MR represent experimental, spontaneous, and maximum calcein release, respectively. Spontaneous release is the fluorescence emitted by target cells incubated in media alone (i.e., in the absence of effector cells), while maximum release is determined by lysing target cells with an equal volume of 10% SDS.
[0324] As shown in Figure 5B, NK cells from MC38 tumor-bearing mice treated with mCD40L trimer-Fc 340F2 demonstrated an increased ability to kill YAC-1 target cells as compared to cells from mice treated with control. NK cells from MC38 tumor-bearing mice treated with an anti-mCD40 antibody also had an increased ability to kill target cells but to a smaller extent than 340F2.
[0325] Antigen-specific cytotoxic T-cells are critical to the adaptive immune system.
CD8+ cytotoxic cell activity in mice treated with mCD40L trimer-Fc 340F2 was assessed by measuring the cytotoxic activity of CD8+ T-cells on tumor targets. Cells were harvested from the spleens of the MC38 tumor-bearing mice described above in Example 4. A CD8+ T-cell specific MHC class I tumor peptide sequence is not known for the MC38 cell line, therefore the MC38 cells were used as stimulators. MC38 cells were treated with 25μg/ml mitomycin C (Sigma-Aldrich) for 30 minutes at 37°C, washed, and resuspended at 107 cells/ml in RPMI-1640 media containing 10% FCS, 2mM L- glutamine, and antibiotics. Splenocytes were co-cultured with the mitomycin-treated MC38 cells in the presence of IL-2 (2ng/ml), incubated for 5 days at 37°C, harvested, counted, and used in cytotoxicity assays as described above. Calcein AM-labeled MC38 cells were used as targets at an effectontarget ratio of 25 : 1. Calcein release was determined after 4 hours and specific lysis was calculated as described above.
[0326] As shown in Figure 5C, CD8+ cytotoxic cells from MC38 tumor-bearing mice demonstrated an increased ability to kill MC38 target cells when the mice had been treated with mCD40L trimer-Fc 340F2 as compared to cells from mice treated with anti- CD40 antibody or control.
[0327] These results suggest that a CD40L trimer-Fc molecule can increase both NK cell and antigen-specific cytotoxic T-cell activity and therefore enhance anti-tumor immune responses, consistent with increased IL-2 and IFN-gamma production. This increased cytotoxic activity may be due to a direct or indirect effect of CD40L-Fc, i.e., increasing APC activity, B-cell maturation, and/or inhibiting the effect of suppressor cells.
Example 7
In vivo tumor growth inhibition by single chain CD40L trimer-Fc protein
[0328] The murine colon tumor line CT26. WT was implanted subcutaneously (30,000 cells/mouse) in Balb/c mice and allowed to grow to an average size of 113 mm3. Mice were treated with lOmg/kg of single chain mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F4 (SEQ ID NO:79), mCD40L trimer-Fc 340F7 (SEQ ID NO:77), anti-mCD40 antibody, or a control antibody (n = 10 per group). Mice were dosed by intraperitoneal injection on days 12, 15, and 18. All groups of mice were euthanized at day 19, except mice treated with mCD40L trimer-Fc 340F4, which were euthanized on day 15 after the second dose due to low body condition score ("BCS"). The low BCS of 340F4 treated mice was likely due to the high endotoxin levels (6.4 EU/ml) in the 340F4 preparation. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.
[0329] As is shown in Figure 6, treatment with mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F4, mCD40L trimer-Fc 340F7, and anti-mCD40 strongly inhibited and/or prevented tumor growth compared to the control group. At days 14 and 18, tumor size was smaller in all single chain CD40L trimer-Fc molecule treated groups compared to the control group. These results indicate that the 340F2, 340F4 and 340F7 single chain CD40L trimer-Fc molecules have comparable anti-tumor efficacy.
Example 8
Analysis of Serum Liver Enzymes in Single Chain CD40L Trimer-Fc Fusion Polypeptide Treated Mice
[0330] Mouse serum samples were analyzed for liver enzyme panel by IDEXX
Bioresearch. Briefly blood samples were collected in brown micro tubes with Serum Gel with Clotting Activator and allowed to clot by leaving them undisturbed at room temperature for 1 hour. After lhour incubation, samples were centrifuged at 3600rpm for 15 minutes and serum samples were collected. Samples were kept at 4°C and were sent out to IDEXX Bioresearch for the analysis of liver enzyme panel (Test Code: 60405).
[0331] Mice treated with lOmg/kg of single chain mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F7, anti-mCD40 antibody, or a control antibody from Example 7 were euthanized at day 19 and serum was isolated from each animal. From the serum, alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP) levels were determined for each mouse. As shown in Figures 7A-7C, mice treated with mCD40L trimer-Fc 340F7 had lower levels of ALT and ALP than mice treated with mCD40L trimer-Fc 340F2. This result is indicative of less liver damage in mice treated with the integrin binding mutant mCD40L trimer-Fc 340F7 than mice treated with mCD40L trimer-Fc 340F2.
Example 9
[0332] Complete Blood Count Analysis in Single Chain CD40L Trimer-Fc Fusion
Polypeptide Treated Mice
[0333] Mice treated with lOmg/kg of single chain mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F7, anti-mCD40 antibody, or a control antibody from Example 7 were euthanized at day 19 and a complete blood count was performed. As shown in Figures 8 A and 8B, mice treated with mCD40L trimer-Fc 340F7 had lower levels of neutrophils than mice treated with mCD40L trimer-Fc 340F2. Additionally, Figure 8D shows that mice treated with mCD40L trimer-Fc 340F7 had higher levels of lymphocytes than mice treated with mCD40L trimer-Fc 340F2. However, as Figure 8C shows, there was no change in the total amount of white blood cells between the mice treated with mCD40L trimer-Fc 340F2 or the integrin binding mutant mCD40L trimer-Fc 340F7. Lower neutrophil levels detected in mice treated with the integrin binding mutant mCD40L trimer-Fc 340F7 indicate a reduced inflammatory response, which is important for reducing non-specific cellular damage.
Example 10
Cytokine Production in Single Chain CD40L Trimer-Fc Fusion Polypeptide Treated Mice
[0334] Mouse plasma samples were assayed using cytokine and chemokine 25plex (EMD
Millipore) panel by following Luminex assay protocol with adaption of the Drop Array system (Curiox Biosystem). Briefly, the plate was blocked with lOuL 1%BSA/PBS for 30 minutes at room temperature. STD and control were prepared as Luminex assay, 5uL (1/5) bead mix was added to each DropArray wall-less plate, 5uL buffer/well was added to the plasma sample well, and matrix buffer was added to the STDs and control wells. 5uL STD or control samples per well was added with tips changed after each addition. The plate was carefully inserted into shaker and was shaken for 10 seconds @1000RPM. The assay plate was placed on top of the Magnetic stand into a Humidified box and shaken overnight at 4°C. The Wash Plate was washed 3X with DropArray LT washing station MX96 (Curiox Biosystems), the detection ab was added, 5uL/well and incubated on shaker for 60 minutes. 5uL/well of the Streptavidin /PE substrate was added, incubated for 30 minutes with shaking. Plates were washed 3X with DropArray LT washing station MX96, 75uL Sheath fluid was added and the plate was read with the adaptor. Luminex data was analyzed using the EMD Millipore's Milliplex Analyst software. Statistical analysis was performed using the One-Way ANOVA Bonferroni multiple comparison test.
[0335] On day 19, mice treated with lOmg/kg of single chain mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F4, mCD40L trimer-Fc 340F7, anti-mCD40 antibody, or a control antibody from Example 7 were euthanized and serum levels of G-CSF, IFN-gamma, IL- la, IL-Ιβ, IL-2, IL-6, IL-12p40, IFN-gamma-induced protein 10 ("IP-10"), chemokine (C-X-C motif) ligand 1 ("KC"), monocyte chemoattractant protein 1 ("MCP1 "), macrophage inflammatory protein 1-a ("MIP-la"), ΜΙΡΙβ, MIP2, Regulated on
Activation Normal T Cell Expressed and Secreted ("RANTES"), and TNF-alpha were determined. As shown in Figures 9A-90, mice treated with mCD40L trimer-Fc 340F7 had lower levels of pro-inflammatory cytokines including IL-Ιβ, IL-6, TNF-alpha, IP-10, KC, MIP-la than mice treated with mCD40L trimer-Fc 340F2. These results indicate that the integrin binding mutant mCD40L trimer-Fc 340F7-treated mice had lower cytokine production and reduced inflammation than mCD40L trimer-Fc 340F2-treated mice.
Example 11
Liver Histology in Single Chain CD40L Trimer-Fc Fusion Polypeptide Treated Mice
[0336] Mice treated with lOmg/kg of single chain mCD40L trimer-Fc 340F2, mCD40L trimer-Fc 340F4, mCD40L trimer-Fc 340F7, anti-mCD40 antibody, or a control antibody from Example 7 were euthanized and livers were isolated. Liver sections were stained with hematoxylin and eosin following standard protocols.
[0337] As shown in Figure 10A and B, mice treated with mCD40L trimer-Fc 340F7 had large thrombi and multifocally extensive coagulative necrosis. Mice treated with mCD40L trimer-Fc 340F2 had multifocal infiltrates, with and without associated hepatocyte necrosis, and occasional thrombi. Mice treated with anti-mCD40 antibody had multifocally extensive perivascular infiltrates. And mice treated with mCD40L trimer-Fc 340F4 had rare infiltrates. The liver histology of 340F4 treated mice, which were euthanized earlier than the other groups due to low BCS, is consistent with these animals getting sick from the high endotoxin levels in the 340F4 preparation. The liver histology indicates that 340F7 is capable of inducing liver damage at the very robust dose tested.
[0338] Mice treated with single chain integrin binding mutant mCD40L trimer-Fc 340F7 had a lower cytokine response, reduced systemic inflammation, and decreased serum liver enzymes (indicating less liver damage) compared to mice treated with mCD40L trimer-Fc 340F2. As indicated by the liver histology, 340F7 is still capable of inducing liver damage at the very robust dose tested. These results in total indicate that with mCD40L trimer-Fc 340F7 has an improved safety profile, which could enable an improved therapeutic index.
Example 12
Generation of single chain integnn binding mutant human CD40L trimer constructs
[0339] A soluble single chain integrin binding mutant human CD40L trimer-construct will be generated comprising three copies of a fragment of the stalk region and TNF family domain of human CD40L (amino acids 113-261 of SEQ ID NO: 12; also see SEQ ID NO:69 and SEQ ID NO:70). The N-terminal end of this construct will be attached to the C-terminal end of a deglycosylated variant of a human IgGl Fc region (SEQ ID NO:71 with signal sequence and SEQ ID NO: 72 without signal sequence). The deglycosylated variant of the human IgGl Fc region (SEQ ID NO:82) comprises an alanine residue at position 526 of SEQ ID NO:34. The alanine residue replaces the asparagine residue in the native Fc region and results in deglycosylation of the Fc region and reduced effector function of the molecule.
Example 13
In vivo tumor growth inhibition in humanized mice by a single chain integrin binding mutant human CD40L trimer-Fc protein
[0340] A humanized mouse model will be used to study the efficacy of treatment with a single chain integrin binding mutant human CD40L trimer-Fc protein on a human tumor. The humanized mice can be obtained from Jackson Laboratories. These mice can be created by injecting human hematopoietic stem cells (CD34+ cells) into irradiated NSG mice. After 15 weeks, the presence of mature human lymphocytes can be confirmed by flow cytometry. Each mouse will be injected subcutaneously with patient-derived human tumor cells, for example, with melanoma tumor cells (OMP-M9, 75,000 cells/mouse). Tumors will be allowed to grow, for example for 16 days until they reach an average volume of approximately 60mm3. Tumor-bearing mice will be randomized into 2 groups (n = 3 mice per group). Tumor-bearing mice will be treated with either a control protein or a single chain integrin binding mutant human CD40L trimer-Fc protein. Tumor growth will be monitored and tumor volumes will be measured with electronic calipers.
[0341] It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in light thereof will be suggested to person skilled in the art and are to be included within the spirit and purview of this application.
[0342] All publications, patents, patent applications, internet sites, and accession
numbers/database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.
SEQUENCES
[0343] Sequences disclosed in the application are:
Mouse CD40L amino acid sequence (SEQ ID NO: l)
MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLH EDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQ IAAHVVSEA SNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNR EPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNV TEASQVIHRVGFSSFGLLKL
Mouse CD40L extracellular domain amino acid sequence (aa 47-260) (SEQ ID NO: 2)
HRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKK ENSFEMQRGDEDPQIAAHVVSEA SNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREG LYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGG VFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL
Mouse CD40L Stalk Region (SEQ ID NO: 3)
HRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKK ENSFEMQRGDE
Mouse CD40L extracellular domain without stalk region amino acid sequence (SEQ ID NO: 4)
DPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQV FC SNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLCEQQSVHLGGVFELQAGASVF VNVTEASQVIHRVGFSSFGLLKL
Mouse CD40L Stalk - Fragment 1 (SEQ ID NO:5)
MQRGDE
Mouse CD40L extracellular domain with stalk fragment 1 (aa 112-260) (SEQ ID NO:6)
MQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVY TQV FCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLCEQQSVHLGGVFELQ AGASVFVNV EASQVIHRVGFSSFGLLKL
Mouse single chain CD40L trimer amino acid sequence without signal sequence (SEQ ID NO:7)
MQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVY TQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQ AGASVFVNVTEASQVIHRVGFSSFGLLKLMQRGDEDPQIAAHVVSEA SNAASVLQWAKK GYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSER ILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLMQ RGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQ VTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLCEQQSVHLGGVFELQAG ASVFVNVTEASQVIHRVGFSSFGLLKL
340F1 Mouse single chain CD40L trimer-Fc (mIgG2a) with predicted signal sequence underlined (SEQ ID NO: 8)
MEWGYLLEVTSLLAALLLLQRSPIVHASPPCKCPAPNLLGGPSVFI FPPKIKDVLMISLS PIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG KEFKCKVNNKDLPAPIER ISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMV DFMPED IYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKK WVERNSYSCSVVHEGLHNHH T KSFSR PGKGRAMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENG KQLTVKREGLYYVYTQV FCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLC EQQSVHLGGVFELQAGASVFVNV EASQVIHRVGFSSFGLLKLMQRGDEDPQIAAHVVSE ANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPF IVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIH RVGFSSFGLLKLMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQ LTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQ QSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL
340F1 Mouse single chain CD40L trimer-Fc (mIgG2a) without signal sequence (SEQ ID NO:9)
SPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVE VHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSV RAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGS YFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGRAMQRGDEDPQIAAH VVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSS QRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEAS QVIHRVGFSSFGLLKLMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLE NGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQ LCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLMQRGDEDPQIAAHVV SEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQR PFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQV IHRVGFSSFGLLKL
340F2 Mouse single chain CD40L trimer (C term)-Fc (N term) (deglycosylated mIgG2a) with predicted signal sequence underlined (SEQ ID NO: 10)
MEWGYLLEVTSLLAALLLLQRSPIVHASPPCKCPAPNLLGGPSVFI FPPKIKDVLMISLS PIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSG KEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPED IYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHH TTKSFSRTPGKGRAMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENG KQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLC EQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLMQRGDEDPQIAAHVVSE ANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPF IVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIH RVGFSSFGLLKLMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQ LTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQ QSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL
340F2 Mouse single chain CD40L trimer (C term)-Fc (N term) (deglycosylated mIgG2a) without signal sequence
(SEQ ID NO: 11)
SPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVE VHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSV RAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGS YFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGRAMQRGDEDPQIAAH VVSEA SNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSS QRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEAS QVIHRVGFSSFGLLKLMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLE NGKQLTVKREGLYYVYTQV FCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQ LCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLMQRGDEDPQIAAHVV SEA SNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQR PFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQV IHRVGFSSFGLLKL
Human CD40L (TNFSF5) amino acid sequence (SEQ ID NO: 12)
MIETYNQ SPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLH EDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNP QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQV FCSN REASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPGASVFVN VTDPSQVSHGTGFTSFGLLKL
Human CD40L extracellular domain amino acid sequence (aa 47-261) (SEQ ID NO: 13)
HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETK KENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQ GLYYIYAQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLG GVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL
Human CD40L Stalk Region (SEQ ID NO: 14)
HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETK KENSFEMQKGDQ
Human CD40L extracellular domain without stalk region amino acid sequence (SEQ ID NO: 15)
NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFC SNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVF VNVTDPSQVSHGTGFTSFGLLKL
Human CD40L Stalk Region - Fragment 1 (SEQ ID NO: 16)
MQKGDQ
Human CD40L Stalk Region - Fragment 2 (SEQ ID NO: 17)
FEMQKGDQ
Human CD40L Stalk Region - Fragment 3 (SEQ ID NO: 18)
EMQKGDQ
Human CD40L Stalk Region - Fragment 4 (SEQ ID NO: 19)
QKGDQ
Human CD40L Stalk Region - Fragment 5 (SEQ ID NO:20)
KGDQ
Human CD40L TNF homology domain (SEQ ID NO:21)
IAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNR EASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNV TDPSQVSHGTGFTSFGLLKL
Human CD40L extracellular with stalk fragment 1 (aa 1 13-261) (SEQ ID NO:22) MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY
AQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKL
Human CD40L extracellular domain with stalk fragment 2 (aa 1 1 1-261) (SEQ ID NO:23)
FEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYY IYAQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFE LQPGASVFVNV DPSQVSHGTGF SFGLLKL
Human CD40L extracellular domain with stalk fragment 3 (aa 1 12-261) (SEQ ID NO: 24)
EMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYI YAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFEL QPGASVFVNVTDPSQVSHGTGFTSFGLLKL
Human CD40L extracellular domain with stalk fragment 4 (aa 1 14-261) (SEQ ID NO:25)
QKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYA QVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQP GASVFVNVTDPSQVSHGTGFTSFGLLKL
Human CD40L extracellular domain with stalk fragment 5 (aa 1 15-261) (SEQ ID NO: 26)
KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKL
Human single chain CD40L trimer amino acid sequence with signal sequence underlined (SEQ ID NO:27)
MEWGYLLEVTSLLAALLLLQRSPIVHAMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKG DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGAS VFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL
Human single chain CD40L trimer amino acid sequence without signal sequence (SEQ ID NO:28)
MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKL
Human single chain CD40L trimer-Fc (hlgGl) amino acid sequence with signal sequence underlined (SEQ ID NO:29)
MEWGYLLEVTSLLAALLLLQRSPIVHAMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKG DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGAS VFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT PPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human single chain CD40L trimer-Fc (hlgGl) amino acid sequence without signal sequence (SEQ ID NO:30)
MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKT SVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKLDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK QVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK
Human single chain CD40L trimer-Fc (hIgG2) amino acid sequence with signal sequence underlined (SEQ ID NO:31)
MEWGYLLEVTSLLAALLLLQRSPIVHAMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKG DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPGAS VFVNV DPSQVSHGTGF SFGLLKLMQKGDQNPQIAAHVISEASSKT SVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLVERKSC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human single chain CD40L trimer-Fc (hIgG2) amino acid sequence without signal sequence (SEQ ID NO:32)
MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKLVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK
Human single chain CD40L trimer-Fc (deglycosylated hIgG2 ) amino acid sequence with signal sequence underlined (SEQ ID NO:33)
MEWGYLLEVTSLLAALLLLQRSPIVHAMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKG DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPGAS VFVNV DPSQVSHGTGF SFGLLKLMQKGDQNPQIAAHVISEASSKT SVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AAN HSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGF SFGLLKLVERKSC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human single chain CD40L trimer-Fc (deglycosylated hIgG2) amino acid sequence without signal sequence (SEQ ID NO:34)
MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKT SVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPG ASVFVNV DPSQVSHGTGF SFGLLKLVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK
Human single chain CD40L trimer (2 stalks regions) amino acid sequence with signal sequence underlined (SEQ ID NO:35)
MEWGYLLEVTSLLAALLLLQRSPIVHANPQIAAHVISEASSKTTSVLQWAEKGYYTMSNN LVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANT HSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQI AAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNRE ASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVT DPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLV TLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHS SAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL
Human single chain CD40L trimer (2 stalks regions) amino acid sequence without signal sequence (SEQ ID NO:36)
NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFC SNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVF VNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMS NNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAA NTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNP QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSN REASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVN VTDPSQVSHGTGFTSFGLLKL
Human CD40L anchor region and extracellular domain amino acid sequence (SEQ ID NO:37)
IFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLL NCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAE KGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFE RILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Human single chain CD40L trimer nucleotide sequence without signal sequence (SEQ ID NO:38)
ATGCAAAAAGGGGATCAGAATCCTCAAATTGCAGCACATGTCATATCTGAGGCCAGCTCA AAAACAACATCTGTGCTCCAGTGGGCTGAAAAAGGA AC ACACCATGAGCAACAACCTC GTTACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTAT A ATC AT GCCCAAGTCACCTTCTGTTCCAATCGGGAAGCATCTTCACAAGCTCCATTTATCGCCAGC CTCTGCCTCAAGTCCCCCGGTCGGTTCGAGAGAATCCTCCTCAGAGCTGCAAATACCCAC TCTTCCGCCAAACCTTGCGGGCAACAATCCATTCACCTCGGAGGAGTTTTTGAACTGCAA CCAGGGGCTTCTGTGTTTGTCAATGTGACTGATCCAAGCCAAGTGTCTCATGGAACTGGC TTCACTTCCTTTGGCCTCCTCAAACTCATGCAGAAAGGGGACCAAAACCCCCAGATAGCC GCTCACGTTATTTCCGAAGCAAGCTCAAAAACAACATCTGTGCTCCAGTGGGCTGAAAAA GGATACTACACCATGAGCAACAACCTCGTTACCCTGGAGAACGGAAAGCAACTCACTGTG AAGCGGCAGGGGCTGTACTACATATACGCACAAGTGACTTTTTGCAGCAACAGGGAGGCA TCCTCTCAGGCCCCTTTCATTGCCAGCCTCTGCCTGAAGTCCCCCGGTAGATTCGAGAGA ATCCTCCTCAGAGCTGCAAATACCCACTCCTCCGCAAAACCCTGTGGCCAGCAGAGCATC CATCTGGGCGGCGTGTTCGAGCTCCAGCCTGGGGCCTCCGTCTTCGTGAACGTCACCGAC CCTTCCCAAGTCAGCCACGGCACTGGCTTCACATCCTTTGGCCTCCTCAAACTCATGCAA AAAGGCGATCAGAATCCTCAAATTGCTGCACATGTCATTTCCGAAGCCTCATCCAAAACT ACCTCCGTCCTGCAATGGGCCGAGAAGGGGTAT A ACAATGTCAAATAACCTGGTTACT CTGGAAAACGGCAAACAGCTCACTGTTAAGCGCCAAGGTCTCTAC A A A ATGCACAA GTTACTTTCTGTTCAAATCGCGAAGCATCATCACAAGCACCATTTATAGCATCACTCTGT CTCAAGTCACCAGGTCGCTTTGAACGCATACTGCTCCGCGCAGCAAATACTCACTCATCA GCAAAACCATGCGGTCAACAATCAATACACCTCGGTGGTGTTTTTGAGCTCCAACCAGGC GCTTCAGTTTTTGTTAATGTTACTGATCCATCACAAGTTTCACATGGTACAGGTTTCACT TCATTTGGTCTGCTCAAACTCTAATAG
Human IgGl Fc region (SEQ ID NO: 39)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human IgGl Fc region (SEQ ID NO: 40)
KSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK IS KAKGQPREPQVYTLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human IgGl Fc region (SEQ ID NO:41)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human IgG2 Fc region (SEQ ID NO: 42)
CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human IgG2 Fc region (SEQ ID NO:43)
VERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human IgG2 Fc region variant (mutation for deglycosylation) (SEQ ID NO: 44)
CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human IgG2 Fc region (mutation for deglycosylation) (SEQ ID NO:45)
VERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human IgGl Heavy chain constant region (SEQ ID NO: 46)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human IgG2 Heavy chain constant region (SEQ ID NO: 47)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKT PPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK
Human IgG3 Heavy chain constant region (SEQ ID NO:48)
ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSC DTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESSGQPENNYNT PPMLDSDGSFFLYSKLTVDKSRWQQGNI FSCSVMHE ALHNRFTQKSLSLSPGK
Human IgG4 Heavy chain constant region (SEQ ID NO: 49)
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISR PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT PPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK
Linker (SEQ ID NO: 50)
GGGSGGG
Linker (SEQ ID NO:51)
ESGGGGVT
Linker (SEQ ID NO:52) LESGGGGVT
Linker (SEQ ID NO: 53)
GRAQVT
Linker (SEQ ID NO:54)
WRAQVT
Linker (SEQ ID NO: 55)
ARGRAQVT
FLAG Tag (SEQ ID NO: 56)
DYKDDDDK
Human single chain CD40L trimer-Fc (deglycosylated hlgGl) amino acid sequence with signal sequence underlined (SEQ ID NO: 57)
MEWGYLLEVTSLLAALLLLQRSPIVHAMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKG DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPGAS VFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AAN HSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGF SFGLLKLDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT PPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human single chain CD40L trimer-Fc (deglycosylated hlgGl) amino acid sequence without signal sequence (SEQ ID NO: 58)
MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKT SVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKLDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK QVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK
Human integrin-binding region mutant CD40L Stalk Region - Fragment 1 (SEQ ID NO: 59)
MQKADQ
Human integrin-binding region mutant CD40L Stalk Region - Fragment 2 (SEQ ID NO: 60)
FEMQKADQ
Human integrin-binding region mutant CD40L Stalk Region - Fragment 3 (SEQ ID NO: 61)
EMQKADQ Human integrin-binding region mutant CD40L Stalk Region - Fragment 4 (SEQ ID NO: 62)
QKADQ
Human integrin-binding region mutant CD40L Stalk Region - Fragment 5 (SEQ ID NO:63)
KADQ
Human integrin-binding region mutant CD40L extracellular with stalk fragment 1 (aa 113-261) (SEQ ID NO: 64)
MQKADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKL
Human integrin-binding region mutant CD40L extracellular domain with stalk fragment 2 (aa 111-261) (SEQ ID NO:65)
FEMQKADQNPQIAAHVISEASSKT SVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYY IYAQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFE LQPGASVFVNV DPSQVSHGTGF SFGLLKL
Human integrin-binding region mutant CD40L extracellular domain with stalk fragment 3 (aa 112-261) (SEQ ID NO:66)
EMQKADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYI YAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFEL QPGASVFVNVTDPSQVSHGTGFTSFGLLKL
Human integrin-binding region mutant CD40L extracellular domain with stalk fragment 4 (aa 114-261) (SEQ ID NO:67)
QKADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYA QVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQP GASVFVNVTDPSQVSHGTGFTSFGLLKL
Human integrin-binding region mutant CD40L extracellular domain with stalk fragment 5 (aa 115-261) (SEQ ID NO:68)
KADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKL
Human single chain integrin-binding region mutant CD40L trimer amino acid sequence with signal sequence underlined (SEQ ID NO: 69)
MEWGYLLEVTSLLAALLLLQRSPIVHAMQKADQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKA DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGAS VFVNVTDPSQVSHGTGFTSFGLLKLMQKADQNPQIAAHVISEASSKTTSVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL
Human single chain integrin-binding region mutant CD40L trimer amino acid sequence without signal sequence (SEQ ID NO:70)
MQKADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKADQNPQIAAHVISEASSKTTSVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKL
Human single chain integrin-binding region mutant CD40L trimer Fc (deglycosylated hlgGl) amino acid sequence with signal sequence underlined (SEQ ID NO: 71)
MEWGYLLEVTSLLAALLLLQRSPIVHAMQKADQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKA DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPGAS VFVNV DPSQVSHGTGF SFGLLKLMQKADQNPQIAAHVISEASSKT SVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AAN HSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGF SFGLLKLDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT PPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Human single chain integrin-binding region mutant CD40L trimer Fc (deglycosylated hlgGl) amino acid sequence without signal sequence (SEQ ID NO: 72)
MQKADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKADQNPQIAAHVISEASSKT SVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKLDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK QVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK
Mouse integrin-binding region mutant CD40L Stalk - Fragment 1 (SEQ ID NO:73)
MQRADE
Mouse integrin-binding region mutant CD40L extracellular domain with stalk fragment 1 (aa 1 12-260) (SEQ ID NO:74)
MQRADEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVY TQV FCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLCEQQSVHLGGVFELQ AGASVFVNVTEASQVIHRVGFSSFGLLKL
Mouse single chain integrin-binding region mutant CD40L trimer amino acid sequence without signal sequence (SEQ ID NO:75)
MQRADEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVY TQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQ AGASVFVNVTEASQVIHRVGFSSFGLLKLMQRADEDPQIAAHVVSEA SNAASVLQWAKK GYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSER ILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLMQ RADEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQ VTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAG ASVFVNVTEASQVIHRVGFSSFGLLKL 340F7Mouse single chain integrin-binding region mutant CD40L trimer-Fc (deglycosylated mIgG2a) with predicted signal sequence underlined (SEQ ID NO: 76)
MEWGYLLEVTSLLAALLLLQRSPIVHAMQRADEDPQIAAHVVSEA SNAASVLQWAKKGY YTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERIL LKAAN HSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLMQRA DEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVT FCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLCEQQSVHLGGVFELQAGAS VFVNV EASQVIHRVGFSSFGLLKLMQRADEDPQIAAHVVSEA SNAASVLQWAKKGYYT MKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLK AAN HSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLDKTHTC KCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQ HREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER ISKPKGSVRAPQ VYVLPPPEEEM KKQVTLTCMVTDFMPEDIYVEWTNNGK ELNYK TEPVLDSDGSYFMY SKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
340F7 Mouse single chain integrin-binding region mutant CD40L trimer-Fc (deglycosylated mIgG2a) without signal sequence (SEQ ID NO:77)
MQRADEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVY TQV FCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLCEQQSVHLGGVFELQ AGASVFVNV EASQVIHRVGFSSFGLLKLMQRADEDPQIAAHVVSEA SNAASVLQWAKK GYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSER ILLKAAN HSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLMQ RADEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQ V FCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLCEQQSVHLGGVFELQAG ASVFVNV EASQVIHRVGFSSFGLLKLDK HTCKCPAPNLLGGPSVFI FPPKIKDVLMIS LSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWM SGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMP EDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKK WVERNSYSCSVVHEGLHN HHTTKSFSRTPGK
340F4 Mouse single chain CD40L trimer (N term)-Fc (C term) (deglycosylated mIgG2a) with predicted signal sequence underlined (SEQ ID NO:78)
MEWGYLLEVTSLLAALLLLQRSPIVHAMQRGDEDPQIAAHVVSEA SNAASVLQWAKKGY YTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERIL LKAAN HSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLMQRG DEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVT FCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLCEQQSVHLGGVFELQAGAS VFVNV EASQVIHRVGFSSFGLLKLMQRGDEDPQIAAHVVSEA SNAASVLQWAKKGYYT MKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLK AAN HSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLDKTHTC KCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTA QTQ HREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER ISKPKGSVRAPQ VYVLPPPEEEM KKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYK TEPVLDSDGSYFMY SKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
340F4 Mouse single chain CD40L trimer (N term)-Fc (C term) (deglycosylated mIgG2a) without signal sequence (SEQ ID NO:79)
MQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVY TQV FCSNREPSSQRPFIVGLWLKPSSGSERILLKAA THSSSQLCEQQSVHLGGVFELQ AGASVFVNVTEASQVIHRVGFSSFGLLKLMQRGDEDPQIAAHVVSEA SNAASVLQWAKK GYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSER ILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLMQ RGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQ VTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAG ASVFVNV EASQVIHRVGFSSFGLLKLDK HTCKCPAPNLLGGPSVFI FPPKIKDVLMIS LSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWM SGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMP EDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKK WVERNSYSCSVVHEGLHN HHTTKSFSRTPGK
Human single chain integrin-binding region mutant CD40L trimer (2 stalk region) amino acid sequence without signal sequence (SEQ ID NO: 80)
MQKADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKADQNPQIAAHVISEASSKT SVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKL
Human single chain integrin-binding region mutant CD40L trimer Fc (deglycosylated hIgG2) amino acid sequence without signal sequence (SEQ ID NO: 81)
MQKADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQV FCSNREASSQAPFIASLCLKSPGRFERILLRAA THSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKADQNPQIAAHVISEASSKTTSVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KADQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKLVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK
Human IgGl Fc region variant (mutation for deglycosylation) (SEQ ID NO: 82)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

What is claimed is:
1. A polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
2. A single chain fusion human CD40 ligand (CD40L) polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
3. The polypeptide of claim 1 or claim 2, wherein the extracellular domain of human
CD40L or a fragment thereof has a lower binding affinity to integrin alpha (lib) beta 3 compared to a corresponding extracellular domain of human CD40L or a fragment thereof not comprising the mutation.
4. The polypeptide of any one of claims 1 to 3, wherein the integrin-binding region of
CD40L comprises the KGD motif (residues 115-117 of SEQ ID NO: 12).
5. The polypeptide of any one of claims 1 to 4, wherein the integrin-binding region of
CD40L consists of the KGD motif (residues 115-117 of SEQ ID NO: 12).
6. The polypeptide of any one of claims 1 to 5, wherein the mutation comprises the
substitution, insertion, or deletion of at least one residue in the integrin-binding region.
7. The polypeptide of any one of claims 1 to 6, wherein the mutation comprises the
substitution or deletion of 1, 2, or 3 residues in the integrin-binding region.
8. The polypeptide of any one of claims 1 to 7, wherein the mutation in the integrin-binding region comprises
(a) a D to E substitution in the KGD motif (residues 115-117 of SEQ ID NO: 12); and/or
(b) a G to A substitution in the KGD motif (residues 115-117 of SEQ ID NO: 12).
9. The polypeptide of any one of claims 1 to 8, wherein the fragment of the stalk region of CD40L is selected from the group consisting of: MQKGDQ (SEQ ID NO: 16),
FEMQKGDQ (SEQ ID NO: 17), EMQKGDQ (SEQ ID NO: 18), QKGDQ (SEQ ID
NO: 19), and KGDQ (SEQ ID NO:20), wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
10. The polypeptide of any one of claims 1 to 9, wherein the fragment of the stalk region comprises an amino acid sequence selected from the group consisting of: MQKADQ (SEQ ID NO:59), FEMQKADQ (SEQ ID NO:60), EMQKADQ (SEQ ID NO:61), QKADQ (SEQ ID NO:62), and KADQ (SEQ ID NO:63).
11. The polypeptide of any one of claims 1 to 10, wherein at least two of the first, second, or third copies of the extracellular domain or a fragment thereof comprise the fragment of the stalk region.
12. The polypeptide of any one of claims 1 to 11, wherein the second and third copies of the extracellular domain or a fragment thereof comprise the fragment of the stalk region.
13. The polypeptide of any one of claims 1 to 12, wherein each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the fragment of the stalk region.
14. The polypeptide of any one of claims 1 to 13, wherein the polypeptide does not comprise an exogenous peptide linker between the extracellular domains or fragments thereof of CD40L.
15. The polypeptide of any one of claims 1 to 14, wherein at least one of the copies of the extracellular domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 15.
16. The polypeptide of any one of claims 1 to 15, wherein at least one of the copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin- binding region.
17. The polypeptide of any one of claims 1 to 16, wherein at least one of the copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:64.
18. The polypeptide of any one of claims 1 to 17, wherein each of the first, second, or third copies of the extracellular domain or fragment thereof comprises the amino acid sequence of SEQ ID NO: 15.
19. The polypeptide of any one of claims 1 to 18, wherein each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:22, wherein the fragment of the stalk region comprises a mutation in the integrin-binding region.
20. The polypeptide of any one of claims 1 to 19, wherein each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO: 15, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68.
21. The polypeptide of any one of claims 1 to 20, wherein each of the first, second, and third copies of the extracellular domain or a fragment thereof comprises the amino acid sequence of SEQ ID NO:64.
22. The polypeptide of any one of claims 1 to 21, which comprises a polypeptide having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:70 or 80.
23. The polypeptide of any one of claims 1 to 22, which comprises a polypeptide having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:70 or 80.
24. The polypeptide of any one of claims 1 to 23, which comprises the amino acid sequence of SEQ ID NO:70 or 80.
25. The polypeptide of any one of claims 1 to 24, which further comprises a non-CD40L polypeptide.
26. The polypeptide of any one of claims 1 to 25, wherein the first or third copy of the
extracellular domain of human CD40L or a fragment thereof is directly linked to the non- CD40L polypeptide.
27. The polypeptide of any one of claims 1 to 25, wherein the first or third copy of the
extracellular domain of human CD40L or a fragment thereof is connected to the non- CD40L polypeptide by a linker.
28. The polypeptide of any one of claims 1 to 27, wherein the first copy of the extracellular domain is linked to the carboxy-terminal end of the non-CD40L polypeptide.
29. The polypeptide of any one of claims 1 to 27, wherein the third copy of the extracellular domain is linked to the amino-terminal end of the non-CD40L polypeptide.
30. The polypeptide of any one of claims 25 to 29, wherein the non-CD40L polypeptide comprises an Fc region.
31. The polypeptide of claim 30, wherein the Fc region is a human Fc region.
32. The polypeptide of any one of claims 30 to 31, wherein the Fc region is from a human IgGl, IgG2, IgG3, or IgG4 immunoglobulin.
33. The polypeptide of any one of claims 30 to 32, wherein the Fc region is selected from the group consisting of: SEQ ID NOs:39-43.
34. The polypeptide of any one of claims 30 to 33, wherein the Fc region is deglycosylated.
35. The polypeptide of claim 34, wherein the Fc region comprises a mutation in the N-linked glycosylation site of the CH2 domain.
36. The polypeptide of claim 35, wherein the mutation is a substitution or deletion of the asparagine corresponding to residue 77 of SEQ ID NO:39.
37. The polypeptide of claim 36, wherein the mutation is an alanine substitution of the
asparagine corresponding to residue 77 of SEQ ID NO:39.
38. The polypeptide of claim 35, wherein the mutation is a substitution or deletion of the asparagine corresponding to residue 79 of SEQ ID NO:43.
39. The polypeptide of claim 38, wherein the mutation is an alanine substitution of the
asparagine corresponding to residue 79 of SEQ ID NO:43.
40. The polypeptide of claim 34, wherein the Fc region comprises SEQ ID NO:82.
41. The polypeptide of claim 34, wherein the Fc region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:44 and SEQ ID NO:45.
42. The polypeptide of claim 34, which comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:72.
43. The polypeptide of claim 42, which comprises a an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:72.
44. The polypeptide of claim 43, which comprises the amino acid sequence of SEQ ID NO:72.
45. The polypeptide of claim 34, which comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:81.
46. The polypeptide of claim 45, which comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:81.
47. The polypeptide of claim 46, which comprises the amino acid sequence of SEQ ID NO:81.
48. The polypeptide of any one of claims 25 to 47, wherein the non-CD40L polypeptide comprises an immunoglobulin heavy chain.
49. The polypeptide of claim 48, wherein the immunoglobulin heavy chain is linked to an immunoglobulin light chain.
50. The polypeptide of claim 49, wherein the immunoglobulin heavy chain and
immunoglobulin light chain form an antigen-binding site.
51. The polypeptide of any one of claims 25 to 50, wherein the non-CD40L polypeptide comprises a single chain antibody or a Fab.
52. An agent comprising the polypeptide of any one of claims 1 to 51.
53. A homodimeric agent comprising the polypeptide of any one of claims 1 to 52.
A heterodimeric agent comprising the polypeptide of any one of claims 1 to 52.
The agent of any one of claims 52 to 54, wherein the agent
(a) activates CD40; and/or
(b) induces CD40 activity.
56. The agent of any one of claims 52 to 54, wherein the agent
(a) has an improved therapeutic index compared to a reference agent;
(b) induces reduced systemic inflammation compared to a reference agent;
(c) induces lower serum liver enzyme level compared to a reference agent; and/or
(d) induces lower pro-inflammatory cytokine release than a reference agent, wherein the reference agent and the agent comprise the same polypeptide except that the reference agent comprises a polypeptide comprising a stalk region that does not comprise a mutation in the integrin-binding region.
57. The agent of claim 56, wherein reduced systemic inflammation is indicated by lower levels of blood neutrophils.
58. The agent of claim 57, wherein the blood neutrophil level is at least about 20%, 30%>, 40%, 50%, 60%, or 70% lower.
59. The agent of claim 57, wherein the blood neutrophil level is from about 20% to about 70%) lower, from about 20% to about 60%> lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50%) to about 70%) lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower.
60. The agent of claim 57, wherein the blood neutrophil level is about 20% lower, about 30%> lower, about 40% lower, about 50% lower, about 60%> lower, or about 70% lower.
61. The agent of claim 56, wherein the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP).
62. The agent of claim 61, wherein the liver enzyme level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
63. The agent of claim 61, wherein the liver enzyme level is from about 20% to about 70% lower, from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20%) to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50% lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40%) to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70%) lower, from about 50% to about 60% lower, or from about 60% to about 70% lower.
64. The agent of claim 61, wherein the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
65. The agent of 56, wherein the pro-inflammatory cytokine is selected from the group
consisting of: IL-Ιβ, IL-6, TNF-alpha, IP-10, KC, and ΜΙΡ-Ια.
66. The agent of claim 57, wherein the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower
67. The agent of claim 57, wherein the pro-inflammatory cytokine release is from about 20% to about 70%) lower, from about 20% to about 60% lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30%> to about 60%> lower, from about 30%> to about 50%) lower, from about 30%> to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50%) to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower.
The agent of claim 57, wherein the pro-inflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
A bispecific agent comprising:
a) the polypeptide of any one of claims 1 to 52; and
b) an antigen-binding site from an antibody.
The bispecific agent of claim 69, wherein the agent is a homodimer or a heterodimer.
A bispecific agent comprising:
a) the polypeptide of any one of claims 1 to 52, and
b) an immune response stimulating agent or functional fragment thereof, or an antibody or functional fragment thereof.
The bispecific agent of claim 71, wherein the agent is a homodimer or a heterodimer.
A heterodimeric bispecific agent comprising:
a) a first arm comprising:
(i) a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40 ligand (CD40L) or a fragment thereof capable of binding CD40, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises a fragment of the stalk region of CD40L, and wherein the stalk region comprises a mutation in the integrin-binding region, and
(ii) a Fc region; and b) a second arm comprising an antigen-binding site from an antibody.
74. The bispecific agent of any one of claims 69 to 73, wherein the Fc region is
deglycosylated.
75. The bispecific agent of any one of claims 69 to 74, wherein each of the first, second, and third copies comprises the amino acid sequence of SEQ ID NO: 15, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, or SEQ ID NO:68.
76. The bispecific agent of any one of claims 69 to 75, wherein the first arm comprises the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:80.
77. The bispecific agent of any one of claims 69 to 76, wherein the first arm comprises the amino acid sequence of SEQ ID NO:72.
78. The bispecific agent of any one of claims 69 to 76, wherein the first arm comprises the amino acid sequence of SEQ ID NO:81.
79. The bispecific agent of any one of claims 69 to 76, wherein the single chain fusion
polypeptide is directly linked to the Fc region.
80. The bispecific agent of any one of claims 69 to 76 wherein the single chain fusion
polypeptide is connected to the Fc region by a linker.
81. The bispecific agent of any one of claims 69 to 80, wherein the Fc region is from a human IgGl, IgG2, IgG3, or IgG4 immunoglobulin.
82. The bispecific agent of any one of claims 69 to 81, wherein the Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:39-45 and 82.
83. The bispecific agent of claim 82, wherein the Fc region comprises the amino acid sequence of SEQ ID NO:44 or SEQ ID NO:45.
84. The bispecific agent of claim 82, wherein the Fc region comprises the amino acid
sequence of SEQ ID NO:82.
85. The bispecific agent of any one of claims 69 to 84, wherein the first arm comprises a first CH3 domain and the second arm comprises a second CH3 domain, each of which is modified to promote formation of heterodimers.
86. The bispecific agent of claim 85, wherein the first and second CH3 domains are modified based upon electrostatic effects.
87. The bispecific agent of claim 85 or claim 86, wherein the first arm comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:47, wherein the amino acids are replaced with glutamate or aspartate, and the second arm comprises a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO:47, wherein the amino acids are replaced with lysine.
88. The bispecific agent of claim 85 or claim 86, wherein the first arm comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO:47, wherein the amino acids are replaced with lysine, and the second arm comprises a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:47, wherein the amino acids are replaced with glutamate or aspartate.
89. The bispecific agent of claim 86, wherein the first and second CH3 domains are modified using a knobs-into-holes technique.
90. The bispecific agent any one of claims 69 to 89, wherein the antigen-binding site specifically binds a tumor antigen.
91. The bispecific agent of any one of claims 69 to 89, wherein the antigen-binding site
specifically binds PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM-3, or B7-H4.
92. The bispecific agent of any one of claims 69 to 91, which comprises an immune response stimulating agent.
93. The bispecific agent of any one of claims 69 to 92, which comprises an antibody.
94. The bispecific agent of any one of claims 69 to 93, wherein the antibody specifically binds to PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, or B7-H4.
95. The polypeptide or agent of any one of claims 1 to 94, which increases cell-mediated immunity.
96. The polypeptide or agent of any one of claims 1 to 95, which increases antigen-presenting cell (APC) activity.
97. The polypeptide or agent of any one of claims 1 to 96, which increases T-cell activity.
98. The polypeptide or agent of any one of claims 1 to 97, which increases cytolytic T-cell (CTL) activity.
99. The polypeptide or agent of any one of claims 1 to 98, which increases natural killer (NK) activity.
100. The polypeptide or agent of any one of claims 1 to 99, which decreases or inhibits
regulatory T-cell (Treg) activity.
101. The polypeptide or agent of any one of claims 1 to 100, which decreases or inhibits myeloid-derived suppressor cell (MDSC) activity.
102. The polypeptide or agent of any one of claims 1 to 101, which increases an effective
immune response without causing substantial side effects and/or immune-based toxicities.
103. The polypeptide or agent of any one of claims 1 to 102, which increases an effective
immune response without causing cytokine release syndrome (CRS) or a cytokine storm.
104. A cell producing the polypeptide or agent of any one of claims 1 to 103.
105. A composition comprising the polypeptide or agent of any one of claims 1 to 103.
106. A pharmaceutical composition comprising the polypeptide or agent of any one of claims 1 to 103 and a pharmaceutically acceptable carrier.
107. A polynucleotide comprising a nucleotide sequence that encodes the polypeptide or agent of any one of claims 1 to 103.
108. A polynucleotide comprising a nucleotide sequence that encodes a polypeptide or agent of any one of claims 1 to 103 and a signal sequence.
109. A vector comprising the polynucleotide of claim 107 or claim 108.
110. An isolated cell comprising the polynucleotide of claim 107 or claim 108 or the vector of claim 109.
111. A method of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
112. The method of claim 111, wherein the immune response is against a tumor or cancer.
113. A method of inhibiting the growth of a tumor, comprising contacting a tumor or tumor cell with an effective amount of the polypeptide or agent of any one of claims 1 to 103.
114. A method of inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
115. The method of claim 113 or claim 114, wherein the tumor is selected from the group consisting of colorectal tumor, colon tumor, ovarian tumor, pancreatic tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor.
116. A method of treating cancer in a subject, comprising administering to the subject a
therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
117. The method of claim 116, wherein the cancer is selected from the group consisting of colorectal cancer, colon cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, head and neck cancer, lymphoma and leukemia.
118. A method of increasing T-cell activity in a subject, comprising administering to the
subject a therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
119. A method of increasing antigen-presenting cell (APC) activity in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
A method of increasing dendritic cell activity in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
121. A method of increasing cytolytic T-cell (CTL) activity in a subject, comprising
administering to the subject a therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
A method of increasing natural killer (NK) activity in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
A method of decreasing or inhibiting regulatory T-cell (Treg) activity in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
124. A method of decreasing or inhibiting myeloid-derived suppressor cell (MDSC) activity in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide or agent of any one of claims 1 to 103.
The method of any one of claims 111 to 124, which further comprises administering least one additional therapeutic agent.
126. The method of claim 125, wherein the additional therapeutic agent is a chemotherapeutic agent.
127. The method of claim 125, wherein the additional therapeutic agent is an antibody.
128. The method of claim 127, wherein the additional therapeutic agent is an anti-PD-1
antibody, an anti-PD-Ll antibody, an anti-CTLA-4 antibody, an anti-TIGIT antibody, or an anti-B7-H4 antibody.
The method of claim 125, wherein the additional therapeutic agent is an immune response stimulating agent.
The method of claim 129, wherein the immune response stimulating agent is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM- CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4- IBB ligand, anti-CD3 antibody, anti- CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-Ll antibody, anti- LAG-3 antibody, and anti-TEVI-3 antibody.
The method of any one of claims 1 11 to 130, wherein the administration of the polypeptide or agent of any one of claims 1 to 103
(a) induces reduced systemic inflammation compared to a reference polypeptide or reference agent;
(b) induces lower serum liver enzyme level compared to a reference polypeptide or reference agent; and/or
(c) induces lower pro-inflammatory cytokine release than a reference polypeptide or reference agent,
wherein the reference polypeptide or reference agent and the polypeptide or agent comprise the same polypeptide except that the reference polypeptide or reference agent comprise a polypeptide comprising a stalk region that does not comprise a mutation in the integrin-binding region.
The method of claim 131, wherein reduced systemic inflammation is indicated by lower levels of blood neutrophils.
The method of claim 132, wherein the blood neutrophil level is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
134. The method of claim 131, wherein the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP).
135. The method of claim 134, wherein the liver enzyme level is at least about 20%, 30%>, 40%, 50%, 60%, or 70% lower.
136. The method of claim 134, wherein the liver enzyme level is from about 20% to about 70%) lower, from about 20% to about 60%> lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50%) to about 70%) lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower.
137. The method of claim 134, wherein the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
138. The method of claim 131, wherein the pro-inflammatory cytokine is selected from the group consisting of: IL-Ιβ, IL-6, T F-alpha, IP-10, KC, and ΜΙΡ-Ια.
139. The method of claim 138, wherein the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
140. The method of claim 138, wherein the pro-inflammatory cytokine release is from about 20%) to about 70%) lower, from about 20% to about 60% lower, from about 20% to about 50%) lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30%) to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60% lower, or from about 60% to about 70% lower.
141. The method of claim 138, wherein the pro-inflammatory cytokine release is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
142. The method of any one of claims 111 to 141, wherein the subject is human.
143. A method of improving the therapeutic index of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40- binding fragment thereof, wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region.
144. A method of decreasing the toxicity of a human CD40 agonist polypeptide comprising at least one copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the method comprises introducing a mutation into the integrin-binding region of the CD40L stalk region.
145. The method of claim 143 or claim 144, wherein the human CD40 agonist polypeptide comprises three copies of the extracellular domain of human CD40L or a CD40-binding fragment thereof.
146. The method of any one of claims 143 to 145, wherein the improving the therapeutic index or decreasing the toxicity comprises
(a) induction of reduced systemic inflammation compared to the human CD40 agonist;
(b) induction of lower serum liver enzyme level compared to the human CD40 agonist; and/or
(c) induction of lower pro-inflammatory cytokine compared to the human CD40 agonist.
147. The method of claim 146, wherein the liver enzyme is selected from the group consisting of: alanine aminotransferase (ALT), aspartate aminotransferase (ASP), and alkaline phosphatase (ALP).
148. The method of claim 146, wherein the liver enzyme level is at least about 20%, 30%>, 40%, 50%, 60%, or 70% lower.
149. The method of claim 146, wherein the liver enzyme level is from about 20% to about 70%) lower, from about 20% to about 60%> lower, from about 20% to about 50% lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30%) to about 70%) lower, from about 30% to about 60% lower, from about 30% to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70% lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50%) to about 70%) lower, from about 50% to about 60% lower, or from about 60% to about 70%) lower.
150. The method of claim 146, wherein the liver enzyme level is about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
151. The method of claim 146, wherein the pro-inflammatory cytokine is selected from the group consisting of: IL-Ιβ, IL-6, T F-alpha, IP-10, KC, and ΜΙΡ-Ια.
152. The method of claim 146, wherein the pro-inflammatory cytokine release is at least about 20%, 30%, 40%, 50%, 60%, or 70% lower.
153. The method of claim 146, wherein the pro-inflammatory cytokine release is from about 20%) to about 70%) lower, from about 20% to about 60% lower, from about 20% to about 50%) lower, from about 20% to about 40% lower, from about 20% to about 30% lower, from about 30% to about 70% lower, from about 30% to about 60% lower, from about 30%) to about 50%) lower, from about 30% to about 40% lower, from about 40% to about 70%) lower, from about 40% to about 60% lower, from about 40% to about 50% lower, from about 50% to about 70% lower, from about 50% to about 60%> lower, or from about 60% to about 70% lower.
The method of claim 146, wherein the pro-inflammatory cytokine release is about 20% lower, about 30%> lower, about 40% lower, about 50% lower, about 60% lower, or about 70% lower.
155. An improved human CD40 agonist produced by the method of claim 143 or claim 144.
156. A method of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of the improved human CD40 agonist of claim 155.
157. The method of claim 145, wherein the immune response is against a tumor or cancer.
158. A method of inhibiting the growth of a tumor, comprising contacting a tumor or tumor cell with an effective amount of the improved human CD40 agonist of claim 155.
A method of inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of the improved human CD40 agonist of claim 155.
160. A method of treating cancer in a subject, comprising administering to the subject a
therapeutically effective amount of the improved human CD40 agonist of claim 155.
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