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EP4646432A1 - Anticorps anti-il-18bp - Google Patents

Anticorps anti-il-18bp

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Publication number
EP4646432A1
EP4646432A1 EP24704650.1A EP24704650A EP4646432A1 EP 4646432 A1 EP4646432 A1 EP 4646432A1 EP 24704650 A EP24704650 A EP 24704650A EP 4646432 A1 EP4646432 A1 EP 4646432A1
Authority
EP
European Patent Office
Prior art keywords
seq
sequence
nos
antibody
sequences comprise
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24704650.1A
Other languages
German (de)
English (en)
Inventor
Robert A. Horlick
Helen Toni JUN
Magdalena S. WILLEN
Christine M. CHIDESTER
Mark G. BARRETT
David J. King
Deborah A. WITHERDEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Lassen Therapeutics Inc
Original Assignee
Lassen Therapeutics Inc
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Filing date
Publication date
Application filed by Lassen Therapeutics Inc filed Critical Lassen Therapeutics Inc
Publication of EP4646432A1 publication Critical patent/EP4646432A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • Interleukin- 18 is an immune-stimulatory cytokine with antitumor activity. It plays pivotal roles in linking inflammatory immune responses and tumor progression. Indeed, recombinant human IL- 18 has been evaluated as a cancer immunotherapeutic agent. However, this approach has not worked, at least in part because of a feedback loop in humans where administration of IL-18 leads to the induction of increased IL-18BP production, neutralizing the administered IL-18 cytokine (see, for example, Robertson et al., Clinical Cancer Res. 12:4265- 4273, 2006).
  • IL-18BP is a high-affinity IL-18 decoy receptor that is frequently upregulated in tumors. Studies have implicated IL-18BP as a secreted immune checkpoint and a barrier to IL- 18 immunotherapy (see, for example, Zhou et al., Nature. 583(7817):609-614, 2020). IL-18BP inhibits the pro-inflammatory activity of IL-18 by sequestering it away from the cell-surface receptor. The affinity of IL- 18 for IL-18BP is higher than that of IL- 18 for IL- 18 receptor, and IL-18BP is frequently present in amounts in excess of IL-18, ensuring tight regulation.
  • IL-18BP has also been shown to balance Thl and Th2 immune responses, among others, and plays a critical role in autoimmune diseases (see, for example, Park et al., Biomedicines. 10(7): 1750, 2022). Thus, there is a need in the art for agents that specifically modulate the activity of IL- 18BP.
  • the present disclosure relates to antibodies that bind to interleukin- 18 binding protein (IL-18BP) and related compositions, which may be used in any of a variety of therapeutic and diagnostic methods, including the treatment or diagnosis of cancers and other diseases.
  • IL-18BP interleukin- 18 binding protein
  • aspects of the present disclosure include an isolated antibody, or an antigen binding fragment thereof, which binds to interleukin- 18 binding protein (IL-18BP), wherein at least one antibody, or antigen binding fragment thereof, comprises: a heavy chain variable region (VH) that comprises complementary determining region VHCDRI, VHCDR2, and VHCDR3 sequences selected from Table Al and variants thereof which specifically bind to IL-18BP; and a light chain variable region (VL) that comprises complementary determining region VLCDRI, VLCDR2, and VLCDR3 sequences selected from Table Al and variants thereof which specifically bind to IL-18BP.
  • VH heavy chain variable region
  • VHCDRI complementary determining region
  • VHCDR2 VHCDR3 sequences selected from Table Al and variants thereof which specifically bind to IL-18BP
  • VL light chain variable region
  • the VHCDRI, VHCDR2, and VHCDR3 sequences comprise TFXIX2X 3 X 4 X 5 H, IX6X7X8X9X10X11X12X13X14X15AQKFQG, and X16X17X18X19X20X21X22DY, respectively
  • the VLCDRI, VLCDR2, and VLCDR3 sequences comprise X23X24X25X26X27X28X29X30WX31A, X32X33X34X35X36X37X38, and QX39X40X41SFPYX42, respectively (see Table Ell for the definition of “X” residues).
  • the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 4-6, respectively;
  • the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 25-27, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 28- 30, respectively;
  • the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 31-33, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 34- 36, respectively;
  • the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 34-39, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 40- 42, respectively;
  • the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2, optionally wherein the VH has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations in the framework regions.
  • the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2, optionally wherein the VL has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations in the framework regions.
  • the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 277
  • the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 278
  • the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 279
  • the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 280
  • the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 285
  • the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 286
  • the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or
  • aspects of the present disclosure also include, an isolated antibody, or antigen binding fragment thereof, that binds to an epitope that comprises 197 and V153 according to the numbering of UniProt: 095998 (alternatively, 195 and V151 as defined by SEQ ID NO: 1; or 167 and V123 as defined by SEQ ID NO: 2).
  • the isolated antibody, or an antigen binding fragment thereof binds to interleukin- 18 binding protein (IL-18BP) at an epitope that comprises 197 and V153 according to the numbering of UniProt: 095998 (alternatively, 195 and V151 as defined by SEQ ID NO: 1; or 167 and V123 as defined by SEQ ID NO: 2).
  • IL-18BP interleukin- 18 binding protein
  • the VHCDRI, VHCDR2, and VHCDR3 sequences comprise TFXIX2X 3 X 4 X 5 H, IX6X7X8X9X10X11X12X13X14X15AQKFQG, and X16X17X18X19X20X21X22DY, respectively
  • the VLCDR1, VLCDR2, and VLCDR3 sequences comprise X23X24X25X26X27X28X29X30WX31A, X32X33X34X35X36X37X38, and QX39X40X41 SFPYX42, respectively (see Table Ell for the definition of “X” residues).
  • VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 4-6, respectively; or the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 265-267, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 268- 270, respectively.
  • the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 277
  • the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 278
  • the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 279
  • the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 280
  • the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 367
  • the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 368.
  • aspects of the present disclosure also include an isolated antibody, or an antigen binding fragment thereof, wherein the antibody binds to interleukin- 18 binding protein (IL- 18BP) and wherein the antibody competes with IL-18 for the binding of IL-18BP.
  • the present disclosure provides an antibody which binds to IL-18BP and interferes with the binding of IL- 18 to IL-18BP.
  • the present disclosure provides an antibody which binds to IL-18BP and is an IL-18BP antagonist, which antagonizes the binding activity between IL-18BP and IL-18.
  • the present disclosure provides an antibody which binds to the preformed IL-18-IL-18BP complex.
  • the present disclosure provides an antibody which binds to free IL-18BP.
  • the antibody or an antigen binding fragment thereof binds to a conformational epitope of IL-18BP.
  • the conformational epitope thereof comprises two or more amino acid residues selected from the group consisting of K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372.
  • the conformational epitope thereof comprises the amino acid residues of K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372.
  • the antibody or an antigen binding fragment thereof binds to a linear epitope of IL-18BP. In some embodiments, the isolated antibody or antigen binding fragment thereof binds to the binding interface between IL- 18 and a mature form of IL-18BP. In some embodiments, the isolated antibody or antigen binding fragment thereof binds the amino acid residues R61, Y69 and R131 of SEQ ID NO: 372.
  • the isolated antibody or antigen binding fragment thereof comprises a VH comprising a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 333, and a VL comprising a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 334.
  • the isolated antibody, or antigen binding fragment thereof binds to human IL-18BP and cynomolgus IL-18BP but does not bind (specifically or substantially) to mouse IL-18BP. In some embodiments, the isolated antibody, or antigen binding fragment thereof, binds to human IL-18BP, cynomolgus IL-18BP, and mouse IL-18BP. In some embodiments, the isolated antibody, or antigen binding fragment thereof, binds to the binding interface between IL- 18 and a mature form of IL-18BP.
  • the isolated antibody, or antigen binding fragment thereof binds to IL-18BP with a binding affinity that is stronger than the binding affinity between IL-18 and IL-18BP (KD ⁇ 650 pM), optionally a binding affinity of about 1 pm to about 650 pm, or about or less than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 300, 400, 500, 600, or 650 pM.
  • the isolated antibody, or antigen binding fragment thereof is an IL-18BP antagonist, which antagonizes the binding activity between IL-18BP and IL-18.
  • the isolated antibody, or antigen binding fragment thereof blocks the inhibitory activity of IL-18BP towards IL- 18, and thereby increases IL-18-mediated signaling, including induction of IFN-gamma, CXCL10, and/or TNFa.
  • the isolated antibody, or antigen binding fragment thereof comprises an IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3, and IgG4), or IgM Fc domain, optionally a human Fc domain, or a hybrid and/or variant thereof.
  • the isolated antibody, or antigen binding fragment thereof comprises an IgG Fc domain with high effector function in humans, optionally an IgGl or IgG3 Fc domain. In some embodiments, the isolated antibody, or antigen binding fragment thereof, comprises an IgG Fc domain with low effector function in humans, optionally an IgG2 or IgG4 Fc domain. In some embodiments, the isolated antibody, or antigen binding fragment thereof, is a monoclonal antibody. In some embodiments, the isolated antibody, or antigen binding fragment thereof, is a humanized antibody.
  • the isolated antibody, or antigen binding fragment thereof is selected from an Fv fragment, a single chain Fv (scFv) polypeptide, an adnectin, an anticalin, an aptamer, an avimer, a camelid antibody, a designed ankyrin repeat protein (DARPin), a minibody, a nanobody, and a unibody.
  • Fv fragment a single chain Fv (scFv) polypeptide
  • adnectin an anticalin
  • an aptamer an avimer
  • a camelid antibody a designed ankyrin repeat protein (DARPin)
  • DARPin ankyrin repeat protein
  • isolated polynucleotides encoding an isolated anti IL-18BP antibody, or antigen binding fragment thereof, described herein, an expression vector comprising the isolated polynucleotide, and an isolated host cell comprising the vector.
  • isolated polynucleotide encoding an anti-IL-18BP antibody described herein. For instance, provided herein is a first polynucleotide encoding a VH region of an antibody disclosed herein and a second polynucleotide encoding a VL region of an antibody disclosed herein.
  • compositions comprising an isolated anti IL-18BP antibody, or antigen binding fragment thereof, described herein, and a pharmaceutically-acceptable carrier.
  • the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis with respect to the at least one antibody or antigen binding fragment and is substantially aggregate- and endotoxin-free.
  • the composition is a sterile, injectable solution, optionally suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration.
  • the disease or condition is a cancer or tumor or proliferative disease or disorder, optionally a proliferative disease or disorder selected from a lymphoproliferative disorder, a myeloproliferative disorder, proliferative enteritis, proliferative diabetic retinopathy, and a proliferative kidney disease.
  • the cancer or tumor expresses or overexpresses IL-18BP and/or IL-18, or the proliferative disease or disorder is associated with increased expression of IL-18BP and/or IL-18.
  • the cancer is selected from one or more of bone cancer, prostate cancer, melanoma (e.g., metastatic melanoma), pancreatic cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, hairy cell leukemias, acute lymphoblastic leukemias), lymphoma (e.g., non-Hodgkin’s lymphomas, Hodgkin’s lymphoma), hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lympho
  • Certain embodiments comprise administering the pharmaceutical composition (with the anti-IL18BP antibody, or antigen binding fragment thereof) in combination with IL-18. Some embodiments comprise administering the pharmaceutical composition (with the anti- IL18BP antibody, or antigen binding fragment thereof) in combination with an immune checkpoint modulatory agent selected from an antagonist of an inhibitory immune checkpoint molecule and an agonist of a stimulatory immune checkpoint molecule.
  • the immune checkpoint modulatory agent is a polypeptide, optionally an antibody or antigen binding fragment thereof or a ligand, or a small molecule.
  • the inhibitory immune checkpoint molecule is selected from one or more of Programmed Death-Ligand 1 (PD- Ll), Programmed Death 1 (PD-1), Programmed Death-Ligand 2 (PD-L2), Cytotoxic T- Lymphocyte-Associated protein 4 (CTLA-4), Indoleamine 2,3 -dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3), V-domain Ig suppressor of T cell activation (VISTA), B and T Lymphocyte Attenuator (BTLA), CD 160, Herpes Virus Entry Mediator (HVEM), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).
  • PD- Ll Programmed Death-Ligand 1
  • PD-1 Programmed Death 1
  • PD-L2 Programmed Death-Ligand 2
  • CTL-4 Cytotoxic T- Lymph
  • the antagonist is a PD-L1 and/or PD-L2 antagonist optionally selected from one or more of an antibody or antigen binding fragment or small molecule that specifically binds thereto, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), optionally wherein the cancer is selected from one or more of colorectal cancer, melanoma, breast cancer, non-small-cell lung carcinoma, bladder cancer, and renal cell carcinoma; the antagonist is a PD-1 antagonist optionally selected from one or more of an antibody or antigen binding fragment or small molecule that specifically binds thereto, nivolumab, pembrolizumab, MK-3475, AMP -224, AMP-514PDR001, and pidilizumab, optionally wherein the PD-1 antagonist is nivolumab and the cancer is optionally selected from one or more of Hodgkin’s lympho
  • Particular embodiments comprise administering the pharmaceutical composition (with the anti-IL18BP antibody, or antigen binding fragment thereof) in combination with at least one chemotherapeutic agent.
  • the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti -microtubule agent.
  • the alkylating agent is selected from one or more of nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide , and busulfan), nitrosoureas (optionally N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mytomycin, and diaziquone (AZQ)), cisplatins and derivatives thereof (optionally carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine); the anti-metabolite is selected from one or more of nitrogen mustards (optionally mech
  • the disease or condition is a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the disease or condition is an infectious disease.
  • the infectious disease is selected from viral, bacterial, fungal (optionally yeast), and protozoal infections.
  • Certain embodiments comprise administering the pharmaceutical composition (with the anti-IL18BP antibody, or antigen binding fragment thereof) in combination with IL-18.
  • the IL-18 and IL-18BP are mouse IL-18 and IL-18BP. In some embodiments, the IL-18 and IL-18BP are human IL-18 and IL-18BP.
  • the hypo-IL-18 fusion protein comprises, in an N- to C-terminal orientation, a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and IL-18BP. In some embodiments, the hypo-IL-18 fusion protein comprises an amino acid sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence from Table SI
  • hypo-IL-18 fusion protein comprising, in an N- to C-terminal orientation, a signal peptide, IL- 18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and IL-18BP.
  • the hypo-IL-18 fusion protein comprises, consists, or consists essentially of an amino acid sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence from Table SI
  • the immune response is an IL-18 mediated immune response.
  • the IL- 18 mediated immune response comprises induction of IFN- gamma, CXCL10, and/or TNFa in the subject in need thereof.
  • FIG. 1 shows a genealogy of the antibodies of the present disclosure.
  • FIG. 2 shows an alignment of cynomolgus (SEQ ID NO: 373), human (SEQ ID NO: 371), and mouse (SEQ ID NO: 375) IL-18BP orthologs.
  • the signal peptide (not present in the mature peptide) is underlined.
  • the four regions marked with a down arrow (J,) indicate amino acids that are identical in human and mouse but different in cynomolgus IL-18BP.
  • FIGS. 3 A-3B show the design of hypo-IL- 18.
  • FIG. 3 A shows a schematic representation of the hypo-IL- 18 expression cassette. Landmarks in the gene from N’ to C’ terminal include: osteonectin signal peptide, human IL-18 coding region, a flexible gly-ser linker interrupted by a Tobacco Etch Virus (TEV) protease cleavage site, human IL-18BP coding sequence, and 6x HIS tag.
  • FIG. 3B shows a view of the crystal structure-derived model of human IL-18 in complex with Ectromelia virus IL-18BP (as indicated). The IL-18BP N- and IL- 18 C-termini are indicated with a box and arrow.
  • FIG. 3 A shows a schematic representation of the hypo-IL- 18 expression cassette. Landmarks in the gene from N’ to C’ terminal include: osteonectin signal peptide, human IL-18 coding region, a flexible gly-ser linker interrupted by a Tob
  • mAbl l91 (R&D Systems) is a commercially available mouse anti-human IL-18BP neutralizing antibody that was used as a positive control in this experiment (cat. no. mabl 191).
  • FIG. 5 shows anti-mIL-18BP mAbs tested in a murine IL-18 reporter assay.
  • FIG. 6 shows anti-mIL-18BP mAbs tested in a mouse splenocyte assay. There was no clear upper limit plateau; consequently, IC50 values were not determinable.
  • FIGS. 7A-7B show the anti -turn or activity of mIL-18 in combination with SA51d (SA0051d) in established MC38 colon tumors.
  • FIG. 7B shows that the combination of mIL-18 + SA51d was able to significantly inhibit growth by day 15 compared to the vehicle control (p ⁇ 0.0001).
  • FIGS. 8A-8B show an assessment of mAbs in IL-18-mediated HEK293 reporter assay.
  • FIG. 8A shows pre-incubation of mAb with IL-18BP;
  • FIG. 8B shows preincubation of IL- 18 with IL-18BP.
  • FIG. 9 shows the effect of mAbs on IFNy secretion in KG-1 cells. Complex alone indicates addition of IL-18BP and mAb in the absence of IL-18.
  • FIGS. 10A-10B show induction of IFNY response in human (10A) and cyno (10B) PBMCs by high affinity mAbs.
  • FIGS. 11A-11B show induction of IFNY response in PBMC from precomplexed IL- 18/IL-18BP by high affinity mAbs.
  • FIG. 12 shows the cocrystal structural model of IL-18/IL-18BP complex.
  • the location of the four differences each in IL-18BP and in IL- 18 shared by human and mouse but not by cyno are shown in darker font (V75M, I97M, R113Q, V153M) and lighter font (V47I, T99A, KI 15R, F170Y), respectively.
  • I98M and V153M are vicinal and present at the binding interface, while V75M and R113Q are not near the active site.
  • FIG. 13 shows mAh binding to human IL-18BP with two orthologous cyno amino acid replacements.
  • the quality of the measured kinetics is summarized in the associated Table as for no binding, and 1 to 4 +’s for varying degrees of binding.
  • FIG. 14 shows the conformational epitope of SA64a as determined by XL-MS mass spectrometry. Partial sequence of IL-18BP is shown with residues that were cross-linked to SA64a indicated as the epitope.
  • FIG. 16 shows a spider plot of the efficacy of an anti-mouse IL-18BP antibody in an MC38 syngeneic tumor model.
  • the results demonstrate significantly improved efficacy using anti-mouse- PD-1 + anti-mouse IL-18BP (SA51d, also SA0051d) + mIL-18 compared to antimouse PD-1 alone or anti-mouse PD-1 + mIL-18.
  • FIG. 17 shows IL-18 (left) and lENy (right) levels in MC38 tumors from anti-IL-18BP treated mice compared with vehicle treated animals. Tumors were harvested at the endpoint of the efficacy study and assessed for proinflammatory cytokines by ELISA.
  • FIG. 18 shows relative NK cell marker (left) and Granzyme B (right) expression in MC38 tumors harvested at the endpoint of the efficacy study as assessed by qPCR. Markers of NK cell number and activity are increased in response to anti-IL-18BP and further increased with anti -PD-1 combination therapy.
  • FIGS. 19A-B shows the efficacy of an anti-mouse IL-18BP antibody in an MC38 syngeneic tumor rechallenge model. The results demonstrate a durable response in 11 of 12 animals upon rechallenge.
  • FIG. 20 shows CXCL10 (left) and CCL2 (right) production in human PBMC in response to high affinity mAbs. CXCL10 and CCL2 are secreted upon addition of IL-12+ IL-18 and enhanced with mAb addition as assessed by ELISA.
  • FIGS. 21A-E shows the efficacy of anti-mouse IL-18BP antibody SA0051d in the EMT6 mouse syngeneic tumor model.
  • FIG. 21 A shows the survival of mice bearing EMT6 tumors treated with anti-IL-18BP antibody SA0051d is significantly enhanced, and this is further improved in combination with anti -mouse PD-1 antibody.
  • FIG. 21B-E shows spider plots monitoring tumor growth in each animal. Treatment with SA0051d reduced tumor growth, and in combination with anti -PD-1 tumor regression was observed in some animals.
  • FIGS. 22A-E shows the efficacy of anti-mouse IL-18BP antibody SA0051d in the E0771 mouse syngeneic tumor model.
  • FIG. 22A shows mean tumor growth in each group and
  • FIG. 22B-E shows the spider plots monitoring tumor growth in each animal.
  • Treatment with SA0051d significantly reduced tumor growth as did treatment with the combination of SA0051d and anti -mouse PD-1 antibody.
  • the present disclosure relates to antibodies, and antigen binding fragments thereof, which specifically bind to interleukin- 18 binding protein (IL-18BP), for example, human IL- 18BP, in particular antibodies having epitopic specificity and improved characteristics.
  • IL-18BP interleukin- 18 binding protein
  • Some embodiments include specific humanized antibodies and fragments thereof capable of binding to IL-18BP, blocking or reducing the inhibitory binding of IL-18BP to its ligand IL- 18, and thereby increasing IL- 18 mediated downstream signaling.
  • an anti-IL- 18BP antibody, or antigen binding fragment thereof is an IL-18BP antagonist or inhibitor.
  • the IL-18BP antagonist antibodies described herein are useful in the treatment and prevention of various diseases and conditions, such as cancers and others. Some embodiments thus relate to the use of anti-IL-18BP antibodies, or antigen binding fragments thereof, for the diagnosis, assessment, and treatment of diseases and conditions, including those associated with IL-18 and/or IL-18BP activity or aberrant expression thereof.
  • “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • An antigen may have one or more epitopes.
  • the term “antigen” includes substances that are capable, under appropriate conditions, of inducing an immune response to the substance and of reacting with the products of the immune response.
  • an antigen can be recognized by antibodies (humoral immune response) or sensitized T-lymphocytes (T helper or cell- mediated immune response), or both.
  • Antigens can be soluble substances, such as toxins and foreign proteins, or particulates, such as bacteria and tissue cells; however, only the portion of the protein or polysaccharide molecule known as the antigenic determinant (epitopes) combines with the antibody or a specific receptor on a lymphocyte. More broadly, the term “antigen” includes any substance to which an antibody binds, or for which antibodies are desired, regardless of whether the substance is immunogenic. For such antigens, antibodies can be identified by recombinant methods, independently of any immune response.
  • an “antagonist” refers to an agent (e.g., antibody) that interferes with or otherwise reduces the physiological action of another agent or molecule. In some instances, the antagonist specifically binds to the other agent or molecule. Included are full and partial antagonists.
  • An “agonist” refers to an agent (e.g., antibody) that increases or enhances the physiological action of another agent or molecule. In some instances, the agonist specifically binds to the other agent or molecule. Included are full and partial agonists.
  • amino acid is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics.
  • Naturally- occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example.
  • Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art.
  • Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moi eties on the amino acid or by derivatization of the amino acid.
  • Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid.
  • Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.
  • antibody encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as dAb, Fab, Fab’, F(ab’)2, Fv), single chain (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion with an antigen binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site or fragment (epitope recognition site) of the required specificity. Certain features and characteristics of antibodies (and antigen binding fragments thereof) are described in greater detail herein.
  • an antibody or antigen binding fragment can be of essentially any type.
  • an antibody is an immunoglobulin molecule capable of specific binding to a target, such as an immune checkpoint molecule, through at least one epitope recognition site, located in the variable region of the immunoglobulin molecule.
  • an antigen binding fragment refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and/or light chain that binds to the antigen of interest.
  • an antigen binding fragment of the herein described antibodies may comprise 1, 2, 3, 4, 5, or all 6 CDRs of a VH and VL sequence from antibodies that bind to a target molecule.
  • an antigen binding fragment of the present disclosure comprises all 6 CDRs of the VH and VL sequences of an antibody disclosed herein.
  • an antibody or antigen binding fragment thereof specifically binds to a target molecule, for example, an IL-18BP polypeptide or an epitope or complex thereof, with an equilibrium dissociation constant that is about or ranges from about ⁇ 10' 7 M to about 10' 8 M. In some embodiments, the equilibrium dissociation constant is about or ranges from about ⁇ 10' 9 M to about ⁇ 10' 10 M.
  • an antibody or antigen binding fragment thereof has an affinity (KD or ECso) for a target molecule (to which it specifically binds) of about, at least about, or less than about, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.
  • KD or ECso affinity for a target molecule (to which it specifically binds) of about, at least about, or less than about, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.
  • a molecule such as a polypeptide or antibody is said to exhibit “specific binding” or “preferential binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell, substance, or particular epitope than it does with alternative cells or substances, or epitopes.
  • An antibody “specifically binds” or “preferentially binds” to a target molecule or epitope if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances or epitopes, for example, by a statistically significant amount.
  • one member of the pair of molecules that exhibit specific binding has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and/or polar organization of the other member of the pair of molecules.
  • the members of the pair have the property of binding specifically to each other.
  • an antibody that specifically or preferentially binds to a specific epitope is an antibody that binds that specific epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds to other epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
  • Immunological binding generally refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example by way of illustration and not limitation, as a result of electrostatic, ionic, hydrophilic and/or hydrophobic attractions or repulsion, steric forces, hydrogen bonding, van der Waals forces, and other interactions.
  • the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (KD) of the interaction, wherein a smaller KD represents a greater affinity.
  • Immunological binding properties of selected polypeptides can be quantified using methods well known in the art.
  • One such method entails measuring the rates of antigen binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions.
  • both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
  • the ratio of Koff /Kon enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant KD.
  • affinity includes the equilibrium constant for the reversible binding of two agents and is expressed as KD or EC50.
  • Affinity of a binding protein to a ligand such as affinity of an antibody for an epitope can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM).
  • the term “avidity” refers to the resistance of a complex of two or more agents to dissociation after dilution.
  • affinity is expressed in the terms of the half maximal effective concentration (EC50), which refers to the concentration of an agent, such as an antibody, or an anti-IL-18BP antibody, as disclosed herein, which induces a response halfway between the baseline and maximum after a specified exposure time.
  • EC50 half maximal effective concentration
  • Antibodies can be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific for a polypeptide of interest can be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto. Also included are methods that utilize transgenic animals such as mice to express human antibodies.
  • Antibodies can also be generated or identified by the use of phage display or yeast display libraries (see, e.g., U.S. Patent No. 7,244,592; Chao et al., Nature Protocols. 1 :755-768, 2006).
  • HuCAL Human Combinatorial Antibody Library
  • human libraries designed with human-donor-sourced fragments encoding a lightchain variable region, a heavy-chain CDR-3, synthetic DNA encoding diversity in heavy-chain CDR-1, and synthetic DNA encoding diversity in heavy-chain CDR-2.
  • Other libraries suitable for use will be apparent to persons skilled in the art.
  • antibodies and antigen binding fragments thereof as described herein include a heavy chain and a light chain CDR set, respectively interposed between a heavy chain and a light chain framework region (FR) set which provide support to the CDRs and define the spatial relationship of the CDRs relative to each other.
  • CDR set refers to the three hypervariable regions of a heavy or light chain V region. Proceeding from the N-terminus of a heavy or light chain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3” respectively.
  • An antigen binding site therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region.
  • a polypeptide comprising a single CDR (e.g., a CDR1, CDR2 or CDR3) is referred to herein as a “molecular recognition unit.” Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with bound antigen, wherein the most extensive antigen contact is with the heavy chain CDR3. Thus, the molecular recognition units are primarily responsible for the specificity of an antigen binding site.
  • FR set refers to the four flanking amino acid sequences which frame the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the V region into the antigen binding site, particularly the FR residues directly adjacent to the CDRs. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, most V region sequences contain an internal disulfide loop of around 90 amino acid residues. When the V regions fold into a binding-site, the CDRs are displayed as projecting loop motifs which form an antigen binding surface.
  • immunoglobulin variable domains may be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof.
  • monoclonal antibodies which refer to a homogeneous antibody population wherein the monoclonal antibody is comprised of amino acids (naturally occurring and non-naturally occurring) that are involved in the selective binding of an epitope.
  • the term “monoclonal antibody” encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv), variants thereof, fusion proteins comprising an antigen binding portion, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding fragment (epitope recognition site) of the required specificity and the ability to bind to an epitope. It is not intended to be limited as regards the source of the antibody or the manner in which it is made (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals). The term includes whole immunoglobulins as well as the fragments etc. described above under the definition of “antibody.”
  • the proteolytic enzyme papain preferentially cleaves IgG molecules to yield several fragments, two of which (the F(ab) fragments) each comprise a covalent heterodimer that includes an intact antigen binding site.
  • the enzyme pepsin is able to cleave IgG molecules to provide several fragments, including the F(ab’)2 fragment which comprises both antigen binding sites.
  • An Fv fragment for use according to certain embodiments can be produced by preferential proteolytic cleavage of an IgM, and on rare occasions of an IgG or IgA immunoglobulin molecule. Fv fragments are, however, more commonly derived using recombinant techniques known in the art.
  • the Fv fragment includes a non-covalent VH::VL heterodimer including an antigen binding site which retains much of the antigen recognition and binding capabilities of the native antibody molecule (Inbar et al., PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980).
  • Fvs are stabilized by other means, for example, incorporation of at least one disulfide bond (Worn & Pluckthun, J. Mol. Biol. 305, 989-1010, 2001)
  • single chain Fv (scFV) antibodies are contemplated.
  • Kappa bodies Ill et al., Prot. Eng. 10:949-57, 1997
  • minibodies Martin et al., EMBO J 13:5305-9, 1994
  • diabodies Holliger et al., PNAS 90: 6444-8, 1993
  • Janusins Traunecker et al., EMBO J 10: 3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl. 7:51-52, 1992
  • a single chain Fv (scFv) polypeptide is a covalently linked VH::VL heterodimer which is expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide- encoding linker.
  • Huston et al. PNAS USA. 85(16):5879-5883, 1988.
  • a number of methods have been described to discern chemical structures for converting the naturally aggregated — but chemically separated — light and heavy polypeptide chains from an antibody V region into an scFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen binding site. See, e.g., U.S. Pat. Nos.
  • the antibodies or antigen binding fragments described herein are in the form of a “diabody.”
  • Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, the two domains being linked (e.g., by a peptide linker) but unable to associate with each other to form an antigen binding site: antigen binding sites are formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94/13804).
  • a dAb fragment of an antibody consists of a VH domain (Ward et al., Nature 341 :544-546, 1989).
  • Diabodies and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO94/13804; and Holliger et al., PNAS USA. 90:6444-6448, 1993)).
  • Minibodies comprising a scFv joined to a CH3 domain are also included (see Hu et al., Cancer Res. 56:3055-3061, 1996). See also Ward et al., Nature. 341 :544-546, 1989; Bird et al., Science. 242:423-426, 1988; Huston et al., PNAS USA. 85:5879-5883, 1988);
  • bispecific antibodies may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), e.g., prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
  • Bispecific diabodies as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against antigen X, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by a number of methods (Brinkman & Kontermann, mAbs 9: 182-212, 2017) including knobs-into-holes engineering (Ridgeway et al., Protein Eng. 9:616-621, 1996).
  • the antibodies or antigen binding fragments described herein are in the form of a UniBody®.
  • a UniBody® is an IgG4 antibody with the hinge region removed (see GenMab Utrecht, The Netherlands; see also, e.g., US20090226421). This antibody technology creates a stable, smaller antibody format with an anticipated longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and thus do not interact with the immune system. Fully human IgG4 antibodies may be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments having distinct stability properties relative to the corresponding intact IgG4 (GenMab, Utrecht).
  • the UniBody® Halving the IgG4 molecule leaves only one area on the UniBody® that can bind to cognate antigens (e.g., disease targets) and the UniBody® therefore binds univalently to only one site on target cells. For certain cancer cell surface antigens, this univalent binding may not stimulate the cancer cells to grow as may be seen using bivalent antibodies having the same antigen specificity, and hence UniBody® technology may afford treatment options for some types of cancer that may be refractory to treatment with conventional antibodies.
  • the small size of the UniBody® can be a great benefit when treating some forms of cancer, allowing for better distribution of the molecule over larger solid tumors and potentially increasing efficacy.
  • the antibodies and antigen binding fragments described herein are in the form of a nanobody.
  • Nanobodies are encoded by single genes and are efficiently produced in almost all prokaryotic and eukaryotic hosts, for example, E. coli (see U.S. Pat. No. 6,765,087), molds (for example Aspergillus or Trichoderma) and yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia (see U.S. Pat. No. 6,838,254).
  • the production process is scalable and multi-kilogram quantities of nanobodies have been produced.
  • Nanobodies may be formulated as a ready -to-use solution having a long shelf life.
  • the Nanoclone method (see WO 06/079372) is a proprietary method for generating Nanobodies against a desired target, based on automated high-throughput selection of B-cells.
  • the antibodies or antigen binding fragments described herein are in the form of an aptamer (see, e.g., Ellington et al., Nature. 346, 818-22, 1990; and Tuerk et al., Science. 249, 505-10, 1990, incorporated by reference).
  • aptamers included nucleic acid aptamers (e.g., DNA aptamers, RNA aptamers) and peptide aptamers.
  • Nucleic acid aptamers refer generally to nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalent method, such as SELEX (systematic evolution of ligands by exponential enrichment), to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. See, e.g., U.S. Patent Nos. 6,376,190; and 6,387,620, incorporated by reference.
  • Peptide aptamers typically include a variable peptide loop attached at both ends to a protein scaffold, a double structural constraint that typically increases the binding affinity of the peptide aptamer to levels comparable to that of an antibody’s (e.g., in the nanomolar range).
  • variable loop length may be composed of about 10-20 amino acids (including all integers in between), and the scaffold may include any protein that has good solubility and compacity properties.
  • Certain exemplary embodiments utilize the bacterial protein Thioredoxin-A as a scaffold protein, the variable loop being inserted within the reducing active site (-Cys-Gly-Pro-Cys- loop in the wild protein), with the two cysteines lateral chains being able to form a disulfide bridge.
  • Methods for identifying peptide aptamers are described, for example, in U.S. Application No. 2003/0108532, incorporated by reference.
  • Peptide aptamer selection can be performed using different systems known in the art, including the yeast two- hybrid system.
  • the antibodies or antigen binding fragments described herein are in the form of an avimer.
  • Avimers refer to multimeric binding proteins or peptides engineered using in vitro exon shuffling and phage display. Multiple binding domains are linked, resulting in greater affinity and specificity compared to single epitope immunoglobulin domains. See, e.g., Silverman et al., Nature Biotechnology. 23: 1556-1561, 2005; U.S. Patent No. 7,166,697; and U.S. Application Nos. 2004/0175756, 2005/0048512, 2005/0053973, 2005/0089932 and 2005/0221384, incorporated by reference.
  • the antibodies or antigen binding fragments described herein are in the form of an adnectin.
  • Adnectins refer to a class of targeted biologies derived from human fibronectin, an abundant extracellular protein that naturally binds to other proteins. See, e.g., U.S. Application Nos. 2007/0082365; 2008/0139791; and 2008/0220049, incorporated by reference.
  • Adnectins typically consists of a natural fibronectin backbone, as well as the multiple targeting domains of a specific portion of human fibronectin. The targeting domains can be engineered to enable an adnectin to specifically recognize an IL-18BP polypeptide or an epitope thereof.
  • the antibodies or antigen binding fragments described herein are in the form of an anticalin.
  • Anticalins refer to a class of antibody mimetics that are typically synthesized from human lipocalins, a family of binding proteins with a hypervariable loop region supported by a structurally rigid framework. See, e.g., U.S. Application No. 2006/0058510.
  • Anticalins typically have a size of about 20 kDa.
  • Anticalins can be characterized by a barrel structure formed by eight antiparallel P-strands (a stable P-barrel scaffold) that are pairwise connected by four peptide loops and an attached a-helix. In certain aspects, conformational deviations to achieve specific binding are made in the hypervariable loop region(s). See, e.g., Skerra, FEBS J. 275:2677-83, 2008, incorporated by reference.
  • the antibodies or antigen binding fragments described herein are in the form of a designed ankyrin repeat protein (DARPin).
  • DARPins include a class of nonimmunoglobulin proteins that can offer advantages over antibodies for target binding in drug discovery and drug development.
  • DARPins are ideally suited for in vivo imaging or delivery of toxins or other therapeutic payloads because of their favorable molecular properties, including small size and high stability. The low-cost production in bacteria and the rapid generation of many target-specific DARPins make the DARPin approach useful for drug discovery.
  • DARPins can be easily generated in multispecific formats, offering the potential to target an effector DARPin to a specific organ or to target multiple receptors with one molecule composed of several DARPins. See, e.g., Stumpp et al., Curr Opin Drug Discov Devel. 10: 153-159, 2007; U.S. Application No. 2009/0082274; and PCT/EP2001/10454, incorporated by reference.
  • heavy chain dimers such as antibodies from camelids and sharks.
  • Camelid and shark antibodies comprise a homodimeric pair of two chains of V-like and C-like domains (neither has a light chain). Since the VH of a heavy chain dimer IgG in a camelid does not have to make hydrophobic interactions with a light chain, the region in the heavy chain that normally contacts a light chain is changed to hydrophilic amino acid residues in a camelid. VH domains of heavy-chain dimer IgGs are called VHH domains.
  • Shark Ig-NARs comprise a homodimer of one variable domain (termed a V-NAR domain) and five C-like constant domains (C-NAR domains).
  • camelids the diversity of antibody repertoire is determined by the complementary determining regions (CDR) 1, 2, and 3 in the VH or VHH regions.
  • the CDR3 in the camelid VHH region is characterized by its relatively long length averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9): 1129). This is in contrast to CDR3 regions of antibodies of many other species.
  • the CDR3 of mouse VH has an average of 9 amino acids.
  • the antibodies or antigen binding fragments thereof are humanized. These embodiments refer to a chimeric molecule, generally prepared using recombinant techniques, having an antigen binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin.
  • the antigen binding site may comprise either complete variable domains fused onto constant domains or only the CDRs (entire or in part) grafted onto appropriate framework regions in the variable domains.
  • Epitope binding sites may be wild type or modified by one or more amino acid substitutions.
  • variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs.
  • CDRs complementarity-determining regions
  • FRs framework regions
  • the variable regions can be “reshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified.
  • humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In some embodiments, only some of the CDR sequences are grafted from the nonhuman antibody (Bowers et al., J. Biol. Chem. 288:7688- 7696, 2013).
  • humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.
  • the antibodies are “chimeric” antibodies.
  • a chimeric antibody is comprised of an antigen binding fragment of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody.
  • the Fc domain or heterologous Fc domain is of human origin.
  • the Fc domain or heterologous Fc domain is of mouse origin.
  • the heterologous Fc domain may be from a different Ig class from the parent antibody, including IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3, and IgG4), and IgM.
  • the heterologous Fc domain may be comprised of CH2 and CH3 domains from one or more of the different Ig classes.
  • the antigen binding fragment of a chimeric antibody may comprise only one or more of the CDRs of the antibodies described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibodies described herein), or may comprise an entire variable domain (VL, VH or both).
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • coding sequence is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene.
  • non-coding sequence refers to any nucleic acid sequence that does not directly contribute to the code for the polypeptide product of a gene.
  • effector function in the context of antibodies refers to the ability of that antibody to engage with other arms of the immune system, including for example, the activation of the classical complement pathway, or through engagement of Fc receptors.
  • Complement dependent pathways are primarily driven by the interaction of Clq with the Cl complex with clustered antibody Fc domains.
  • Antibody dependent cellular cytotoxicity is primarily driven by the interaction of Fc receptors (FcRs) on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) which bind to the Fc region of an IgG which itself is bound to a target cell.
  • Fc receptors are key immune regulatory receptors connecting the antibody mediated (humoral) immune response to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcyR (IgG), FcsRI (IgE), FcaRI (IgA), FcpR (IgM) and FcSR (IgD). There are at least three classes of receptors for human IgG found on leukocytes: CD64 (FcyRI), CD32 (FcyRIIa, FcyRIIb and FcyRIIc) and CD 16 (FcyRIIIa and FcyRIIIb).
  • FcyRI is classed as a high affinity receptor (nanomolar range KD) while FcyRII and FcyRIII are low to intermediate affinity (micromolar range KD).
  • Fc binding Upon Fc binding a signaling pathway is triggered which results in the secretion of various substances, such as lytic enzymes, perforin, granzymes and tumor necrosis factor, which mediate in the destruction of the target cell.
  • the level of ADCC effector function various for human IgG subtypes. Although this is dependent on the allotype and specific FcvR, in simple terms ADCC effector function is “high” for human IgGl and IgG3, and “low” for IgG2 and IgG4.
  • endotoxin free or “substantially endotoxin free” relates generally to compositions, solvents, and/or vessels that contain at most trace amounts (e.g., amounts having no clinically adverse physiological effects to a subject) of endotoxin, and preferably undetectable amounts of endotoxin.
  • Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically gram-negative bacteria, although endotoxins may be found in gram-positive bacteria, such as Listeria monocytogenes .
  • LPS lipopolysaccharides
  • LOS lipo-oligo-saccharides
  • a depyrogenation oven may be used for this purpose, as temperatures in excess of 300 °C are typically required to break down most endotoxins. For instance, based on primary packaging material such as syringes or vials, the combination of a glass temperature of 250 °C and a holding time of 30 minutes is often sufficient to achieve a 3 log reduction in endotoxin levels.
  • Other methods of removing endotoxins are contemplated, including, for example, chromatography and filtration methods, as described herein and known in the art.
  • Endotoxins can be detected using routine techniques known in the art.
  • the Limulus Amoebocyte Lysate assay which utilizes blood from the horseshoe crab, is a very sensitive assay for detecting presence of endotoxin.
  • very low levels of LPS can cause detectable coagulation of the limulus lysate due a powerful enzymatic cascade that amplifies this reaction.
  • Endotoxins can also be quantitated by enzyme-linked immunosorbent assay (ELISA).
  • endotoxin levels may be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mg of active compound.
  • 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.
  • epitope includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor.
  • An epitope includes a region of an antigen that is bound by an antibody.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and may in certain embodiments have specific three-dimensional structural characteristics, and/or specific charge characteristics. Epitopes can be contiguous or non-contiguous in relation to the primary structure of the antigen, for example, an IL-18BP polypeptide.
  • an epitope comprises, consists, or consists essentially of about, at least about, or no more than about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids (i.e., a linear epitope) or non-contiguous amino acids (i.e., conformational epitope) of a reference sequence (see, e.g., Table Bl) or target molecule described herein.
  • An “epitope” includes that portion of an antigen or other macromolecule capable of forming a binding interaction that interacts with the variable region binding pocket of a binding protein. Such binding interaction can be manifested as an intermolecular contact with one or more amino acid residues of a CDR.
  • Antigen binding can involve a CDR3 or a CDR3 pair.
  • An epitope can be a linear peptide sequence (i.e., “continuous”) or can be composed of noncontiguous amino acid sequences (i.e., “conformational” or “discontinuous”).
  • a binding protein can recognize one or more amino acid sequences; therefore an epitope can define more than one distinct amino acid sequence.
  • Epitopes recognized by binding protein can be determined by peptide mapping and sequence analysis techniques well known to one of skill in the art.
  • a “cryptic epitope” or a “cryptic binding site” is an epitope or binding site of a protein sequence that is not exposed or substantially protected from recognition within an unmodified polypeptide, but is capable of being recognized by a binding protein of a denatured or proteolyzed polypeptide. Amino acid sequences that are not exposed, or are only partially exposed, in the unmodified polypeptide structure are potential cryptic epitopes. If an epitope is not exposed, or only partially exposed, then it is likely that it is buried within the interior of the polypeptide.
  • Candidate cryptic epitopes can be identified, for example, by examining the three- dimensional structure of an unmodified polypeptide.
  • EC50 half maximal effective concentration
  • concentration of an agent e.g., antibody
  • the EC50 of a graded dose response curve therefore represents the concentration of a compound at which 50% of its maximal effect is observed.
  • EC50 also represents the plasma concentration required for obtaining 50% of a maximum effect in vivo.
  • EC90 refers to the concentration of an agent or composition at which 90% of its maximal effect is observed. The “EC90” can be calculated from the “EC50” and the Hill slope, or it can be determined from the data directly, using routine knowledge in the art.
  • the EC50 of an agent is less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200 or 500 nM.
  • an agent will have an EC5O value of about InM or less.
  • Immune response means any immunological response originating from immune system, including responses from the cellular and humeral, innate and adaptive immune systems.
  • Exemplary cellular immune cells include for example, lymphocytes, macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, dendritic cells, mast cells, monocytes, and all subsets thereof.
  • Cellular responses include for example, effector function, cytokine release, phagocytosis, efferocytosis, translocation, trafficking, proliferation, differentiation, activation, repression, cell-cell interactions, apoptosis, etc.
  • Humeral responses include for example IgG, IgM, IgA, IgE, responses and their corresponding effector functions.
  • the “half-life” of an agent such as an antibody can refer to the time it takes for the agent to lose half of its pharmacologic, physiologic, or other activity, relative to such activity at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point.
  • “Half-life” can also refer to the time it takes for the amount or concentration of an agent to be reduced by half of a starting amount administered into the serum or tissue of an organism, relative to such amount or concentration at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point.
  • the half-life can be measured in serum and/or any one or more selected tissues.
  • modulating and “altering” include “increasing,” “enhancing” or “stimulating,” as well as “decreasing”, “reducing”, or “inhibiting”, typically in a statistically significant or a physiologically significant amount or degree relative to a control.
  • An “increased,” “stimulated” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times (e.g., 500, 1000 times) (including all integers and ranges in between e.g., 1.5, 1.6, 1.7.
  • a “decreased” or “reduced” or “inhibited” amount is typically a “statistically significant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease (including all integers and ranges in between) in the amount produced by no composition (e.g., the absence of an agent) or a control composition.
  • polypeptide “protein” and “peptide” are used interchangeably and mean a polymer of amino acids not limited to any particular length.
  • enzyme includes polypeptide or protein catalysts. The terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences.
  • polypeptide or “protein” means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non- covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally- occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence.
  • the polypeptide is a “recombinant” polypeptide, produced by recombinant cell that comprises one or more recombinant DNA molecules, which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the cell.
  • polynucleotide and “nucleic acid” includes mRNA, RNA, cRNA, cDNA, and DNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • isolated DNA and “isolated polynucleotide” and “isolated nucleic acid” refer to a molecule that has been isolated free of total genomic DNA of a particular species.
  • an isolated DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Also included are non-coding polynucleotides (e.g., primers, probes, oligonucleotides), which do not encode a polypeptide. Also included are recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phage, viruses, and the like.
  • Additional coding or non-coding sequences may, but need not, be present within a polynucleotide described herein, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
  • a polynucleotide or expressible polynucleotides regardless of the length of the coding sequence itself, may be combined with other sequences, for example, expression control sequences.
  • “Expression control sequences” include regulatory sequences of nucleic acids, or the corresponding amino acids, such as promoters, leaders, enhancers, introns, recognition motifs for RNA, or DNA binding proteins, polyadenylation signals, terminators, internal ribosome entry sites (IRES), secretion signals, subcellular localization signals, and the like, which have the ability to affect the transcription or translation, or subcellular, or cellular location of a coding sequence in a host cell. Exemplary expression control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
  • a “promoter” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3’ direction) coding sequence.
  • the promoter sequence is bounded at its 3’ terminus by the transcription initiation site and extends upstream (5’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI) can be found within a promoter sequence, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters can often, but not always, contain “TATA” boxes and “CAT” boxes.
  • Prokaryotic promoters contain Shine- Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • promoters including constitutive, inducible and repressible promoters, from a variety of different sources are well known in the art.
  • Representative sources include for example, viral, mammalian, insect, plant, yeast, and bacterial cell types), and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available on line or, for example, from depositories such as the ATCC as well as other commercial or individual sources.
  • Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3’ or 5’ direction).
  • Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter.
  • Inducible promoters include the Tet system, (US Patents 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad. Sci.
  • An “expressible polynucleotide” includes a cDNA, RNA, mRNA or other polynucleotide that comprises at least one coding sequence and optionally at least one expression control sequence, for example, a transcriptional and/or translational regulatory element, and which can express an encoded polypeptide upon introduction into a cell, for example, a cell in a subject.
  • adenoviral vectors include adenoviral vectors, herpes virus vectors, vaccinia virus vectors, adeno-associated virus (AAV) vectors, and retroviral vectors.
  • AAV adeno-associated virus
  • retroviral vector is a derivative of a murine or avian retrovirus, or is a lentiviral vector.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • SIV BIV
  • HIV Rous Sarcoma Virus
  • a number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • the vector may be made target specific.
  • Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a protein. Illustrative targeting may be accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector.
  • the expressible polynucleotide is a modified RNA or modified mRNA polynucleotide, for example, a non-naturally occurring RNA analog.
  • the modified RNA or mRNA polypeptide comprises one or more modified or non-natural bases, for example, a nucleotide base other than adenine (A), guanine (G), cytosine (C), thymine (T), and/or uracil (U).
  • the modified mRNA comprises one or more modified or non-natural intemucleotide linkages.
  • RNA polynucleotides for delivering an encoded therapeutic polypeptide are described, for example, in Kormann et al., Nat Biotechnol. 29: 154-7, 2011; and U.S. Application Nos. 2015/0111248; 2014/0243399; 2014/0147454; and 2013/0245104, which are incorporated by reference in their entireties.
  • isolated polypeptide or protein referred to herein means that a subject protein (1) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or non-covalent interaction) with portions of a protein with which the “isolated protein” is associated in nature, (6) is operably associated (by covalent or non-covalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature.
  • Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, or may be of synthetic origin, or any combination thereof.
  • the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).
  • the “purity” of any given agent (e.g., antibody) in a composition may be defined.
  • certain compositions may comprise an agent that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure on a protein basis or a weight-weight basis, including all decimals and ranges in between, as measured, for example and by no means limiting, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.
  • HPLC high performance liquid chromatography
  • reference sequence refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the Sequence Listing.
  • Certain embodiments include biologically active “variants” and “fragments” of the polypeptides (e.g., antibodies) described herein, and the polynucleotides that encode the same. “Variants” contain one or more substitutions, additions, deletions, and/or insertions relative to a reference polypeptide or polynucleotide (see, e.g., the Tables and the Sequence Listing).
  • a variant polypeptide or polynucleotide comprises an amino acid or nucleotide sequence with at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity or similarity or homology to a reference sequence, as described herein, and substantially retains the activity of that reference sequence.
  • sequences that consist of or differ from a reference sequences by the addition, deletion, insertion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150 or more amino acids or nucleotides and which substantially retain the activity of that reference sequence.
  • the additions or deletions include C-terminal and/or N-terminal additions and/or deletions.
  • sequence identity or, for example, comprising a “sequence at least 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys,
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al., Nucl. Acids Res. 25:3389, 1997.
  • solubility refers to the property of an agent (e.g., antibody) provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg/ml, etc.), molarity, molality, mole fraction or other similar descriptions of concentration.
  • the maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent.
  • solubility is measured at physiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8).
  • solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaPCU).
  • solubility is measured at relatively lower pH (e.g., pH 6.0) and relatively higher salt (e.g., 500mM NaCl and lOmM NaPC ).
  • solubility is measured in a biological fluid (solvent) such as blood or serum.
  • the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25 °C) or about body temperature (37 °C).
  • an agent has a solubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/ml at room temperature or at 37 °C.
  • a “subject” or a “subject in need thereof’ or a “patient” or a “patient in need thereof’ includes a mammalian subject such as a human subject.
  • “Substantially” or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantity.
  • Statistical significance it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
  • “Therapeutic response” refers to improvement of symptoms (whether or not sustained) based on administration of one or more therapeutic agents.
  • terapéuticaally effective amount is the amount of an agent (e.g., anti-IL-18BP antibody, immunotherapy agent) needed to elicit the desired biological response following administration.
  • an agent e.g., anti-IL-18BP antibody, immunotherapy agent
  • treatment of a subject (e.g., a mammal, such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of a disease.
  • Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
  • prophylactic treatments which can be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset.
  • “Treatment” or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.
  • wild-type refers to a gene or gene product (e.g., a polypeptide) that is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.
  • an antibody or antigen binding fragment thereof modulates (e.g., interferes with, antagonizes, inhibits) binding of IL-18BP to its ligand, interleukin 18 (IL-18).
  • an antibody or antigen binding fragment thereof is characterized by or comprises a heavy chain variable region (VH) that comprises complementary determining region VHCDRI, VHCDR2, and VHCDR3 sequences, and a light chain variable region (VL) that comprises complementary determining region VLCDRI, VLCDR2, and VLCDR3 sequences.
  • VH heavy chain variable region
  • VHCDRI complementary determining region
  • VHCDR2 complementary determining region
  • VL light chain variable region
  • Exemplary VH, VHCDRI, V H CDR2, V H CDR3, VL, VLCDRI, VLCDR2, and VLCDR3 sequences are provided in Table Al and Table A2 below.
  • an antibody or antigen binding fragment thereof comprises a VH sequence that comprises complementary determining region VHCDRI, VHCDR2, and VHCDR3 sequences selected from Table Al and variants thereof which bind to IL-18BP; and a VL sequence that comprises complementary determining region VLCDRI, VLCDR2, and VLCDR3 sequences selected from Table Al and variants thereof which bind to IL-18BP.
  • an antibody comprises a VH sequence that comprises a VHCDRI, a VHCDR2, and a VHCDR3 sequence and a VL sequence that comprises a VLCDRI, a VLCDR2, and a VLCDR3 sequence, wherein all of the CDR sequences are from a single named antibody (e.g., SAOla) in Table Al.
  • the CDR sequences are as follows: the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 4-6, respectively; the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 25-27, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 28- 30, respectively; the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 31-33, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 34- 36, respectively; the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 37-39, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 40-
  • an antibody or antigen binding fragment thereof comprises CDR consensus sequences, for example, wherein the VHCDRI, VHCDR2, and VHCDR3 sequences comprise TFX1X2X 3 X 4 X 5 H, IX6X7X8X9X10X11X12X13X14X15AQKFQG, and X16X17X18X19X20X21X22DY, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise X23X24X25X26X27X28X29X30WX31A, X32X33X34X35X36X37X38, and QX39X40X41SFPYX42, respectively (see Table Ell for the definition of “X” residues).
  • exemplary variants bind to IL- 18BP and have 1, 2, or 3 total alterations in any one or more of the individual CDRs, for example, any one or more the VHCDRI, VHCDR2, VHCDR3, VLCDRI, VLCDR2, and/or VLCDR3 sequences described herein.
  • exemplary “alterations” include amino acid substitutions, additions, and deletions.
  • VH and VL sequences are provided in Table A2 below.
  • an antibody, or antigen binding fragment thereof binds to IL-18BP and comprises a VH sequence and a corresponding VL sequence selected from Table A2.
  • the VH comprises a sequence least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2, including, for example, wherein the VH has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations in one or more framework regions.
  • the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2, including, for example, wherein the VL has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations in one or more framework regions.
  • the VH comprises a sequence least 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2 and the VL comprises a sequence at least 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2 and is from the same single named antibody (e.g., SAOla) as the VH region.
  • the VH comprises a sequence least 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2 and the VL comprises a sequence at least 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2 and is from the same single named antibody (e.g. SAOla) as the VH region, wherein any alterations are not found in the CDRs as underlined in Table A2.
  • SAOla single named antibody
  • an antibody may comprise VH and VL sequences that are at least 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identical to the respective sequences from a single named antibody (e.g., SAOla) in Table A2, wherein the antibody comprises the CDRs of said single named antibody (e.g., SAOla) as recited in Table Al.
  • SAOla single named antibody
  • the VH and VL of an antibody or antigen binding fragment are as follows: the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 277, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 278; the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 279, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 280; the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 285, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 286; the VH comprises a sequence at least 80, 85
  • variants thereof that bind to IL-18BP for example, variants having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations in one or more framework regions of any one or more of the foregoing VH and/or VL sequences.
  • exemplary “alterations” include amino acid substitutions, additions, and deletions.
  • an antibody or antigen binding fragment thereof binds to IL-18BP.
  • an antibody or an antigen binding fragment thereof binds to human IL-18BP, cynomolgus IL-18BP, and/or mouse IL-18BP, or a region or fragment or epitope thereof.
  • Human interleukin- 18-binding protein or IL-18BP, is encoded by the IL18BP gene (see Gene ID: 10068; and UniProt: 095998) and has at least three isoforms.
  • an antibody of the disclosure binds to isoform A of IL-18BP.
  • an antibody of the disclosure binds to isoform B of IL-18BP.
  • an antibody of the disclosure binds to both isoform A and isoform C of IL-18BP.
  • an antibody of the disclosure binds to all isoforms of IL-18BP. It is an inhibitor of early Thl cytokine responses and the proinflammatory cytokine IL-18.
  • IL-18BP binds to IL- 18, inhibits the binding of IL- 18 to its receptor, and thereby inhibits IL- 18- induced IFN-gamma production, among other IL- 18 signaling activities.
  • the amino acid sequences of the human, cynomolgus, and mouse IL-18BP isoforms are provided in Table Bl below (see also FIG. 2 for an alignment).
  • an antibody or antigen binding fragment thereof binds to a mature IL-18BP sequence in Table Bl, for example, at a region excluding the signal peptide (underlined).
  • an antibody or antigen binding fragment thereof binds to an epitope that comprises the IL-18 binding interface of the mature form of IL-18BP.
  • an antibody or antigen binding fragment thereof binds to an epitope that comprises 197 and VI 53 according to the numbering of UniProt: 095998 (alternatively, 195 and V151 as defined by SEQ ID NO: 1; or 167 and V123 as defined by SEQ ID NO: 2).
  • an antibody binds to a conformational epitope of mature IL- 18BP sequence of SEQ ID NO: 372 (mature human Isoform A).
  • an antibody of the disclosure binds to at least two residues selected from the group consisting of K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372.
  • an antibody of the disclosure binds to residues K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372.
  • such an antibody comprises the VH of a sequence comprising at least 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identity to SEQ ID NO: 333, and the VL of a sequence comprising at least 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identity to SEQ ID NO: 334.
  • the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 169-171, respectively
  • the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 172-174, respectively.
  • an antibody binds to an epitope that comprises the IL-18 binding interface of the mature form of IL-18BP. Residues on IL-18BP which interact with IL- 18 were identified as: R61, Y69, S75, H79, T116, SI 19 and R131. In exemplary embodiments, an antibody of the disclosure binds to residues R61, Y69 and R131, which are also recognized by IL-18.
  • such an antibody comprises the VHCDR1, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 169-171, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 172-174, respectively.
  • an antibody binds to a linear epitope of mature IL-18BP sequence of SEQ ID NO: 372 (mature human Isoform A).
  • an antibody has ortholog specificity or ortholog cross-reactivity for IL-18BP. For instance, in certain embodiments, an antibody binds to human IL-18BP and cynomolgus IL-18BP but does not bind (specifically or substantially) to mouse IL-18BP. In some embodiments, an antibody binds to human IL-18BP, cynomolgus IL-18BP, and mouse IL-18BP.
  • an antibody or antigen binding fragment thereof has ortholog specificity or ortholog cross-reactivity for IL-18BP.
  • an antibody or antigen binding fragment thereof binds to human IL-18BP and cynomolgus IL-18BP but does not bind (specifically or substantially) to mouse IL-18BP.
  • an antibody or antigen binding fragment thereof binds to human IL-18BP, cynomolgus IL-18BP, and mouse IL-18BP (for example, SAOla, SA51d, SA45a, SA54a, SA55a, SA56a, SA57a, SA59a, SA60a, SA61a, SA62a, SA63a, SA65a, SA73a, SA77a, SA64a, SA66a).
  • an antibody or antigen binding fragment thereof binds to human IL-18BP with a binding affinity that is stronger than the binding affinity between IL-18 and IL- 18BP (KD ⁇ 650 pM for human IL-18 and IL-18BP; or specifically measured as 655 ⁇ 136 pM).
  • an antibody or antigen binding fragment thereof binds to human IL-18BP with a binding affinity of about 1 pM to about 10 pM to about 600 pM or 65 pM, or about, at least about, or less than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 300, 400, 500, 600, or 650 pM, or optionally with an affinity that ranges from about 1 pM to about 600 pM, 1 pM to about 500 pM, 1 pM to about 400 pM, 1 pM to about 300 pM, about 1 pM to about 200 pM, about 1 pM to about 100 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 p
  • the KD may be determined by the biolayer interferometry (BLI) assay described herein.
  • binding kinetic measurements may be taken on a Fortebio (now Sartorius) Octet RED96e instrument by loading mAbs onto anti-human constant domain (AHC) biosensors (ForteBio) in lOx kinetics buffer consisting of PBS containing 0.1% BSA, 0.02% Tween 20 for 90-120 s to achieve a spectral shift value between 0.8 to 1.2 nm. Association may then be carried out in the presence of a 2-fold dilution series of hIL-18BP and allowed to proceed for 90-120 s. Dissociation may be measured for 300 to 1200 s. Dilution series may start at 100 nM for weaker variants or 10 nM for the most potent mAbs.
  • an antibody, or antigen binding fragment thereof is an IL-18BP antagonist. In some instances, an antibody, or antigen binding fragment thereof, antagonizes the binding and/or signaling activity between IL-18BP and its ligand, IL-18. In some embodiments, an antibody, or antigen binding fragment thereof, antagonizes or reduces the binding and/or signaling activity between IL-18BP and IL-18 by about or at least about 10-1000% (e.g., about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more), for example, in a cell-based assay.
  • 10-1000% e.g., about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more
  • the antagonistic anti-IL- 18BP antibody blocks the inhibitory activity of IL-18BP towards IL-18, and thereby increases IL-18-mediated signaling, for example, IL-18-mediated induction of IFN-gamma, CXCL10, and TNFa.
  • IL-18BP for instance, human IL-18BP
  • IL- 18 e.g. recombinant human IL- 18
  • the resulting solution may then be added to IL-18 Reporter HEK 293 cells that respond to exogenously added IL-18 by expressing an NF-KB/AP-1 -inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene.
  • SEAP embryonic alkaline phosphatase
  • Another potential assay involves the measurement of derepression of IFNy expression by anti-IL-18BP mAbs in KG-1 cells.
  • IL-18BP may be pre-blocked with a serial dilution of antibodies, IL-18 may then be added to the mixture, and this mixture may be added to the KG-1 cells and incubated.
  • Secreted IFN-y can then be measured according to conventional means, such as ELISA, and the test antibody’s effect compared to the effect of suitable controls, such as isotype control antibodies. Further details are disclosed in the materials and methods section herein.
  • test antibody for incubation of PBMCs with the test antibody, IL-12, and IL- 18 and the measurement of IFNy and/or CCL2 by conventional means.
  • the test antibody’s effect may be compared to the effect of suitable controls, such as isotype control antibodies. Further details are disclosed in the materials and methods section herein.
  • a further assay involves the incubation of NK cells and pre-complexed hIL-18/hIL-18BP, followed by the addition of IL-12, and then serial dilutions of the test antibody.
  • the test antibody for effect compared to the effect of suitable controls, such as isotype control antibodies. Further details are disclosed in the materials and methods section herein.
  • Certain embodiments include methods of screening an anti-IL-18BP antibody or antigen binding fragment thereof for the ability to block or inhibit binding between IL- 18 and IL-18BP, comprising (a) determining binding affinity of the antibody or antigen binding fragment thereof for (i) IL-18BP alone, and (ii) a hypo-IL-18 fusion protein, wherein the hypo- IL-18 fusion protein comprises IL- 18 fused to IL-18BP via a flexible linker (and an optional protease cleavage site in between), wherein the IL-18 portion of the fusion protein is bound to the IL-18BP portion of the fusion protein and sterically blocks the IL-18 binding site of the IL- 18BP portion of the fusion protein; (b) comparing the binding affinity of (i) to the binding affinity of (ii); and (c) identifying or selecting the antibody or antigen binding fragment thereof as being able to block or inhibit binding between IL- 18 and IL-18BP if the binding affinity of (i) is significantly stronger than the binding affinity (
  • Certain embodiments comprise (c) identifying or selecting the antibody or antigen binding fragment thereof as being able to block or inhibit binding between IL-18 and IL-18BP if the binding affinity of (i) is about or at least about 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, or lOOOx or more stronger than the binding affinity (ii).
  • the IL-18 and IL-18BP are mouse IL-18 and IL-18BP.
  • the IL-18 and IL-18BP are human IL-18 and IL-18BP.
  • the hypo-IL-18 fusion protein comprises, in an N- to C-terminal orientation, a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and IL-18BP.
  • the hypo-IL-18 fusion protein comprises an amino acid sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence from Table SI
  • the binding interactions can be detected and quantified using a variety of routine methods, including Biacore® assays (for example, with appropriately tagged soluble reagents, bound to a sensor chip), FACS analyses with cells expressing IL-18BPr on the cell surface (either native, or recombinant), immunoassays, fluorescence staining assays, ELISA assays, and microcalorimetry approaches such as ITC (Isothermal Titration Calorimetry).
  • Biacore® assays for example, with appropriately tagged soluble reagents, bound to a sensor chip
  • FACS analyses with cells expressing IL-18BPr on the cell surface either native, or recombinant
  • immunoassays fluorescence staining assays
  • ELISA assays either native, or recombinant
  • microcalorimetry approaches such as ITC (Isothermal Titration Calorimetry).
  • anti-IL-18BP antibodies may be assessed using a variety of methods known to the skilled person affinity /binding assays (for example, surface plasmon resonance, competitive inhibition assays); cytotoxicity assays, cell viability assays, cell proliferation or differentiation assays, cancer cell and/or tumor growth inhibition using in vitro or in vivo models.
  • affinity /binding assays for example, surface plasmon resonance, competitive inhibition assays
  • cytotoxicity assays for example, cell viability assays, cell proliferation or differentiation assays, cancer cell and/or tumor growth inhibition using in vitro or in vivo models.
  • Other assays may test the ability of antibodies described herein to modulate (e.g., inhibit) IL-18BP and/or IL-18- mediated responses.
  • the antibodies described herein may also be tested for in vitro and in vivo efficacy.
  • Such assays may be performed using well-established protocols known to the skilled person (see e.g., Current Protocols in Molecular Biology (
  • the Fc region of an antibody, or antigen binding fragment thereof comprises, consists, or consists essentially an IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3, and IgG4), or IgM Fc domain, optionally a human Fc domain, or a hybrid and/or variant thereof.
  • the Fc region comprises, consists, or consists essentially of the Fc from human IgGl or IgG4 (see, e.g., Allberse and Schuurman, Immunology. 105:9-19, 2002), or a fragment or variant thereof.
  • an antibody or antigen binding fragment thereof comprises variant or otherwise modified Fc region(s), including those having altered properties or biological activities relative to wild-type Fc region(s).
  • modified Fc regions include those having mutated sequences, for instance, by substitution, insertion, deletion, or truncation of one or more amino acids relative to a wild-type sequence, hybrid Fc polypeptides composed of domains from different immunoglobulin classes/subclasses, Fc polypeptides having altered glycosylation/sialylation patterns, and Fc polypeptides that are modified or derivatized, for example, by biotinylation (see, e.g., US Application No.
  • FcRs e.g., FcyRI, FcyRIIa, FcyRIIb, FcyRIIc, FcyRIIIa, FcyRIIIb, FcRn
  • pharmacokinetic properties e.g., stability or half-life, bioavailability, tissue distribution, volume of distribution, concentration, elimination rate constant, elimination rate, area under the curve (AUC), clearance, Cmax, Tmax, Cmin, fluctuation
  • an antibody or antigen binding fragment thereof comprises a hybrid Fc region, for example, an Fc region that comprises a combination of Fc domains (e.g., hinge, CH2, CH3, CH4) from immunoglobulins of different species (e.g., human, mouse), different Ig classes, and/or different Ig subclasses. Also included are antibodies or antigen binding fragments thereof that comprise derivatized or otherwise modified Fc regions.
  • the Fc region is modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like, for instance, relative to a wildtype or naturally-occurring Fc region.
  • the Fc region comprises wildtype or native glycosylation patterns, or alternatively, it comprises increased glycosylation relative to a native form, decreased glycosylation relative to a native form, or it is entirely deglycosylated.
  • decreased glycosylation of an Fc region reduces binding to the Clq region of the first complement component Cl, a decrease in ADCC-related activity, and/or a decrease in CDC-related activity.
  • Certain embodiments thus employ a deglycosylated or aglycosylated Fc region. See, e.g., WO 2005/047337 for the production of exemplary aglycosylated Fc regions.
  • Fc region glycoform is generated by substituting the Q295 position with a cysteine residue (see, e.g., U.S. Application No. 2010/0080794), according to the Kabat et al. numbering system. Certain embodiments include Fc regions where about 80-100% of the glycoprotein in Fc region comprises a mature core carbohydrate structure that lacks fucose (see, e.g., U.S. Application No. 2010/0255013).
  • Some embodiments include Fc regions that are optimized by substitution or deletion to reduce the level of fucosylation, for instance, to increase affinity for FcyRI, FcyRIa, or FcyRIIIa, and/or to improve phagocytosis by FcyRIIa-expressing cells (see U.S. Application Nos. 2010/0249382 and 2007/0148170).
  • an Fc region of an antibody or antigen binding fragment thereof may comprise oligomannose-type N-glycans, and optionally have one or more of the following: increased ADCC effector activity, increased binding affinity for FcyRIIIA (and certain other FcRs), similar or increased binding specificity for the target of the IL-18BP polypeptide, similar or higher binding affinity for the target of the IL-18BP polypeptide, and/or similar or lower binding affinity for mannose receptor, relative to a corresponding Fc region that contains complex-type N-glycans (see, e.g., U.S. Application No. 2007/0092521 and U.S. Patent No. 7,700,321).
  • enhanced affinity of Fc regions for FcyRs has been achieved using engineered glycoforms generated by expression of antibodies in engineered or variant cell lines (see, e.g., Umana et al., Nat Biotechnol. 17:176- 180, 1999; Davies et al., Biotechnol Bioeng. 74:288-294, 2001; Shields et al., J Biol Chem. 277:26733-26740, 2002; Shinkawa et al., 2003, J Biol Chem. 278:3466-3473, 2003; and U.S. Application No. 2007/0111281).
  • Certain Fc region glycoforms comprise an increased proportion of N-gly coside bond type complex sugar chains, which do not have the 1 -position of fucose bound to the 6-position of N-acetylglucosamine at the reducing end of the sugar chain (see, e.g., U.S. Application No. 2010/0092997).
  • Particular embodiments may include IgG Fc region that is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by an a-2,6 linkage, optionally where the Fc region has a higher anti-inflammatory activity relative to a corresponding, wild-type Fc region (see U.S. Application No.
  • Certain variant, fragment, hybrid, or otherwise modified Fc regions of an antibody or antigen binding fragment thereof may have altered binding to one or more FcRs, and/or corresponding changes to effector function, relative to a corresponding, wild-type Fc sequence (e.g., same species, same Ig class, same Ig subclass). For instance, such Fc regions may have increased binding to one or more of Fey receptors, Fea receptors, Fes receptors, and/or the neonatal Fc receptor, relative to a corresponding, wild-type Fc sequence.
  • FcRs e.g., same species, same Ig class, same Ig subclass
  • variant, fragment, hybrid, or modified Fc regions may have decreased binding to one or more of Fey receptors, Fea receptors, Fes receptors, and/or the neonatal Fc receptor, relative to a corresponding, wild-type Fc sequence.
  • Specific FcRs are described elsewhere herein.
  • an antibody comprises an Fc domain, comprising one or more mutations to increase binding to one or more of Fey receptors, Fea receptors, Fes receptors, and/or the neonatal Fc receptor, relative to a corresponding, wild-type Fc sequence.
  • an antibody comprises an IgGl or IgG3 Fc domain, comprising one or more mutations to increase binding to one or more of Fey receptors, Fea receptors, Fes receptors, and/or the neonatal Fc receptor, relative to a corresponding, wild-type Fc sequence.
  • an antibody comprises an Fc domain, comprising one or more mutations to increase effector function.
  • the antibody comprises an Fc domain selected from a human IgGl and IgG3, comprising one or more mutations to increase effector function.
  • an antibody is a blocking antibody that comprises an Fc domain with high effector activity.
  • the blocking antibody comprises an Fc domain selected from a human IgGl and IgG3, comprising one or more mutations to increase effector function.
  • an antibody is a partial -blocking antibody that comprises an Fc domain with high effector activity.
  • a partial-blocking antibody comprises an Fc domain selected from a human IgGl and IgG3, comprising one or more mutations to increase effector function.
  • an antibody is a nonblocking antibody that comprises an Fc domain with high effector activity.
  • the non-blocking antibody comprises an Fc domain selected from a human IgGl or IgG3, comprising one or more mutations to increase effector function.
  • an antibody comprises an Fc domain, comprising one or more mutations to decrease binding to one or more of Fey receptors, Fea receptors, Fes receptors, and/or the neonatal Fc receptor, relative to a corresponding, wild-type Fc sequence.
  • an antibody comprises an IgGl or IgG3 Fc domain, comprising one or more mutations to decrease binding to one or more of Fey receptors, Fea receptors, Fes receptors, and/or the neonatal Fc receptor, relative to a corresponding, wild-type Fc sequence.
  • an antibody comprises an Fc domain, comprising one or more mutations to decrease effector function.
  • an antibody comprises an Fc domain selected from a human IgG2 and IgG4, comprising one or more mutations to decrease effector function.
  • an antibody is a blocking antibody comprising an Fc domain with low effector activity.
  • the blocking antibody comprises an Fc domain selected from a human IgG2 and IgG4, comprising one or more mutations to decrease effector function.
  • an antibody is a partial -blocking antibody comprising an Fc domain with low effector activity.
  • the partial-blocking antibody comprises an Fc domain selected from a human IgG2 and IgG4, comprising one or more mutations to decrease effector function.
  • an antibody is a non-blocking antibody comprising an Fc domain with low effector activity.
  • the nonblocking antibody comprises an Fc domain selected from a human IgG2 and IgG4, comprising one or more mutations to decrease effector function.
  • Fc variants having altered (e.g., increased, decreased) effector function/FcR binding can be found, for example, in U.S. Pat. Nos. 5,624,821 and 7,425,619; U.S. Application Nos. 2009/0017023, 2009/0010921, and 2010/0203046; and WO 2000/42072 and WO 2004/016750.
  • Certain examples include human Fc regions having a one or more substitutions at position 298, 333, and/or 334, for example, S298A, E333 A, and/or K334A (based on the numbering of the EU index of Kabat et al.), which have been shown to increase binding to the activating receptor FcyRIIIa and reduce binding to the inhibitory receptor FcyRIIb. These mutations can be combined to obtain double and triple mutation variants that have further improvements in binding to FcRs.
  • Certain embodiments include a S298A/E333A/K334A triple mutant, which has increased binding to FcyRIIIa, decreased binding to FcyRIIb, and increased ADCC (see, e.g., Shields et al., J Biol Chem. 276:6591-6604, 2001; and Presta et al., Biochem Soc Trans. 30:487-490, 2002). See also engineered Fc glycoforms that have increased binding to FcRs, as disclosed in Umana et al., supra; and U.S. Patent No. 7,662,925.
  • Some embodiments include Fc regions that comprise one or more substitutions selected from 434S, 252Y/428L, 252Y/434S, and 428L/434S (see U.S. Application Nos. 2009/0163699 and 20060173170), based on the EU index of Kabat et al. Some embodiments include Fc regions that comprise one or more substitutions selected from L234A, L235A, and G237A (see U.S. Application No. 17/779,425), based on the EU index of Kabat et al. Some embodiments include Fc regions that comprise substitutions at L234A and L235A, based on the EU index of Kabat et al.
  • Some embodiments include Fc regions that comprise substitutions at L234A and G237A, based on the EU index of Kabat et al. Some embodiments include Fc regions that comprise substitutions at L235A and G237A, based on the EU index of Kabat et al. Some embodiments include Fc regions that comprise substitutions at L234A, L235A, and G237A, based on the EU index of Kabat et al. Some embodiments include Fc regions that comprise one or more substitutions selected from M252Y, S254T, and T256E, based on the EU index of Kabat et al.
  • Some embodiments include Fc regions that comprise one or more substitutions selected from M428L and N434S, based on the EU index of Kabat et al.
  • the above-mentioned Fc substitutions are to an Fc domain selected from a human IgGl, IgG2, IgG3 and IgG4.
  • the above-mentioned Fc substitutions are to a human IgGl Fc domain.
  • the above-mentioned Fc substitutions are to a human IgG2 Fc domain.
  • the above-mentioned Fc substitutions are to a human IgG3 Fc domain.
  • the above-mentioned Fc substitutions are to a human IgG4 Fc domain.
  • the antibody of the disclosure comprises the Fc substitutions disclosed herein and the VH and VL sequences that are at least 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identical to the respective sequences from a single named antibody (e.g., SAOla) in Table A2, wherein the antibody comprises the CDRs of said single named antibody (e.g., SAOla) as recited in Table Al.
  • Certain variant, fragment, hybrid, or modified Fc regions may have altered effector functions, relative to a corresponding, wild-type Fc sequence.
  • such Fc regions may have increased complement fixation or activation, increased Clq binding affinity, increased CDC-related activity, increased ADCC-related activity, and/or increased ADCP -related activity, relative to a corresponding, wild-type Fc sequence.
  • such Fc regions may have decreased complement fixation or activation, decreased Clq binding affinity, decreased CDC-related activity, decreased ADCC-related activity, and/or decreased ADCP-related activity, relative to a corresponding, wild-type Fc sequence.
  • an Fc region may comprise a deletion or substitution in a complement-binding site, such as a Clq- binding site, and/or a deletion or substitution in an ADCC site. Examples of such deletions/substitutions are described, for example, in U.S. Patent No. 7,030,226.
  • Many Fc effector functions, such as ADCC can be assayed according to routine techniques in the art. (see, e.g., Zuckerman et al., CRC Crit Rev Microbiol. 7: 1-26, 1978).
  • Useful effector cells for such assays includes, but are not limited to, natural killer (NK) cells, macrophages, and other peripheral blood mononuclear cells (PBMC).
  • NK natural killer
  • PBMC peripheral blood mononuclear cells
  • certain Fc effector functions may be assessed in vivo, for example, by employing an animal model described in Clynes et al. PNAS. 95:652-656, 1998.
  • variant hybrid, or modified Fc regions may have altered stability or half-life relative to a corresponding, wild-type Fc sequence. In certain embodiments, such Fc regions may have increased half-life relative to a corresponding, wild-type Fc sequence. In other embodiments, variant hybrid, or modified Fc regions may have decreased half-life relative to a corresponding, wild-type Fc sequence. Half-life can be measured in vitro (e.g., under physiological conditions) or in vivo, according to routine techniques in the art, such as radiolabeling, ELISA, or other methods.
  • In vivo measurements of stability or half-life can be measured in one or more bodily fluids, including blood, serum, plasma, urine, or cerebrospinal fluid, or a given tissue, such as the liver, kidneys, muscle, central nervous system tissues, bone, etc.
  • modifications to an Fc region that alter its ability to bind the FcRn can alter its half-life in vivo.
  • Assays for measuring the in vivo pharmacokinetic properties e.g., in vivo mean elimination half-life
  • non-limiting examples of Fc modifications that alter its binding to the FcRn are described, for example, in U.S. Pat. Nos. 7,217,797 and 7,732,570; and U.S. Application Nos. US 2010/0143254 and 2010/0143254.
  • modifications to alter stability or half-life include substitutions/deletions at one or more of amino acid residues selected from 251-256, 285-290, and 308-314 in the CH2 domain, and 385-389 and 428-436 in the CH3 domain, according to the numbering system of Kabat et al. 5ee U.S. Application No. 2003/0190311.
  • variant hybrid, or modified Fc regions may have altered solubility relative to a corresponding, wild-type Fc sequence. In certain embodiments, such Fc regions may have increased solubility relative to a corresponding, wild-type Fc sequence. In other embodiments, variant hybrid, or modified Fc regions may have decreased solubility relative to a corresponding, wild-type Fc sequence. Solubility can be measured, for example, in vitro (e.g., under physiological conditions) according to routine techniques in the art. Exemplary solubility measurements are described elsewhere herein.
  • Variant Fc regions can also have one or more mutated hinge regions, as described, for example, in U.S. Application No. 2003/0118592.
  • one or more cysteines in a hinge region can be deleted or substituted with a different amino acid.
  • the mutated hinge region can comprise no cysteine residues, or it can comprise 1, 2, or 3 fewer cysteine residues than a corresponding, wild-type hinge region.
  • an Fc region having a mutated hinge region of this type exhibits a reduced ability to dimerize, relative to a wild-type Ig hinge region.
  • an antibody or antigen binding fragment thereof has a biological half-life at about pH 7.4, at about a physiological pH, at about 25 °C or room temperature, and/or at about 37 °C or human body temperature (e.g., in vivo, in serum, in a given tissue, in a given species such as rat, mouse, monkey, or human), of about or at least about 30 minutes, about 1 hour, about 2 hour, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 48 hours, about 50 hours, about 60 hours, about 70 hours, about 72 hours, about 80 hours, about 84 hours, about 90 hours, about 96 hours, about 120 hours, or about
  • an antibody or antigen binding fragment thereof has a T m of about or at least about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 °C. In some embodiments, an antibody or antigen binding fragment thereof has a T m of about 65 °C or greater, for example, in PBS (phosphate buffered saline).
  • an antibody or antigen binding fragment thereof conjugated to one or more cytotoxic or chemotherapeutic agents conjugated to one or more cytotoxic or chemotherapeutic agents.
  • cytotoxic or chemotherapeutic agents include, without limitation, alkylating agents, anti-metabolites, anthracyclines, anti-tumor antibiotics, platinums, type I topoisomerase inhibitors, type II topoisomerase inhibitors, vinca alkaloids, and taxanes.
  • cytotoxic or chemotherapeutic agents include, without limitation, cyclophosphamide, cilengitide, lomustine (CCNU), melphalan, procarbazine, carmustine (BCNU), enzastaurin, busulfan, daunorubicin, doxorubicin, gefitinib, erlotinib idarubicin, temozolomide, epirubicin, mitoxantrone, bleomycin, cisplatin, carboplatin, oxaliplatin, camptothecins, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, temsirolimus, everolimus, vincristine, vinblastine, vinorelbine, vindesine, CT52923, paclitaxel, imatinib, dasatinib, sorafenib, pazopan
  • cytotoxic or chemotherapeutic agents include alkylating agents such as thiotepa, cyclophosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as car
  • a herein-disclosed antibody is conjugated or operably linked to a radioisotope to form a radioconjugate and/or macrocyclic chelators useful for conjugating radiometal ions.
  • radioactive isotopes are available for the production of radioconjugate antibodies. Examples include, but are not limited to 90 Y, 123 I, 125 I, 131 I, 186 Re, 188 Re, 211 At, and 212 Bi.
  • the macrocyclic chelator is 1,4,7,10- tetraazacyclododecane-N,N’,N”,N”’ -tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
  • DOTA 1,4,7,10- tetraazacyclododecane-N,N’,N”,N”’ -tetraacetic acid
  • linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman etal., 1999, Nucl. Med. Biol. 26:943-50.
  • the antibody is linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
  • the antibody is entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatinmicrocapsules and poly-(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules
  • the antibodies or antigen binding fragments thereof can be used in any of the compositions, methods, and/or kits described herein, and combined with one or more of the additional agents described herein.
  • Certain embodiments relate to methods of treating, ameliorating the symptoms of, and/or reducing the progression of, a disease or condition in a subject in need thereof, comprising administering to the subject an antibody or antigen binding fragment thereof that binds to IL-18BP, as described herein, or a pharmaceutical composition comprising the same. Also included are methods of stimulating an immune response in a subject in need thereof, for example, an IL- 18 mediated immune response, comprising administering to the subject a pharmaceutical composition described herein.
  • the antibody or antigen binding fragment thereof antagonizes the binding/signaling activity between IL-18BP and its ligand, IL- 18, and thereby increases IL-18-mediated signaling or activity (for example, increased induction of IFN-gamma, CXCL10, and/or TNFa).
  • the disease or condition is a cancer or tumor, or an infectious disease.
  • the disease is any disease where activation of the immune system may be beneficial.
  • the disease or condition is a cancer or tumor or other proliferative disease or disorder, such as a lymphoproliferative disorder, a myeloproliferative disorder, proliferative enteritis, proliferative diabetic retinopathy, or a proliferative kidney disease.
  • the cancer or tumor expresses or overexpresses IL-18BP, IL- 18, or both.
  • the proliferative disease or disorder is associated with increased expression of IL-18BP, IL- 18, or both.
  • the cancer is a primary cancer. In some instances, the cancer is a metastatic cancer.
  • Certain embodiments thus include methods of treating, reducing the severity of, or preventing a cancer in a patient in need thereof, comprising administering to the patient a composition described herein, including wherein the antibody or antigen binding fragment thereof is an IL-18BP antagonist, thereby treating, reducing the severity of, or preventing the cancer.
  • Exemplary cancers include, without limitation, bone cancer, prostate cancer, melanoma (e.g., metastatic melanoma), pancreatic cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, hairy cell leukemias, acute lymphoblastic leukemias), lymphoma (e.g., non-Hodgkin’s lymphomas, Hodgkin’s lymphoma), hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphom
  • an antibody, antigen binding fragment thereof, or pharmaceutical composition of the present disclosure for use as a medicament.
  • An antibody, fragment thereof, or pharmaceutical composition of the present disclosure may be for use in any method of treatment disclosed herein.
  • an antibody, fragment thereof, or pharmaceutical composition of the present disclosure may be for use in a method of treating, ameliorating the symptoms of, and/or reducing the progression of any disease or disorder disclosed herein, such as a cancer or tumor or other proliferative disease or disorder, such as a lymphoproliferative disorder, a myeloproliferative disorder, proliferative enteritis, proliferative diabetic retinopathy, or a proliferative kidney disease.
  • combination therapies for instance, which comprise administering a pharmaceutical composition described herein (comprising an anti-IL-18BP antibody or antigen binding fragment thereof) in combination with one or more additional therapeutic agents, for example, immune-stimulating agents, immune checkpoint modulatory agents, and/or chemotherapeutic agents.
  • the additional therapeutic agent comprises IL- 18, including human IL- 18 (or a functional variant or fragment thereof).
  • the additional therapeutic agent comprises an immune checkpoint modulatory agent.
  • immune checkpoint modulatory agents include “antagonists” or “inhibitors” of one or more inhibitory immune checkpoint molecules, and “agonists” of one or more stimulatory immune checkpoint molecules.
  • immune checkpoint molecules are components of the immune system that either turn up a signal (costimulatory molecules) or turn down a signal, the targeting of which has therapeutic potential in cancer because cancer cells can perturb the natural function of immune checkpoint molecules (see, e.g., Sharma and Allison, Science. 348:56-61, 2015; Topalian et al., Cancer Cell. 27:450- 461, 2015; Pardoll, Nature Reviews Cancer. 12:252-264, 2012).
  • the immune checkpoint modulatory agent e.g., antagonist, agonist
  • the immune checkpoint modulatory agent is a polypeptide or peptide.
  • the terms “peptide” and “polypeptide” are used interchangeably herein, however, in certain instances, the term “peptide” can refer to shorter polypeptides, for example, polypeptides that consist of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids, including all integers and ranges (e.g., 5-10, 8-12, 10-15) in between.
  • Polypeptides and peptides can be composed of naturally-occurring amino acids and/or non- naturally occurring amino acids, as described herein
  • Antibodies are also included as polypeptides.
  • the immune checkpoint modulatory polypeptide agent is an “antibody or antigen binding fragment thereof’, as described herein.
  • the agent is or comprises a “ligand,” for example, a natural ligand, of the immune checkpoint molecule.
  • a “ligand” refers generally to a substance or molecule that forms a complex with a target molecule (e.g., biomolecule) to serve a biological purpose, and includes a “protein ligand,” which generally produces a signal by binding to a site on a target molecule or target protein.
  • a target molecule e.g., biomolecule
  • protein ligand which generally produces a signal by binding to a site on a target molecule or target protein.
  • modified ligands for example, protein ligands that are fused to a pharmacokinetic modifier, for example, an Fc region derived from an immunoglobulin.
  • polypeptide binding properties of polypeptides can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990).
  • a polypeptide specifically binds to a target molecule, for example, an immune checkpoint molecule or an epitope thereof, with an equilibrium dissociation constant that is about or ranges from about ⁇ 10' 7 to about 10' 8 M.
  • the equilibrium dissociation constant is about or ranges from about ⁇ 10' 9 M to about ⁇ 1O' 10 M.
  • the polypeptide has an affinity (Kd or ECso) for a target described herein (to which it specifically binds) of about, at least about, or less than about, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.
  • affinity Kd or ECso
  • a target described herein to which it specifically binds
  • the agent is a “small molecule,” which refers to an organic compound that is of synthetic or biological origin (biomolecule), but is typically not a polymer.
  • Organic compounds refer to a large class of chemical compounds whose molecules contain carbon, typically excluding those that contain only carbonates, simple oxides of carbon, or cyanides.
  • a “biomolecule” refers generally to an organic molecule that is produced by a living organism, including large polymeric molecules (biopolymers) such as peptides, polysaccharides, and nucleic acids as well, and small molecules such as primary secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins, and hormones.
  • a “polymer” refers generally to a large molecule or macromolecule composed of repeating structural units, which are typically connected by covalent chemical bond.
  • a small molecule has a molecular weight of about or less than about 1000-2000 Daltons, typically between about 300 and 700 Daltons, and including about or less than about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 500, 650, 600, 750, 700, 850, 800, 950, 1000 or 2000 Daltons.
  • a small molecule specifically binds to a target, for example, an immune checkpoint molecule, with a binding affinity (Kd or ECso) of about, at least about, or less than about, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM.
  • the immune checkpoint modulatory agent is an antagonist or inhibitor of one or more inhibitory immune checkpoint molecules.
  • Exemplary inhibitory immune checkpoint molecules include Programmed Death-Ligand 1 (PD-L1), Programmed Death-Ligand 2 (PD-L2), Programmed Death 1 (PD-1), Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4), Indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3 -dioxygenase (TDO), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3), V-domain Ig suppressor of T cell activation (VISTA), B and T Lymphocyte Attenuator (BTLA), CD160, and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).
  • P-L1 Programmed Death-Ligand 1
  • PD-L2 Programmed Death-Ligand 2
  • PD-1 Programmed Death 1
  • CTL-4 Cytotoxic T-Lymphocyte-Associated protein 4
  • IDO Indoleamine 2,3-dioxy
  • the agent is a PD-1 (receptor) antagonist or inhibitor, the targeting of which has been shown to restore immune function in the tumor environment (see, e.g., Phillips et al., Int Immunol. 27:39-46, 2015).
  • PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells.
  • PD-1 interacts with two ligands, PD-L1 and PD-L2.
  • PD-1 functions as an inhibitory immune checkpoint molecule, for example, by reducing or preventing the activation of T-cells, which in turn reduces autoimmunity and promotes self-tol erance.
  • the inhibitory effect of PD-1 is accomplished at least in part through a dual mechanism of promoting apoptosis in antigen specific T-cells in lymph nodes while also reducing apoptosis in regulatory T cells (suppressor T cells).
  • Some examples of PD-1 antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to PD-1 and reduces one or more of its immune- suppressive activities, for example, its downstream signaling or its interaction with PD-L1.
  • PD-1 antagonists or inhibitors include the antibodies nivolumab, pembrolizumab, PDR001, MK-3475, AMP -224, AMP-514, and pidilizumab, and antigen binding fragments thereof (see, e.g., U.S. Patent Nos. 8,008,449; 8,993,731; 9,073,994;
  • the agent is a PD-L1 antagonist or inhibitor.
  • PD-L1 is one of the natural ligands for the PD-1 receptor.
  • General examples of PD-L1 antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to PD-L1 and reduces one or more of its immune-suppressive activities, for example, its binding to the PD-1 receptor.
  • Specific examples of PD-L1 antagonists include the antibodies atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), and antigen binding fragments thereof (see, e.g., U.S. Patent Nos. 9,102,725; 9,393,301; 9,402,899; 9,439,962).
  • the agent is a PD-L2 antagonist or inhibitor.
  • PD-L2 is one of the natural ligands for the PD-1 receptor.
  • General examples of PD-L2 antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to PD-L2 and reduces one or more of its immune-suppressive activities, for example, its binding to the PD-1 receptor.
  • the agent is an IDO antagonist or inhibitor, or a TDO antagonist or inhibitor.
  • IDO and TDO are tryptophan catabolic enzymes with immune-inhibitory properties.
  • IDO is known to suppress T-cells and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumor angiogenesis.
  • General examples of IDO and TDO antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to IDO or TDO (see, e.g., Flatten et al., Front Immunol. 5: 673, 2014) and reduces or inhibits one or more immune-suppressive activities.
  • IDO antagonists or inhibitors include indoximod (NLG-8189), 1-methyl-tryptophan (1MT), P- Carboline (norharmane; 9H-pyrido[3,4-b]indole), rosmarinic acid, and epacadostat (see, e.g., Sheridan, Nature Biotechnology. 33:321-322, 2015).
  • TDO antagonists or inhibitors include 680C91 and LM10 (see, e.g., Pilotte et al., PNAS USA. 109:2497-2502, 2012).
  • the agent is a TIM-3 antagonist or inhibitor.
  • T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3) is expressed on activated human CD4+ T-cells and regulates Thl and Thl7 cytokines.
  • TIM-3 also acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9.
  • TIM-3 contributes to the suppressive tumor microenvironment and its overexpression is associated with poor prognosis in a variety of cancers (see, e.g., Li et al., Acta Oncol. 54: 1706-13, 2015).
  • General examples of TIM-3 antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to TIM-3 and reduces or inhibits one or more of its immune-suppressive activities.
  • LAG3 also maintains CD8+ T-cells in a tolerogenic state and combines with PD-1 to maintain CD8 T-cell exhaustion.
  • LAG-3 antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to LAG-3 and inhibits one or more of its immune-suppressive activities. Specific examples include the antibody BMS-986016, and antigen binding fragments thereof.
  • the agent is a BTLA antagonist or inhibitor.
  • B- and T- lymphocyte attenuator (BTLA; CD272) expression is induced during activation of T-cells, and it inhibits T-cells via interaction with tumor necrosis family receptors (TNF-R) and B7 family of cell surface receptors.
  • BTLA is a ligand for tumor necrosis factor (receptor) superfamily, member 14 (TNFRSF14), also known as herpes virus entry mediator (HVEM).
  • BTLA-HVEM complexes negatively regulate T-cell immune responses, for example, by inhibiting the function of human CD8+ cancer-specific T-cells (see, e.g., Derre et al., J Clin Invest 120: 157-67, 2009).
  • BTLA antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to BTLA-4 and reduce one or more of its immune-suppressive activities.
  • the agent is an HVEM antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to HVEM and interferes with its interaction with BTLA or CD 160.
  • HVEM antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to HVEM, optionally reduces the HVEM/BTLA and/or HVEM/CD160 interaction, and thereby reduces one or more of the immune-suppressive activities of HVEM.
  • the agent is a CD 160 antagonist or inhibitor, for example, an antagonist or inhibitor that specifically binds to CD 160 and interferes with its interaction with HVEM.
  • CD 160 antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to CD 160, optionally reduces the CD160/HVEM interaction, and thereby reduces or inhibits one or more of its immune- suppressive activities.
  • the agent is a TIGIT antagonist or inhibitor.
  • T cell Ig and ITIM domain are a co-inhibitory receptor that is found on the surface of a variety of lymphoid cells, and suppresses antitumor immunity, for example, via Tregs (Kurtulus et al., J Clin Invest. 125:4053-4062, 2015).
  • TIGIT antagonists or inhibitors include an antibody or antigen binding fragment or small molecule that specifically binds to TIGIT and reduce one or more of its immune-suppressive activities (see, e.g., Johnston et al., Cancer Cell. 26:923-37, 2014).
  • the immune checkpoint modulatory agent is an agonist of one or more stimulatory immune checkpoint molecules.
  • stimulatory immune checkpoint molecules include 0X40, CD40, Glucocorticoid-Induced TNFR Family Related Gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and Herpes Virus Entry Mediator (HVEM).
  • the agent is an 0X40 agonist.
  • 0X40 CD 134) promotes the expansion of effector and memory T cells, and suppresses the differentiation and activity of T- regulatory cells (see, e.g., Croft et al., Immunol Rev. 229: 173-91, 2009).
  • 0X40 signaling influences both T-cell activation and survival, it plays a key role in the initiation of an anti-tumor immune response in the lymph node and in the maintenance of the anti-tumor immune response in the tumor microenvironment.
  • 0X40 agonists include an antibody or antigen binding fragment or small molecule or ligand that specifically binds to 0X40 and increases one or more of its immunostimulatory activities.
  • the agent is a CD40 agonist.
  • CD40 is expressed on antigen- presenting cells (APC) and some malignancies. Its ligand is CD40L (CD 154).
  • APC antigen- presenting cells
  • ligation results in upregulation of costimulatory molecules, potentially bypassing the need for T-cell assistance in an antitumor immune response.
  • CD40 agonist therapy plays an important role in APC maturation and their migration from the tumor to the lymph nodes, resulting in elevated antigen presentation and T cell activation.
  • Anti-CD40 agonist antibodies produce substantial responses and durable anticancer immunity in animal models, an effect mediated at least in part by cytotoxic T-cells (see, e.g., Johnson et al. Clin Cancer Res.
  • CD40 agonists include an antibody or antigen binding fragment or small molecule or ligand that specifically binds to CD40 and increases one or more of its immunostimulatory activities.
  • Specific examples include sotigalilmab, CP-870,893, dacetuzumab, Chi Lob 7/4, ADC-1013, CD40L, rhCD40L, and antigen binding fragments thereof.
  • the agent is a GITR agonist.
  • Glucocorticoid-Induced TNFR family Related gene increases T cell expansion, inhibits the suppressive activity of Tregs, and extends the survival of T-effector cells.
  • GITR agonists have been shown to promote an anti -tumor response through loss of Treg lineage stability (see, e.g., Schaer et al., Cancer Immunol Res. 1 :320-31, 2013). These diverse mechanisms show that GITR plays an important role in initiating the immune response in the lymph nodes and in maintaining the immune response in the tumor tissue. Its ligand is GITRL.
  • GITR agonists include an antibody or antigen binding fragment or small molecule or ligand that specifically binds to GITR and increases one or more of its immunostimulatory activities.
  • Specific examples include GITRL, INCAGN01876, DTA-1, MEDI1873, and antigen binding fragments thereof.
  • the agent is a CD137 agonist.
  • CD137 (4-1BB) is a member of the tumor necrosis factor (TNF) receptor family, and crosslinking of CD137 enhances T-cell proliferation, IL-2 secretion, survival, and cytolytic activity.
  • CD137-mediated signaling also protects T-cells such as CD8+ T-cells from activation-induced cell death.
  • CD137 agonists include an antibody or antigen binding fragment or small molecule or ligand that specifically binds to CD137 and increases one or more of its immunostimulatory activities. Specific examples include the CD137 (or 4-1BB) ligand (see, e.g., Shao and Schwarz, J Leukoc Biol. 89:21-9, 2011) and the antibody utomilumab, including antigen binding fragments thereof.
  • the agent is a CD27 agonist. Stimulation of CD27 increases antigen-specific expansion of naive T cells and contributes to T-cell memory and long-term maintenance of T-cell immunity. Its ligand is CD70.
  • the targeting of human CD27 with an agonist antibody stimulates T-cell activation and antitumor immunity (see, e.g., Thomas et al., Oncoimmunology. 2014;3:e27255. doi: 10.4161/onci.27255; and He et al ., J Immunol.
  • CD27 agonists include an antibody or antigen binding fragment or small molecule or ligand that specifically binds to CD27 and increases one or more of its immunostimulatory activities.
  • Specific examples include CD70 and the antibodies varlilumab and CDX-1127 (1F5), including antigen binding fragments thereof.
  • the agent is a CD28 agonist.
  • CD28 is constitutively expressed CD4+ T cells some CD8+ T cells.
  • Its ligands include CD80 and CD86, and its stimulation increases T-cell expansion.
  • General examples of CD28 agonists include an antibody or antigen binding fragment or small molecule or ligand that specifically binds to CD28 and increases one or more of its immunostimulatory activities. Specific examples include CD80, CD86, the antibody TAB08, and antigen binding fragments thereof.
  • the agent is CD226 agonist.
  • CD226 is a stimulating receptor that shares ligands with TIGIT, and opposite to TIGIT, engagement of CD226 enhances T-cell activation (see, e.g., Kurtulus et al., J Clin Invest. 125:4053-4062, 2015; Bottino et al., J Exp Med. 1984:557-567, 2003; and Tahara-Hanaoka et al., Int Immunol. 16:533-538, 2004).
  • General examples of CD226 agonists include an antibody or antigen binding fragment or small molecule or ligand (e.g., CD112, CD 155) that specifically binds to CD226 and increases one or more of its immunostimulatory activities.
  • the agent is an HVEM agonist.
  • Herpesvirus entry mediator also known as tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is a human cell surface receptor of the TNF-receptor superfamily.
  • HVEM is found on a variety of cells including T-cells, APCs, and other immune cells. Unlike other receptors, HVEM is expressed at high levels on resting T-cells and down-regulated upon activation. It has been shown that HVEM signaling plays a crucial role in the early phases of T-cell activation and during the expansion of tumor-specific lymphocyte populations in the lymph nodes.
  • General examples of HVEM agonists include an antibody or antigen binding fragment or small molecule or ligand that specifically binds to HVEM and increases one or more of its immunostimulatory activities.
  • the additional therapeutic agent comprises a chemotherapeutic agent, for example, small molecule chemotherapeutic agents.
  • chemotherapeutic agents include alkylating agents, anti-metabolites, cytotoxic antibiotics, topoisomerase inhibitors (type 1 or type II), and anti -microtubule agents, among others.
  • alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide , and busulfan), nitrosoureas (e.g., N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mytomycin, and diaziquone (AZQ)), cisplatins and derivatives thereof (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine).
  • nitrogen mustards e.g., mech
  • anti-metabolites include anti-folates (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5 -fluorouracil and capecitabine), deoxynucleoside analogues (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine);
  • cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycins, mitomycin C, mitoxantrone), bleo
  • topoisomerase inhibitors examples include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin.
  • anti -microtubule agents include taxanes (e.g., paclitaxel and docetaxel) and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine).
  • taxanes e.g., paclitaxel and docetaxel
  • vinca alkaloids e.g., vinblastine, vincristine, vindesine, vinorelbine
  • the methods and compositions described herein are sufficient to result in tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least a 10%, 20%, 30%, 40%, 50% or greater decrease in tumor mass, or by altered (e.g., decreased with statistical significance) scan dimensions.
  • the methods and compositions described herein reduce the growth rate (e.g., in vivo or in vitro, including cancer cells isolated from a biopsy or other sample and grown in vitro) of the cancer by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to an untreated control.
  • the growth rate e.g., in vivo or in vitro, including cancer cells isolated from a biopsy or other sample and grown in vitro
  • the methods and compositions described herein reduce the growth rate (e.g., in vivo or in vitro, including cancer cells isolated from a biopsy or other sample and grown in vitro) of the cancer by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to an untreated control.
  • the methods and compositions described herein reduce cancer cell initiation, migration, adhesion, invasiveness, and/or metastasis by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to an untreated control.
  • the methods and compositions described herein reduce angiogenesis in the tumor environment by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to an untreated control.
  • the disease or condition is a myelodysplastic syndrome (MDS) (see, for example, Wang et al., Blood. 140 (Supplement 1): 12297, 2022), for example, for which antagonizing IL-18BP represents a viable approach.
  • MDS refers to a group of cancers in which immature blood cells in the bone marrow do not mature, and as a result, do not develop into healthy blood cells.
  • Certain embodiments thus include methods of treating, reducing the severity of, or preventing an MDS in a patient in need thereof, comprising administering to the patient a composition described herein, including wherein the antibody or antigen binding fragment thereof is an IL-18BP antagonist, thereby treating, reducing the severity of, or preventing the MDS.
  • the disease or condition is an infectious disease.
  • the infectious disease is selected from viral (see, for example, Vecchie et al., J Cell Physiol. 236(3): 1638-1657, 2021), bacterial (see, for example, Kinoshita et al., Ann Surg. 240(2): 313-20, 2004), fungal (for example, yeast), and protozoal infections.
  • Some embodiments thus include methods of treating, reducing the severity of, or preventing an infectious disease in a patient in need thereof, comprising administering to the patient a composition described herein, including wherein the antibody or antigen binding fragment thereof is an IL-18BP antagonist, thereby treating, reducing the severity of, or preventing the infectious disease.
  • the methods and compositions described herein increase median survival time of a subject by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certain embodiments, the methods and compositions described herein increase median survival time of a subject by 1 year, 2 years, 3 years, or longer. In some embodiments, the methods and compositions described herein increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer. In certain embodiments, the methods and compositions described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer.
  • the methods and compositions described herein are sufficient to result in stable disease. In certain embodiments, the methods and compositions described herein are sufficient to result in clinically relevant reduction in symptoms of a particular disease indication known to the skilled clinician.
  • compositions comprising an antibody or antigen binding fragment thereof, as described herein, and a pharmaceutically- acceptable carrier.
  • a pharmaceutical carrier may be liquid, semi-liquid or solid.
  • Solutions or suspensions used for parenteral, intradermal, intraocular, subcutaneous, direct instillation into the bladder, or topical application may include, for example, a sterile diluent (such as water), saline solution (e.g., phosphate buffered saline; PBS), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methyl parabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates).
  • a sterile diluent such as water
  • saline solution e.g., phosphate buffered saline; PBS
  • fixed oil polyethylene glycol, glycerin, propylene glycol or other synthetic solvent
  • antimicrobial agents such
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • the therapeutic or pharmaceutical compositions can be prepared by combining an agent-containing composition with an appropriate physiologically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • an appropriate physiologically acceptable carrier such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • suitable excipients such as salts, buffers and stabilizers may, but need not, be present within the composition.
  • Administration may be achieved by a variety of different routes, including oral, parenteral, nasal, intravenous, intraocular, intradermal, intramuscular, subcutaneous, installation into the bladder, or topical. Preferred modes of administration depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.
  • Carriers can include, for example, pharmaceutically- or physiologically-acceptable carriers, excipients, or stabilizers that are non-toxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.
  • physiologically-acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, histidine, and/or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as polysorbate 20 (TWEENTM) polyethylene glycol (PEG), and poloxamers (PLURONICSTM), and the like.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • one or more agents can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate)microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the particle(s) or liposomes may further comprise other therapeutic or diagnostic agents.
  • the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated.
  • a pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.
  • Typical routes of administering these and related therapeutic or pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, ocular, rectal, vaginal, and intranasal.
  • parenteral as used herein includes subcutaneous injections, intravenous, instillation into the bladder, intramuscular, intrastemal injection or infusion techniques.
  • Therapeutic or pharmaceutical compositions according to certain embodiments of the present disclosure are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a subject or patient.
  • compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described agent in aerosol form may hold a plurality of dosage units.
  • composition to be administered will typically contain a therapeutically effective amount of an agent described herein, for treatment of a disease or condition of interest.
  • a therapeutic or pharmaceutical composition can be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) can be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalation administration.
  • the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid. Certain embodiments include sterile, injectable solutions.
  • the pharmaceutical composition may be formulated into a powder, granule, gel, compressed tablet, pill, capsule, chewing gum, wafer or the like.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • a liquid carrier such as polyethylene glycol or oil.
  • the therapeutic or pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, gel, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • the liquid therapeutic or pharmaceutical compositions may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Physiological saline is a preferred adjuvants
  • Physiological saline
  • a liquid therapeutic or pharmaceutical composition intended for either parenteral, intraocular, or oral administration should contain an amount of an agent such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the agent of interest in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain between about 4% and about 75% of the agent of interest. In certain embodiments, therapeutic or pharmaceutical compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the agent of interest prior to dilution.
  • the therapeutic or pharmaceutical compositions may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a therapeutic or pharmaceutical composition for topical administration.
  • the composition may include a transdermal patch or iontophoresis device.
  • compositions may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.
  • the therapeutic or pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • the therapeutic or pharmaceutical compositions in solid or liquid form may include a component that binds to agent and thereby assists in the delivery of the compound. Suitable components that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.
  • the therapeutic or pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.
  • compositions described herein may be prepared with carriers that protect the agents against rapid elimination from the body, such as time release formulations or coatings.
  • carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art.
  • compositions may be prepared by methodology well known in the pharmaceutical art.
  • a therapeutic or pharmaceutical composition intended to be administered by injection may comprise one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non- covalently interact with the agent so as to facilitate dissolution or homogeneous suspension of the agent in the aqueous delivery system.
  • the therapeutic or pharmaceutical compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., ⁇ 0.07 mg) to about 100 mg/kg (i.e., ⁇ 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., ⁇ 0.7 mg) to about 50 mg/kg (i.e., ⁇ 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., ⁇ 70 mg) to about 25 mg/kg (i.e., ⁇ 1.75 g).
  • the therapeutically effective dose is administered on a weekly, bi-weekly, or monthly basis. In specific embodiments, the therapeutically effective dose is administered on a weekly, bi-weekly, or monthly basis, for example, at a dose of about 1-10 or 1-5 mg/kg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg.
  • kits comprising (a) an antibody or antigen binding fragment thereof that binds to IL-18BP, as described herein; and optionally (b) at least one additional therapeutic agent.
  • kits comprising (a) an antibody or antigen binding fragment thereof that binds to IL-18BP, as described herein; and optionally (b) at least one additional therapeutic agent.
  • kits comprising (a) an antibody or antigen binding fragment thereof that binds to IL-18BP, as described herein; and optionally (b) at least one additional therapeutic agent.
  • kits are in separate therapeutic compositions.
  • (a) and (b) are in the same therapeutic composition.
  • kits herein may also include one or more additional therapeutic agents or other components suitable or desired for the indication being treated, or for the desired diagnostic application.
  • the kits herein can also include one or more syringes or other components necessary or desired to facilitate an intended mode of delivery (e.g., stents, implantable depots, etc.).
  • a patient care kit contains separate containers, dividers, or compartments for the composition(s) and informational material(s).
  • the composition(s) can be contained in a bottle, vial, or syringe, and the informational material(s) can be contained in association with the container.
  • the separate elements of the kit are contained within a single, undivided container.
  • the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.
  • the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of an antibody and optionally at least one additional therapeutic agent.
  • the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of an antibody and optionally at least one additional therapeutic agent.
  • the containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight.
  • the patient care kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device.
  • a device suitable for administration of the composition e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device.
  • the device is an implantable device that dispenses metered doses of the agent(s).
  • methods of providing a kit e.g., by combining the components described herein.
  • Certain embodiments include methods and related compositions for expressing and purifying an anti-IL-18BP antibody or antigen binding fragment thereof described herein.
  • Such recombinant anti-IL-18BP antibodies can be conveniently prepared using standard protocols as described for example in Sambrook, et aL, (1989, supra), in particular Sections 16 and 17; Ausubel et al. , (1994, supra), in particular Chapters 10 and 16; and Coligan et al. , Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.
  • anti-IL-18BP antibodies may be prepared by a procedure including one or more of the steps of: (a) preparing a construct that comprises a polynucleotide sequence which encodes an anti-IL-18BP antibody heavy chain and/or light chains, and which is operably linked to a regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the anti-IL-18BP antibody; and (d) isolating the anti-IL-18BP from the host cell.
  • Certain embodiments thus include polynucleotides that encode an anti -IL-18BP antibody or antigen binding fragment thereof described herein, including vectors comprising said polynucleotides, and host cells comprising the polynucleotides and/or vectors.
  • a nucleotide sequence encoding an anti-IL-18BP, or a functional equivalent may be inserted into appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of expression vector/host systems are known and may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cell and more specifically human cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with virus expression vectors (e.g., baculovirus)
  • control elements or “regulatory sequences” present in an expression vector are those non-translated regions of the vector— enhancers, promoters, 5’ and 3’ untranslated regions- -which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used.
  • a number of expression vectors may be selected depending upon the use intended for the expressed polypeptide. For example, when large quantities are needed, vectors which direct high level expression of fusion proteins that are readily purified may be used.
  • vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the polypeptide of interest may be ligated into the vector in frame with sequences for the aminoterminal Met and the subsequent 7 residues of 0-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem.
  • pGEX Vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • Certain embodiments may employ E. coli- a&Q expression systems (see, e.g., Structural Genomics Consortium et al., Nature Methods . 5: 135-146, 2008). These and related embodiments may rely partially or totally on ligation-independent cloning (LIC) to produce a suitable expression vector.
  • protein expression may be controlled by a T7 RNA polymerase (e.g., pET vector series).
  • T7 RNA polymerase e.g., pET vector series
  • These and related embodiments may utilize the expression host strain BL21(DE3), a XDE3 lysogen of BL21 that supports T7-mediated expression and is deficient in Ion and ompT proteases for improved target protein stability.
  • expression host strains carrying plasmids encoding tRNAs rarely used in E. coli such as ROSETTATM (DE3) and Rosetta 2 (DE3) strains.
  • Cell lysis and sample handling may also be improved using reagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER® Protein Extraction Reagent.
  • BENZONASE® nuclease and BUGBUSTER® Protein Extraction Reagent.
  • auto-inducing media can improve the efficiency of many expression systems, including high-throughput expression systems.
  • Media of this type e.g., OVERNIGHT EXPRESSTM Autoinduction System gradually elicit protein expression through metabolic shift without the addition of artificial inducing agents such as IPTG.
  • Particular embodiments employ hexahistidine tags (such as those sold under the trademark HIS»TAG® fusions), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques.
  • clinical grade proteins can be isolated from E. coli inclusion bodies, without or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006).
  • affinity tags see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006.
  • certain embodiments may employ a cold-shock induced E. coli high-yield production system, because over-expression of proteins in Escherichia coli at low temperature improves their solubility and stability (see, e.g., Qing et al., Nature Biotechnology.
  • high-density bacterial fermentation systems For example, high cell density cultivation of Ralstonia eutropha allows protein production at cell densities of over 150 g/L, and the expression of recombinant proteins at titers exceeding 10 g/L.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH
  • Pichia pandoris expression systems see, e.g., Li et al., Nature Biotechnology. 24, 210 - 215, 2006; and Hamilton et al., Science, 301 : 1244, 2003.
  • Certain embodiments include yeast systems that are engineered to selectively glycosylate proteins, including yeast that have humanized N-glycosylation pathways, among others (see, e.g., Hamilton et al., Science.
  • sequences encoding polypeptides may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 3:17-311 (1987)).
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi et al., EMBO J. 3 : 1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ.
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, e.g., Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).
  • An insect system may also be used to express a polypeptide of interest.
  • Autographa califomica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia cells.
  • the sequences encoding the polypeptide may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses may then be used to infect, for example, S.
  • frugiperda cells or Trichoplusia cells in which the polypeptide of interest may be expressed (Engelhard et al., Proc. Natl. Acad. Sci. U.S.A. 91 :3224-3227 (1994)). Also included are baculovirus expression systems, including those that utilize SF9, SF21, and Tni cells (see, e.g., Murphy and Piwnica-Worms, Curr Protoc Protein Sci. Chapter 5:Unit 5.4, 2001). Insect systems can provide post-translation modifications that are similar to mammalian systems.
  • a number of viral -based expression systems are generally available.
  • sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non- essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. U.S.A. 81 :3655-3659 (1984)).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • CHO Chinese hamster ovary
  • DHFR-CHO cells Urlaub et al., PNAS USA 77:4216 (1980)
  • myeloma cell lines such as NSO and Sp2/0.
  • CHO Chinese hamster ovary
  • myeloma cell lines such as NSO and Sp2/0.
  • Certain preferred mammalian cell expression systems include CHO and HEK293-cell based expression systems.
  • Mammalian expression systems can utilize attached cell lines, for example, in T-flasks, roller bottles, or cell factories, or suspension cultures, for example, in IL and 5L spinners, 5L, 14L, 40L, 100L and 200L stir tank bioreactors, or 20/50L and 100/200L WAVE bioreactors, among others known in the art.
  • RNA polymerase typically utilize purified RNA polymerase, ribosomes, tRNA and ribonucleotides; these reagents may be produced by extraction from cells or from a cell-based expression system.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding a polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, et al., Results Probl. Cell Differ. 20: 125-162 (1994)).
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function.
  • Different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition to bacterial cells, which have or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.
  • cell lines which stably express a polynucleotide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type. Transient production, such as by transient transfection or infection, can also be employed. Exemplary mammalian expression systems that are suitable for transient production include HEK293 and CHO-based systems.
  • Any number of selection systems may be used to recover transformed or transduced cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 :223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfir which confers resistance to methotrexate (Wigler et al., PNAS USA.
  • npt which confers resistance to the aminoglycosides, neomycin and G-418 (Colbere- Garapin et al., J. Mol. Biol. 150: 1-14 (1981)); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. U.S.A.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • YFP fluorescent protein
  • anthocyanins P-glucuronidase and its substrate GUS
  • luciferase and its substrate luciferin being widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (see, e.g., Rhodes et al., Methods Mol. Biol. 55: 121-131 (1995)).
  • high-throughput protein production systems or micro-production systems. Certain aspects may utilize, for example, hexa-histidine fusion tags for protein expression and purification on metal chelate-modified slide surfaces or MagneHis Ni-Particles (see, e.g., Kwon et al., BMC Biotechnol. 9:72, 2009; and Lin et al., Methods Mol Biol. 498: 129- 41, 2009)). Also included are high-throughput cell-free protein expression systems (see, e.g., Sitaraman et al., Methods Mol Biol. 498:229-44, 2009). These and related embodiments can be used, for example, to generate microarrays of antibodies which can then be used for screening libraries to identify antibodies and antigen binding domains that interact with the IL-18BP polypeptide(s) of interest.
  • a wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • the sequences, or any portions thereof may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • Certain specific embodiments utilize serum free cell expression systems. Examples include HEK293 cells and CHO cells that can grow in serum free medium (see, e.g., Rosser et al., Protein Expr. Purif. 40:237-43, 2005; and U.S. Patent number 6,210,922).
  • An antibody, or antigen binding fragment thereof, produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides may be designed to contain signal sequences which direct secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane.
  • Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will facilitate purification and/or detection of soluble proteins.
  • cleavable and non-cleavable affinity purification and epitope tags such as avidin, FLAG tags, poly-histidine tags (e.g., 6xHis), cMyc tags, V5-tags, glutathione S-transferase (GST) tags, and others.
  • the protein produced by a recombinant cell can be purified and characterized according to a variety of techniques known in the art.
  • Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC).
  • FPLC fast protein liquid chromatography
  • HPLC high-pressure liquid chromatography
  • Exemplary chemistries for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration, reverse-phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. Also included are analytical methods such as SDS-PAGE (e.g., Coomassie, silver stain), immunoblot, Bradford, and ELISA, which may be utilized during any step of the production or purification process, typically to measure the purity of the protein composition.
  • affinity purification e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G
  • gel filtration e.g., reverse-phase, ceramic HYPERD® ion exchange chromatography
  • HIC hydrophobic interaction columns
  • analytical methods such as SDS-PAGE (e.
  • concentrated solutions of anti-IL-18BP antibodies comprise proteins at a concentration of about 5 mg/mL; or about 8 mg/mL; or about 10 mg/mL; about 15 mg/mL; or about 20 mg/mL or more.
  • compositions are substantially monodisperse, for example, where the anti-IL-18BP antibody exists primarily (i.e., at least about 90%, or greater) in one apparent molecular weight form when assessed, for example, by size exclusion chromatography, dynamic light scattering, and/or analytical ultracentrifugation.
  • the compositions have a purity (on a protein basis) of at least about 90%, or in some aspects at least about 95% purity, or in some embodiments, at least about 98% purity. Purity can be determined via any routine analytical method as known in the art.
  • the compositions have a high molecular weight aggregate content of less than about 10%, less than about 5%, less than about 3%, less than about 1%. High molecular weight aggregate content can be determined by a variety of analytical techniques including for example, by size exclusion chromatography, dynamic light scattering, and/or analytical ultracentrifugation.
  • concentration approaches contemplated herein include lyophilization, which is typically employed when the solution contains few soluble components other than the protein of interest. Lyophilization is often performed after HPLC run, and can remove most or all volatile components from the mixture. Also included are ultrafiltration techniques, which typically employ one or more selective permeable membranes to concentrate a protein solution. The membrane allows water and small molecules to pass through and retains the protein; the solution can be forced against the membrane by mechanical pump, gas pressure, or centrifugation, among other techniques.
  • the anti-IL-18BP antibodies, reagents, or related agents have a purity of at least about 90%, as measured according to routine techniques in the art.
  • an anti-IL-18BP composition has a purity of at least about 95%.
  • an anti-IL-18BP antibody composition has a purity of at least about 97% or 98% or 99%.
  • anti-IL-18BP antibodies can be of lesser purity, and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured overall or in relation to selected components, such as other proteins, e.g., purity on a protein basis.
  • Purified antibodies can also be characterized according to their biological characteristics. Binding affinity and binding kinetics can be measured according to a variety of techniques known in the art, such as Biacore® and related technologies that utilize surface plasmon resonance (SPR), an optical phenomenon that enables detection of unlabeled interactants in real time. SPR-based biosensors can be used in determination of active concentration, screening and characterization in terms of both affinity and kinetics.
  • SPR surface plasmon resonance
  • the presence or levels of one or more canonical or non-canonical biological activities can be measured according to cell-based assays, including those that utilize a cellular binding partner of a selected anti-IL-18BP antibody, which is functionally coupled to a readout or indicator, such as a fluorescent or luminescent indicator of biological activity, as described herein.
  • a readout or indicator such as a fluorescent or luminescent indicator of biological activity
  • a composition is substantially endotoxin free, including, for example, about or at least about 95% endotoxin free, about or at least about 99% endotoxin free, or about or at least about 99.99% endotoxin free.
  • endotoxins can be detected according to routine techniques in the art, as described herein.
  • a composition is made from a eukaryotic cell such as a mammalian or human cell in substantially serum free media.
  • a composition has an endotoxin content of less than about 10 EU/mg of antibody, or less than about 5 EU/mg of antibody, less than about 3 EU/mg of antibody, or less than about 1 EU/mg of antibody.
  • a composition comprises less than about 10% wt/wt high molecular weight aggregates, or less than about 5% wt/wt high molecular weight aggregates, or less than about 2% wt/wt high molecular weight aggregates, or less than about or less than about 1% wt/wt high molecular weight aggregates.
  • Protein-based analytical assays and methods which can be used to assess, for example, protein purity, size, solubility, and degree of aggregation, among other characteristics.
  • Protein purity can be assessed a number of ways. For instance, purity can be assessed based on primary structure, higher order structure, size, charge, hydrophobicity, and glycosylation.
  • methods for assessing primary structure include N- and C-terminal sequencing and peptide-mapping (see, e.g., Allen et al., Biologicals. 24:255-275, 1996)).
  • methods for assessing higher order structure include circular dichroism (see, e.g., Kelly et al., Biochim Biophys Acta.
  • Hydrophobicity can be assessed, for example, by reverse-phase HPLC and hydrophobic interaction chromatography HPLC. Glycosylation can affect pharmacokinetics (e.g., clearance), conformation or stability, receptor binding, and protein function, and can be assessed, for example, by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.
  • pharmacokinetics e.g., clearance
  • conformation or stability e.g., conformation or stability
  • receptor binding e.g., and protein function
  • NMR nuclear magnetic resonance
  • certain embodiments include the use of SEC-HPLC to assess protein characteristics such as purity, size (e.g., size homogeneity) or degree of aggregation, and/or to purify proteins, among other uses.
  • SEC also including gel-filtration chromatography (GFC) and gel-permeation chromatography (GPC) refers to a chromatographic method in which molecules in solution are separated in a porous material based on their size, or more specifically their hydrodynamic volume, diffusion coefficient, and/or surface properties. The process is generally used to separate biological molecules, and to determine molecular weights and molecular weight distributions of polymers.
  • a biological or protein sample (such as a protein extract produced according to the protein expression methods provided herein and known in the art) is loaded into a selected size-exclusion column with a defined stationary phase (the porous material), preferably a phase that does not interact with the proteins in the sample.
  • the stationary phase is composed of inert particles packed into a dense three- dimensional matrix within a glass or steel column.
  • the mobile phase can be pure water, an aqueous buffer, an organic solvent, or a mixture thereof.
  • the stationary-phase particles typically have small pores and/or channels which only allow molecules below a certain size to enter.
  • More recent techniques for analyzing protein purity include, without limitation, the LabChip GXII, an automated platform for rapid analysis of proteins and nucleic acids, which provides high throughput analysis of titer, sizing, and purity analysis of proteins.
  • clinical grade proteins such as protein fragments and antibodies can be obtained by utilizing a combination of chromatographic materials in at least two orthogonal steps, among other methods (see, e.g., Therapeutic Proteins: Methods and Protocols. Vol. 308, Eds., Smales and James, Humana Press Inc., 2005).
  • protein agents e.g., antibodies and antigen binding fragments
  • Protein solubility assays are also included. Such assays can be utilized, for example, to determine optimal growth and purification conditions for recombinant production, to optimize the choice of buffer(s), and to optimize the choice of antibodies or antigen binding fragments thereof. Solubility or aggregation can be evaluated according to a variety of parameters, including temperature, pH, salts, and the presence or absence of other additives. Examples of solubility screening assays include, without limitation, microplate-based methods of measuring protein solubility using turbidity or other measure as an end point, high-throughput assays for analysis of the solubility of purified recombinant proteins (see, e.g., Stenvall et al., Biochim Biophys Acta.
  • Antibodies with increased solubility can be identified or selected for according to routine techniques in the art, including simple in vivo assays for protein solubility (see, e.g., Maxwell et al., Protein Sci. 8: 1908-11, 1999).
  • Protein solubility and aggregation can also be measured by dynamic light scattering techniques.
  • Aggregation is a general term that encompasses several types of interactions or characteristics, including soluble/insoluble, covalent/noncovalent, reversible/irreversible, and native/denatured interactions and characteristics.
  • the presence of aggregates is typically considered undesirable because of the concern that aggregates may cause an immunogenic reaction (e.g., small aggregates), or may cause adverse events on administration (e.g., particulates).
  • Dynamic light scattering refers to a technique that can be used to determine the size distribution profile of small particles in suspension or polymers such as proteins in solution.
  • This technique also referred to as photon correlation spectroscopy (PCS) or quasielastic light scattering (QELS), uses scattered light to measure the rate of diffusion of the protein particles. Fluctuations of the scattering intensity can be observed due to the Brownian motion of the molecules and particles in solution.
  • This motion data can be conventionally processed to derive a size distribution for the sample, wherein the size is given by the Stokes radius or hydrodynamic radius of the protein particle. The hydrodynamic size depends on both mass and shape (conformation). Dynamic scattering can detect the presence of very small amounts of aggregated protein ( ⁇ 0.01% by weight), even in samples that contain a large range of masses.
  • certain embodiments include the use of dynamic light scattering to analyze the solubility and/or presence of aggregates in a sample that contains an antibody of the present disclosure.
  • Embodiment 1-1 An isolated antibody, or an antigen binding fragment thereof, which binds to interleukin- 18 binding protein (IL-18BP), wherein the at least one antibody, or antigen binding fragment thereof, comprises: a heavy chain variable region (VH) that comprises complementary determining region VHCDRI, VHCDR2, and VHCDR3 sequences selected from Table Al and variants thereof which specifically bind to IL-18BP; and a light chain variable region (VL) that comprises complementary determining region VLCDRI, VLCDR2, and VLCDR3 sequences selected from Table Al and variants thereof which specifically bind to IL-18BP.
  • VH heavy chain variable region
  • VHCDRI complementary determining region
  • VHCDR2 VHCDR3 sequences selected from Table Al and variants thereof which specifically bind to IL-18BP
  • VL light chain variable region
  • Embodiment 1-2 The isolated antibody, or antigen binding fragment thereof, of embodiment 1-1, wherein: the VHCDRI, VHCDR2, and VHCDR3 sequences comprise TFXIX2X 3 X 4 X 5 H, IX6X7X8X9X10X11X12X13X14X15AQKFQG, and X16X17X18X19X20X21X22DY, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise X23X24X25X26X27X28X29X30WX31A, X32X33X34X35X36X37X38, and QX39X40X41SFPYX42, respectively (see Table Ell for the definition of “X” residues); the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 4- 6, respectively;
  • Embodiment 1-3 The isolated antibody, or antigen binding fragment thereof, of embodiment 1-1 or 1-2, wherein the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2, optionally wherein the VH has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations in the framework regions.
  • Embodiment 1-4 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-3, wherein the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence selected from Table A2, optionally wherein the VL has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 alterations in the framework regions.
  • Embodiment 1-5 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-4, wherein: the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 277, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 278; the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 279, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 280; the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 285, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 2
  • Embodiment 1-6 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-5, wherein the antibody binds to an epitope that comprises 197 and V153 according to the numbering of UniProt: 095998 (alternatively, 195 and V151 as defined by SEQ ID NO: 1; or 167 and V123 as defined by SEQ ID NO: 2).
  • Embodiment 1-7 An isolated antibody, or an antigen binding fragment thereof, wherein the antibody binds to interleukin- 18 binding protein (IL-18BP) at an epitope that comprises 197 and V153 according to the numbering of UniProt: 095998 (alternatively, 195 and V151 as defined by SEQ ID NO: 1; or 167 and V123 as defined by SEQ ID NO: 2).
  • IL-18BP interleukin- 18 binding protein
  • Embodiment 1-8 The isolated antibody, or antigen binding fragment thereof, of embodiment 1-7, wherein: the VHCDRI, VHCDR2, and VHCDR3 sequences comprise TFXIX2X 3 X 4 X 5 H, IX6X7X8X9X10X11X12X13X14X15AQKFQG, and X16X17X18X19X20X21X22DY, respectively, and the VLCDR1, VLCDR2, and VLCDR3 sequences comprise X23X24X25X26X27X28X29X30WX31A, X32X33X34X35X36X37X38, and QX39X40X41SFPYX42, respectively (see Table Ell for the definition of “X” residues); the VHCDRI, VHCDR2, and VHCDR3 sequences comprise SEQ ID NOs: 1-3, respectively, and the VLCDRI, VLCDR2, and VLCDR3 sequences comprise SEQ ID NOs: 4- 6, respectively; or
  • Embodiment 1-9 The isolated antibody, or antigen binding fragment thereof, of embodiment 1-8, wherein: the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 277, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 278; the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 279, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 280; or the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 367, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 368
  • Embodiment I- 10 An isolated antibody, or an antigen binding fragment thereof, wherein the antibody binds to interleukin- 18 binding protein (IL-18BP) and wherein the antibody interferes with the binding of IL- 18 to IL-18BP.
  • IL-18BP interleukin- 18 binding protein
  • Embodiment 1-11 The isolated antibody, or antigen binding fragment thereof, of embodiment I- 10, wherein the antibody binds to a conformational epitope of IL-18BP.
  • Embodiment 1-12 The isolated antibody, or antigen binding fragment thereof, of embodiment I- 10 or 1-11, wherein the conformational epitope of IL-18BP comprises two or more amino acid residues selected from the group consisting of K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372.
  • Embodiment 1-13 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments I- 10 to 1-12, wherein the conformational epitope of IL-18BP comprises the amino acid residues of K32, T40, S60, R61, S66, Y69, R91, R93, T96, K102, R131, and H132 of SEQ ID NO: 372.
  • Embodiment 1-14 The isolated antibody, or antigen binding fragment thereof, of embodiment I- 10, wherein the antibody binds to a linear epitope of IL-18BP.
  • Embodiment 1-15 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments I- 10 to 1-14, wherein the antibody binds to the binding interface between IL- 18 and a mature form of IL-18BP.
  • Embodiment 1-16 The isolated antibody, or antigen binding fragment thereof, of embodiment 1-15, wherein the antibody binds the amino acid residues R61, Y69 and R131 of SEQ ID NO: 372.
  • Embodiment 1-17 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-12 to 1-16, wherein: the VH comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 333, and the VL comprises a sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to SEQ ID NO: 334.
  • Embodiment 1-18 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-17, wherein the antibody binds to human IL-18BP and cynomolgus IL-18BP but does not bind (specifically or substantially) to mouse IL-18BP.
  • Embodiment 1-19 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-17, wherein the antibody binds to human IL-18BP, cynomolgus IL- 18BP, and mouse IL-18BP.
  • Embodiment 1-20 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-19, wherein the antibody binds to the binding interface between IL- 18 and a mature form of IL-18BP.
  • Embodiment 1-21 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-20, which binds to IL-18BP with a binding affinity that is stronger than the binding affinity between IL- 18 and IL-18BP (KD ⁇ 650 pM), optionally a binding affinity of about 1 pm to about 650 pm, or about or less than about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 300, 400, 500, 600, or 650 pM.
  • Embodiment 1-22 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-21, which is an IL-18BP antagonist, which antagonizes the binding activity between IL-18BP and IL- 18.
  • Embodiment 1-2 The isolated antibody, or antigen binding fragment thereof, of embodiment 1-22, wherein the antibody blocks the inhibitory activity of IL-18BP towards IL- 18, and thereby increases IL-18-mediated signaling, including induction of IFN-gamma, CXCL10, and/or TNFa.
  • Embodiment 1-24 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-8, comprising an IgA (including subclasses IgAl and IgA2), IgD, IgE, IgG (including subclasses IgGl, IgG2, IgG3, and IgG4), or IgM Fc domain, optionally a human Fc domain, or a hybrid and/or variant thereof.
  • IgA including subclasses IgAl and IgA2
  • IgD including subclasses IgAl and IgA2
  • IgG including subclasses IgGl, IgG2, IgG3, and IgG4
  • IgM Fc domain optionally a human Fc domain, or a hybrid and/or variant thereof.
  • Embodiment 1-25 The isolated antibody, or antigen binding fragment thereof, of embodiment 1-24, comprising an IgG Fc domain with high effector function in humans, optionally an IgGl or IgG3 Fc domain.
  • Embodiment 1-26 The isolated antibody, or antigen binding fragment thereof, of embodiment 1-24, comprising an IgG Fc domain with low effector function in humans, optionally an IgG2 or IgG4 Fc domain.
  • Embodiment 1-27 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-26, which is a monoclonal antibody.
  • Embodiment 1-28 The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-27, which is a humanized antibody.
  • Embodiment 1-29. The isolated antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-28, which is selected from an Fv fragment, a single chain Fv (scFv) polypeptide, an adnectin, an anticalin, an aptamer, an avimer, a camelid antibody, a designed ankyrin repeat protein (DARPin), a minibody, a nanobody, and a unibody.
  • Fv fragment a single chain Fv (scFv) polypeptide
  • adnectin an anticalin
  • an aptamer an avimer
  • a camelid antibody a designed ankyrin repeat protein (DARPin)
  • DARPin ankyrin repeat protein
  • Embodiment 1-30 An isolated polynucleotide encoding the isolated anti IL-18BP antibody, or antigen binding fragment thereof, according to any one of embodiments 1-1 to 1-29, an expression vector comprising the isolated polynucleotide, or an isolated host cell comprising the vector.
  • Embodiment 1-3 A pharmaceutical composition, comprising the isolated anti IL- 18BP antibody, or antigen binding fragment thereof, of any one of embodiments 1-1 to 1-29, and a pharmaceutically-acceptable carrier.
  • Embodiment 1-32 The pharmaceutical composition of embodiment 1-31, wherein the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% on a protein basis with respect to the at least one antibody or antigen binding fragment and is substantially aggregate- and endotoxin-free.
  • Embodiment 1-33 The pharmaceutical composition of embodiment 1-31 or 1-32, wherein the composition is a sterile, injectable solution, optionally suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration.
  • Embodiment 1-34 A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of any one of embodiments 1-31 to 1-33.
  • Embodiment 1-35 The method of embodiment 1-34, wherein the disease or condition is a cancer or tumor or proliferative disease or disorder, optionally a proliferative disease or disorder selected from a lymphoproliferative disorder, a myeloproliferative disorder, proliferative enteritis, proliferative diabetic retinopathy, and a proliferative kidney disease.
  • a proliferative disease or disorder selected from a lymphoproliferative disorder, a myeloproliferative disorder, proliferative enteritis, proliferative diabetic retinopathy, and a proliferative kidney disease.
  • Embodiment 1-36 The method of embodiment 1-35, wherein the cancer or tumor expresses or overexpresses IL-18BP and/or IL- 18, or wherein the proliferative disease or disorder is associated with increased expression of IL-18BP and/or IL-18.
  • Embodiment 1-37 The method of embodiment 1-35 or 1-36, wherein the cancer is selected from one or more of bone cancer, prostate cancer, melanoma (e.g., metastatic melanoma), pancreatic cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (e.g., lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, hairy cell leukemias, acute lymphoblastic leukemias), lymphoma (e.g., non-Hodgkin’s lymphomas, Hodgkin’s lymphoma), hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary a
  • Embodiment 1-39 The method of any one of embodiments 1-35 to 1-38, comprising administering the pharmaceutical composition (with the anti-IL18BP antibody, or antigen binding fragment thereof) in combination with an immune checkpoint modulatory agent selected from an antagonist of a inhibitory immune checkpoint molecule and an agonist of a stimulatory immune checkpoint molecule.
  • an immune checkpoint modulatory agent selected from an antagonist of a inhibitory immune checkpoint molecule and an agonist of a stimulatory immune checkpoint molecule.
  • Embodiment 1-40 The method of embodiment 1-39, wherein the immune checkpoint modulatory agent is a polypeptide, optionally an antibody or antigen binding fragment thereof or a ligand, or a small molecule.
  • the immune checkpoint modulatory agent is a polypeptide, optionally an antibody or antigen binding fragment thereof or a ligand, or a small molecule.
  • Embodiment 1-4 The method of embodiment 1-39 or 1-40, wherein the inhibitory immune checkpoint molecule is selected from one or more of Programmed Death-Ligand 1 (PD- Ll), Programmed Death 1 (PD-1), Programmed Death-Ligand 2 (PD-L2), Cytotoxic T- Lymphocyte-Associated protein 4 (CTLA-4), Indoleamine 2,3 -dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO), T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3), V-domain Ig suppressor of T cell activation (VISTA), B and T Lymphocyte Attenuator (BTLA), CD 160, Herpes Virus Entry Mediator (HVEM), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT).
  • PD- Ll Programmed Death-Ligand 1
  • PD-1 Programmed Death 1
  • PD-L2 Programmed
  • Embodiment 1-42 The method of embodiment 1-41, wherein: the antagonist is a PD-L1 and/or PD-L2 antagonist optionally selected from one or more of an antibody or antigen binding fragment or small molecule that specifically binds thereto, atezolizumab (MPDL3280A), avelumab (MSB0010718C), and durvalumab (MEDI4736), optionally wherein the cancer is selected from one or more of colorectal cancer, melanoma, breast cancer, non-small-cell lung carcinoma, bladder cancer, and renal cell carcinoma; the antagonist is a PD-1 antagonist optionally selected from one or more of an antibody or antigen binding fragment or small molecule that specifically binds thereto, nivolumab, pembrolizumab, MK-3475, AMP-224, AMP-514PDR001, and pidilizumab, optionally wherein the PD-1 antagonist is nivolumab and the cancer is optionally selected from one
  • Embodiment 1-43 The method of embodiment 1-39 or 1-40, wherein the stimulatory immune checkpoint molecule is selected from one or more of 0X40, CD40, Glucocorticoid- Induced TNFR Family Related Gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and Herpes Virus Entry Mediator (HVEM).
  • the stimulatory immune checkpoint molecule is selected from one or more of 0X40, CD40, Glucocorticoid- Induced TNFR Family Related Gene (GITR), CD137 (4-1BB), CD27, CD28, CD226, and Herpes Virus Entry Mediator (HVEM).
  • Embodiment 1-44 The method of embodiment 1-43, wherein: the agonist is an 0X40 agonist optionally selected from one or more of an antibody or antigen binding fragment or small molecule or ligand that specifically binds thereto, 0X86, Fc-OX40L, and GSK3174998; the agonist is a CD40 agonist optionally selected from one or more of an antibody or antigen binding fragment or small molecule or ligand that specifically binds thereto, CP- 870,893, dacetuzumab, Chi Lob 7/4, ADC-1013, and rhCD40L, and wherein the cancer is optionally selected from one or more of melanoma, pancreatic carcinoma, mesothelioma, and hematological cancers optionally lymphoma such as Non-Hodgkin’s lymphoma; the agonist is a GITR agonist optionally selected from one or more of an antibody or antigen binding fragment or small molecule or ligand that
  • Embodiment 1-45 The method of any one of embodiments 1-35 to 1-44, comprising administering the pharmaceutical composition (with the anti-IL18BP antibody, or antigen binding fragment thereof) in combination with at least one chemotherapeutic agent.
  • Embodiment 1-46 The method of embodiment 1-45, wherein the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent.
  • the at least one chemotherapeutic agent is selected from one or more of an alkylating agent, an anti-metabolite, a cytotoxic antibiotic, a topoisomerase inhibitor (type 1 or type II), and an anti-microtubule agent.
  • Embodiment 1-47 The method of embodiment 1-46, wherein: the alkylating agent is selected from one or more of nitrogen mustards (optionally mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide , and busulfan), nitrosoureas (optionally N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (optionally dacarbazine, mitozolomide, and temozolomide), aziridines (optionally thiotepa, mytomycin, and diaziquone (AZQ)), cisplatins and derivatives thereof (optionally carboplatin and oxaliplatin), and non-classical alkylating agents (optionally procarbazine and hexamethylmelamine); the alkylating
  • Embodiment 1-48 The method of embodiment 1-34, wherein the disease or condition is a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • Embodiment 1-49 The method of embodiment 1-34, wherein the disease or condition is an infectious disease.
  • Embodiment 1-50 The method of embodiment 1-49, wherein the infectious disease is selected from viral, bacterial, fungal (optionally yeast), and protozoal infections.
  • Embodiment 1-51 The method of any one of embodiments 1-48 to 1-50, comprising administering the pharmaceutical composition (with the anti-IL18BP antibody, or antigen binding fragment thereof) in combination with IL- 18.
  • Embodiment 1-52 A method of screening an anti -IL-18BP antibody or antigen binding fragment thereof for the ability to block or inhibit binding between IL- 18 and IL-18BP, comprising a) determining binding affinity of the antibody or antigen binding fragment thereof for i) IL-18BP alone, and ii) a hypo-IL-18 fusion protein, wherein the hypo-IL-18 fusion protein comprises IL- 18 fused to IL-18BP via a flexible linker (and an optional protease cleavage site in between), wherein the IL-18 portion of the fusion protein is bound to the IL-18BP portion of the fusion protein and sterically blocks the IL- 18 binding site of the IL- 18BP portion of the fusion protein; b) comparing the binding affinity of (i) to the binding affinity of (ii); and c) identifying or selecting the antibody or antigen binding fragment thereof as being able to block or inhibit binding between IL- 18 and IL-18BP if the binding affinity of (i) is significantly stronger
  • Embodiment 1-53 The method of embodiment 1-52, wherein the IL- 18 and IL-18BP are mouse IL- 18 and IL-18BP.
  • Embodiment 1-54 The method of embodiment 1-52, wherein the IL- 18 and IL-18BP are human IL- 18 and IL-18BP.
  • Embodiment 1-55 The method of any one of embodiments 1-52 to 1-54, wherein the hypo-IL-18 fusion protein comprises, in an N- to C-terminal orientation, a signal peptide, IL-18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and IL-18BP.
  • Embodiment 1-56 The method of embodiment 1-55, wherein the hypo-IL-18 fusion protein comprises an amino acid sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence from Table SI.
  • a hypo-IL-18 fusion protein comprising, in an N- to C-terminal orientation, a signal peptide, IL- 18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and IL-18BP.
  • Embodiment 1-58 The hypo-IL-18 fusion protein of embodiment 1-57, comprising an amino acid sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence from Table SI [0333]
  • Embodiment 1-59 A method of stimulating an immune response in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of any one of any one of embodiments 31-33.
  • Embodiment 1-60 The method of embodiment 1-59, wherein the immune response is an IL- 18 mediated immune response.
  • Embodiment 1-61 The method of embodiment 1-60, wherein the IL- 18 mediated immune response comprises induction of IFN-gamma, CXCL10, and/or TNFa in the subject in need thereof.
  • mice were immunized with an alternating schedule of human IL-18BP and cynomolgus monkey IL-18BP with either his or Fc tags, while titers were tested with human, cyno, or mouse IL-18BP using the other tag in each case, to avoid detection of tag specific antibodies.
  • antigen specific B cells from the appropriate mice were isolated as single cells using the Berkeley Lights Beacon instrument using procedures recommended by the manufacturer as described (Mullen et al., Antibody Therapeutics. 4(3): 185-196, 2021). Immunoglobulin gene sequences from antigen specific B cells were obtained and used to generate recombinant antibodies using established methodology.
  • SAOla is a human IgGl antibody while SA51d is a mouse IgG2a antibody.
  • PCR was performed with high-fidelity DNA Polymerase (Q5, New England Biolabs) according to the manufacturer’s protocols.
  • Parental plasmids (both heavy and light chains) were diluted to 10 ng/pL and 1 pL was used as template for each 50 pL reaction.
  • OE-PCR overlap extension PCR
  • Gibson cloning Gibson cloning.
  • OE-PCR fragments were amplified with corresponding forward and reverse primers containing restriction sites (Agel-Nhel for heavy chain, Sbfl-Mfel for light chain) and column purified (Qiagen PCR purification kit). Restriction digests were carried out using high fidelity enzymes (New England Biolabs) and fragments were ligated using T4 DNA ligase (New England Biolabs, Cat # M0202L) into appropriate vectors for heavy and light chain. Empty heavy chain vector contains the majority of the human IgGl constant region with an engineered Nhel site (created by altering wobble positions) 12 amino acids into the constant region.
  • Light chain empty vector contains the majority of the human Kappa constant region with an engineered Mfel site 18 amino acids into the constant region. Gibson Cloning was achieved using the fragments with the same empty vectors (Gibson Assembly® Master Mix Kit, New England Biolabs Cat # ES261 IL). Inserts were normalized to 1 ng/pL and a total of 2 ng of insert DNA is used (1 ng per fragment). 10 pL reaction volume was made up of 5 pL of Gibson Master Mix and QS with purified water. The reaction was incubated at 50 °C for 15-60 minutes.
  • Ligation products from either OE-PCR or Gibson assembly were transformed into competent E. coli (Monserate Biotechnology, San Diego) by adding 2-5 pL of the ligation reaction mix to the cells and incubating on ice for 5 minutes. Cells were heat shocked at 42 °C for 30 seconds and placed on ice. 250 pL of SOC media (BioPioneer, San Diego or Teknova, San Diego) was added and tubes were incubated at 37 °C, 200 rpm for 1 hour. 100 pL of culture was plated onto antibiotic selection plates (BioPioneer, San Diego or Teknova, San Diego), and incubated at 37 °C overnight.
  • Plates were sent for colony sequencing of the antibody genes (Genewiz or Eton) where 24-48 clones per plate are sequenced. Sequences were analyzed with SnapGene Software (GSL Biotech) against the reference sequence which is an in silico cloning of the library. Clones were picked based on sequence alignment and amino acids that are encoded by the mutation primer, and used to generate individual plasmid mini-preps.
  • Expi293F cells from the Expi293 Expression System Kit (Thermo Fisher, cat. no. A14635) were grown in Expi293F expression medium (cat. no. A1435101). Cells were grown to a density of 3-6xl0 6 cells/mL and then counted using a hemocytometer. Plasmid DNA (1.0 ug per 1.0 mL of culture) was diluted in Opti-MEM Reduced Serum Medium (RSM) (cat. no. 31985062). Values of Opti-MEM RSM were taken from manufacturer’s recommendations for transfections.
  • RSM Opti-MEM Reduced Serum Medium
  • Expifectamine 293 reagent was diluted in Opti-MEM RSM and incubated for 5 min. at room temperature before mixing with diluted plasmid DNA. This mixture was left to incubate for 10-20 minutes at room temperature. While the expifectamine/plasmid DNA complex was incubating, Expi293F cells were diluted to a density of 3xl0 6 cells/mL and added to Erlenmeyer flasks of desired volume (BioPioneer, DGFPC0125S for 125 mL flask).
  • the expifectamine/plasmid DNA complex was then slowly transferred to a shaker flask with Expi293F cells, and the flasks were placed in a shaking incubator with a 25 mm orbital throw (Infors-HT Multitron) at 37 °C, 8% CO2, 125 rpm. 18-22 h post transfection, ExpiFectamine 293 Transfection Enhancer 1 (#100013863) and 2 (#A14350-01) were added to the cells, and the cells were returned to the shaking incubator. Cells were then left to incubate for 4 additional days, then spun down at 4000 x g for 20 minutes in a refrigerated centrifuge and 0.22 um filtered prior to purification.
  • Antibodies were purified using 5 mL HiTrap Mab Select SuRe (Protein A) columns on an AKTA Explorer FPLC system. Columns were first cleared of any residual bound protein by the addition of 50 mL of 0.1 M glycine, pH 3.0 (elution buffer) followed by 50 mL of 50 mM glycine, 50 mM glycinate pH 8 (binding/washing buffer). Antibodies (25 - 400 mL) were loaded onto column at 5 mL/min and further washed with 25 mL equilibration/wash buffer until UV reading reached baseline.
  • MAbs were subsequently eluted by using a 25 mL linear gradient of 0- 100% elution buffer for 2 min at 5 mL/min. Antibody elution was monitored by absorbance at 280 nm. Peak fractions were collected and pooled in a 15 mL conical tube. Material was then buffer exchanged into storage buffer (PBS, pH 7.4) using PD10 columns (Cytiva cat. no. 17085101), and subsequently filter sterilized (GenClone Syringe Filters, cat. no. 25-244 attached to BD 5 mL [cat. no. 309646] and 20 mL [cat. no. 302830] BD Luer-LokTM syringes) into a 15 mL conical tube and used for subsequent characterization assays.
  • storage buffer PBS, pH 7.4
  • SEC-HPLC Size -exclusion HPLC
  • SEC-HPLC was performed on a 5 pm particle size, 7.8 mm I.D. X 30 cm TSKgel G3000SWXL and run isocratically using 50 mM sodium phosphate, 200 mM arginine pH 6.8 at a flow rate of 1 mL/minute on an Agilent 1100 HPLC. Detection was at 280 nm using a diode array detector and peaks were integrated using Agilent ChemStation software.
  • SEC- HPLC standards used to calibrate the column consisted of bovine thyroglobulin, bovine IgG, chicken albumin, bovine ribonuclease A and p-aminobenzoic acid (Sigma Aldrich #69385).
  • Binding kinetic measurements were taken on a Fortebio (now Sartorius) Octet RED96e instrument. mAbs were loaded onto anti-human constant domain (AHC) biosensors (ForteBio) in lOx kinetics buffer consisting of PBS containing 0.1% BSA, 0.02% Tween 20 for 90-120 s to achieve a spectral shift value between 0.8 to 1.2 nm. Association was carried out in the presence of a 2-fold dilution series of hIL-18BP and was typically allowed to proceed for 90-120 s; dissociation was generally measured for 300 to 1200 s.
  • AHC anti-human constant domain
  • Dilution series started at 100 nM for weaker variants or 10 nM for the most potent mAbs.
  • Cross-reactivity to cyno IL-18BP and mouse IL-18BP was determined using the same methodology with the appropriate species’ IL-18BP.
  • IL-18 Reporter HEK 293 cells from InvivoGen (hkb-hmill8) respond to exogenously added IL- 18 by expressing an NF-KB/AP-1 -inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene.
  • SEAP embryonic alkaline phosphatase
  • Cells were counted, spun down at 200 x g for 5 min., and resuspended in complete DMEM media without selection at 2.5xl0 5 cells/mL. Cells were then plated at 25,000 cells in 100 pL/well in a 96 well plate (Genesee Scientific, 25-109). Plated cells were then placed in an incubator at 37 °C, 5% CO2. Test antibodies solutions were prepared at 90 pg/mL in selection free DMEM and incubated with human IL-18BP at 120 ng/mL for 30 min. at room temperature.
  • a 0.6 ng/mL stock of recombinant human IL-18 (SinoBiological, 10119-HNCE) was then prepared and added to the antibody/IL-18BP complex after incubation. The resulting solution was immediately added to the cells, giving a final concentration of 15 pg/mL test antibody, 20 ng/mL human IL-18BP, and 0.1 ng/mL human IL-18. Cells were left to incubate for 18-22 hours at 37 °C, 5% CO2.
  • PBMC Assays for human IL-18 activity Human peripheral blood mononuclear cells (PBMC) obtained from the San Diego Blood Bank were seeded into 96 well flat bottom plates (GenClone, cat. no. 25-109) at 2 x 10 5 cells/well or round bottom plates (GenClone, cat. no. 25- 221) at 1.7 x 10 5 cells/well in RPMI+GlutaMAX (Gibco cat. no. 61870036) containing 100 U penicillin, 100 pg streptomycin (Gibco cat. no. 10378016) and 10% FBS (RPMIc). Test antibodies were added to wells at 4x final concentration in a volume of 50 pL.
  • 50 pL recombinant human IL- 12 (R&D system, cat. no. 219-IL-005) was then added at 4 ng/mL followed by 50 pL recombinant human IL- 18 (Sino Biological, cat. no.10119-HNCE) at 8 ng/mL for final concentrations of 1 ng/mL and 2 ng/mL, respectively.
  • 50 pL recombinant human IL- 12 (R&D system, cat. no. 219-IL-005) was then added at 4 ng/mL followed by 50 pL recombinant human IL- 18 (Sino Biological, cat. no.10119-HNCE) at 8 ng/mL for final concentrations of 1 ng/mL and 2 ng/mL, respectively.
  • cyno PBMCs iQ Biosciences, cat. no. IQB-MnPB102
  • cells were seeded at 1.7 x 10 5 cells/
  • rhesus macaque IL-18 (R&D Systems cat. no. 2548-RM-025/CF [note that the amino acid IL-18 sequence for rhesus and cynomolgus are identical]) was added at a final concentration of 2 ng/mL. All dilutions were made in RPMIc. Cells were incubated for 48 h at 37 °C, 5% CO2. At 48 h, a 50 pL aliquot of supernatant was removed from each well and assayed for the presence of IFNy using a DuoSet ELISA kit (R&D Systems, cat. no.
  • IL- 18 Reporter Assay Mouse IL- 18 Reporter Assay.
  • IL- 18 Reporter HEK 293 cells (Invivogen, hkb-hmill8) were grown in complete DMEM Media (10% HI FBS, 1% PS) with lx HEK-Blue Selection (Invivogen, hb-sel). Cells were carefully rinsed twice with lx PBS and lifted with lx PBS at 37 °C for 5 minutes. Cells were counted, spun down at 200 x g for 5 minutes, and resuspended in complete DMEM media without selection at 2.5E 5 cells/mL.
  • Test antibodies solutions were prepared at 90 pg/mL in selection free DMEM and incubated with mouse IL-18BP at 120 ng/mL for 30 minutes at room temperature. A 60 ng/mL stock of recombinant mouse IL- 18 was then prepared and added to the antibody/IL-18BP complex after incubation. The resulting solution was immediately added to the cells, giving a final concentration of 15 pg/mL test antibody, 20 ng/mL mouse IL-18BP, and 10 ng/mL mouse IL-18.
  • Mouse Splenocyte Assay -Murine Splenocyte Isolation Mouse spleens were added to a 10 mm petri dish, and 1 mL of enzyme-free dissociation buffer (Gibco, cat. no. 13151014) was added per spleen. Spleens were then mashed with the back of a 1 mL syringe until dissociated. This solution was passed through a 70 pm cell strainer, the petri dish was washed with RPMI 10% FBS lx P/S, and this was also passed through the cell strainer. Cells were spun down at 300xg for 5 min, supernatant was removed, and lx RBC Lysis Buffer (Biolegend, cat. no.
  • Murine Splenocyte mIL-18BP Inhibition Assay Mouse splenocytes were thawed into RPMI 10% HI FBS + Pen-strep. Cells were counted and seeded at 110k cells/well into the center 60 wells of a 96-well u-bottom plate. 9-point serial dilutions of antibodies were made and mlL- 18 at 0.1 ng/mL final concentration (R&D Systems, cat. no. 9139-IL-010), and mIL-12 at 10 ng/mL final concentration (R&D Systems, cat. no. 419-ML-010) were added to this to make a 2x solution.
  • mIFNy was measured using a mouse IFN-gamma DuoSet ELISA (R&D Systems, cat. no. DY485) on a Nunc MaxiSorp Flat-Bottom Plate (Invitrogen, cat. no. 44-2404- 21) as per manufacturer’s protocol, and absorbance at 450 nm was measured on a SpectraMax iD5 plate reader.
  • ATX-GK mice were immunized with human and cynomolgus monkey IL-18BP. Titers to human, cyno, and mouse IL-18BP were measured by ELISA and mice with high titers were selected for isolation of antibody secreting B cells using the Berkeley Lights Beacon Optofluidic System (Mullen, 2021). Screening identified single cells secreting antibodies which crossreacted to human and cyno IL-18BP and some which also bound mouse IL-18BP. In addition, a novel screen was designed to identify cells secreting antibodies which were unable to recognize a form of IL-18BP in which the active site has been blocked, termed “hypo-IL-18”. This enabled the screen to identify putative ligand blocking antibodies.
  • IL- 18 displays high affinity for the binding protein with a KD of less than 1 nM (Kim et al., PNAS 97(3): 1190-1195, 2000; Kimura et al., Allergol Int. 57(4):367-76, 2008), it might not be possible for mAbs with affinities in the nM affinity range, such as those typically isolated from antigen specific B cells, to antagonize the IL-18/IL-18BP interaction. Therefore, to assess the potential blocking abilities of newly-identified mAb candidates, the novel chimeric protein “hypo-IL-18” was generated.
  • Hypo-IL-18 is comprised of human or mouse IL-18 tethered to their respective IL-18BPs but separated with a flexible linker peptide (shown schematically in FIGS. 3A-3B).
  • the sequence of hypo-IL-18 is provided in Table SI below.
  • the rational for creating this molecule is that the IL-18BP active site will be blocked since its tethered ligand will be unable to dissociate.
  • mAbs which recognize the active binding site of the BP will be sterically hindered from recognizing hypo-IL-18, while most nonblocking antibodies will be able to bind to IL-18BP and to hypo-IL-18 equally.
  • a TEV protease cleavage site was also included in the design to provide the ability to separate the hIL-18 from its binding protein.
  • hypo-IL- 18, joining the C-terminus of IL-18 to the N-terminus of IL- 18BP was made based on a crystal structure (Protein Data Bank [PDB] structure 3F62), which included human IL- 18 in complex with the Ectromelia virus IL-18BP. Because Orthopoxviruses, including Ectromelia, encode functional IL-18BP homologs that exhibit 17-34% amino acid identity to the mammalian orthologous IL-18BP (Calderara, 2001), it was felt that the 3F62 crystal structure would be instructive in the design of the hypo-IL-18 construct.
  • PDB Protein Data Bank
  • Certain embodiments thus include a hypo-IL- 18 fusion protein, comprising, in an N- to C-terminal orientation, a signal peptide, IL- 18, a first flexible linker, a protease cleavage site (optionally a TEV protease cleavage site), a flexible linker, and IL-18BP; wherein the IL-18 portion of the fusion protein is bound to the IL-18BP portion of the fusion protein and sterically blocks the IL-18 binding site of the IL-18BP portion of the fusion protein.
  • the hypo-IL-18 fusion protein comprises, consist, or consists essentially of an amino acid sequence at least 80, 85, 90, 95, 97, 98, 99, or 100% identical to a sequence from Table SI. Also included are nucleic acid molecules encoding the hypo-IL- 18 fusion protein.
  • Antibodies of interest were also tested for activity in a cell-based reporter assay.
  • This assay uses an engineered cell line where IL-18 signaling results in the secretion of alkaline phosphatase, which is readily measured.
  • IL-18 induction of alkaline phosphatase is inhibited by the addition of IL-18BP, and this inhibition is alleviated by the addition of antibodies to IL- 18BP provided they are capable of blocking IL-18BP/IL-18 binding.
  • Table E2 several antibodies were capable of blocking the IL-18/IL-18BP interaction and allowing induction of an IL- 18 driven response.
  • the antibodies that resulted in activity in this assay were consistent with those unable to bind hypo-IL-18, verifying the screening approach used to isolate the antibodies of interest.
  • KG-1 is a human bone marrow derived macrophage cell line that responds to IL- 18 by producing IFNy.
  • the addition of IL-18BP blocks the ability of IL-18 to induce IFNy expression, thereby repressing the response.
  • the further addition of a neutralizing anti-IL-18BP antibody which binds to IL-18BP disrupting the interaction with IL-18, therefore liberates IL-18 to induce ITNy. This can also be termed derepression.
  • FIG. 4 shows the ability of the initial set of antibodies to effect derepression of IFNy in KG-1 cells. There is an apparent rank order potency among the mAb candidates with SA12a > SA09a > SAlOa > SAOla.
  • SAOla was prioritized for optimization (see Table E3).
  • SAOla was derived from an Alloy ATX-GK mouse and was a human IgG.
  • the initial screen of the combinatorial library identified two mAbs that measured with very high affinity for mIL-18BP by BLI, named SA51d and SA52d. These two mAbs were reexpressed and retested for full binding kinetics, yielding KD values of ⁇ 50 pM and 140, respectively. Although both mAbs also measured as off-scale by BLI vs hIL-18BP, the affinity vs cyno IL-18BP was approximately 2 orders of magnitude weaker, indicating these entities were not preferred for further human preclinical development.
  • the screen also identified a subset of amino acid substitutions that specifically improved binding to both human and cyno IL-18BP. Consequently, these changes were recombined in various combinations for further analysis.
  • A99S greatly improves binding to the cyno binding protein but somewhat worsens recognition of the human ortholog.
  • Table E4 shows the variants leading to improved binding to human and cyno IL-18BP
  • Parenthetical (N) values indicate how many data points from independently run experiments are included in the average; ⁇ indicates S.E.M.; NT, not tested; ND, not determined. In cases where a mAb was tested only twice, both values are provided. Cyno/human provides the ratio difference between the two species.
  • IL-18 activity is inhibited by added murine IL-18BP, and this inhibition is relieved by high affinity neutralizing mAbs to IL-18BP.
  • affinity neutralizing mAbs to IL-18BP As shown in FIG. 5, the improvement in affinity correlated with improved activity in this assay. SAOla demonstrated activity with an IC50 of 19.8 nM, whereas the matured mAbs SA51d and SA52d generated IC50 values >100 fold more potent - 0.14 and 0.19 nM, respectively.
  • the anti-mouse IL-18BP mAbs were tested in an additional bioassay using mouse splenocytes.
  • Mouse splenocytes release IFNy in response to IL- 18, which is inhibited by the addition of IL-18BP.
  • Active mAbs relieve this inhibition restoring production of IFNy.
  • FIG. 6 shows that the SAOla, SA51d, and SA52d significantly increased ITNy production in this assay.
  • SA51d which had high affinity and high functional potency in both reporter and splenocyte assays, was considered optimal for in vivo evaluation.
  • Treatments included PBS vehicle control, mIL-18 (0.32 mg/kg), SA0051d (10 mg/kg), or mIL-18 (0.32 mg/kg) + SA0051d (10 mg/kg).
  • MAbs with high affinity for human and cyno IL-18BP were further characterized to identify those suitable for further clinical development.
  • This included assessment of antibody binding characteristics for example, anti-human IL-18BP that blocks IL-18BP mediated neutralization of IL-18 signaling; human IgGl / kappa; cross-reacts with cyno IL-18BP; high affinity with a KD to human IL-18BP that is lower than that of IL-18 to IL-18BP, no detectable non-specific binding or binding to homologous proteins
  • functional properties for example, blocks IL- 18 binding to IL-18BP; neutralizes IL-18BP, thus antagonizing its suppression of IL- 18 mediated induction of IFNy
  • the tested mAbs were able to bind IL-18BP, relieve inhibition of IL- 18 and allow IL- 18 to signal in the reporter assay (resulting in the secretion of the reporter protein alkaline phosphatase).
  • the order of potency correlated with binding affinity for the blocking mAbs; that is, the highest affinity mAbs were most potent in this assay.
  • FIG. 9 shows that the tested mAbs were also showed high potency in the KG-1 assay.
  • Table E7 provides the EC50 values.
  • IL-18 in conjunction with IL-12, induces IFNY production by multiple cell types in PBMCs, including NK cells and T cells.
  • IL-18BP is endogenously produced by PBMCs and upregulated by IFNy, providing a negative feedback loop that suppresses the IFNy response.
  • the addition of a neutralizing anti-IL-18BP antibody would bind endogenous IL-18BP and prevent the negative feedback loop, resulting in induction of IFNy production.
  • the ability of a set of mAbs to compete with IL-18 for binding to IL-18BP and allow IL-18-mediated induction of IFNy was tested in healthy donor PBMC in vitro.
  • IL-18 concentrations were only enough to provide a low level IFNy response in PBMC, likely due to inhibition by endogenous IL-18BP.
  • FIGS. 10A-10B the addition of mAbs removed this inhibition, resulting in a dose-dependent increase in IFNy production.
  • human (10A) and cyno (10B) PBMC were used to test activity of the antibodies as well as cross-reactivity.
  • the epitope of mAb SA64a on IL-18BP was determined by cross-linking/high resolution mass spectrometry methodology developed by CovalX AG (Pimenova et al., 2008, J. Mass Spectrometry 43: 185).
  • human IL-18BP was allowed to bind to SA64a and crosslinked with a heterobifunctional linker.
  • the resulting complexes were digested with 5 different proteases (trypsin, chymotrypsin, ASP-N, elastase and thermolysin) and the resulting peptides, cross-linked or not, were analyzed by high-resolution mass spectrometry.
  • Residues on IL-18BP which interact with IL-18 were identified as: R61, Y69, S75, H79, T116, SI 19 and R131.
  • the sequence of mature IL-18BP is shown below with the residues interacting with SA64a and IL- 18 highlighted as indicated.
  • Residues found to interact with IL- 18 are highlighted in bold and underlined. Residues that form the epitope of SA64a are highlighted in bold in italic font. The residues R61, Y69 and R131 are recognized both by IL- 18 and SA64a and are shown in bold, italic and underlined.
  • CDR consensus sequences from the SAOla antibody were identified from the foregoing combinatorial library screening data (see FIG. 15).
  • the consensus sequence binding variants are summarized in Table E10 and Table Ell, which show their relative position within the CDRs and each amino acid variant that retained or improved binding.
  • mice were randomized into 6 separate groups (n 10) where the group mean tumor size was 36 mm 3 .
  • Treatments were administered every three days (q3d) by intraperitoneal (i.p.) injection for up to 7 total doses for animals that survived the length of the study. Animals were removed from the study for a variety of reasons, including reaching the permitted size limit or observation of moribund behavior as outlined by the study guidelines.
  • Treatments included PBS vehicle control, anti-mPD-1 (5 mg/kg), anti-mPD-1 (5 mg/kg) + SA0051d (10 mg/kg), anti- mPD-1 (5 mg/kg) + mIL-18 (0.32 mg/kg), mIL18 (0.32 mg/kg) + SA0051d (10 mg/kg), or anti- mPD-1 (5 mg/kg) + mIL-18 (0.32 mg/kg) + SA0051d (10 mg/kg).
  • Tumors were measured twice a week by caliper measurement and averaged to calculate mean tumor growth inhibition for each group.
  • results demonstrate improved efficacy with mouse-PD-1 + anti-mouse IL18BP (SA51d) + mIL-18 compared to anti-mouse PD-1 alone or anti-mouse PD-1 + mIL-18.
  • Anti-human IL-18BP downstream cell-based activity The ability of anti-human IL-18BP to allow IL-18 induced production of additional cytokines from human PBMC was tested.
  • Human PBMC were isolated and tested as described in Example 1.
  • the LEGENDplex Human Essential Immune Response Panel multiplex assay BioLegend cat# 740930 was used. Samples were diluted 1 : 50 in assay buffer and measured according to manufacturer's instructions with the exception that the volumes of all samples and reagents were halved and samples were mixed by pipetting at each step of the protocol, as well as shaking at 450 rpm, during each incubation.
  • Treatments included PBS vehicle control, anti-PD-1 (5 mg/kg, q2w x 3), SA0051d (10 mg/kg q2w), or anti-PD-1 (5 mg/kg q2w x 3) + SA51d (10 mg/kg q2w). Animals were inspected daily, and tumors were measured twice a week by caliper measurement. Tumor sizes were measured over time as shown for individual animals in FIGS. 21B-E.
  • results demonstrated that treatment with SA0051d improved survival of the animals compared to control (p ⁇ 0.0001), whereas treatment with anti-PD-1 alone did not. Treatment with the combination of SA0051d with anti-PD-1 further improved survival and resulted in tumor regression in some animals.
  • This example demonstrates that anti-IL-18BP has singleagent anti -tumor activity, which is compatible with treatment with anti-PD-1.

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Abstract

L'invention concerne des anticorps et des fragments de liaison à l'antigène de ceux-ci qui se lient à la protéine de liaison à l'interleukine 18 (IL-18BP) et des compositions associées, qui peuvent être utilisées dans l'une quelconque d'une variété de méthodes thérapeutiques ou diagnostiques, y compris le traitement ou le diagnostic de cancers et d'autres maladies.
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Family Cites Families (73)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
DE3883899T3 (de) 1987-03-18 1999-04-22 Sb2, Inc., Danville, Calif. Geänderte antikörper.
US5091513A (en) 1987-05-21 1992-02-25 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US5132405A (en) 1987-05-21 1992-07-21 Creative Biomolecules, Inc. Biosynthetic antibody binding sites
US6765087B1 (en) 1992-08-21 2004-07-20 Vrije Universiteit Brussel Immunoglobulins devoid of light chains
DK0672142T3 (da) 1992-12-04 2001-06-18 Medical Res Council Multivalente og multispecifikke bindingsproteiner samt fremstilling og anvendelse af disse
ES2162863T3 (es) 1993-04-29 2002-01-16 Unilever Nv Produccion de anticuerpos o fragmentos (funcionalizados) de los mismos derivados de inmunoglobulinas de cadena pesada de camelidae.
US5464758A (en) 1993-06-14 1995-11-07 Gossen; Manfred Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters
US5814618A (en) 1993-06-14 1998-09-29 Basf Aktiengesellschaft Methods for regulating gene expression
DE19742706B4 (de) 1997-09-26 2013-07-25 Pieris Proteolab Ag Lipocalinmuteine
AUPP221098A0 (en) 1998-03-06 1998-04-02 Diatech Pty Ltd V-like domain binding molecules
US6210922B1 (en) 1998-11-30 2001-04-03 National Research Council Of Canada Serum free production of recombinant proteins and adenoviral vectors
US7115396B2 (en) 1998-12-10 2006-10-03 Compound Therapeutics, Inc. Protein scaffolds for antibody mimics and other binding proteins
KR101077001B1 (ko) 1999-01-15 2011-10-26 제넨테크, 인크. 효과기 기능이 변화된 폴리펩티드 변이체
US6387620B1 (en) 1999-07-28 2002-05-14 Gilead Sciences, Inc. Transcription-free selex
AU2001289284A1 (en) 2000-04-04 2001-10-15 Enanta Pharmaceuticals, Inc. Methods for identifying peptide aptamers capable of altering a cell phenotype
US7029872B2 (en) 2000-06-28 2006-04-18 Glycofi, Inc Methods for producing modified glycoproteins
JP5291279B2 (ja) 2000-09-08 2013-09-18 ウニヴェルジテート・チューリッヒ 反復モジュールを含む反復タンパク質の集合体
US6376190B1 (en) 2000-09-22 2002-04-23 Somalogic, Inc. Modified SELEX processes without purified protein
FR2814642B1 (fr) 2000-10-03 2005-07-01 Ass Pour Le Dev De La Rech En Souris transgenique pour la recombinaison ciblee mediee par la cre-er modifiee
US6596541B2 (en) 2000-10-31 2003-07-22 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
DK1355919T3 (da) 2000-12-12 2011-03-14 Medimmune Llc Molekyler med længere halveringstider, sammensætninger og anvendelser deraf
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20050053973A1 (en) 2001-04-26 2005-03-10 Avidia Research Institute Novel proteins with targeted binding
US20050048512A1 (en) 2001-04-26 2005-03-03 Avidia Research Institute Combinatorial libraries of monomer domains
US20050089932A1 (en) 2001-04-26 2005-04-28 Avidia Research Institute Novel proteins with targeted binding
WO2002088171A2 (fr) 2001-04-26 2002-11-07 Avidia Research Institute Banques combinatoires de domaines monomeres
US20040175756A1 (en) 2001-04-26 2004-09-09 Avidia Research Institute Methods for using combinatorial libraries of monomer domains
US6900292B2 (en) 2001-08-17 2005-05-31 Lee-Hwei K. Sun Fc fusion proteins of human erythropoietin with increased biological activities
WO2003025020A1 (fr) 2001-09-13 2003-03-27 Institute For Antibodies Co., Ltd. Procede pour creer une banque d'anticorps de chameaux
ATE430580T1 (de) 2001-10-25 2009-05-15 Genentech Inc Glycoprotein-zusammensetzungen
US8093357B2 (en) 2002-03-01 2012-01-10 Xencor, Inc. Optimized Fc variants and methods for their generation
US7244592B2 (en) 2002-03-07 2007-07-17 Dyax Corp. Ligand screening and discovery
EP1498491A4 (fr) 2002-04-09 2006-12-13 Kyowa Hakko Kogyo Kk Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc gamma iiia
EP1537878B1 (fr) 2002-07-03 2010-09-22 Ono Pharmaceutical Co., Ltd. Compositions immunostimulantes
US20090017023A1 (en) 2002-08-14 2009-01-15 Macrogenics, Inc. FcGammaRIIB Specific Antibodies and Methods of Use Thereof
DK1534335T4 (en) 2002-08-14 2015-10-05 Macrogenics Inc FCGAMMARIIB-SPECIFIC ANTIBODIES AND PROCEDURES FOR USE THEREOF
US7217797B2 (en) 2002-10-15 2007-05-15 Pdl Biopharma, Inc. Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis
JP2007531707A (ja) 2003-10-15 2007-11-08 ピーディーエル バイオファーマ， インコーポレイテッド ＩＧの重鎖定常領域の位置２５０、３１４および／または４２８の変異誘発によるＦｃ融合タンパク質血清半減期の改変
BRPI0416262B1 (pt) 2003-11-05 2022-04-12 Roche Glycart Ag Anticorpo anti-cd20 humano tipo ii humanizado, seu método de produção, seus usos, bem como polinucleotídeo isolado, vetor de expressão e composição farmacêutica
WO2005063815A2 (fr) 2003-11-12 2005-07-14 Biogen Idec Ma Inc. Variants de polypeptides de liaison au recepteur fc$g(g) et procede apparentes
JP4762904B2 (ja) 2003-11-13 2011-08-31 ハンミ ホールディングス カンパニー リミテッド 免疫グロブリン不変領域の量産方法
US20080220049A1 (en) 2003-12-05 2008-09-11 Adnexus, A Bristol-Myers Squibb R&D Company Compositions and methods for intraocular delivery of fibronectin scaffold domain proteins
US7462697B2 (en) 2004-11-08 2008-12-09 Epitomics, Inc. Methods for antibody engineering
EP2325206B1 (fr) 2004-11-12 2014-03-19 Xencor, Inc. Variants de fc avec une liaison altérée à fcrn
US8367805B2 (en) 2004-11-12 2013-02-05 Xencor, Inc. Fc variants with altered binding to FcRn
WO2006079372A1 (fr) 2005-01-31 2006-08-03 Ablynx N.V. Procede de generation de sequences a domaine variable d'anticorps a chaine lourde
EP1888649A2 (fr) 2005-05-09 2008-02-20 GlycArt Biotechnology AG Molecules de liaison a l'antigene possedant des zones fc modifiees et une liaison alteree aux recepteurs fc
EP2418278A3 (fr) 2005-05-09 2012-07-04 Ono Pharmaceutical Co., Ltd. Anticorps monoclonaux humains pour mort programmée 1 (PD-1) et procédés de traitement du cancer à l'aide d'anticorps anti-PD-1 seuls ou combinés à d'autres formulations immunothérapeutiques
KR101607288B1 (ko) 2005-07-01 2016-04-05 이. 알. 스퀴부 앤드 선즈, 엘.엘.씨. 예정 사멸 리간드 1 (피디-엘1)에 대한 인간 모노클로날 항체
DK1931709T3 (en) 2005-10-03 2017-03-13 Xencor Inc FC VARIETIES WITH OPTIMIZED FC RECEPTOR BINDING PROPERTIES
PL1945665T3 (pl) 2005-10-21 2012-02-29 Genzyme Corp Leki oparte na przeciwciałach z ulepszoną aktywnością adcc
US20080206246A1 (en) 2006-04-05 2008-08-28 Ravetch Jeffrey V Polypeptides with enhanced anti-inflammatory and decreased cytotoxic properties and relating methods
US10155816B2 (en) 2005-11-28 2018-12-18 Genmab A/S Recombinant monovalent antibodies and methods for production thereof
EP2011870A4 (fr) 2006-04-14 2010-09-15 Medical & Biol Lab Co Ltd Polypeptide mutant doté d'une fonction d'effecteur
WO2008048545A2 (fr) 2006-10-16 2008-04-24 Medimmune, Llc. Molécules ayant des demi-vies réduites, compositions et leurs utilisations
WO2009097017A2 (fr) 2007-09-18 2009-08-06 The Jackson Laboratory Modifications d'anticorps et de protéines de fusion fc présentant une persistance accrue ou une stabilité pharmacocinétique in vivo et leurs procédés d'utilisation
CA2736816C (fr) 2008-09-12 2018-05-22 Isis Innovation Limited Anticorps specifiques de pd-1 et leurs utilisations
HUE030807T2 (en) 2008-09-26 2017-05-29 Dana Farber Cancer Inst Inc Human anti-PD-1, anti-PD-L1 and anti-PD-L2 antibodies and their applications
EP3192811A1 (fr) 2009-02-09 2017-07-19 Université d'Aix-Marseille Anticorps pd-1 et pd-l1 et leurs utilisations
CN102892786B (zh) 2010-03-11 2016-03-16 Ucb医药有限公司 Pd-1抗体
TW201134488A (en) 2010-03-11 2011-10-16 Ucb Pharma Sa PD-1 antibodies
US8822663B2 (en) 2010-08-06 2014-09-02 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
SI2791160T1 (sl) 2011-12-16 2022-07-29 Modernatx, Inc. Sestave modificirane MRNA
US10501512B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides
JOP20200308A1 (ar) * 2012-09-07 2017-06-16 Novartis Ag جزيئات إرتباط il-18
SMT202200337T1 (it) 2012-11-26 2022-09-14 Modernatx Inc Rna modificato al livello del terminale
RS63571B9 (sr) 2013-09-13 2023-02-28 Beigene Switzerland Gmbh Anti-pd1 antitela i njihova primena kao terapeutska i dijagnostička sredstva
US9209965B2 (en) 2014-01-14 2015-12-08 Microsemi Semiconductor Ulc Network interface with clock recovery module on line card
TWI681969B (zh) 2014-01-23 2020-01-11 美商再生元醫藥公司 針對ｐｄ-１的人類抗體
KR20200066623A (ko) * 2017-09-06 2020-06-10 예일 유니버시티 인터류킨-18 변이체 및 사용 방법
CN117279950B (zh) * 2022-01-28 2025-08-08 和径医药科技（上海）有限公司 一种靶向il-18bp的抗体及其应用
MX2024011278A (es) * 2022-03-15 2024-09-25 Compugen Ltd Anticuerpos antagonistas de il-18bp y su uso en monoterapia y terapia de combinacion en el tratamiento del cancer.

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