US20190048089A1 - Antagonistic Antibodies Specifically Binding Human CD40 and Methods of Use - Google Patents

Antagonistic Antibodies Specifically Binding Human CD40 and Methods of Use Download PDF

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US20190048089A1
US20190048089A1 US15/763,673 US201615763673A US2019048089A1 US 20190048089 A1 US20190048089 A1 US 20190048089A1 US 201615763673 A US201615763673 A US 201615763673A US 2019048089 A1 US2019048089 A1 US 2019048089A1
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antibody
nos
antigen
antibodies
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Holger Babbe
Nathan Felix
Johan Fransson
Paul Kim
Michael Scully
Hong Zhou
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Janssen Biotech Inc
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
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    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • 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
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
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    • 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
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to antagonistic antibodies specifically binding human CD40, polynucleotides encoding the antibodies or antigen-binding fragments thereof, and methods of making and using the foregoing.
  • the cell surface CD40 molecule is a member of the tumor necrosis factor receptor superfamily (TNFR) and a key regulator in both innate and adaptive immune responses.
  • CD40 is constitutively expressed on antigen presenting cells, in particular B-cells, dendritic cells and macrophages, but can also be found on fibroblasts, synoviocytes, smooth muscle cells, endothelial cells and epithelial cells.
  • CD40 The natural ligand of CD40, designated CD154 or CD40L, is mainly expressed on activated T lymphocytes and platelets.
  • the interaction of CD40 with CD40L on T cells induces both humoral and cell-mediated immune responses.
  • CD40 regulates this ligand-receptor pair to activate B cells and other antigen-presenting cells (APC) including dendritic cells (DCs), driving T cell activation.
  • APC antigen-presenting cells
  • DCs dendritic cells
  • activation of CD40 on B cells induces B cell proliferation, somatic hypermutation, differentiation into antibody secreting cells and isotype switching in germinal centers of secondary lymphoid organs.
  • cytokine production e.g.
  • IL-6 IL-6, IL-10, IL-12, TNF- ⁇
  • adhesion molecules and costimulatory receptors e.g. ICAM, CD23, CD80 and CD86
  • MHC class I MHC class II
  • TAP transporter B lymphocytes
  • Antibodies that modulate the CD40/CD40L interaction are of interest in treating diseases such as inflammatory diseases, including autoimmune diseases.
  • the invention provides for an isolated antagonistic antibody or an antigen binding portion thereof specifically binding human CD40 of SEQ ID NO: 1, comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 5, a HCDR2 of SEQ ID NO: 61, a HCDR3 of SEQ ID NO: 62, a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 63, a LCDR2 of SEQ ID NO: 9 and a LCDR3 of SEQ ID NO: 10.
  • HCDR heavy chain complementarity determining region
  • LCDR light chain complementarity determining region
  • the invention also provides for an isolated antagonistic antibody or an antigen binding portion thereof specifically binding human CD40 of SEQ ID NO: 1, comprising certain HCDR1, HCDR2, HCDR3, LCDR1, LCDR3, LCDR3, VH, VL, HC and/or LC sequences.
  • the invention also provides for a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the invention and a pharmaceutically acceptable carrier.
  • the invention also provides for an immunoconjugate comprising the antibody of the invention linked to a therapeutic agent or an imaging agent.
  • the invention also provides for an isolated polynucleotide encoding
  • the invention also provides for an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NOs: 13, 14, 28, 29, 30, 31, 32, 33, or 34.
  • the invention also provides for an isolated polynucleotide encoding the heavy chain of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.
  • the invention also provides for an isolated polynucleotide encoding the light chain of SEQ ID NOs: 77 or 78.
  • the invention also provides for an isolated polynucleotide encoding the heavy chain of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46 and a light chain of SEQ ID NOs: 77 or 78.
  • the invention also provides for an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 77 or 78.
  • the invention also provides for a vector comprising the polynucleotide of the invention.
  • the invention also provides for a host cell comprising the vector of the invention.
  • the invention also provides for a method of producing an antagonistic antibody or an antigen binding portion thereof specifically binding human CD40 of SEQ ID NO: 1, comprising culturing the host cell of the invention in conditions wherein the antibody is expressed, and isolating the antibody.
  • the invention also provides for a method of treating a subject having an inflammatory disease, comprising administering to the subject in need thereof the isolated antibody of the invention for a time sufficient to treat the inflammatory disease.
  • the invention also provides for the antibody of the invention for use in therapy.
  • the invention also provides for an anti-idiotypic antibody binding to the antibody of the invention.
  • the invention also provides for a kit comprising the antibody of the invention.
  • FIG. 1 shows that C40B16 as wild-type IgG1 demonstrated comparable minimal agonism when compared to Fc effector silent Antibody D.
  • Agonism was assessed in HEK-BlueTM CD40L NF- ⁇ B activation assay.
  • FIG. 2 shows that C40B16 does not induce agonism in an assay measuring antibody-mediated IL-12p40 production by human dendritic cells (DC), whereas ASKP-1240, CFZ533 and BMS-986090 induce IL-12p40 production.
  • IL-12p40 production was evaluated at 6 different antibody concentrations, (350, 110, 35, 11, 3.5, and 1.1 nM) each represented by a separate column for each antibody in the Figure.
  • DC+CD40L positive control.
  • DC only negative control.
  • FIG. 3A shows that 500 ng/ml concentrations of anti-CD40 antibodies C40B176, C40B179, C40B180 and C40B183 do not induce activation of dendritic cells (DC), whereas 350 nM ASKP-1240 induces IL-12p40 production.
  • DC activation was assessed by IL-12p40 production by DCs in the presence of antibody.
  • PP1B40 IgG1sigma isotype control
  • CNT09412 IgG4_PAA isotype control.
  • FIG. 3B shows that high concentrations of anti-CD40 antibodies C40B176, C40B179, C40B180 and C40B183 do not induce proliferation of B cells, whereas 500 nM ASKP-1240 does.
  • PP1B40 IgG1sigma isotype control
  • CNT09412 IgG4_PAA isotype control. Two separate dose response curves are shown for ASKP-1240.
  • Specific binding or “specifically binds” or “binds” refers to antibody binding to human CD40 with greater affinity than for non-related antigens.
  • the antibody binds to human CD40 with a dissociation constant (K D ) of 1 ⁇ 10 ⁇ 8 M or less, for example 1 ⁇ 10 ⁇ 9 M or less, 1 ⁇ 10 ⁇ 10 M or less, 1 ⁇ 10 ⁇ 11 M or less, or 1 ⁇ 10 ⁇ 12 M or less, typically with a K D that is at least one hundred fold less than its K D for binding to a non-related antigen (for example, BSA, casein).
  • K D dissociation constant
  • the dissociation constant may be measured using standard procedures.
  • Antibodies that specifically bind human CD40 may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (common marmoset, marmoset). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.
  • homologs such as human or monkey
  • Macaca fascicularis cynomolgus, cyno
  • Pan troglodytes chimpanzee, chimp
  • Callithrix jacchus common marmoset, marmoset
  • Antibodies is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen-binding fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity.
  • “Full length antibodies” are comprised of two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds as well as multimers thereof (for example IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CH1, hinge CH2 and CH3).
  • Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL).
  • VL light chain variable region
  • CL light chain constant region
  • the VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • CDR complementarity determining regions
  • CDRs are “antigen binding sites” in an antibody.
  • CDRs may be defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • “Hypervariable regions”, “HVR”, or “HV”, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk, Mol Biol 196:901-17, 1987).
  • the International ImMunoGeneTics (IMGT) database http://www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003.
  • CDR CDR
  • HCDR1 CDR1
  • HCDR2 CDR3
  • LCDR1 CDR2
  • LCDR3 CDR3
  • Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes IgA 1 , IgA 2 , IgG 1 , IgG 2 , IgG 3 and IgG 4 .
  • Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • Antigen-binding fragment refers to a portion of an immunoglobulin molecule that retains the antigen binding properties of the parental full length antibody.
  • Exemplary antigen-binding fragments are as heavy chain complementarity determining regions (HCDR) 1, 2 and/or 3, light chain complementarity determining regions (LCDR) 1, 2 and/or 3, a heavy chain variable region (VH), or a light chain variable region (VL), Fab, F(ab′)2, Fd and Fv fragments as well as domain antibodies (dAb) consisting of either one VH domain or one VL domain.
  • VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs in which the VH/VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate chains, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Pat. Publ. No. WO1998/44001, Int. Pat. Publ. No. WO1988/01649; Int. Pat. Publ. No. WO1994/13804; Int. Pat. Publ. No. WO1992/01047.
  • scFv single chain Fv
  • “Monoclonal antibody” refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well known alterations such as removal of C-terminal lysine from the antibody heavy chain.
  • Monoclonal antibodies typically bind one antigenic epitope, except that multispecific monoclonal antibodies bind two or more distinct antigens or epitopes.
  • Bispecific monoclonal antibodies bind two distinct antigenic epitopes.
  • Monoclonal antibodies may have heterogeneous glycosylation within the antibody population.
  • Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A multispecific antibody, such as a bispecific antibody or a trispecific antibody is included in the term monoclonal antibody.
  • isolated antibody refers to an antibody or an antigen-binding fragment thereof that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding human CD40 is substantially free of antibodies that specifically bind antigens other than human CD40).
  • an isolated antibody specifically binding human CD40 is substantially free of antibodies that specifically bind antigens other than human CD40.
  • the bispecific antibody specifically binds two antigens of interest, and is substantially free of antibodies that specifically bind antigens other that the two antigens of interest.
  • isolated antibody encompasses antibodies that are isolated to a higher purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.
  • Humanized antibodies refers to antibodies in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include intentionally introduced mutations in the framework regions so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.
  • Human antibodies refers to antibodies having heavy and light chain variable regions in which both the framework and the antigen binding site are derived from sequences of human origin. If the antibody contains a constant region or a portion of the constant region, the constant region also is derived from sequences of human origin.
  • a human antibody comprises heavy or light chain variable regions that are derived from sequences of human origin if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes.
  • Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci as described herein.
  • a human antibody typically contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to, for example naturally occurring somatic mutations, intentional introduction of substitutions into the framework or antigen binding site and amino acid changes introduced during cloning and VDJ recombination in non-human animals.
  • a human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.
  • a human antibody may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., J Mol Biol 296:57-86, 2000, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al., J Mol Biol 397:385-96, 2010 and Int. Pat. Publ. No. WO2009/085462.
  • Antibodies in which antigen binding sites are derived from a non-human species are not included in the definition of human antibody.
  • Recombinant includes antibodies and other proteins that are prepared, expressed, created or isolated by recombinant means.
  • Epitope refers to a portion of an antigen to which an antibody specifically binds.
  • Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope may be composed of contiguous and/or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule.
  • Multispecific refers to an antibody that specifically binds at least two distinct antigens or two distinct epitopes within the antigens, for example three, four or five distinct antigens or epitopes.
  • Bispecific refers to an antibody that specifically binds two distinct antigens or two distinct epitopes within the same antigen.
  • the bispecific antibody may have cross-reactivity to other related antigens or can bind an epitope that is shared between two or more distinct antigens.
  • Variant refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions or deletions.
  • Vector refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems.
  • Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system.
  • Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector.
  • the polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.
  • “Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.
  • Polynucleotide refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry.
  • cDNA is a typical example of a polynucleotide.
  • Polypeptide or “protein” refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as “peptides”.
  • CD40 or “huCD40” refers to the human CD40 protein.
  • CD40 is also known as Tumor necrosis factor receptor superfamily member 5 (TNFRSF5), CD40L receptor or CD154 receptor.
  • TNFRSF5 Tumor necrosis factor receptor superfamily member 5
  • CD40L receptor CD154 receptor
  • the amino acid sequence of the full length human CD40 is shown in SEQ ID NO: 1.
  • Human full length CD40 protein is a type I membrane protein with 277 amino acids. The signal sequence spans residues 1-20, the extracellular domain spans residues residues 21-193, the transmembrane domain spans residues 194-215, and the cytoplasmic domain spans residues 216-277 of SEQ ID NO: 1.
  • CD40-ECD refers to the CD40 fragment of residues 21-193 of SEQ ID NO: 1.
  • “Antagonist” or “antagonistic” refers to an antibody that specifically binds human CD40 and inhibits CD40 biological activity in the presence of CD40L in cellular assays such as CD40L-driven human B cell proliferation or CD40L-driven IL-12p40 production by human dendritic cells.
  • the antagonist may inhibit CD40 biological activity in a statistically significant manner when compared to a control sample without the antibody.
  • the antagonistic antibody specifically binding human CD40 may inhibit CD40 biological activity with an IC 50 value of about 1 nM or less.
  • CD40L in the assays may be provided as a soluble form or membrane-bound (e.g. as cells expressing CD40L, such as Jurkat cells).
  • CD40 biological activity refers to a measurable event in a cell occurring as a result of binding of CD40L to CD40 on human cells.
  • CD40 biological activity may be for example proliferation of human B cells or production of IL-12p40 by human dendritic cells, or downstream activation of CD40 signaling pathways.
  • CD40 biological activity may be measured using known methods and methods described herein.
  • “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result or embodiment, “about” means within one standard deviation per practice in the art, or a range of up to 5%, whichever is larger.
  • “In combination with” means that two or more therapeutics can be administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order.
  • Cross-linking refers higher order multimerization of CD40 on cells induced by an antibody specifically binding human CD40 binding to Fc ⁇ RIIb cis or trans, resulting in induction of CD40 agonistic activity.
  • the invention provides antagonistic antibodies specifically binding human CD40, polynucleotides encoding the antibodies, vectors, host cells and methods of using the antibodies.
  • the antibodies of the invention are potent inhibitors of CD40 and have minimal agonistic activity. It is documented that antagonistic anti-CD40 antibodies, albeit being antagonists, can also have agonistic activity as a result of Fc-dependent cross-linking (for example see U.S. Pat. No. 7,537,763) and therefore pose a potential safety risk when administered to subjects in which suppression of CD40 signaling is desired, such as patients with autoimmune disease.
  • Preferable anti-CD40 antibodies for the treatment of conditions in which inhibition of CD40 biological function is desired are hence those that lack agonistic activity, or have minimal agonistic activity.
  • Suitable therapeutic CD40 antibodies would thus be Fc-engineered to abolish Fc ⁇ R binding, and as a result lack Fc-mediated cross-linking and potential for Fc-mediated agonism.
  • effector silent Fc-engineered antibodies are for example ASKP-1240, CFZ533, BMS-986090 and Antibody D (“benchmark antibodies”).
  • ASKP-1240 and CFZ533 are currently in clinical development for inflammatory or autoimmune diseases.
  • the antibodies of the invention demonstrate improved properties when compared to the benchmark antibodies.
  • the antibody C40B16 of the invention has minimal agonistic activity and activates CD40 signaling to a lesser extend when compared to the benchmark antibodies ASKP-1240, CFZ533 and BMS-986090.
  • ASKP-1240, CFZ533 and BMS-986090 are Fc effector silent antibodies, the observed agonism of these antibodies may be epitope-dependent.
  • C40B16 on the contrary is a wild-type IgG1 and therefore possesses neither epitope-dependent nor Fc-dependent agonism.
  • Fc engineered effector silent antibodies C40B176, C40B179, C40B180 and C40B183 of the invention demonstrate only minimal agonism but also up to 10-fold improved potency when compared to Antibody D.
  • the invention provides for an isolated antagonistic antibody or an antigen-binding fragment thereof specifically binding human CD40 of SEQ ID NO: 1, comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 5, a HCDR2 of SEQ ID NO: 61, a HCDR3 of SEQ ID NO: 62, a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 63, a LCDR2 of SEQ ID NO: 9 and a LCDR3 of SEQ ID NO: 10.
  • HCDR heavy chain complementarity determining region
  • LCDR light chain complementarity determining region
  • SEQ ID NOs: 61, 62 and 63 represent the HCDR2, the HCDR3 and the LCDR1, genus sequences of antagonistic antibodies specifically binding CD40, the genera encompassing variants of a parental antibody C40B16 in which putative sites for post-translational modifications have been mutated.
  • the antibodies within the genus are expected to display no shift in epitope, e.g. the antibodies comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 5, 61, 62, 63, 9 and 10 are expected to have similar characteristics when compared to the parental C40B16 antibody.
  • Exemplary such antibodies are antibodies comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 or the VH and the VL amino acid sequences of antibodies C40M141, C40M152, C40M142, C40M153, C40M144, C40M155, C40M148, C40M194, C40M198, C40M197, C40M201 or C40M126 as shown in Table 2 and Table 7.
  • X 1 is N, D or Q
  • X 2 is N, Q or A
  • X 3 is S or A.
  • X 1 is M or L
  • X 1 is C or A.
  • the antibody competes for binding to human CD40 of SEQ ID NO: 1 with an antibody comprising
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody binds to the same epitope on human CD40 of SEQ ID NO: 1 to which the antibody comprising
  • VH heavy chain variable region
  • VL light chain variable region
  • VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 binds to.
  • the antibody binds human CD40 with a dissociation constant (K D ) of about 1.5 ⁇ 10 ⁇ 10 M or less, when the K D is measured using ProteOn XPR36 system at 25° C. in Dulbecco's phosphate buffered saline containing 0.01% polysorbate 20 (PS-20) and 100 ⁇ g/ml bovine serum albumin.
  • K D dissociation constant
  • the antibody inhibits soluble human CD40L-driven human tonsillar B cell proliferation with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M.
  • the antibody inhibits soluble human CD40L-driven production of IL-12p40 by human dendritic cells with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M.
  • the antibody binds human CD40 with a dissociation constant (K D ) of about 1.5 ⁇ 10 ⁇ 10 M or less, when the K D is measured using ProteOn XPR36 system at 25° C. in Dulbecco's phosphate buffered saline containing 0.01% PS-20 and 100 ⁇ g/ml bovine serum albumin, inhibits soluble human CD40L-driven human tonsillar B cell proliferation with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M and inhibits soluble human CD40L-driven production of IL-12p40 by human dendritic cells with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M.
  • K D dissociation constant
  • the antibody is an IgG1, IgG2, IgG3 or IgG4 isotype.
  • the antibody is an IgG1 isotype.
  • the antibody is an IgG2 isotype.
  • the antibody is an IgG3 isotype.
  • the antibody is an IgG4 isotype.
  • the antibody is an IgG1 isotype, optionally comprising a L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody is an IgG1/ ⁇ isotype, optionally comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody is an IgG1/ ⁇ isotype comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody is an IgG4 isotype, optionally comprising a S228P mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4/ ⁇ isotype, optionally comprising the S228P mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4/ ⁇ isotype comprising the S228P mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4 isotype, optionally comprising a S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4/ ⁇ isotype, optionally comprising the S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4/ ⁇ isotype comprising the S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody is a multispecific antibody, such as a bispecific antibody.
  • the antibody is suitable for use in therapy, for example in treating an inflammatory disease.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune disease.
  • the antibody is suitable for use in therapy, for example in treating Addinson's disease.
  • the antibody is suitable for use in therapy, for example in treating an ankylosing spondylitis.
  • the antibody is suitable for use in therapy, for example in treating an atherosclerosis.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune hepatitis.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune diabetes.
  • the antibody is suitable for use in therapy, for example in treating Graves' disease.
  • the antibody is suitable for use in therapy, for example in treating Buillain-Barre syndrome.
  • the antibody is suitable for use in therapy, for example in treating Hashimoto's disease.
  • the antibody is suitable for use in therapy, for example in treating, an idiopathic thrombocytopenia.
  • the antibody is suitable for use in therapy, for example in treating an inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the antibody is suitable for use in therapy, for example in treating a systemic lupus erythematosus.
  • the antibody is suitable for use in therapy, for example in treating a multiple sclerosis.
  • the antibody is suitable for use in therapy, for example in treating a myasthenia gravis.
  • the antibody is suitable for use in therapy, for example in treating a psoriasis.
  • the antibody is suitable for use in therapy, for example in treating an arthritis.
  • the antibody is suitable for use in therapy, for example in treating a scleroderma.
  • the antibody is suitable for use in therapy, for example in treating Sjogren's syndrome.
  • the antibody is suitable for use in therapy, for example in treating a systemic sclerosis.
  • the antibody is suitable for use in therapy, for example in treating a transplantation.
  • the antibody is suitable for use in therapy, for example in treating a kidney transplantation.
  • the antibody is suitable for use in therapy, for example in treating a skin transplantation.
  • the antibody is suitable for use in therapy, for example in treating a bone marrow transplantation.
  • the antibody is suitable for use in therapy, for example in treating a graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • the antibody is suitable for use in therapy, for example in treating a type I diabetes.
  • the antibody is suitable for use in therapy, for example in treating a rheumatoid arthritis.
  • the antibody is suitable for use in therapy, for example in treating a juvenile arthritis.
  • the antibody is suitable for use in therapy, for example in treating a psoriatic arthritis.
  • the antibody is suitable for use in therapy, for example in treating Reiter's syndrome.
  • the antibody is suitable for use in therapy, for example in treating a gouty arthritis.
  • the antibody is suitable for use in therapy, for example in treating Crohn's disease.
  • the antibody is suitable for use in therapy, for example in treating an ulcerative colitis.
  • the antibody is suitable for use in therapy, for example in treating an inflammatory disease in combination with a second therapeutic agent.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune disease in combination with a second therapeutic agent.
  • the antibody is suitable for use in therapy, for example in treating a rheumatoid arthritis in combination with a second therapeutic agent.
  • the antibody is suitable for use in therapy, for example in treating a systemic lupus erythematosus in combination with a second therapeutic agent.
  • the invention also provides for an isolated antagonistic antibody or an antigen-binding fragment thereof specifically binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 5, 6, 7, 8, 9 and 10, respectively.
  • the antibody comprises the VH and the VL of SEQ ID NOs: 11 and 12, respectively.
  • the VH is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 13 and the VL is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 14.
  • the antibody comprises a heavy chain of SEQ ID NO: 35 and a light chain of SEQ ID NO: 47.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 65 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 77.
  • the invention also provides for an isolated antagonistic antibody or an antigen-binding fragment thereof specifically binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 5, 15, 7, 20, 9 and 10, respectively.
  • the antibody comprises the VH and the VL of SEQ ID NOs: 21 and 27, respectively.
  • the VH is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 28 and the VL is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 34.
  • the antibody comprises the heavy chain of SEQ ID NO: 36 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 66 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the antibody comprises the heavy chain of SEQ ID NO: 37 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 67 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the invention also provides for an isolated antagonistic antibody or an antigen-binding fragment thereof specifically binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 5, 16, 7, 20, 9 and 10, respectively.
  • the antibody comprises the VH and the VL of SEQ ID NOs: 22 and 27, respectively.
  • the VH is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 29 and the VL is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 34.
  • the antibody comprises the heavy chain of SEQ ID NO: 38 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 68 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the antibody comprises the heavy chain of SEQ ID NO: 39 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 69 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the invention also provides for an isolated antagonistic antibody or an antigen-binding fragment thereof specifically binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 5, 17, 7, 20, 9 and 10, respectively.
  • the antibody comprises the VH and the VL of SEQ ID NOs: 23 and 27, respectively.
  • the VH is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 30 and the VL is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 34.
  • the antibody comprises the heavy chain of SEQ ID NO: 40 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 70 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the antibody comprises the heavy chain of SEQ ID NO: 41 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 71 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the invention also provides for an isolated antagonistic antibody or an antigen-binding fragment thereof specifically binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 5, 18, 7, 20, 9 and 10, respectively.
  • the antibody comprises the VH and the VL of SEQ ID NOs: 24 and 27, respectively.
  • the VH is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 31 and the VL is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 34.
  • the antibody comprises the heavy chain of SEQ ID NO: 42 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 72 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the invention also provides for an isolated antagonistic or an antigen-binding fragment thereof specifically binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 5, 18, 19, 20, 9 and 10, respectively.
  • the antibody competes for binding to human CD40 of SEQ ID NO: 1 with an antibody comprising
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody binds to the same epitope on human CD40 of SEQ ID NO: 1 to which the antibody comprising
  • VH heavy chain variable region
  • VL light chain variable region
  • VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 binds to.
  • the antibody binds human CD40 with a dissociation constant (K D ) of about 1.5 ⁇ 10 ⁇ 10 M or less, when the K D is measured using ProteOn XPR36 system at 25° C. in Dulbecco's phosphate buffered saline containing 0.01% PS-20 and 100 ⁇ g/ml bovine serum albumin.
  • K D dissociation constant
  • the antibody inhibits soluble human CD40L-driven production of IL-12p40 by human dendritic cells with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M.
  • the antibody binds human CD40 with a dissociation constant (K D ) of about 1.5 ⁇ 10 ⁇ 10 M or less, when the K D is measured using ProteOn XPR36 system at 25° C. in Dulbecco's phosphate buffered saline containing 0.01% PS-20 and 100 ⁇ g/ml bovine serum albumin, inhibits soluble human CD40L-driven human tonsillar B cell proliferation with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M and inhibits soluble human CD40L-driven production of IL-12p40 by human dendritic cells with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M.
  • K D dissociation constant
  • the antibody comprises the VH and the VL of SEQ ID NOs: 25 and 27, respectively.
  • the VH is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 32 and the VL is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 34.
  • the antibody is an IgG1, IgG2, IgG3 or IgG4 isotype.
  • the antibody is an IgG1 isotype.
  • the antibody is an IgG2 isotype.
  • the antibody is an IgG3 isotype.
  • the antibody is an IgG4 isotype.
  • the antibody is an IgG1 isotype, optionally comprising a L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody is an IgG1/ ⁇ isotype, optionally comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody comprises the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 and is an IgG1/ ⁇ isotype, optionally comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody comprises the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 and is an IgG1/ ⁇ isotype comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody is an IgG4 isotype, optionally comprising a S228P mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4/ ⁇ isotype, optionally comprising the S228P mutation when compared to the wild-type IgG4.
  • the antibody comprises the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 and is an IgG4/ ⁇ isotype, optionally comprising the S228P mutation when compared to the wild-type IgG4.
  • the antibody comprises the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 and is an IgG4/ ⁇ isotype comprising the S228P mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4 isotype, optionally comprising a S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4/ ⁇ isotype, optionally comprising the S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody comprises the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 and is an IgG4/ ⁇ isotype, optionally comprising the S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody comprises the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 and is an IgG4/ ⁇ isotype comprising the S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody comprises the heavy chain of SEQ ID NO: 43 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 73 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the antibody comprises the heavy chain of SEQ ID NO: 44 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 74 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the antibody is a multispecific antibody, such as a bispecific antibody.
  • the antibody is suitable for use in therapy, for example in treating an inflammatory disease.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune disease.
  • the antibody is suitable for use in therapy, for example in treating Addinson's disease.
  • the antibody is suitable for use in therapy, for example in treating an ankylosing spondylitis.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune hepatitis.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune diabetes.
  • the antibody is suitable for use in therapy, for example in treating Graves' disease.
  • the antibody is suitable for use in therapy, for example in treating Buillain-Barre syndrome.
  • the antibody is suitable for use in therapy, for example in treating Hashimoto's disease.
  • the antibody is suitable for use in therapy, for example in treating, an idiopathic thrombocytopenia.
  • the antibody is suitable for use in therapy, for example in treating an inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the antibody is suitable for use in therapy, for example in treating a systemic lupus erythematosus.
  • the antibody is suitable for use in therapy, for example in treating a multiple sclerosis.
  • the antibody is suitable for use in therapy, for example in treating a myasthenia gravis.
  • the antibody is suitable for use in therapy, for example in treating a psoriasis.
  • the antibody is suitable for use in therapy, for example in treating an arthritis.
  • the antibody is suitable for use in therapy, for example in treating a scleroderma.
  • the antibody is suitable for use in therapy, for example in treating Sjogren's syndrome.
  • the antibody is suitable for use in therapy, for example in treating a systemic sclerosis.
  • the antibody is suitable for use in therapy, for example in treating a transplantation.
  • the antibody is suitable for use in therapy, for example in treating a kidney transplantation.
  • the antibody is suitable for use in therapy, for example in treating a skin transplantation.
  • the antibody is suitable for use in therapy, for example in treating a bone marrow transplantation.
  • the antibody is suitable for use in therapy, for example in treating a graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • the antibody is suitable for use in therapy, for example in treating a type I diabetes.
  • the antibody is suitable for use in therapy, for example in treating a rheumatoid arthritis.
  • the antibody is suitable for use in therapy, for example in treating a juvenile arthritis.
  • the antibody is suitable for use in therapy, for example in treating a psoriatic arthritis.
  • the antibody is suitable for use in therapy, for example in treating Reiter's syndrome.
  • the antibody is suitable for use in therapy, for example in treating a gouty arthritis.
  • the antibody is suitable for use in therapy, for example in treating Crohn's disease.
  • the antibody is suitable for use in therapy, for example in treating an ulcerative colitis.
  • the antibody is suitable for use in therapy, for example in treating an inflammatory disease, in combination with a second therapeutic agent.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune disease, in combination with a second therapeutic agent.
  • the antibody is suitable for use in therapy, for example in treating a rheumatoid arthritis in combination with a second therapeutic agent.
  • the antibody is suitable for use in therapy, for example in treating a systemic lupus erythematosus in combination with a second therapeutic agent.
  • the invention also provides for an isolated antagonistic antibody or an antigen-binding fragment thereof specifically binding human CD40 of SEQ ID NO: 1, comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 5, 17, 19, 20, 9 and 10, respectively.
  • the antibody competes for binding to human CD40 of SEQ ID NO: 1 with an antibody comprising
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody binds to the same epitope on human CD40 of SEQ ID NO: 1 to which the antibody comprising
  • VH heavy chain variable region
  • VL light chain variable region
  • VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 binds to.
  • the antibody binds human CD40 with a dissociation constant (K D ) of about 1.5 ⁇ 10 ⁇ 10 M or less, when the K D is measured using ProteOn XPR36 system at 25° C. in Dulbecco's phosphate buffered saline containing 0.01% PS-20 and 100 ⁇ g/ml bovine serum albumin.
  • K D dissociation constant
  • the antibody inhibits soluble human CD40L-driven human tonsillar B cell proliferation with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M.
  • the antibody inhibits soluble human CD40L-driven production of IL-12p40 by human dendritic cells with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M.
  • the antibody binds human CD40 with a dissociation constant (K D ) of about 1.5 ⁇ 10 ⁇ 10 M or less, when the K D is measured using ProteOn XPR36 system at 25° C. in Dulbecco's phosphate buffered saline containing 0.01% PS-20 and 100 ⁇ g/ml bovine serum albumin, inhibits soluble human CD40L-driven human tonsillar B cell proliferation with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M and inhibits soluble human CD40L-driven production of IL-12p40 by human dendritic cells with an IC 50 value of less than about 1 ⁇ 10 ⁇ 9 M.
  • K D dissociation constant
  • the antibody comprises the VH and the VL of SEQ ID NOs: 26 and 27, respectively.
  • the VH is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 33 and the VL is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 34.
  • the antibody is an IgG1, IgG2, IgG3 or IgG4 isotype.
  • the antibody is an IgG1 isotype.
  • the antibody is an IgG2 isotype.
  • the antibody is an IgG3 isotype.
  • the antibody is an IgG4 isotype.
  • the antibody is an IgG1 isotype, optionally comprising a L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody is an IgG1/ ⁇ isotype, optionally comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 and is an IgG1/ ⁇ isotype, optionally comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 and is an IgG1/ ⁇ isotype comprising the L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation when compared to the wild-type IgG1.
  • the antibody is an IgG4 isotype, optionally comprising a S228P mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4/ ⁇ isotype, optionally comprising the S228P mutation when compared to the wild-type IgG4.
  • the antibody comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 and is an IgG4/ ⁇ isotype, optionally comprising the S228P mutation when compared to the wild-type IgG4.
  • the antibody comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 and is an IgG4/ ⁇ isotype comprising the S228P mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4 isotype, optionally comprising a S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody is an IgG4/ ⁇ isotype, optionally comprising the S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 and is an IgG4/ ⁇ isotype, optionally comprising the S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 and is an IgG4/ ⁇ isotype comprising the S228P/F234A/L235A mutation when compared to the wild-type IgG4.
  • the antibody comprises the heavy chain of SEQ ID NO: 45 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 75 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the antibody comprises the heavy chain of SEQ ID NO: 46 and the light chain of SEQ ID NO: 48.
  • the heavy chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 76 and the light chain is encoded by a polynucleotide comprising a polynucleotide sequence of SEQ ID NO: 78.
  • the antibody is a multispecific antibody, such as a bispecific antibody.
  • the antibody is suitable for use in therapy, for example in treating an inflammatory disease.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune disease.
  • the antibody is suitable for use in therapy, for example in treating Addinson's disease.
  • the antibody is suitable for use in therapy, for example in treating an ankylosing spondylitis.
  • the antibody is suitable for use in therapy, for example in treating an atherosclerosis.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune hepatitis.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune diabetes.
  • the antibody is suitable for use in therapy, for example in treating Graves' disease.
  • the antibody is suitable for use in therapy, for example in treating Buillain-Barre syndrome.
  • the antibody is suitable for use in therapy, for example in treating Hashimoto's disease.
  • the antibody is suitable for use in therapy, for example in treating, an idiopathic thrombocytopenia.
  • the antibody is suitable for use in therapy, for example in treating an inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the antibody is suitable for use in therapy, for example in treating a systemic lupus erythematosus.
  • the antibody is suitable for use in therapy, for example in treating a multiple sclerosis.
  • the antibody is suitable for use in therapy, for example in treating a myasthenia gravis.
  • the antibody is suitable for use in therapy, for example in treating a psoriasis.
  • the antibody is suitable for use in therapy, for example in treating an arthritis.
  • the antibody is suitable for use in therapy, for example in treating a scleroderma.
  • the antibody is suitable for use in therapy, for example in treating Sjogren's syndrome.
  • the antibody is suitable for use in therapy, for example in treating a systemic sclerosis.
  • the antibody is suitable for use in therapy, for example in treating a transplantation.
  • the antibody is suitable for use in therapy, for example in treating a kidney transplantation.
  • the antibody is suitable for use in therapy, for example in treating a skin transplantation.
  • the antibody is suitable for use in therapy, for example in treating a bone marrow transplantation.
  • the antibody is suitable for use in therapy, for example in treating a graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • the antibody is suitable for use in therapy, for example in treating a type I diabetes.
  • the antibody is suitable for use in therapy, for example in treating a rheumatoid arthritis.
  • the antibody is suitable for use in therapy, for example in treating a juvenile arthritis.
  • the antibody is suitable for use in therapy, for example in treating a psoriatic arthritis.
  • the antibody is suitable for use in therapy, for example in treating Reiter's syndrome.
  • the antibody is suitable for use in therapy, for example in treating a gouty arthritis.
  • the antibody is suitable for use in therapy, for example in treating Crohn's disease.
  • the antibody is suitable for use in therapy, for example in treating an ulcerative colitis.
  • the antibody is suitable for use in therapy, for example in treating an inflammatory disease in combination with a second therapeutic agent.
  • the antibody is suitable for use in therapy, for example in treating an autoimmune disease in combination with a second therapeutic agent.
  • the antibody is suitable for use in therapy, for example in treating a rheumatoid arthritis in combination with a second therapeutic agent.
  • the antibody is suitable for use in therapy, for example in treating a systemic lupus erythematosus in combination with a second therapeutic agent.
  • CD40-ECD-his His-tagged recombinant soluble human CD40
  • 5 ⁇ L of anti-his mAb (10 ⁇ g/mL, R&D Systems, MAB050) is directly coated on MSD HighBind plates for 2 hours at room temperature and then blocked with 5% MSD Blocker A buffer for an additional 2 hours at room temperature.
  • 25 ⁇ L of 10 ⁇ g/mL CD40-ECD-his protein is added to be captured by anti-his mAb.
  • MSD Read Buffer T is diluted with distilled water (4-fold) and dispensed into each well then analyzed with a SECTOR Imager 6000 (Meso Scale Discovery, Gaithersburg, Md.).
  • the test antibody competes for binding to human CD40 with a reference antibody (e.g. an antibody comprising the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, the antibody comprising the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 or the antibody comprising the VH of SEQ ID NO: 26 and the VL of SEQ DI NO: 27) when the test antibody reduces the MDS signal obtained in the above assay using the Ru-labeled reference antibody or a Fab portion thereof by more than 90%.
  • a reference antibody e.g. an antibody comprising the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, the antibody comprising the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 or
  • Antibodies that compete for binding to CD40 with an antibody comprising the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27 or the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27 may be generated by isolating antibodies specifically binding human CD40 using phage display libraries, and screening the generated antibodies for their ability to compete for binding to CD40 with the aforementioned antibodies.
  • the CD40 epitope the antibody of the invention binds to may be resolved for example using hydrogen/deuterium exchange (H/D exchange) or by analyzing a crystal structure of the antibody in complex with CD40.
  • H/D exchange hydrogen/deuterium exchange
  • the epitope residues are those CD40 residues that reside within 4 ⁇ distance or less from any of the antibody CDR residues.
  • CD40 protein is incubated in the presence or absence of the antibody in deuterated water for predetermined times resulting in deuterium incorporation at exchangeable hydrogen atoms which are unprotected by the antibody, followed by protease digestion of the protein and analyses of the peptide fragments using LC-MS.
  • 5 ⁇ L of the test antibody (10 ⁇ g) or 5 ⁇ L of the complex of CD40 and the test antibody (10 & 7.35 ⁇ g, respectively) is incubated with 120 ⁇ L deuterium oxide labeling buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) for 0 sec, 60 sec, 300 sec, 1800 sec, 7200 sec, and 14400 sec.
  • Deuterium exchange is quenched by adding 63 ⁇ L of 5 M guanidine hydrochloride and final pH is 2.5.
  • the quenched sample is subjected to on-column pepsin/protease type XIII digestion and LC-MS analysis.
  • pepsin/protease type XIII digestion 5 ⁇ g of the samples in 125 ⁇ L control buffer (50 mM phosphate, 100 mM sodium chloride at pH 7.4) are denatured by adding 63 ⁇ L of 5 M guanidine hydrochloride (final pH is 2.5) and incubating the mixture for 3 min.
  • the mixture is subjected to on-column pepsin/protease type XIII digestion and the resultant peptides analyzed using an UPLC-MS system comprised of a Waters Acquity UPLC coupled to a Q ExactiveTM Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo).
  • Raw MS data is processed using HDX WorkBench, software for the analysis of H/D exchange MS data.
  • the deuterium levels are calculated using the average mass difference between the deuteriated peptide and its native form (t 0 ).
  • Peptide identification is done through searching MS/MS data against the CD40 sequence with Mascot.
  • the mass tolerance for the precursor and product ions is 20 ppm and 0.05 Da, respectively.
  • CD40 and the test antibody are expressed and purified using standard protocols.
  • the CD40/test antibody complex is incubated overnight at 4° C., concentrated, and separated from the uncomplexed species using size-exclusion chromatography.
  • the complex is crystallized by the vapor-diffusion method from various known test solutions for example solutions containing PEG3350, ammonium citrate and 2-(N-morpholino)ethanesulfonic acid (MES).
  • MES 2-(N-morpholino)ethanesulfonic acid
  • Antibodies binding the same epitope on CD40 as a reference antibody may be generated by isolating antibodies binding CD40 using phage display libraries, selecting those antibodies that compete with the reference antibody for binding to CD40 by 100%, and identifying the antibody epitope by H/D exchange or by X-ray crystallography.
  • mice or rabbits may be immunized using peptides encompassing the epitope residues, and the generated antibodies may be evaluated for their binding within the recited region.
  • the affinity of an antibody to human or cyno CD40 may be determined experimentally using any suitable method.
  • An exemplary method utilizes ProteOn XPR36, Biacore 3000 or KinExA instrumentation, ELISA or competitive binding assays known to those skilled in the art.
  • the measured affinity of a particular antibody to CD40 may vary if measured under different conditions (e.g., osmolarity, pH).
  • affinity and other binding parameters e.g., K D , K on , and Koff
  • K D , K on , and Koff are typically made with standardized conditions and a standardized buffer, such as the buffer described herein.
  • 1 ⁇ 10 5 human tonsil B cells may be cultured in RPMI medium containing glutamax, 10% FBS and 1% Pen/Strep, titrations of each of the antibodies specifically binding human CD40 may be added to the cells, followed by addition of 0.5 ⁇ g/ml soluble human CD154.
  • Cells may be cultured for 48 hours at 37° C., pulsed with 3H-thymidine (1 ⁇ Ci/well) in 50 ⁇ l medium and cultured for 16-18 hours before harvest and counting.
  • human DCs may be generated by culturing purified human monocytes (2.5 ⁇ 10 6 /well 6-well plates) for 6 days in 3 ml RPMI medium containing glutamax, 25 mM HEPES, 10% FBS, 1% Pen/Strep and 50 ng each GM-CSF and IL-4. On day 3, 1 ml of the medium may be removed and replaced with 2 ml fresh medium containing 50 ng/ml each of GM-CSF and IL-4.
  • DCs may be plated into 96-well plates (100,000 cells/well) followed by titrations of each of the antibodies specifically binding human CD40 and addition of 1 ⁇ g/ml of soluble human CD154 to the cultures.
  • Cells may be cultured for 48 hours before collecting and analyzing supernatants for IL-12p40 by MSD.
  • Immune effector properties of the antibodies of the invention may be enhanced or silenced through Fc modifications.
  • Fc effector functions such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. may be provided and/or controlled by modifying residues in the Fc responsible for these activities.
  • the antibody of the invention has reduced binding to Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa or Fc ⁇ RIIIb.
  • “Reduced binding” refers to reduced binding of the antibody of the invention having at least one mutation in the Fc region to an Fc ⁇ receptor (Fc ⁇ R) when compared to the binding of the parental antibody without the mutation to the same Fc ⁇ R. “Reduced binding” may be at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 20,000-fold reduced binding. In practice, antibodies exhibiting “reduced binding” to a particular Fc ⁇ R refer to antibodies that have statistically insignificant effector function mediated by the particular Fc ⁇ R.
  • the antibody of the invention comprises at least one mutation in the Fc region that reduces binding of the antibody to an Fc ⁇ R.
  • the Fc ⁇ R is Fc ⁇ RI, Fc ⁇ RIIa or Fc ⁇ RIIIa or Fc ⁇ RIIIb.
  • the at least one mutation in the Fc region is a L234A mutation, a L235A mutation, a G237A mutation, a P238S mutation, a M252Y mutation, a S254T mutation, a T256E mutation, a H268A mutation, a A330S mutation or a P331S mutation, wherein residue numbering is according to the EU Index.
  • the antibody of the invention comprises a L234A/L235A/G237A/P238S/H268A/A330S/P331S mutation in the Fc region, wherein residue numbering is according to the EU Index.
  • the at least one mutation in the Fc region is a V234A mutation, a G237A mutation, a P238S mutation, a M252Y mutation, a S254T mutation, a T256E mutation, a H268A mutation, a V309L mutation, an A330S mutation or a P331S mutation, wherein residue numbering is according to the EU Index.
  • the antibody of the invention comprises a V234A/G237A/P238S/H268A/V309L/A330S/P331S mutation in the Fc region, wherein residue numbering is according to the EU Index.
  • the antibody of the invention comprises a S228P mutation in the Fc region, wherein residue numbering is according to the EU Index.
  • the antibody of the invention comprises a F234A mutation in the Fc region, wherein residue numbering is according to the EU Index.
  • the antibody of the invention comprises a L235A mutation in the Fc region, wherein residue numbering is according to the EU Index.
  • the antibody of the invention comprises a S228P/F234A/L235A mutation, wherein residue numbering is according to the EU Index.
  • Binding of the antibodies of the invention to Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa and Fc ⁇ RIIIb may be evaluated using recombinant soluble forms or cell-associated forms of the Fc ⁇ receptors. For example, direct or indirect, e.g., competitive binding, measurements may be applied for assessing relative affinities and avidities of the antibodies of the invention to various Fc ⁇ R.
  • a test antibody binding to soluble Fc ⁇ R captured on a plate is evaluated using competitive binding between 1 ⁇ g/ml biotinylated human IgG1 and serial dilutions of the test antibody pre-complexed with antigen.
  • the antibody of the invention comprising at least one mutation in the Fc region has reduced antibody dependent cellular cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis, (“ADCP”) and/or reduced complement dependent cytotoxicity (CDC).
  • ADCC antibody dependent cellular cytotoxicity
  • ADCP reduced antibody-dependent cellular phagocytosis
  • CDC complement dependent cytotoxicity
  • Antibody-dependent cellular cytotoxicity is a mechanism for inducing cell death that depends upon the interaction of antibody-coated target cells with effector cells possessing lytic activity, such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (Fc ⁇ R) expressed on effector cells.
  • effector cells possessing lytic activity, such as natural killer cells, monocytes, macrophages and neutrophils via Fc gamma receptors (Fc ⁇ R) expressed on effector cells.
  • Fc ⁇ R Fc gamma receptors
  • NK cells express Fc ⁇ RIIIa
  • monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIIIa.
  • the antibody may be added to target cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell.
  • Cytolysis is generally detected by the release of label (e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells.
  • label e.g. radioactive substrates, fluorescent dyes or natural intracellular proteins
  • exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells.
  • PBMC peripheral blood mononuclear cells
  • NK cells e.g. CD40.
  • ADCP antibody-dependent cellular phagocytosis
  • monocyte-derived macrophages as effector cells and cells expressing CD40 engineered to express GFP or other labeled molecule as target cells. Effector:target cell ratio may be for example 4:1. Effector cells may be incubated with target cells for 4 hours with or without the test CD40 antibody. After incubation, cells may be detached using accutase. Macrophages may be identified with anti-CD11b and anti-CD14 antibodies coupled to a fluorescent label, and percent phagocytosis may be determined based on % GFP fluorescent in the CD11 + CD14 + macrophages using standard methods.
  • “Complement-dependent cytotoxicity”, or “CDC”, refers to a mechanism for inducing cell death in which an Fc effector domain of a target-bound antibody binds and activates complement component C1q which in turn activates the complement cascade leading to target cell death. Activation of complement may also result in deposition of complement components on the target cell surface that facilitate ADCC by binding complement receptors (e.g., CR3) on leukocytes.
  • CDC of CD40-expressing cells may be measured for example by plating cells expressing CD40 in an appropriate medium, adding anti-CD40 antibodies into the mixture, followed by addition of pooled human serum. After incubation period, percentage (%) lysed cells may be detected as % propidium iodide stained cells in FACS assay using standard methods.
  • Reduced ADCC refers to a statistically significant reduction in ADCC, CDC and/or ADCP mediated by the antibody of the invention comprising at least one mutation in the Fc region when compared to the same antibody without the mutation.
  • ADCC, CDC and/or ADCP such as assays described herein and in assays described in U.S. Pat. No. 8,871,204.
  • variants of the antibodies of the invention comprising the VH or the VL amino acid sequences shown in Table 7 are within the scope of the invention.
  • variants may comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions in the VH and/or the VL that do not adversely affect the characteristics of the antibodies.
  • sequence identity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the antibody VH or the VL amino acid sequence of the invention.
  • the percent identity between two amino acid sequences may be determined using the algorithm of E. Meyers and W. Miller ( Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch ( J. Mol. Biol.
  • the antibody of the invention comprises the VH that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NOs: 11, 21, 22, 23, 24, 25 or 26 and the VL that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VL of SEQ ID NOs: 12 or 27, wherein the antibody exhibits one or more of the following properties:
  • K D dissociation constant
  • the antibody of the invention comprises the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12.
  • the antibody of the invention comprises the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 27.
  • the antibody of the invention comprises the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 27.
  • the antibody of the invention comprises the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 23 and the VL of SEQ ID NO: 27.
  • the antibody of the invention comprises the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 27.
  • the antibody of the invention comprises the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27.
  • the antibody of the invention comprises the VH and the VL which are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27.
  • the antibody of the invention comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, wherein the VH, the VL or both the VH and the VL comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions and the antibody exhibits one or more of the following properties:
  • K D dissociation constant
  • the antibody of the invention comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12, wherein the VH, the VL or both the VH and the VL comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • the antibody of the invention comprises the VH of SEQ ID NO: 21 and the VL of SEQ ID NO: 27, wherein the VH, the VL or both the VH and the VL comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • the antibody of the invention comprises the VH of SEQ ID NO: 22 and the VL of SEQ ID NO: 27, wherein the VH, the VL or both the VH and the VL comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • the antibody of the invention comprises the VH of SEQ ID NO: 23 and the VL of SEQ ID NO: 27, wherein the VH, the VL or both the VH and the VL comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • the antibody of the invention comprises the VH of SEQ ID NO: 24 and the VL of SEQ ID NO: 27, wherein the VH, the VL or both the VH and the VL comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • the antibody of the invention comprises the VH of SEQ ID NO: 25 and the VL of SEQ ID NO: 27, wherein the VH, the VL or both the VH and the VL comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • the antibody of the invention comprises the VH of SEQ ID NO: 26 and the VL of SEQ ID NO: 27, wherein the VH, the VL or both the VH and the VL comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions.
  • Constant modification refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequences.
  • Conservative modifications include amino acid substitutions, additions and deletions.
  • Conservative amino acid substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain.
  • amino acids with acidic side chains e.g., aspartic acid, glutamic acid
  • basic side chains e.g., lysine, arginine, histidine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan
  • aromatic side chains e.g., phenylalanine, tryptophan, histidine, tyrosine
  • aliphatic side chains e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine
  • amide e.g., asparagine, glutamine
  • any native residue in the polypeptide may be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., Acta Physiol. Scand. Suppl. 643:55-67, 1998; Sasaki et al., Adv. Biophys. 35:1-24, 1998).
  • Amino acid substitutions to the antibodies of the invention may be made by well-known methods for example by PCR mutagenesis (U.S. Pat. No. 4,683,195).
  • libraries of variants may be generated using known methods, for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp).
  • NNK random
  • DVK codons which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp).
  • the resulting antibody variants may be tested for their characteristics using assays described herein.
  • variable regions one from a heavy chain and one from a light chain
  • alternative embodiments may comprise single heavy or light chain variable regions.
  • the single variable region may be used to screen for variable domains capable of forming a two-domain specific antigen-binding fragment capable of for example specifically binding to human CD40.
  • the screening may be accomplished by phage display screening methods using for example hierarchical dual combinatorial approach disclosed in Int. Pat. Publ. No. WO1992/01047.
  • Antibodies of the invention may be generated using various technologies. For example, the hybridoma method of Kohler and Milstein, Nature 256:495, 1975 may be used to generate monoclonal antibodies.
  • a mouse or other host animal such as a hamster, rat, rabbit or monkey, is immunized with human, marmoset or cyno CD40 or fragments of CD40, such as soluble form of CD40, followed by fusion of spleen cells from immunized animals with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Colonies arising from single immortalized hybridoma cells are screened for production of antibodies with desired properties, such as specificity of binding, cross-reactivity or lack thereof, and affinity for the antigen.
  • Balb/c mice may be used to generate antibodies.
  • the antibodies made in Balb/c mice and other non-human animals may be humanized using various technologies to generate more human-like sequences.
  • Exemplary humanization techniques including selection of human acceptor frameworks are known and include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), Resurfacing (Padlan, Mol Immunol 28:489-499, 1991), Specificity Determining Residues Resurfacing (U.S. Pat. Publ. No. 20100261620), human-adaptation (or human framework adaptation) (U.S. Pat. Publ. No.
  • CDRs of parental antibodies are transferred onto human frameworks that may be selected based on their overall homology to the parental frameworks, based on framework CDR length, homology or canonical structure information, or a combination thereof.
  • Humanized antibodies may be further optimized to improve their selectivity or affinity to a desired antigen by incorporating altered framework support residues to preserve binding affinity (backmutations) by techniques such as those disclosed as described in Int. Pat. Publ. No. WO90/007861 and in Int. Pat. Publ. No. WO92/22653, or by introducing variation to any of the CDRs to improve for example affinity of the antibody.
  • Transgenic mice carrying human immunoglobulin (Ig) loci in their genome may be used to generate human antibodies against a target protein, and are described in for example Int. Pat. Publ. No. WO90/04036, U.S. Pat. No. 6,150,584, Int. Pat. Publ. No. WO99/45962, Int. Pat. Publ. No. WO02/066630, Int. Pat. Publ. No. WO02/43478, Lonberg et al (1994) Nature 368:856-9; Green et al (1994) Nature Genet. 7:13-21; Green & Jakobovits (1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int. Rev.
  • the endogenous immunoglobulin loci in such mice may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the mouse genome using homologous or non-homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron (http://_www_regeneron_com), Harbour Antibodies (http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc.
  • OMT (http://_www_omtinc_net), KyMab (http://_www_kymab_com), Trianni (http://_www.trianni_com) and Ablexis (http://_www_ablexis_com) may be engaged to provide human antibodies directed against a selected antigen using technology as described above.
  • Human antibodies may be selected from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al., J. Mol Biol 296:57-86, 2000; Krebs et al., J Immunol Meth 254:67-84, 2001; Vaughan et al., Nature Biotechnology 14:309-314, 1996; Sheets et al., PITAS (USA) 95:6157-6162, 1998; Hoogenboom and Winter, J Mol Biol 227:381, 1991; Marks et al., J Mol Biol 222:581, 1991).
  • human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions
  • the antibodies of the invention may be isolated for example from phage display libraries expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al., J Mol Biol 397:385-96, 2010 and Int. Pat. Publ. No. WO09/085462).
  • the libraries may be screened for phage binding to human and/or cyno CD40 and the obtained positive clones may be further characterized, the Fabs isolated from the clone lysates, and expressed as full length IgGs.
  • Such phage display methods for isolating human antibodies are described in for example: U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat.
  • immunogenic antigens and monoclonal antibody production may be performed using any suitable technique, such as recombinant protein production.
  • the immunogenic antigens may be administered to an animal in the form of purified protein, or protein mixtures including whole cells or cell or tissue extracts, or the antigen may be formed de novo in the animal's body from nucleic acids encoding said antigen or a portion thereof.
  • the antibodies of the invention may be human or humanized.
  • the antibody of the invention comprises a VH framework derived from human germline gene VH3_3-23 (SEQ ID NO: 49).
  • the antibody of the invention comprises a VL framework derived from human germline gene VL3_3R (IGLV3-1) (SEQ ID NO: 50).
  • the antibodies of the invention may be of IgA, IgD, IgE, IgG or IgM type.
  • the antibodies of the invention may be of IgG1, IgG2, IgG3, IgG4 type.
  • the antibodies of the invention may further be engineered to generate modified antibodies with similar or altered properties when compared to the parental antibodies.
  • the VH, the VL, the VH and the VL, the constant regions, VH framework, VL framework, or any or all of the six CDRs may be engineered in the antibodies of the invention.
  • the antibodies of the invention may be engineered by CDR grafting.
  • One or more CDR sequences of the antibodies of the invention may be grafted to a different framework sequence.
  • CDR grafting may be done using methods described herein.
  • the antibodies of the invention comprise a VH that comprises the HDCR1 of SEQ ID NO: 5, the HCDR2 of SEQ ID NO: 6, 15, 16, 17 or 18, the HCDR3 of SEQ ID NOs: 7 or 19, and the VL that comprises the LCDR1 of SEQ ID NOs: 8 or 20, the LCDR2 of SEQ ID NO: 9 and/or the LCDR3 of SEQ ID NO: 10, wherein the VH framework is not derived from VH3_3-23 (SEQ ID NO: 49) and the VL framework is not derived from VL3_3R (IGLV3-1) (SEQ ID NO: 50).
  • framework sequences to be used may be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • germline DNA and the encoded protein sequences for human heavy and light chain variable region genes can be found at IMGT®, the international ImMunoGeneTics information System® http://_www-imgt_org.
  • Framework sequences that may be used to replace the existing framework sequences in the antibodies of the invention are those that show the highest percent identity to C40B16, C40B124, C40B135, C40B125, C40B136, C40B127, C40B138, C40B131, C40B176, C40B180, C40B179 or C40B183 VH or VL at amino acid level.
  • the framework sequences of the parental and engineered antibodies may further be modified, for example by backmutations to restore and/or improve binding of the resulting antibody to the antigen as described for example in U.S. Pat. No. 6,180,370.
  • the framework sequences of the parental and engineered antibodies may further be modified by mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and described in further detail in U.S. Pat. Publ. No. 20030153043.
  • the CDR residues of the antibodies of the invention may be mutated to improve one or more binding properties of the antibody of interest.
  • Site-directed mutagenesis or PCR-mediated mutagenesis may be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, may be evaluated in in vitro or in vivo assays as described herein and provided in the Examples.
  • Exemplary substitutions that may be introduced are conservative modifications as discussed supra.
  • typically no more than one, two, three, four or five residues within a CDR region are altered.
  • Antibodies of the invention may be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation or non-naturally occurring covalent modification such as the addition of polyethylene glycol moieties (pegylation) and lipidation. Such modifications may occur in vivo or in vitro.
  • the antibodies of the invention may be conjugated to polyethylene glycol (PEGylated) to improve their pharmacokinetic profiles. Conjugation may be carried out by techniques known to those skilled in the art.
  • Antibodies or antigen-binding fragments thereof of the invention may be modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity or other desirable biological or biophysical property are within the scope of the invention.
  • Stability of an antibody is influenced by a number of factors, including core packing of individual domains that affects their intrinsic stability, protein/protein interface interactions that have impact upon the HC and LC pairing, burial of polar and charged residues, (4) H-bonding network for polar and charged residues; and surface charge and polar residue distribution among other intra- and inter-molecular forces (Worn et al., J Mol Biol 305:989-1010, 2001).
  • Potential structure destabilizing residues may be identified based upon the crystal structure of the antibody or by molecular modeling in certain cases, and the effect of the residues on antibody stability may be tested by generating and evaluating variants harboring mutations in the identified residues.
  • One of the ways to increase antibody stability is to raise the thermal transition midpoint (T m ) as measured by differential scanning calorimetry (DSC).
  • T m thermal transition midpoint
  • DSC differential scanning calorimetry
  • the protein T m is correlated with its stability and inversely correlated with its susceptibility to unfolding and denaturation in solution and the degradation processes that depend on the tendency of the protein to unfold (Remmele et al., Biopharm 13:36-46, 2000).
  • the antibody of the invention is a multispecific antibody.
  • the antibody of the invention is a bispecific antibody.
  • the monospecific antagonistic antibodies specifically binding CD40 of the invention may be engineered into bispecific antibodies which are also encompassed within the scope of the invention.
  • Full length bispecific antibodies may be generated for example using Fab arm exchange (e.g., half-molecule exchange, exchanging one heavy chain-light chain pair) between two monospecific bivalent antibodies by introducing substitutions at the heavy chain CH3 interface in each half molecule to favor heterodimer formation of two antibody half molecules having distinct specificity either in vitro in cell-free environment or using co-expression.
  • the Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains. The heavy chain disulfide bonds in the hinge regions of the parental monospecific antibodies are reduced.
  • the resulting free cysteines of one of the parental monospecific antibodies form an inter heavy-chain disulfide bond with cysteine residues of a second parental monospecific antibody molecule and simultaneously CH3 domains of the parental antibodies release and reform by dissociation-association.
  • the CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization.
  • the resulting product is a bispecific antibody having two Fab arms or half molecules which each bind a distinct epitope.
  • Bispecific antibodies may also be generated using designs such as the Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knob-in-Hole (Genentech), CrossMAbs (Roche) and the electrostatically-induced CH3 interaction (Chugai, Amgen, NovoNordisk, Oncomed), the LUZ-Y (Genentech), the Strand Exchange Engineered Domain body (SEEDbody)(EMD Serono), the Biclonic (Merus) and as DuoBody® Products (Genmab A/S).
  • Triomab quadroma technology may be used to generate full length bispecific antibodies of the invention.
  • Triomab technology promotes Fab arm exchange between two parental chimeric antibodies, one parental mAb having IgG2a and the second parental mAb having rat IgG2b constant regions, yielding chimeric bispecific antibodies.
  • the “knob-in-hole” strategy may be used to generate full length bispecific antibodies. Briefly, selected amino acids forming the interface of the CH3 domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into a heavy chain of an antibody specifically binding a first antigen and an amino acid with a large side chain (knob) is introduced into a heavy chain of an antibody specifically binding a second antigen.
  • a heterodimer is formed as a result of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob”.
  • Exemplary CH3 substitution pairs forming a knob and a hole are (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.
  • CrossMAb technology may be used to generate full length bispecific antibodies of the invention.
  • CrossMAbs in addition to utilizing the “knob-in-hole” strategy to promoter Fab arm exchange, have in one of the half arms the CH1 and the CL domains exchanged to ensure correct light chain pairing of the resulting bispecific antibody (see e.g. U.S. Pat. No. 8,242,247).
  • heterodimerization may be promoted by following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): L351Y_F405A_Y407V/T394W, T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F, L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in U.S. Patent Publ. No. US2012/0149876 or U.S. Patent Publ. No. US2013/0195849.
  • LUZ-Y technology may be utilized to generate bispecific antibodies.
  • a leucine zipper is added into the C terminus of the CH3 domains to drive the heterodimer assembly from parental mAbs that is removed post-purification as described in Wranik et al., (2012) J Biol Chem 287(52): 42221-9.
  • SEEDbody technology may be utilized to generate bispecific antibodies.
  • SEEDbodies have, in their constant domains, select IgG residues substituted with IgA residues to promote heterodimerization as described in U.S. Patent No. US20070287170.
  • Bispecific antibodies may be generated in vitro in a cell-free environment by introducing asymmetrical mutations in the CH3 regions of two monospecific homodimeric antibodies and forming the bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies in reducing conditions to allow disulfide bond isomerization according to methods described in Int. Patent Publ. No. WO2011/131746.
  • the first monospecific bivalent antibody and the second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promoter heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange.
  • Substitutions that may be used are F405L in one heavy chain and K409R in the other heavy chain.
  • the incubation conditions may optimally be restored to non-reducing.
  • Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol.
  • 2-MEA 2-mercaptoethylamine
  • DTT dithiothreitol
  • DTE dithioerythritol
  • glutathione glutathione
  • tris(2-carboxyethyl)phosphine (TCEP) tris(2-carboxyethyl)phosphine
  • L-cysteine L-cysteine
  • beta-mercaptoethanol beta-mercaptoethanol
  • substitutions are typically made at the DNA level to a molecule such as the constant domain of the antibody using standard methods.
  • the antibodies of the invention may be engineered into various well known antibody forms.
  • the bispecific antibodies include recombinant IgG-like dual targeting molecules, wherein the two sides of the molecule each contain the Fab fragment or part of the Fab fragment of at least two different antibodies; IgG fusion molecules, wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment; Fc fusion molecules, wherein single chain Fv molecules or stabilized diabodies are fused to heavy-chain constant-domains, Fc-regions or parts thereof; Fab fusion molecules, wherein different Fab-fragments are fused together; ScFv- and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) wherein different single chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.
  • IgG fusion molecules wherein full length IgG antibodies are fused to an extra Fab fragment or parts of Fab fragment
  • Fc fusion molecules wherein single chain F
  • the invention also provides for an isolated polynucleotide encoding any of the antibody heavy chain variable regions, any of the antibody light chain variable regions, or any of the antibody heavy chains and/or the antibody light chains of the invention.
  • the invention also provides for an isolated polynucleotide encoding the VH of SEQ ID NOs: 11, 21, 22, 23, 24, 25 or 26.
  • the invention also provides for an isolated polynucleotide encoding the VL of SEQ ID NOs: 12 or 27.
  • the invention also provides for an isolated polynucleotide encoding the VH of SEQ ID NOs: 11, 21, 22, 23, 24, 25 or 26 and the VL of SEQ ID NOs: 12 or 27.
  • the invention also provides for an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NOs: 13, 14, 28, 29, 30, 31, 32, 33, or 34.
  • the invention also provides for an isolated polynucleotide encoding the heavy chain of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46.
  • the invention also provides for an isolated polynucleotide encoding the light chain of SEQ ID NOs: 77 or 78.
  • the invention also provides for an isolated polynucleotide encoding the heavy chain of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or 46 and a light chain of SEQ ID NOs: 77 or 78.
  • the invention also provides for an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 77 or 78.
  • polynucleotide sequences encoding the VH or the VL or a fragment thereof of the antibodies of the invention or the heavy chain and the light chain of the antibodies of the invention may be operably linked to one or more regulatory elements, such as a promoter or enhancer, that allow expression of the nucleotide sequence in the intended host cell.
  • the polynucleotide may be a cDNA.
  • the invention also provides for a vector comprising the polynucleotide of the invention.
  • vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the synthetic polynucleotide of the invention into a given organism or genetic background by any means.
  • polynucleotides encoding light and/or heavy chain variable regions of the antibodies of the invention, optionally linked to constant regions are inserted into expression vectors.
  • the light and/or heavy chains may be cloned in the same or different expression vectors.
  • the DNA segments encoding immunoglobulin chains may be operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides.
  • control sequences include signal sequences, promoters (e.g. naturally associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are chosen to be compatible with the host cell chosen to express the antibody.
  • the vector comprises the polynucleotide of SEQ ID NO: 13 and the polynucleotide of SEQ ID NO: 14.
  • the vector comprises the polynucleotide of SEQ ID NO: 28 and the polynucleotide of SEQ ID NO: 34.
  • the vector comprises the polynucleotide of SEQ ID NO: 29 and the polynucleotide of SEQ ID NO: 34.
  • the vector comprises the polynucleotide of SEQ ID NO: 30 and the polynucleotide of SEQ ID NO: 34.
  • the vector comprises the polynucleotide of SEQ ID NO: 31 and the polynucleotide of SEQ ID NO: 34.
  • the vector comprises the polynucleotide of SEQ ID NO: 32 and the polynucleotide of SEQ ID NO: 34.
  • the vector comprises the polynucleotide of SEQ ID NO: 33 and the polynucleotide of SEQ ID NO: 34.
  • the vector comprises the polynucleotide of SEQ ID NO: 65 and the polynucleotide of SEQ ID NO: 77.
  • the vector comprises the polynucleotide of SEQ ID NO: 66 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 67 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 68 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 69 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 70 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 71 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 72 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 73 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 74 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 75 and the polynucleotide of SEQ ID NO: 78.
  • the vector comprises the polynucleotide of SEQ ID NO: 76 and the polynucleotide of SEQ ID NO: 78.
  • Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers such as ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences.
  • Suitable promoter and enhancer elements are known in the art.
  • exemplary promoters include light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • Bacterial pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
  • Eukaryotic pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).
  • the invention also provides for a host cell comprising one or more vectors of the invention.
  • “Host cell” refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell but to the progeny of such a cell, and also to a stable cell line generated from the particular subject cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells or archeal cells.
  • Escherichia coli bacilli, such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella, Serratia , and various Pseudomonas species
  • Other microbes such as yeast
  • Saccharomyces e.g., S. cerevisiae
  • Pichia exemplary yeast host cells.
  • Exemplary eukaryotic cells may be of mammalian, insect, avian or other animal origins.
  • Mammalian eukaryotic cells include immortalized cell lines such as hybridomas or myeloma cell lines such as SP2/0 (American Type Culture Collection (ATCC), Manassas, Va., CRL-1581), NS0 (European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646) and Ag653 (ATCC CRL-1580) murine cell lines.
  • An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196).
  • Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics, Walkersville, Md.), CHO-K1 (ATCC CRL-61) or DG44.
  • the invention also provides for a method of producing the antibody of the invention comprising culturing the host cell of the invention in conditions that the antibody is expressed, and recovering the antibody produced by the host cell.
  • Methods of making antibodies and purifying them are known. Once synthesized (either chemically or recombinantly), the whole antibodies, their dimers, individual light and/or heavy chains, or other antibody fragments such as VH and/or VL, may be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC) purification, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)).
  • a subject antibody may be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99%, or more, pure, e.g., free from contaminants such as cell debris, macromolecules, etc. other than the subject antibody.
  • the invention also provides for a method for producing an antagonistic antibody specifically binding CD40 of SEQ ID NO: 1, comprising:
  • polynucleotides encoding certain VH or VL sequences of the invention may be incorporated into vectors using standard molecular biology methods. Host cell transformation, culture, antibody expression and purification are done using well known methods.
  • Antagonistic antibodies specifically binding CD40 of the invention may be used for the treatment and/or prevention of any condition or disease wherein antagonizing the effects of CD40 may be therapeutically effective and may reduce the symptoms of the disease.
  • Examples thereof include the treatment of inflammatory diseases such as autoimmune diseases wherein the induction of tolerance and/or the suppression of humoral immunity are therapeutically desirable.
  • autoimmune diseases that may be treated with the antibodies of the invention are autoimmune diseases, Addison's disease, an ankylosing spondylitis, an atherosclerosis, an autoimmune hepatitis, an autoimmune diabetes, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, an idiopathic thrombocytopenia, an inflammatory bowel disease (IBD), a systemic lupus erythematosus, a multiple sclerosis, a myasthenia gravis, a psoriasis, an arthritis, a scleroderma, Sjogren's syndrome, a systemic sclerosis, a transplantation, a kidney transplantation, a skin transplantation, a bone marrow transplantation, a graft versus host disease (GVHD), a type I diabetes, a rheumatoid arthritis, a juvenile arthritis, a psoriatic arthritis, Reiter's syndrome, an ankylosing
  • the invention also provides for a method of treating an arthritis, comprising administering a therapeutically effective amount of the antibody of the invention to a subject in need thereof for a time sufficient to treat the arthritis.
  • the arthritis is a juvenile arthritis, a rheumatoid arthritis, a psoriatic arthritis, Reiter's syndrome, an ankylosing spondylitis, or a gouty arthritis.
  • the invention also provides for a method of treating a lupus, comprising administering a therapeutically effective amount of the antibody of the invention to a subject in need thereof for a time sufficient to treat the lupus.
  • the lupus is a systemic lupus erythematosus (SLE) or a cutaneous lupus erythematosus (CLE).
  • SLE systemic lupus erythematosus
  • CLE cutaneous lupus erythematosus
  • the subject has lupus nephritis.
  • the subject has a cutaneous lupus erythematosus.
  • the invention also provides for a method of treating an inflammatory bowel disease, comprising administering a therapeutically effective amount of the antibody of the invention to a subject in need thereof for a time sufficient to treat the inflammatory bowel disease.
  • the inflammatory bowel disease is Crohn's disease.
  • the inflammatory bowel disease is an ulcerative colitis.
  • Treatment refers to therapeutic treatment.
  • Subjects in need of treatment include those subjects diagnosed with the disorder or experiencing at least one of the symptoms of the disease.
  • Subjects that may be treated also include those prone to or susceptible to have the disorder, or those in which the disorder is to be prevented.
  • Beneficial or desired clinical results include alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Beneficial treatment result include, in a subject who has received treatment, reduction in the levels of inflammatory cytokines, adhesion molecules, proteases, immunoglobulins, combinations thereof, increased production of anti-inflammatory proteins, a reduction in the number of autoreactive cells, an increase in immune tolerance, inhibition of autoreactive cell survival, and/or a decrease in one or more symptoms mediated by stimulation of CD40-expressing cells by CD154.
  • Clinical response may be assessed using screening techniques such as magnetic resonance imaging (MRI) scan, x-radiographic imaging, computed tomographic (CT) scan, flow cytometry or fluorescence-activated cell sorter (FACS) analysis, histology, gross pathology, and blood chemistry, including but not limited to changes detectable by ELISA, RIA, chromatography, and the like.
  • MRI magnetic resonance imaging
  • CT computed tomographic
  • FACS fluorescence-activated cell sorter
  • the methods of the invention may be used to treat a subject belonging to any animal classification.
  • subjects that may be treated include mammals such as humans, rodents, dogs, cats and farm animals.
  • the antibodies of the invention may be useful in the preparation of a medicament for such treatment, wherein the medicament is prepared for administration in dosages defined herein.
  • the antibodies of the invention may be administered in combination with a second therapeutic agent.
  • the second therapeutic agent may be any known therapy for inflammatory diseases, such as autoimmune diseases, including any agent or combination of agents that are known to be useful, or which have been used or are currently in use for treatment.
  • inflammatory diseases such as autoimmune diseases
  • Such therapies and therapeutic agents include surgery or surgical procedures (e.g. splenectomy, lymphadenectomy, thyroidectomy, plasmapheresis, leukophoresis, cell, tissue, or organ transplantation, intestinal procedures, organ perfusion, and the like), therapy such as steroid therapy and non-steroidal therapy, hormone therapy, cytokine therapy, therapy with dermatological agents (for example, topical agents used to treat skin conditions such as allergies, contact dermatitis, and psoriasis), immunosuppressive therapy, and other anti-inflammatory drugs including monoclonal antibodies.
  • dermatological agents for example, topical agents used to treat skin conditions such as allergies, contact dermatitis, and psoriasis
  • immunosuppressive therapy and other anti-inflammatory drugs including monoclonal antibodies.
  • the second therapeutic agent may be a corticosteroid, an immunosuppressant, a cytotoxic drug, or a B-cell modulator.
  • the antibodies of the invention are administered in combination with a second therapeutic agent.
  • second therapeutic agents are corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), salicylates, hydroxychloroquine, sulfasalazine, cytotoxic drugs, immunosuppressive drugs immunomodulatory antibodies, methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine, tacrolimus (FK506; ProGrafrM), mycophenolate mofetil, and azathioprine (6-mercaptopurine), sirolimus (rapamycin), deoxyspergualin, leflunomide and its malononitriloamide analogs; anti-CTLA4 antibodies and Ig fusions, anti-B lymphocyte stimulator antibodies (e.g., LYMPHOSTAT-BTM) and CTLA4-Ig fusions, anti-CD80 antibodies, anti-T cell antibodies such as anti-CD3 (OK
  • the antibodies of the invention may be administered in combination with a second therapeutic agent simultaneously, sequentially or separately.
  • Treatment effectiveness or RA may be assessed using effectiveness as measured by clinical responses defined by the American College of Rheumatology criteria, the European League of Rheumatism criteria, or any other criteria. See for example, Felson et al. (1995) Arthritis Rheum. 38: 727-35 and van Gestel et al. (1996) Arthritis Rheum. 39: 34-40.
  • the invention also provides for pharmaceutical compositions of the antagonistic antibodies specifically binding CD40 of the invention and a pharmaceutically acceptable carrier.
  • the antibodies the invention may be prepared as pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier.
  • Carrier refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered.
  • Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter.
  • compositions may be sterilized by conventional, well-known sterilization techniques (e.g., filtration).
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc.
  • concentration of the antibodies of the invention in such pharmaceutical compositions may vary widely, i.e., from less than about 0.5%, usually to at least about 1% to as much as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected.
  • Suitable vehicles and formulations including other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.
  • the mode of administration of the antibodies of the invention in the methods of the invention may be any suitable route such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, transmucosal (oral, intranasal, intravaginal, and rectal) or other means appreciated by the skilled artisan, as well known in the art.
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous
  • transmucosal oral, intranasal, intravaginal, and rectal
  • the antibodies of the invention may be administered to the subject by any suitable route, for example parentally by intravenous (i.v.) infusion or bolus injection, intramuscularly or subcutaneously or intraperitoneally.
  • i.v. infusion may be given over for, example, 15, 30, 60, 90, 120, 180, or 240 minutes, or over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.
  • the dose given to the subject is sufficient to alleviate or at least partially arrest the disease being treated (“therapeutically effective amount”) and may be from about 0.005 mg/kg to about 100 mg/kg, for example about 0.05 mg/kg to about 20 mg/kg, about 0.1 mg/kg to about 20 mg/kg, about 1 mg to about 20 mg/kg, about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.
  • a fixed unit dose may also be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose may be based on the patient's surface area, e.g., 500, 400, 300, 250, 200, or 100 mg/m 2 .
  • 1 and 8 doses e.g., 1, 2, 3, 4, 5, 6, 7 or 8
  • the inflammatory disease such as an autoimmune disease such or a rheumatoid arthritis
  • 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be given.
  • the administration of the antibodies of the invention may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or longer. Repeated courses of treatment are also possible, as is chronic administration.
  • the repeated administration may be at the same dose or at a different dose.
  • the antibodies of the invention may be administered at 0.1 mg/kg, at 1 mg/kg, at 5 mg/kg, at 8 mg/kg or at 16 mg/kg at weekly interval for 8 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every two weeks for an additional 16 weeks, followed by administration at 8 mg/kg or at 16 mg/kg every four weeks by intravenous infusion.
  • the antibodies of the invention may be provided by maintenance therapy, such as, e.g., once a week, once a month, once in two months, once in three months, once in four months, once in five months, or once in six months over one or more years.
  • maintenance therapy such as, e.g., once a week, once a month, once in two months, once in three months, once in four months, once in five months, or once in six months over one or more years.
  • the antibodies of the invention may be provided as a daily dosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 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, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
  • 0.1-100 mg/kg such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • the antibodies of the invention may also be administered prophylactically in order to reduce the risk of developing a disease and/or delay the onset of the disease to be treated.
  • An exemplary pharmaceutical composition of the invention for intramuscular injection may be prepared to contain 1 ml sterile buffered water, and between about 1 ng to about 100 mg/kg, e.g. about 50 ng to about 30 mg/kg or about 5 mg to about 25 mg/kg, of the antibody of the invention.
  • An exemplary pharmaceutical composition of the invention for intravenous infusion may be made up to contain about 200 ml of sterile Ringer's solution, and about 8 mg to about 2400 mg, about 400 mg to about 1600 mg, or about 400 mg to about 800 mg of the antibodies of the invention for administration to a 80 kg patient.
  • Methods for preparing parenterally administrable compositions are well known and are described in more detail in, for example, “Remington's Pharmaceutical Science”, 15th ed., Mack Publishing Company, Easton, Pa.
  • “Therapeutically effective amount” of the antibodies of the invention effective in the treatment of a disease may be determined by standard research techniques. For example, in vitro assays may be employed to help identify optimal dosage ranges.
  • the dosage of the antibodies of the invention that may be effective in the treatment of a disease such as the inflammatory disease may be determined by administering the antibodies to relevant animal models well known in the art. Selection of a particular effective dose may be determined (e.g., via clinical trials) by those skilled in the art based upon the consideration of several factors. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known by the skilled artisan.
  • Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the antibodies of the invention may be tested for their efficacy and effective dosage using any of the models described herein.
  • the antibodies of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional protein preparations and well known lyophilization and reconstitution techniques can be employed.
  • the present invention provides for an anti-idiotypic antibody binding to the antibody of the invention.
  • the invention also provides for an anti-idiotypic antibody specifically binding the antibody comprising the VH of SEQ ID NO: 11 and the VL or SEQ ID NO: 12.
  • the invention also provides for an anti-idiotypic antibody specifically binding the antibody comprising the VH of SEQ ID NO: 21 and the VL or SEQ ID NO: 27.
  • the invention also provides for an anti-idiotypic antibody specifically binding the antibody comprising the VH of SEQ ID NO: 22 and the VL or SEQ ID NO: 27.
  • the invention also provides for an anti-idiotypic antibody specifically binding the antibody comprising the VH of SEQ ID NO: 23 and the VL or SEQ ID NO: 27.
  • the invention also provides for an anti-idiotypic antibody specifically binding the antibody comprising the VH of SEQ ID NO: 24 and the VL or SEQ ID NO: 27.
  • the invention also provides for an anti-idiotypic antibody specifically binding the antibody comprising the VH of SEQ ID NO: 25 and the VL or SEQ ID NO: 27.
  • the invention also provides for an anti-idiotypic antibody specifically binding the antibody comprising the VH of SEQ ID NO: 26 and the VL or SEQ ID NO: 27.
  • An anti-idiotypic (Id) antibody is an antibody which recognizes the antigenic determinants (e.g. the paratope or CDRs) of the antibody.
  • the Id antibody may be antigen-blocking or non-blocking.
  • the antigen-blocking Id may be used to detect the free antibody in a sample (e.g. CD40 antibody of the invention described herein).
  • the non-blocking Id may be used to detect the total antibody (free, partially bond to antigen, or fully bound to antigen) in a sample.
  • An Id antibody may be prepared by immunizing an animal with the antibody to which an anti-Id is being prepared.
  • An anti-Id antibody may also be used as an immunogen to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • An anti-anti-Id may be epitopically identical to the original mAb, which induced the anti-Id.
  • Anti-Id antibodies may be varied (thereby producing anti-Id antibody variants) and/or derivatized by any suitable technique.
  • immunoconjugate refers to the antibody of the invention conjugated to one or more heterologous molecule(s).
  • the antibody of the invention is conjugated to one or more cytotoxic agents or an imaging agent.
  • cytotoxic agents include chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and radionuclides.
  • chemotherapeutic agents or drugs growth inhibitory agents
  • toxins e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof
  • radionuclides e.g., radionuclides.
  • the cytotoxic agent may be one or more drugs, such as to a mayatansinoid (see, e.g., U.S. Pat. Nos. 5,208,020, 5,416,06), an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see, e.g., U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298), a dolastatin, a calicheamicin or derivative thereof (see, e.g., U.S. Pat. Nos.
  • a mayatansinoid see, e.g., U.S. Pat. Nos. 5,208,020, 5,416,06
  • an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF)
  • MMAE and MMAF monomethylauristatin drug moieties DE and DF
  • the cytotoxic agent may also be an enzymatically active toxin or fragment thereof, such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain from Pseudomonas aeruginosa
  • ricin A chain modeccin A chain
  • the cytotoxic agent or an imaging agent may also be a radionuclide.
  • radionuclides include Ac-225, At-211, 1-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, Pb-212 and radioactive isotopes of Lu.
  • the radioconjugate When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example Tc-99m or I-123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as I-123, I-131, In-111, F-19, C-13, N-15 or O-17.
  • NMR nuclear magnetic resonance
  • Conjugates of the antibodies of the invention and the heterologous molecule may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine
  • a ricin immunotoxin may be prepared as described in Vitetta et al., (1987) Science 238: 1098.
  • Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, e.g., WO94/11026.
  • the linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell.
  • an acid-labile linker for example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., (1992) Cancer Res 52: 127-131; U.S. Pat. No. 5,208,020) may be used.
  • Conjugates of the antibodies of the invention and the heterologous molecule may be prepared with cross-linker reagents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).
  • cross-linker reagents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB,
  • the invention also provides for an immunoconjugate comprising the antibody specifically binding CD40 of SEQ ID NO: 1 of the invention linked to a therapeutic agent or an imaging agent.
  • the invention also provides for a kit comprising the antagonistic antibody specifically binding human CD40 of the invention.
  • the kit may be used for therapeutic uses and as diagnostic kits.
  • the kit may be used to detect the presence of CD40 in a biological sample.
  • the kit comprises the antagonistic antibody specifically binding human CD40 of the invention and reagents for detecting the antibody.
  • the kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, an antibody to a label or therapeutic agent, or a radioprotective composition; devices or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
  • the kit comprises the antibody of the invention in a container and instructions for use of the kit.
  • the antibody in the kit is labeled.
  • the kit comprises the antibody C40B16, C40B124, C40B135, C40B125, C40B136, C40B127, C40B138, C40B131, C40B176, C40B180, C40B179 or C40B183.
  • the invention also provides for a method of detecting CD40 in a sample, comprising obtaining the sample, contacting the sample with the antibody of the invention, and detecting the antibody bound to CD40 in the sample.
  • the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, cells that are not tissue associated (i.e., free cells), tissues (e.g., surgically resected tumor tissue, biopsies, including fine needle aspiration), histological preparations, and the like.
  • the antibodies of the invention may be detected using known methods. Exemplary methods include direct labeling of the antibodies using fluorescent or chemiluminescent labels, or radiolabels, or attaching to the antibodies of the invention a moiety which is readily detectable, such as biotin, enzymes or epitope tags.
  • Exemplary labels and moieties are ruthenium, 111 In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, poly-histidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes, phenanthridine dyes, rhodamine dyes and Alexafluor® dyes.
  • DTPA 111 In-diethylenetriaminepentaacetic acid
  • HIS tag poly-histidine
  • acridine dyes cyanine dyes
  • fluorone dyes oxazin dyes
  • phenanthridine dyes phenanthridine dyes
  • rhodamine dyes Alexafluor® dyes.
  • the antibodies of the invention may be used in a variety of assays to detect CD40 in the sample.
  • exemplary assays are western blot analysis, radioimmunoassay, surface plasmon resonance, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, and immunohistochemistry, fluorescence-activated cell sorting (FACS) or ELISA assay.
  • Human monocytes were isolated from either frozen/fresh PBMC using CD14 negative isolation kit per manufacturer's protocol (MACS Miltenyi). Cyno monocytes were isolated from fresh PBMC using CD14 positive isolation kit per manufacturer's protocol (MACS Miltenyi). To generate DCs, monocytes were cultured for 5 days in complete RPMI medium (Invitrogen) in the presence of 100 ng/ml human GM-CSF and human IL-4 (Peprotech) and medium was replenished every 2 days. On day 5, DCs were stimulated with 100 ng/ml LPS (Sigma) for 24 hours.
  • Raji cells were obtained from ATCC and HEK-BlueTM CD40L NF- ⁇ B cell line was obtained from Invivogen. Cells were cultured in complete RPMI medium per company's recommendation. Staining was done as described above for binding assay in primary human and cyno DCs.
  • Human DCs were generated by culturing purified human monocytes (2.5 ⁇ 10 6 /well 6-well plates) for 6 days in 3 ml RPMI medium containing glutamax, 25 mM HEPES, 10% FBS, 1% Pen/Strep and 50 ng each GM-CSF and IL-4. On day 3, 1 ml of the medium was removed and replaced with 2 ml fresh medium containing 50 ng/ml each of GM-CSF and IL-4. For agonist assays, day 6 DCs were plated into 96-well plates (100,000 cells/well) followed by titrations of each of the CD40 antibodies.
  • day 6 DCs were plated into 96-well plates (100,000 cells/well) followed by titrations of each of the CD40 antibodies and addition of 1 ⁇ g/ml of soluble human CD154 (R&D Systems) to the cultures.
  • cells were cultured for 48 hours before collecting and analyzing supernatants for IL-12p40 by MSD (according to manufacturer's directions).
  • Jurkat D1.1 (ATCC) antagonist assays day 6 DCs were plated into 96-well plates (10,000 cells/well) followed by titrations of each of the CD40 antibodies, after which irradiated Jurkat D1.1 cells (1,000 rads; 100,000/well) were added to the cultures. Cells were cultured for 24 hours before collecting and analyzing supernatants for IL-12p40 by MSD.
  • Human tonsillar B cells were plated into 96-well plates (100,000 cells/well) in RPMI medium containing glutamax, 10% FBS and 1% Pen/Strep.
  • agonist assays titrations of each of the CD40 antibodies were added to the cells and cultured for 48 hours.
  • antagonist assays titrations of each of the CD40 antibodies were added to the cells, followed by addition of 0.5 ⁇ g/ml soluble human CD154 (R&D Systems). Cells were cultured for 48 hours at 37 C.
  • titrations of each of the CD40 antibodies were added to the cells along with IL-21 (100 ng/ml final, Thermo Fisher Scientific)), after which irradiated Jurkat D1.1. cells (5,000 rads; 100,000/well) were added to the cultures and incubated at 37 C for 48 hours. For all assays, after 48 hours cells were pulsed with 3H-thymidine (1 uCi/well) in 50 ⁇ l medium and cultured for 16-18 hours before harvest and counting.
  • Cyno spleen cells were plated into 96-well plates (100,000 cells/well) in RPMI medium containing glutamax, 10% FBS and 1% Pen/Strep. Titrations of each of the CD40 antibodies were added to the cells, followed by addition of 0.5 ⁇ g/ml soluble human CD154 (R&D Systems). Cells were cultured for 48 hours at 37 C. After 48 hours, cells were pulsed with 3H-thymidine (1 uCi/well) in 50 ⁇ l medium and cultured for 16-18 hours before harvest and counting.
  • HEK-BlueTM CD40L cell lines stably express human CD40 and NF- ⁇ B-inducible secreted embryonic alkaline phosphatase (SEAP). Activation of CD40 on HEK-BlueTM CD40L cells induce downstream signaling events leading to activation of NF- ⁇ B and secretion of SEAP, which can be measured by QUANTI-BlueTM substrate conversion. These cells were used to assess the ability of the antibodies to either block (antagonize) CD40-CD154 interaction or activate (agonize) CD40.
  • SEAP NF- ⁇ B-inducible secreted embryonic alkaline phosphatase
  • HEK-BlueTM CD40L cells (Invivogen), which were maintained according to the vendor's protocol, were seeded into 96 well tissue culture plates in 100 ⁇ l volume (2.5 ⁇ 10 4 cells/well). The assay plates were covered and incubated overnight (37° C., 5% CO 2 ) to allow the cells to recover. On the following day, a 4 ⁇ solution of rhCD154-ECD-His (80 ng/ml final concentration) and 4 ⁇ solution of CD40 antibodies, or Fabs, (1-25 ⁇ g/ml final concentration) were pre-mixed and the resulting 2 ⁇ solution added to the 96 well assay plates containing the cells (200 ⁇ l/well final volumes).
  • HEK-BlueTM CD40L cells were seeded as described above and recovered overnight. On the following day, 2 ⁇ CD40 antibody solutions (1-25 ⁇ g/ml final concentration) were added to the plate at 100 ⁇ l/well and incubated overnight (37° C., 5% CO 2 ). After 16-24 h incubation (37° C., 5% CO 2 ), 40 ⁇ l aliquots of the supernatants were analyzed as described above.
  • Antibodies were generated using transgenic rats expressing human immunoglobulin loci, the OmniRat®; OMT, Inc.
  • the OmniRat® endogenous immunoglobulin loci are replaced by human Ig ⁇ and Ig ⁇ loci and a chimeric human/rat IgH locus with V, D and J segments of human origin linked to the rat C H locus.
  • the IgH locus contains 22 human V H s, all human D and J H segments in natural configuration linked to the rat C H locus.
  • Generation and characterization of the OmniRat® is described in Osborn, et al. J Immunol 190: 1481-1490, 2013; and Int. Pat. Publ. No. WO 14/093908.
  • C40B16 was generated by using the OmniRat® and identified having antagonist activity in the HEK-BlueTM CD40L NF- ⁇ B activation assay based on the criteria that an antagonist had a signal that is lower than the 3 ⁇ standard deviation of the mean signal of HEK-BlueTM CD40L cells treated with rhCD154-ECD-his alone.
  • C40B16 VH and VL were sequenced using standard methods and the framework sequences compared to the closest germline gene sequences in order to identify potential immunogenicity risks.
  • C40B16 VH amino acid sequence was most homologous to IGHV3-23 (SEQ ID NO: 49) with 2 amino acid changes in the framework.
  • C40B16 VL framework was identical to that of IGLV3-1 (SEQ ID NO: 50).
  • variable regions of C40B16 were engineered to reduce possible immunogenicity and/or developability risk(s) by generating mutations at positions R43, H82, N52, S54 and/or M108 in the VH (residue numbering according to SEQ ID NO: 11) and at position C33 in the VL (residue numbering according to SEQ ID NO: 12) to eliminate potential heterogeneity caused by unpaired cysteines.
  • the generated VH/VL domains were cloned as effector silent Fc isoforms IgG1sigma or IgG4PAA.
  • IgG1sigma contains mutations L234A, L235A, G237A, P238S, H268A, A330S, and P331S when compared to the wild-type IgG1.
  • IgG4PAA contains mutations S228P, F234A and L235A when compared to the wild-type IgG4. Residue numbering is according to the EU Index.
  • Table 3 shows the generated antibodies and introduced mutations when compared to the parental C40B16 mAb. A C33A mutation was engineered in the VL of all generated antibodies when compared to the parental C40B16 VL.
  • Table 4 shows the HCDR1 and the HCDR2 amino acid sequences of the antibodies.
  • Table 5 shows the HCDR3 and the LCDR1 amino acid sequences of the antibodies.
  • Table 6 shows the LCDR2 and the LCDR3 amino acid sequences of the antibodies.
  • Table 7 shows the SEQ ID NOs: of the amino acid and cDNA sequences of the VH and the VL regions of the antibodies.
  • Table 8 shows the SEQ ID NOs: of the amino acid sequences of the heavy chains and the light chains of the antibodies.
  • Table 9 shows the amino acid or polynucleotide sequences corresponding to SEQ ID NOs: 11-14, 21-52 and 65-78.
  • HCDR1 HCDR2 SEQ SEQ ID mAb Sequence ID NO: Sequence NO: C40B16 SYAMS 5 TINNSGGGTYYADSVKG 6 C40B124 SYAMS 5 TIDNAGGGTYYADSVKG 15 C40B135 SYAMS 5 TIDNAGGGTYYADSVKG 15 C40B125 SYAMS 5 TIQNAGGGTYYADSVKG 16 C40B136 SYAMS 5 TIQNAGGGTYYADSVKG 16 C40B127 SYAMS 5 TINQAGGGTYYADSVKG 17 C40B138 SYAMS 5 TINQAGGGTYYADSVKG 17 C40B131 SYAMS 5 TINAAGGGTYYADSVKG 18 C40B176 SYAMS 5 TINAAGGGTYYADSVKG 18 C40B180 SYAMS 5 TINAAGGGTYYADSVKG 18 C40B179 SYAMS 5 TINQAGGGTYYADSVKG 17 C40B183 SYAMS 5 TINQAGGGTYYADSVKG 17 C40B
  • the parental C40B16 and the engineered variants were tested for their antagonistic activity in a spectrum of assays including ability of the antibodies to inhibit human soluble CD40L (sCD40L) or membrane-bound CD40L (mCD40L)-mediated proliferation of human or cyno B cells, and inhibition of IL-12p40 production by human DCs.
  • the experiments were conducted according to protocols described in Example 1.
  • Membrane-bound CD40L was provided on Jurkat cells in the assays.
  • Table 10 shows the IC 50 values for inhibition of soluble or membrane-bound CD40L-driven B cell proliferation.
  • Table 11 shows the IC 50 values for inhibition of soluble or membrane-bound CD40L-driven IL-12p40 production by human dendritic cells.
  • Affinity of the antibodies to human CD40 was assessed using Surface Plasmon Resonance (SPR) using a ProteOn XPR36 system (BioRad).
  • SPR Surface Plasmon Resonance
  • a biosensor surface was prepared by coupling anti-Human IgG Fc (Jackson cat#109-005-098) to the modified alginate polymer layer surface of a GLC chip (BioRad, Cat#176-5011) using the manufacturer instructions for amine-coupling chemistry. Approximately 5000 RU (response units) of mAbs were immobilized. The kinetic experiments were performed at 25° C. in running buffer (DPBS+0.01% P20+100 ⁇ g/ml BSA).
  • the collected data were processed using ProteOn Manager software. First, the data was corrected for background using inter-spots. Then, double reference subtraction of the data was performed by using the buffer injection for analyte injections. The kinetic analysis of the data was performed using a Langmuir 1:1 binding model. The results were reported in the format of Ka (On-rate), Kd (Off-rate) and K D (equilibrium dissociation constant).
  • Example 4 Antagonistic Antibodies Specifically Binding Human CD40 are Potent Inhibitors of CD40/CD40L Pathway and Display Minimal Agonistic Activity
  • C40B176, C40B179, C40B180 and C40B183 were benchmarked against anti-CD40 antibodies currently in clinical developments.
  • the antibodies used in comparisons were: Astellas/Kirin ASKP-1240 (HC SEQ ID NO: 53, LC SEQ ID NO: 54, Fc silent IgG4 with S228P and L235E mutations), Antibody D (HC SEQ ID NO: 55, LC SEQ ID NO: 56, Fc silent IgG1 with L234A and L235A mutations; described in U.S. Pat. No.
  • Table 13 shows the IC 50 values for inhibition of soluble or membrane-bound CD40L-driven human or cyno B cell proliferation for the tested antibodies.
  • Table 14 shows the IC 50 values for inhibition of soluble or membrane-bound CD40L-driven IL-12p40 production by human dendritic cells (DC) and the affinity kinetics for each mAb.
  • mAbs C40B176, C40B179, C40B180 and C40B183 exhibited comparable potency when compared to the best performing benchmark mAb ASKP-1240.
  • Possible undesired agonistic activity of the mAbs was assessed using HEK-BlueTM CD40L NF- ⁇ B activation assay and IL12p40 production by dendritic cells as readouts without cross-linking using experimental protocols as described in Example 1.
  • the parental antibody C40B16 on wild-type IgG1 was evaluated.
  • FIG. 1 shows the dose response curves of the NF- ⁇ B reporter assay.
  • FIG. 2 shows the antibody-mediated IL12p40 production by DCs using antibody concentrations between 350 nM and 1.1 nM.
  • ASKP-1240, CFZ533 and BMS-986090 may exhibit V-region driven agonism as the agonism was demonstrated in effector silent Fc.
  • Agonism was also assessed at 500 ng/ml concentration of antibodies in DC or B-cell agonistic assays, comparing ASKP-1240 and C40B176, C40B179, C40B180 and C40B183. At higher antibody concentrations, ASKP-1240 induced IL-12p40 production by DCs ( FIG. 3A ) and proliferation of B cells ( FIG. 3B ), whereas C40B176, C40B179, C40B180 and C40B183 did not.
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Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EA037882B1 (ru) * 2015-09-30 2021-05-31 Янссен Байотек, Инк. Агонистические антитела, специфически связывающие человеческий cd40, и способы их применения
CA3026880A1 (en) 2016-06-08 2017-12-14 Paul Foster Treatment of igg4-related diseases with anti-cd19 antibodies crossbinding to cd32b
WO2017220988A1 (en) 2016-06-20 2017-12-28 Kymab Limited Multispecific antibodies for immuno-oncology
EP3589324A1 (en) * 2017-03-03 2020-01-08 Janssen Biotech, Inc. Co-therapy comprising a small molecule csf-1r inhibitor and an agonistic antibody that specifically binds cd40 for the treatment of cancer
CN110621697B (zh) 2017-05-25 2023-06-27 百时美施贵宝公司 拮抗性cd40单克隆抗体及其用途
JP2020521458A (ja) * 2017-05-25 2020-07-27 ブリストル−マイヤーズ スクイブ カンパニーＢｒｉｓｔｏｌ−Ｍｙｅｒｓ Ｓｑｕｉｂｂ Ｃｏｍｐａｎｙ 改変ＩｇＧ１ Ｆｃドメインおよび該ドメインと抗ＣＤ４０ドメイン抗体の融合物
AU2018278321A1 (en) * 2017-06-02 2019-11-21 Pfizer Inc. Chimeric antigen receptors targeting FLT3
EA202090791A1 (ru) * 2017-09-19 2020-06-17 Мэб Дискавери Гмбх Агонистические антитела против cd40
KR20200074993A (ko) * 2017-11-03 2020-06-25 노파르티스 아게 쇼그렌 증후군 치료에 사용하기 위한 항-cd40 항체
AR117091A1 (es) 2018-11-19 2021-07-07 Bristol Myers Squibb Co Anticuerpos monoclonales antagonistas contra cd40 y sus usos
WO2020207470A1 (zh) 2019-04-10 2020-10-15 南开大学 抗cd40抗体及其用途
CA3164129A1 (en) 2019-12-20 2021-06-24 Amgen Inc. Mesothelin-targeted cd40 agonistic multispecific antibody constructs for the treatment of solid tumors
WO2021195326A1 (en) * 2020-03-26 2021-09-30 Vanderbilt University Human monoclonal antibodies to severe acute respiratory syndrome coronavirus 2 (sars-cov-2)
WO2022002065A1 (zh) * 2020-06-30 2022-01-06 百奥泰生物制药股份有限公司 抗cd40抗体或抗原结合片段及其应用
US20240109973A1 (en) * 2020-12-16 2024-04-04 Memorial Sloan Kettering Cancer Center Cd40 binding molecules and uses thereof
CN117295764A (zh) * 2021-06-28 2023-12-26 江苏恒瑞医药股份有限公司 抗cd40抗体、其抗原结合片段及医药用途
WO2023009891A2 (en) * 2021-07-30 2023-02-02 Janssen Biotech, Inc. Materials and methods of making or using il-23r binding proteins
WO2023020475A1 (en) * 2021-08-16 2023-02-23 Utc Therapeutics (Shanghai) Co., Ltd. Cd40-targetting antibodies and uses thereof
TW202330600A (zh) * 2021-08-24 2023-08-01 大陸商江蘇恆瑞醫藥股份有限公司 Fap/cd40 結合分子及其醫藥用途

Family Cites Families (122)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US226A (en) 1837-06-03 Samuel goss
US7709A (en) 1850-10-08 Chas S Gaylord Improved spring-grapple
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5247069A (en) 1986-06-13 1993-09-21 Oncogen Ligands and methods for augmenting B-cell proliferation
DE3785186T2 (de) 1986-09-02 1993-07-15 Enzon Lab Inc Bindungsmolekuele mit einzelpolypeptidkette.
US5606040A (en) 1987-10-30 1997-02-25 American Cyanamid Company Antitumor and antibacterial substituted disulfide derivatives prepared from compounds possessing a methyl-trithio group
US5770701A (en) 1987-10-30 1998-06-23 American Cyanamid Company Process for preparing targeted forms of methyltrithio antitumor agents
US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
GB8823869D0 (en) 1988-10-12 1988-11-16 Medical Res Council Production of antibodies
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
IL162181A (en) 1988-12-28 2006-04-10 Pdl Biopharma Inc A method of producing humanized immunoglubulin, and polynucleotides encoding the same
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5427908A (en) 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
US6172197B1 (en) 1991-07-10 2001-01-09 Medical Research Council Methods for producing members of specific binding pairs
US6255458B1 (en) 1990-08-29 2001-07-03 Genpharm International High affinity human antibodies and human antibodies against digoxin
LU91067I2 (fr) 1991-06-14 2004-04-02 Genentech Inc Trastuzumab et ses variantes et dérivés immuno chimiques y compris les immotoxines
ES2136092T3 (es) 1991-09-23 1999-11-16 Medical Res Council Procedimientos para la produccion de anticuerpos humanizados.
WO1993011236A1 (en) 1991-12-02 1993-06-10 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5397703A (en) 1992-07-09 1995-03-14 Cetus Oncology Corporation Method for generation of antibodies to cell surface molecules
EP1834671A3 (en) * 1992-07-09 2013-05-22 Novartis Vaccines and Diagnostics, Inc. A method for generation of antibodies to cell surface molecules
EP1005870B1 (en) 1992-11-13 2009-01-21 Biogen Idec Inc. Therapeutic application of chimeric antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5635483A (en) 1992-12-03 1997-06-03 Arizona Board Of Regents Acting On Behalf Of Arizona State University Tumor inhibiting tetrapeptide bearing modified phenethyl amides
JP3720353B2 (ja) 1992-12-04 2005-11-24 メディカル リサーチ カウンシル 多価および多重特異性の結合タンパク質、それらの製造および使用
US5780588A (en) 1993-01-26 1998-07-14 Arizona Board Of Regents Elucidation and synthesis of selected pentapeptides
ES2211884T3 (es) 1993-10-01 2004-07-16 Immunex Corporation Anticuerpos contra el cd40.
US5773001A (en) 1994-06-03 1998-06-30 American Cyanamid Company Conjugates of methyltrithio antitumor agents and intermediates for their synthesis
AUPO591797A0 (en) 1997-03-27 1997-04-24 Commonwealth Scientific And Industrial Research Organisation High avidity polyvalent and polyspecific reagents
US5714586A (en) 1995-06-07 1998-02-03 American Cyanamid Company Methods for the preparation of monomeric calicheamicin derivative/carrier conjugates
US5712374A (en) 1995-06-07 1998-01-27 American Cyanamid Company Method for the preparation of substantiallly monomeric calicheamicin derivative/carrier conjugates
US6051228A (en) 1998-02-19 2000-04-18 Bristol-Myers Squibb Co. Antibodies against human CD40
US6818749B1 (en) 1998-10-31 2004-11-16 The United States Of America As Represented By The Department Of Health And Human Services Variants of humanized anti carcinoma monoclonal antibody cc49
US6737056B1 (en) 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US6946129B1 (en) 1999-06-08 2005-09-20 Seattle Genetics, Inc. Recombinant anti-CD40 antibody and uses thereof
PT1242438E (pt) 1999-12-29 2007-02-28 Immunogen Inc Agentes citotóxicos compreendendo dixorrubicinas e daunorrubicinas modificadas e seu uso terapêutico
EP1253942A4 (en) 2000-02-01 2004-06-16 Tanox Inc CD40-BINDING AND APC-ACTIVATING MOLECULES
CA2399388A1 (en) 2000-02-11 2001-08-16 Michael J. Lenardo Identification of a domain in the tumor necrosis factor receptor family that mediates pre-ligand receptor assembly and function
WO2001083755A2 (en) 2000-04-28 2001-11-08 La Jolla Institute For Allergy And Immunology Human anti-cd40 antibodies and methods of making and using same
WO2002028481A2 (en) * 2000-10-02 2002-04-11 Chiron Corporation Methods of therapy for b-cell malignancies using antagonist anti-cd40 antibodies
US6596541B2 (en) 2000-10-31 2003-07-22 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
KR100857943B1 (ko) 2000-11-30 2008-09-09 메다렉스, 인코포레이티드 인간 항체의 제조를 위한 형질전환 트랜스염색체 설치류
EP2011802A3 (en) 2001-04-27 2009-04-15 Kyowa Hakko Kirin Co., Ltd. Anti-CD40 monoclonal antibody
EP1283053A1 (en) 2001-08-09 2003-02-12 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Inhibitors of HER3 activity
WO2003029296A1 (en) * 2001-10-02 2003-04-10 Chiron Corporation Human anti-cd40 antibodies
AR039067A1 (es) 2001-11-09 2005-02-09 Pfizer Prod Inc Anticuerpos para cd40
US20040110226A1 (en) 2002-03-01 2004-06-10 Xencor Antibody optimization
US7262012B2 (en) 2002-05-17 2007-08-28 Alligator Bioscience Ab Method for in vitro molecular evolution of protein function using varied exonuclease digestion in two polynucleotide populations
IL149820A0 (en) 2002-05-23 2002-11-10 Curetech Ltd Humanized immunomodulatory monoclonal antibodies for the treatment of neoplastic disease or immunodeficiency
WO2005007800A2 (ja) * 2003-07-18 2005-01-27 Mochida Pharm Co Ltd 抗血小板膜糖蛋白質ⅵモノクローナル抗体
HN2004000285A (es) * 2003-08-04 2006-04-27 Pfizer Prod Inc ANTICUERPOS DIRIGIDOS A c-MET
US20050202531A1 (en) 2003-11-03 2005-09-15 Compugen Ltd. CD40 splice variants, compositions for making and methods of using the same
WO2005044854A2 (en) * 2003-11-04 2005-05-19 Chiron Corporation Antagonist anti-cd40 monoclonal antibodies and methods for their use
KR101128777B1 (ko) * 2003-11-04 2012-04-13 조마 테크놀로지 리미티드 다발성 골수종을 치료하기 위한 길항제 항-ｃｄ４０단클론성 항체의 용도
US8277810B2 (en) * 2003-11-04 2012-10-02 Novartis Vaccines & Diagnostics, Inc. Antagonist anti-CD40 antibodies
SG195524A1 (en) 2003-11-06 2013-12-30 Seattle Genetics Inc Monomethylvaline compounds capable of conjugation to ligands
CA2551008C (en) * 2003-12-25 2013-10-01 Kirin Beer Kabushiki Kaisha Anti-cd40 antibody mutants
RU2006141632A (ru) 2004-04-27 2008-06-10 Новартис Вэксинес Энд Дайэгностикс, Инк. (Us) Антагонистические моноклональные анти-cd40-антитела и способы их применения
EP3342782B1 (en) 2004-07-15 2022-08-17 Xencor, Inc. Optimized fc variants
AU2005282700A1 (en) 2004-09-02 2006-03-16 Genentech, Inc. Heteromultimeric molecules
WO2006081139A2 (en) * 2005-01-26 2006-08-03 Abgenix, Inc. Antibodies against interleukin-1 beta
EP3050963B1 (en) 2005-03-31 2019-09-18 Chugai Seiyaku Kabushiki Kaisha Process for production of polypeptide by regulation of assembly
CA2607147C (en) 2005-05-09 2018-07-17 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1 (pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics
CA2609269C (en) 2005-05-26 2014-08-05 Seattle Genetics, Inc. Humanized anti-cd40 antibodies and their methods of use
DE102005028778A1 (de) 2005-06-22 2006-12-28 SUNJÜT Deutschland GmbH Mehrlagige Folie mit einer Barriere- und einer antistatischen Lage
WO2007053661A2 (en) * 2005-11-01 2007-05-10 Novartis Ag Uses of anti-cd40 antibodies
PT1999154E (pt) 2006-03-24 2013-01-24 Merck Patent Gmbh Domínios proteicos heterodiméricos modificados
BRPI0710826A2 (pt) * 2006-04-21 2011-08-23 Novartis Ag composições farmacêuticas de anticorpo anti-cd40 antagonista
EP1854810A1 (en) 2006-05-09 2007-11-14 PanGenetics B.V. Deimmunized antagonistic anti-human CD40 monoclonal antibody from the ch5D12 antibody
DK2395018T3 (en) * 2006-06-06 2016-04-25 Crucell Holland Bv HUMAN BINDING MOLECULES with killer activity against staphylococci and uses thereof
JP2009541275A (ja) 2006-06-22 2009-11-26 ノボ・ノルデイスク・エー／エス 二重特異性抗体の生産
US20090074711A1 (en) 2006-09-07 2009-03-19 University Of Southhampton Human therapies using chimeric agonistic anti-human cd40 antibody
CN102123712B (zh) * 2006-12-13 2014-03-19 默沙东公司 使用igf1r抑制剂治疗癌症的方法
US20080226635A1 (en) 2006-12-22 2008-09-18 Hans Koll Antibodies against insulin-like growth factor I receptor and uses thereof
NZ579262A (en) * 2007-02-23 2012-04-27 Baylor Res Inst Therapeutic applications of activation of human antigen-presenting cells through dectin-1
WO2008150494A1 (en) 2007-05-30 2008-12-11 Xencor, Inc. Methods and compositions for inhibiting cd32b expressing cells
CN104945508B (zh) 2007-06-18 2019-02-22 默沙东有限责任公司 针对人程序性死亡受体pd-1的抗体
JP2010535032A (ja) 2007-07-31 2010-11-18 メディミューン，エルエルシー 多重特異性エピトープ結合性タンパク質およびその用途
US8748356B2 (en) 2007-10-19 2014-06-10 Janssen Biotech, Inc. Methods for use in human-adapting monoclonal antibodies
AU2008343589A1 (en) 2007-12-19 2009-07-09 Centocor Ortho Biotech Inc. Design and generation of human de novo pIX phage display libraries via fusion to pIX or pVII, vectors, antibodies and methods
US20090162359A1 (en) 2007-12-21 2009-06-25 Christian Klein Bivalent, bispecific antibodies
US9266967B2 (en) 2007-12-21 2016-02-23 Hoffmann-La Roche, Inc. Bivalent, bispecific antibodies
US8242247B2 (en) 2007-12-21 2012-08-14 Hoffmann-La Roche Inc. Bivalent, bispecific antibodies
US8227577B2 (en) 2007-12-21 2012-07-24 Hoffman-La Roche Inc. Bivalent, bispecific antibodies
AU2009206506B2 (en) 2008-01-23 2013-01-10 Xencor, Inc. Optimized CD40 antibodies and methods of using the same
US8008445B2 (en) 2008-03-03 2011-08-30 Dyax Corp. Metalloproteinase 9 binding proteins
US20100261620A1 (en) 2008-10-14 2010-10-14 Juan Carlos Almagro Methods of Humanizing and Affinity-Maturing Antibodies
CN103415534A (zh) 2009-03-10 2013-11-27 贝勒研究院 靶向抗原呈递细胞的癌症疫苗
CA2759146C (en) 2009-04-20 2017-06-13 Kyowa Hakko Kirin Co., Ltd. Agonist anti-cd40 antibody
CA2759233C (en) 2009-04-27 2019-07-16 Oncomed Pharmaceuticals, Inc. Method for making heteromultimeric molecules
WO2011021146A1 (en) * 2009-08-20 2011-02-24 Pfizer Inc. Osteopontin antibodies
JP5998060B2 (ja) 2010-03-04 2016-09-28 マクロジェニクス，インコーポレーテッド Ｂ７−ｈ３と反応性のある抗体、その免疫学的に活性なフラグメントおよびその使用
RS60612B1 (sr) * 2010-03-31 2020-08-31 Boehringer Ingelheim Int Anti-cd40 antitela
MX352929B (es) 2010-11-05 2017-12-13 Zymeworks Inc DISEÑO DE ANTICUERPOS HETERODIMÉRICOS ESTABLES CON MUTACIONES EN EL DOMINIO Fc.
AR083847A1 (es) * 2010-11-15 2013-03-27 Novartis Ag Variantes de fc (fragmento constante) silenciosas de los anticuerpos anti-cd40
CA3167037A1 (en) * 2010-12-20 2012-06-28 The Rockefeller University Modulating agonistic tnfr antibodies
HUE033205T2 (en) 2010-12-23 2017-11-28 Janssen Biotech Inc Active, protease-resistant antibody-FC mutant
CN106279401A (zh) 2011-03-11 2017-01-04 贝丝以色列女执事医疗中心 Cd40片段及其用途
CA2833636A1 (en) 2011-04-20 2012-10-26 Amplimmune, Inc. Antibodies and other molecules that bind b7-h1 and pd-1
SG194701A1 (en) 2011-04-29 2013-12-30 Apexigen Inc Anti-cd40 antibodies and methods of use
GB201115280D0 (en) 2011-09-05 2011-10-19 Alligator Bioscience Ab Antibodies, uses and methods
SG10201610288TA (en) * 2011-10-13 2017-02-27 Bristol Myers Squibb Co Antibody polypeptides that antagonize cd40l
WO2013063702A1 (en) 2011-11-04 2013-05-10 Zymeworks Inc. Stable heterodimeric antibody design with mutations in the fc domain
UY34487A (es) * 2011-12-05 2013-07-31 Novartis Ag Anticuerpos para receptor de factor de crecimiento epidérmico 3(her3)
WO2013124297A1 (en) * 2012-02-22 2013-08-29 U3 Pharma Gmbh Combination of hb-egf binding protein and egfr inhibitor
MY175224A (en) 2012-03-15 2020-06-16 Janssen Biotech Inc Human anti-cd27 antibodies, methods, and uses
US8781132B2 (en) * 2012-03-19 2014-07-15 Motorola Solutions, Inc. Method and device for managing encrypted group rekeying in a radio network link layer encryption system
EP2878166A1 (en) 2012-07-24 2015-06-03 Telefonaktiebolaget L M Ericsson (Publ) Apparatus and method for dynamically selecting a random access response window value for use with random access procedures in a network
DK2889377T3 (da) 2012-08-24 2020-03-30 Chugai Pharmaceutical Co Ltd Fc?RIIb-Specifik Fc-regionsvariant
JP6693745B2 (ja) * 2012-10-30 2020-05-13 アペクシジェン， インコーポレイテッド 抗ｃｄ４０抗体および使用方法
WO2014078866A2 (en) * 2012-11-19 2014-05-22 Xencor, Inc. Engineered immunoglobulins with extended in vivo half-life
PT3653049T (pt) 2012-12-14 2023-11-14 Omniab Inc Polinucleótidos que codificam anticorpos de roedores com idiótipos humanos e animais que os compreendem
US10329350B2 (en) * 2012-12-26 2019-06-25 Industrial Technology Research Institute Method for producing a multivalent fab fragment with collagen-like peptide
EA201500741A1 (ru) 2013-01-10 2016-01-29 Генмаб Б.В. ВАРИАНТЫ Fc-ОБЛАСТИ IGG1 ЧЕЛОВЕКА И ИХ ПРИМЕНЕНИЕ
WO2014126254A1 (ja) * 2013-02-18 2014-08-21 協和発酵キリン株式会社 膜結合型タンパク質発現ベクター
CA2913977C (en) 2013-05-31 2022-11-29 Sorrento Therapeutics, Inc. Antigen binding proteins that bind pd-1
GB201322583D0 (en) 2013-12-19 2014-02-05 Alligator Bioscience Ab Antibodies
WO2015134988A1 (en) * 2014-03-07 2015-09-11 Bristol-Myers Squibb Company Method of using antibody polypeptides that antagonize cd40 to treat ibd
WO2015133882A1 (ko) * 2014-03-07 2015-09-11 사회복지법인 삼성생명공익재단 ScFv 항체 라이브러리, 이의 제조방법 및 이를 이용한 ScFv 항체 스크리닝 방법
US20170233485A1 (en) 2014-08-18 2017-08-17 Biogen Ma Inc. Anti-cd40 antibodies and uses thereof
EA037882B1 (ru) * 2015-09-30 2021-05-31 Янссен Байотек, Инк. Агонистические антитела, специфически связывающие человеческий cd40, и способы их применения

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