WO2011123428A1 - Antibody-based depletion of antigen-presenting cells and dendritic cells - Google Patents
Antibody-based depletion of antigen-presenting cells and dendritic cells Download PDFInfo
- Publication number
- WO2011123428A1 WO2011123428A1 PCT/US2011/030294 US2011030294W WO2011123428A1 WO 2011123428 A1 WO2011123428 A1 WO 2011123428A1 US 2011030294 W US2011030294 W US 2011030294W WO 2011123428 A1 WO2011123428 A1 WO 2011123428A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- seq
- cells
- fragment
- hla
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/4965—Non-condensed pyrazines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/50—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
- 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
- A61K47/68—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
- 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
- A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/50—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
- 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
- A61K47/68—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
- 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
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/50—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
- 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
- A61K47/68—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
- 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
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6815—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/50—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
- 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
- A61K47/68—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
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/50—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
- 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
- A61K47/68—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
- 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
- A61K47/6881—Cluster-antibody conjugates, i.e. the modifying agent consists of a plurality of antibodies covalently linked to each other or of different antigen-binding fragments covalently linked to each other
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/50—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
- 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
- A61K47/68—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
- 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
- A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
- A61K47/6885—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy the conjugate or the polymer being a starburst, a dendrimer, a cascade
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2851—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 lectin superfamily, e.g. CD23, CD72
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3007—Carcino-embryonic Antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
- C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/522—CH1 domain
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/734—Complement-dependent cytotoxicity [CDC]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/75—Agonist effect on antigen
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
- C12N2310/3513—Protein; Peptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention concerns compositions and methods of use of antibodies, antibody fragments, immunoconjugates and/or other targeting molecules for treatment of immune dysfunction diseases, including but not limited to graft- versus-host disease (GVHD) and organ transplant rejection.
- the compositions and methods relate to use of anti-CD74 and/or anti-HLA-DR antibodies, immunoconjugates or fragments thereof to deplete antigen-presenting cells (APCs), such as dendritic cells (DCs).
- APCs antigen-presenting cells
- DCs dendritic cells
- administration of the therapeutic compositions results in significant depletion of myeloid DCs type 1 (mDCl) and type 2 (mDC2) and mild depletion of B cells, without significant depletion of plasmacytoid DCs (pDCs), monocytes or T cells.
- mDCl myeloid DCs type 1
- mDC2 type 2
- B cells without significant depletion of plasmacytoid DCs (pDCs), monocytes or T cells.
- administration of the therapeutic compositions depletes all subsets of APCs, including mDCs, pDCs, B cells and monocytes, without significant depletion of T cells.
- administration of the therapeutic compositions suppresses proliferation of allo- reactive T cells, while preserving cytomegalovirus (CMV)-specific, CD8 + memory T cells.
- CMV cytomegalovirus
- the compositions and methods provide a novel conditioning regimen for maximally preventing acute graft-versus-host disease (aGVHD) without altering preexisting anti-viral immunity.
- Allogeneic hematopoietic stem cell transplantation is a curative therapy for many hematological malignancies, but is frequently followed by aGVHD, the leading cause of mortality and morbidity in allo-HSCT patients (Socie & Blazar, Blood 114, 4327- 4336, 2009).
- the major initiator of aGVHD is host antigen-presenting cells (APCs) that are residual after preparative conditioning (Shlomchik et al. Science 285:412-415, 1999;
- GVHD Despite the use of non-myeloablative or reduced-intensity conditioning regimens, GVHD remains a major and life-threatening complication for allo-HSCT (Landfried, et al. Curr Opin Oncol 21 :S39-S41, 2009). It is well documented that among residual host APCs the critical subset for initiating aGVHD is dendritic cells (DCs) (Duffher et al. J Immunol 172:7393-7398, 2004; Durakovic et al. J Immunol 177:4414-4425, 2006).
- DCs dendritic cells
- Either host myeloid DCs (mDCs) or plasmacytoid DCs (pDCs) alone are sufficient to induce GVHD (Koyama et al. Blood 1 13:2088-2095, 2009).
- Donor APCs, especially mDCs also contribute to the development of GVHD (Matte et al. Nat Med 10:987-992, 2004; Markey et al. Blood 113:5644-5649, 2009).
- Depletion of DCs has been an effective approach to reduce or abrogate GVHD (Merad et al. Nat Med 10:510-517, 2004; Zhang et al. J Immunol 169:71 11- 8, 2002; Wilson et al. J Exp Med 206:387-398, 2009).
- B cells and monocytes are two other major subsets of circulating APCs.
- B cells are involved in the pathogenesis of acute and chronic GVHD (Shimabukuro-Vornhagen et al. Blood 1 14:4919-4927, 2009), and that B-cell depleting therapy is effective in prevention and treatment of GVHD (Alousi et al. Leuk Lymphoma 51 :376-389, 2010).
- the anti-CD20 antibody, rituximab when included in the conditioning regimen, reduces the incidence of aGVHD (Christopeit et al. Blood
- Monocytes may also be involved in the pathogenesis of GVHD, since higher numbers of blood monocytes before conditioning are associated with greater risk of aGVHD (Arpinati et al. Biol Blood Marrow Transplant 13:228-234, 2007).
- the proteosome inhibitor, bortezomib which efficiently depletes monocytes (Arpinati et al. Bone Marrow Transplant 43:253-259, 2009), is active in controlling acute and chronic GVHD (Sun et al. Proc Natl Acad Sci USA 101 :8120-8125, 2004).
- the present invention concerns improved compositions and methods of use of antibodies against APCs in general and DCs in particular for the treatment of aGVHD.
- a variety of antigens associated with dendritic cells are known in the art, including but not limited to CD209 (DC-SIGN), CD34, CD74, CD205, TLR 2 (toll-like receptor 2), TLR 4, TLR 7, TLR 9, BDCA-2, BDCA-3, BDCA-4, and HLA-DR.
- CD209 DC-SIGN
- CD34 CD74
- CD205 toll-like receptor 2
- TLR 4 toll-like receptor 2
- TLR 4 toll-like receptor 2
- TLR 4 TLR 7 TLR 9
- BDCA-2 BDCA-3
- BDCA-4 BDCA-4
- HLA-DR HLA-DR
- the antibodies or fragments thereof of use are targeted to CD74 or HLA-DR, the skilled artisan will realize that antibodies against other DC-associated antigens can be used within the scope of the claimed
- Antibodies against CD74 and HLA-DR include the anti-CD74 hLLl antibody (milatuzumab) and the anti-HLA-DR antibody hL243 (also known as IMMU-1 14) (Berkova et al., 2010, Expert Opin. Investig. Drugs 19:141-49; Burton et al, 2004, Clin Cancer Res 10:6605-11; Chang et al., 2005, Blood 106:4308-14; Griffiths et al., 2003, Clin Cancer Res 9:6567-71 ; Stein et al., 2007, Clin Cancer Res 13:5556s-63s; Stein et al., 2010, Blood 115:5180-90).
- the anti-CD74 antibody is an hLLl antibody (also known as milatuzumab) that comprises the light chain complementarity-determining region (CDR) sequences CDRl (RSSQSLVHRNGNTYLH; SEQ ID NO:l), CDR2 (TVSNRFS; SEQ ID NO:2), and CDR3 (SQSSHVPPT; SEQ ID NO:3) and the heavy chain variable region CDR sequences CDRl (NYGVN; SEQ ID NO:4), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO: 5), and CDR3 (SRGKNEAWFAY; SEQ ID NO:6).
- CDR light chain complementarity-determining region
- a humanized LL1 (hLLl) anti-CD74 antibody suitable for use is disclosed in U.S. Patent No. 7,312,318, incorporated herein by reference from Col. 35, line 1 through Col. 42, line 27 and FIG. 1 through FIG. 4.
- other known and/or commercially available anti-CD74 antibodies may be utilized, such as LS-B1963, LS- B2594, LS-B1859, LS-B2598, LS-C5525, LS-C44929, etc. (LSBio, Seattle, WA); LN2 (BIOLEGEND®, San Diego, CA); ⁇ .1, SPM523, LN3, CerCLIP.l (ABCAM®,
- the anti-CD74 antibody may be selected such that it competes with or blocks binding to CD74 of an LL1 antibody comprising the light chain CDR sequences CDRl
- the anti-CD74 antibody may bind to the same epitope of CD74 as an LL1 antibody.
- anti-HLA-DR antibodies are also known in the art and any such known antibody or fragment thereof may be utilized.
- the anti- HLA-DR antibody is an hL243 antibody (also known as IMMU-114) that comprises the heavy chain CDR sequences CDRl (NYGMN, SEQ ID NO:7), CDR2
- a humanized L243 anti-HLA-DR antibody suitable for use is disclosed in U.S. Patent No. 7,612,180, incorporated herein by reference from Col. 46, line 45 through Col. 60, line 50 and FIG. 1 through FIG. 6.
- anti- HLA-DR antibodies such as 1D10 (apolizumab) (Kostelny et al., 2001, Int J Cancer 93:556-65); MS-GPC-1, MS-GPC-6, MS-GPC-8, MS-GPC-10, etc. (U.S. Patent No.
- the anti-HLA-DR antibody may be selected such that it competes with or blocks binding to HLA-DR of an L243 antibody comprising the heavy chain CDR sequences CDRl (NYGMN, SEQ ID NO:7), CDR2 (WINTYTREPTYADDFKG, SEQ ID NO:8), and CDR3 (DITAVVPTGFDY, SEQ ID NO:9) and the light chain CDR sequences CDRl
- the anti- HLA-DR antibody may bind to the same epitope of HLA-DR as an L243 antibody.
- anti-CD74 and/or anti-HLA-DR antibodies or fragments thereof may be used as naked antibodies, alone or in combination with one or more therapeutic agents.
- the antibodies or fragments may be utilized as immunoconjugates, attached to one or more therapeutic agents.
- immunoconjugates see, e.g., U.S. Patent Nos. 4,699,784; 4,824,659; 5,525,338; 5,677,427; 5,697,902; 5,716,595; 6,071 ,490; 6,187,284; 6,306,393; 6,548,275; 6,653,104; 6,962,702; 7,033,572; 7,147,856; and
- Therapeutic agents may be selected from the group consisting of a radionuclide, a cytotoxin, a
- chemotherapeutic agent a drug, a pro-drug, a toxin, an enzyme, an immunomodulator, an anti-angiogenic agent, a pro-apoptotic agent, a cytokine, a hormone, an oligonucleotide molecule (e.g., an antisense molecule or a gene) or a second antibody or fragment thereof.
- the therapeutic agent may be selected from the group consisting of aplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib, bryostatin-1 , busulfan, calicheamycin, camptothecin, 10-hydroxycamptothecin, carmustine, Celebrex, chlorambucil, cisplatin, irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin,
- the therapeutic agent may comprise a radionuclide selected from the group consisting of 103m Rh, 103 Ru, 105 Rh, 105 Ru, 107 Hg, 109 Pd, 109 Pt, u l Ag, n , In, 1 13m In, 119 Sb, U C, 121m Te, 122m Te, 125 I, 125m Te, 126 I, 131 1, 133 1, 13 N, 142 Pr, 143 Pr, ,49 Pm, 152 Dy, 153 Sm, 15 0, 16 1Ho, ,61 Tb, 165 Tm, l66 Dy, 166 Ho, 167 Tm, 168 Tm, 169 Er, 169 Yb, 177 Lu, 186 Re, ,88 Re, 189m Os, 189 Re, 192 Ir, 194 Ir, 197 Pt, 198 Au, 199 Au, 01 T1, 203 Hg, 211 At, 211 Bi, 2, 1 Pb, 212 Bi, 212 P
- the therapeutic agent may be an enzyme selected from the group consisting of malate dehydrogenase, staphylococcal nuclease, delta- V-steroid isomerase, yeast alcohol
- dehydrogenase alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
- An immunomodulator of use may be selected from the group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and combinations thereof.
- Exemplary immunomodulators may include IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL- 21 , interferon-a, interferon- ⁇ , interferon- ⁇ , G-CSF, GM-CSF, and mixtures thereof.
- anti-angiogenic agents may include angiostatin, endostatin, basculostatin, canstatin, maspin, anti-VEGF binding molecules, anti-placental growth factor binding molecules, or anti-vascular growth factor binding molecules.
- the antibody or fragment may comprise one or more chelating moieties, such as NOTA, DOT A, DTP A, TETA, Tscg-Cys, or Tsca-Cys.
- the chelating moiety may form a complex with a therapeutic or diagnostic cation, such as Group II, Group III, Group IV, Group V, transition, lanthanide or actinide metal cations, Tc, Re, Bi, Cu, As, Ag, Au, At, or Pb.
- the antibody or fragment thereof may be a human, chimeric, or humanized antibody or fragment thereof.
- a humanized antibody or fragment thereof may comprise the complementarity-determining regions (CDRs) of a murine antibody and the constant and framework (FR) region sequences of a human antibody, which may be substituted with at least one amino acid from corresponding FRs of a murine antibody.
- a chimeric antibody or fragment thereof may include the light and heavy chain variable regions of a murine antibody, attached to human antibody constant regions.
- the antibody or fragment thereof may include human constant regions of IgGl, IgG2a, IgG3, or IgG4.
- the anti-CD74 and/or anti-HLA-DR complex may be formed by a technique known as dock-and-lock (DNL) (see, e.g., U.S. Patent Nos.
- DNL dock-and-lock
- the DNL technique takes advantage of the specific and high-affinity binding interaction between a dimerization and docking domain (DDD) sequence derived from the regulatory subunit of human cAMP -dependent protein kinase (PKA) and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins.
- DDD dimerization and docking domain
- PKA human cAMP -dependent protein kinase
- AD anchor domain
- the DDD and AD peptides may be attached to any protein, peptide or other molecule.
- the DNL technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences.
- the standard DNL complex comprises a trimer with two DDD-linked molecules attached to one AD-linked molecule
- variations in complex structure allow the formation of dimers, trimers, tetramers, pentamers, hexamers and other multimers.
- the DNL complex may comprise two or more antibodies, antibody fragments or fusion proteins which bind to the same antigenic determinant or to two or more different antigens.
- the DNL complex may also comprise one or more other effectors, such as a cytokine or PEG moiety.
- a method for treating and/or diagnosing a disease or disorder that includes administering to a patient a therapeutic and/or diagnostic composition that includes any of the aforementioned antibodies or fragments thereof.
- the composition is administered to the patient intravenously, intramuscularly or subcutaneously at a dose of 20- 5000 mg.
- the disease or disorder is an immune dysregulation disease, an autoimmune disease, organ-graft rejection or graft- versus-host disease. More preferably, the disease is aGVHD.
- Exemplary autoimmune diseases include acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thrombo
- granulomatosis membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, or fibrosing alveolitis.
- administration of the anti-CD74 and/or anti- HLA-DR antibodies or fragments thereof can deplete all subsets of APCs, but not T cells, from human peripheral blood mononuclear cells (PBMCs), including myeloid DCs (mDCs), plasmacytoid DCs (pDCs), B cells, and monocytes.
- PBMCs peripheral blood mononuclear cells
- mDCs myeloid DCs
- pDCs plasmacytoid DCs
- B cells and monocytes.
- monocytes preferably, the antibodies or fragments suppress the proliferation of allo-reactive T cells in mixed leukocyte cultures while preserving CMV-specific, CD8 + memory T cells.
- FIG. 1 Milatuzumab, but not its Fab fragment fusion protein, selectively depletes myeloid DCs in human PBMCs.
- Human PBMCs were incubated with 5 ⁇ / ⁇ milatuzumab, control antibodies, or medium only, for 3 days. The effect of each treatment on APC subsets was evaluated by co-staining the cells with PE-labeled anti-CD 14 and anti- CD 19, in combination with APC-labeled anti-BDCA-1, for analysis of mDCl, or a mixture of FITC-labeled anti-BDCA-2 and APC-labeled anti-BDCA-3 for simultaneous analysis of mDC2 and pDCs, respectively. 7-AAD was added before flow cytometric analyses.
- PBMCs were gated to exclude the debris and dead cells on the basis of their forward and side scatter characteristics. The subpopulations of PBMCs were gated as follows: monocytes,
- FIG. 2 Milatuzumab does not alter CD86 expression on APC subsets, or IFN- ⁇ primed, LPS-stimulated, IL-12 production by PBMCs.
- PBMCs were incubated with PBS, hMN-14, or milatuzumab, and stimulated with IFN- ⁇ (100 ng/ml) for 18 h, followed by LPS (10 ⁇ g/ml) for an additional 24 h.
- the cells and the supernatants were collected for assessment of CD86 expression (FIG. 2A) and IL-12 production (FIG. 2B), respectively.
- the cells were stained with PE-conjugated anti-CD 19 and anti-CD 14, APC-conjugated anti- BDCA-1, and Alexa Fluor 488-conjugated anti-CD86 antibodies.
- B cells, monocytes, mDCl , and non-B lymphocytes were gated according to the same strategy as described in the legend to FIG. 1.
- Data are shown as the means ⁇ SD of the geo-mean fluorescence intensity of CD86 expression in different cell subsets, in triplicates from two donors.
- the IL-12 concentration in the supernatants was measured by ELISA, and the data are shown as the means ⁇ SD of the OD4 5 onm in triplicates from two donors.
- FIG. 3 Milatuzumab reduces T-cell proliferation in allo-MLR.
- CFSE-labeled PBMCs from two different donors were mixed and incubated with different antibodies at 5 ⁇ g/ml for 11 days, and the cells were harvested and analyzed by flow cytometry. The proliferating cells were quantitated by measuring the CFSE low cell frequencies.
- FIG. 3A Representative data from one combination of stimulator/responder PBMCs are shown in (FIG. 3A), and the statistical analysis of all combinations is shown in (FIG. 3B).
- Error bars, SD, n 10 stimulator/responder combinations. **, P ⁇ .05; and *** P ⁇ .01 vs. hMN-14. **, P ⁇ .05 vs. hLLl.
- FIG. 4 Anti-HLA antibody IMMU-114 depletes all subsets of human PBMCs.
- Human PBMCs were incubated with 5 ⁇ g/ml IMMU-114, control antibodies (hMN-14 and rituximab), or medium only, for 3 days.
- the effect of each treatment on APC subsets was evaluated by co-staining the cells with PE-labeled anti-CD 14 and anti-CD 19, in combination with APC-labeled anti-BDCA-1 or anti-BDCA-2, for analysis of mDCl and pDCs, respectively; or a mixture of FITC-labeled anti-BDCA-2 and APC-labeled anti-BDCA-3 for analysis of mDC2.
- 7-AAD was added before flow cytometric analyses.
- PBMCs were gated to exclude debris and dead cells on the basis of their forward and side scatter characteristics.
- the subpopulations of PBMCs were gated as follows: monocytes, CD14 + SSC medium ; B cells, CD19 + SSC
- FIG. 5 IMMU-114 is cytotoxic to purified mDCl, mDC2, or pDCs.
- mDCl, mDC2, and pDCs were isolated from human PBMCs using magnetic beads, and treated for 2 days with IMMU-114 or control antibody hMN-14, followed by 7-AAD staining for flow cytometry analysis of cell viability of mDCl (FIG. 5 A), pDCs (FIG. 5B), and mDC2 (FIG. 5C).
- the numbers represent the percentages of live cells in the acquired total events. Data shown are representative of 2 donors.
- FIG. 6 IMMU-114 reduces T-cell proliferation in allo-MLR cultures.
- FIG. 7 hL243, but not hLLl, depletes plasmacytoid DCs (pDC) in human PBMCs.
- Human PBMCs were incubated with different antibodies or control at 5 ⁇ g/ml, in the absence or presence of GM-CSF (280 U/ml) and IL-3 (5 ng/ml). 3 days later, the cells were stained with APC-labeled BDCA-2 antibody and PerCp-labeled HLA-DR antibody.
- pDCs were defined as BDCA-2+ cells.
- FIG. 7 A Effect of hL243 on BDCA-2 + cells in PBMCs in the absence of GM/CSF/IL-3.
- Y-axis shows BDCA-2 + cells in PBMCs (%).
- FIG. 7B Effect of hL243 on HLA-DR + /BDCA-2 + cells in PBMCs in the absence of GM/CSF/IL-3.
- Y-axis shows HLA-DR + /BDCA-2 + cells in PBMCs (%).
- FIG. 7C Effect of hL243 on BDCA-2 + cells in PBMCs in the presence of GM/CSF/IL-3.
- Y-axis shows BDCA- 2 + cells in PBMCs (%).
- an "antibody” refers to a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., antigen-binding) portion of an immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., antigen-binding) portion of an immunoglobulin molecule.
- immunologically active i.e., antigen-binding
- immunoglobulin molecule like an antibody fragment.
- an "antibody fragment” is a portion of an antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, single domain antibodies (DABs or VHHs) and the like, including half-molecules of IgG4 (van der Neut Kolfschoten et al. (Science 2007; 317(14 Sept): 1554-1557).
- an antibody fragment binds with the same antigen that is recognized by the intact antibody.
- an anti-CD74 antibody fragment binds with an epitope of CD74.
- the term "antibody fragment” also includes isolated fragments consisting of the variable regions, such as the "Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("scFv proteins").
- a "chimeric antibody” is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule are derived from those of a human antibody.
- the constant domains of the chimeric antibody may be derived from that of other species, such as a cat or dog.
- a “humanized antibody” is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains.
- the constant domains of the antibody molecule are derived from those of a human antibody.
- a "human antibody” is, for example, an antibody obtained from transgenic mice that have been genetically engineered to produce human antibodies in response to antigenic challenge.
- elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
- the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).
- a fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. (See, e.g., McCafferty et al., Nature 348:552-553 (1990) for the production of human antibodies and fragments thereof in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors).
- antibody variable domain genes are cloned in- frame into either a major or minor coat protein gene of a filamentous bacteriophage, and displayed as functional antibody fragments on the surface of the phage particle.
- the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B cell.
- Phage display can be performed in a variety of formats, for their review, see, e.g. Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993). Human antibodies may also be generated by in vitro activated B cells. (See, U.S. Pat. Nos. 5,567,610 and 5,229,275).
- a "therapeutic agent” is an atom, molecule, or compound that is useful in the treatment of a disease.
- therapeutic agents include but are not limited to antibodies, antibody fragments, drugs, toxins, enzymes, nucleases, hormones,
- immunomodulators antisense oligonucleotides, chelators, boron compounds, photoactive agents, dyes and radioisotopes.
- a "diagnostic agent” is an atom, molecule, or compound that is useful in diagnosing a disease.
- useful diagnostic agents include, but are not limited to, radioisotopes, dyes, contrast agents, fluorescent compounds or molecules and enhancing agents (e.g., paramagnetic ions).
- the diagnostic agents are selected from the group consisting of radioisotopes, enhancing agents, and fluorescent compounds.
- an “immunoconjugate” is a conjugate of an antibody, antibody fragment, antibody fusion protein, bispecific antibody or multispecific antibody with an atom, molecule, or a higher-ordered structure (e.g., with a carrier, a therapeutic agent, or a diagnostic agent).
- a “naked antibody” is an antibody that is not conjugated to any other agent.
- antibody fusion protein is a recombinantly produced antigen-binding molecule in which an antibody or antibody fragment is linked to another protein or peptide, such as the same or different antibody or antibody fragment or a DDD or AD peptide.
- the fusion protein may comprise a single antibody component, a multivalent or multispecific combination of different antibody components or multiple copies of the same antibody component.
- the fusion protein may additionally comprise an antibody or an antibody fragment and a therapeutic agent. Examples of therapeutic agents suitable for such fusion proteins include immunomodulators and toxins.
- One preferred toxin comprises a ribonuclease (RNase), preferably a recombinant RNase.
- a “multispecific antibody” is an antibody that can bind simultaneously to at least two targets that are of different structure, e.g., two different antigens, two different epitopes on the same antigen, or a hapten and/or an antigen or epitope.
- a “multivalent antibody” is an antibody that can bind simultaneously to at least two targets that are of the same or different structure. Valency indicates how many binding arms or sites the antibody has to a single antigen or epitope; i.e., monovalent, bivalent, trivalent or multivalent. The multivalency of the antibody means that it can take advantage of multiple interactions in binding to an antigen, thus increasing the avidity of binding to the antigen.
- Specificity indicates how many antigens or epitopes an antibody is able to bind; i.e., monospecific, bispecific, trispecific, multispecific.
- a natural antibody e.g., an IgG
- Multispecific, multivalent antibodies are constructs that have more than one binding site of different specificity. For example, a diabody, where one binding site reacts with one antigen and the other with another antigen.
- bispecific antibody is an antibody that can bind simultaneously to two targets which are of different structure.
- Bispecific antibodies bsAb and bispecific antibody fragments (bsFab) may have at least one arm that specifically binds to, for example, an APC and/or DC antigen or epitope and at least one other arm that binds to a different antigen or epitope.
- the second arm may bind to a different APC or DC antigen or it may bind to a targetable conjugate that bears a therapeutic or diagnostic agent.
- a variety of bispecific antibodies can be produced using molecular engineering.
- the CD74 antigen is an epitope of the major histocompatibility complex (MHC) class II antigen invariant chain, Ii, present on the cell surface and taken up in large amounts of up to 8xl0 6 molecules per cell per day (Hansen et al, 1996, Biochem. J., 320: 293-300). CD74 is present on the cell surface of B-lymphocytes, monocytes and histocytes, human B- lymphoma cell lines, melanomas, T-cell lymphomas and a variety of other tumor cell types. (Hansen et al., 1996, Biochem.
- MHC major histocompatibility complex
- CD74 associates with ⁇ / ⁇ chain MHC II heterodimers to form MHC II ⁇ complexes that are involved in antigen processing and presentation to T cells (Dixon et al., 2006, Biochemistry 45:5228-34; Loss et al., 1993, J Immunol 150:3187-97; Cresswell et al., 1996; Cell 84:505-7).
- CD74 plays an important role in cell proliferation and survival. Binding of the CD74 ligand, macrophage migration inhibitory factor (MIF), to CD74 activates the MAP kinase cascade and promotes cell proliferation (Leng et al., 2003, J Exp Med 197:1467-76). Binding of MIF to CD74 also enhances cell survival through activation of NF- ⁇ and Bcl-2 (Lantner et al., 2007, Blood 110:4303-11).
- MIF macrophage migration inhibitory factor
- milatuzumab a humanized anti-CD74 antibody
- mDCl myeloid DC type 1
- mDC2 type 2
- pDCs plasmacytoid DCs
- monocytes monocytes
- T cells human peripheral blood mononuclear cells
- milatuzumab linked to an irrelevant protein domain and by the failure of milatuzumab to kill purified mDCl or mDC2 in the absence of PBMCs.
- Milatuzumab suppressed allogenic T- cell proliferation in mixed leukocyte cultures, but preserved CMV-specific CD8 + T cells.
- HLA-DR human leukocyte antigen-DR
- MHC major histocompatibility complex
- HLA-DR molecules The most widely recognized function of HLA molecules is the presentation of antigen in the form of short peptides to the antigen receptor of T lymphocytes.
- signals delivered via HLA-DR molecules contribute to the functioning of the immune system by up- regulating the activity of adhesion molecules, inducing T-cell antigen counterreceptors, and initiating the synthesis of cytokines.
- humanized anti-HLA-DR antibody can deplete all subsets of APCs, but not T cells, from human peripheral blood mononuclear cells (PBMCs), including myeloid DCs (mDCs), plasmacytoid DCs (pDCs), B cells, and monocytes.
- PBMCs peripheral blood mononuclear cells
- mDCs myeloid DCs
- pDCs plasmacytoid DCs
- B cells B cells
- monocytes monocytes.
- purified mDCs or pDCs were still killed efficiently by IMMU- 114, suggesting that IMMU-114 depletes these APCs in PBMCs independently of antibody- dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).
- ADCC antibody- dependent cellular cytotoxicity
- CDC complement-dependent cytotoxicity
- IMMU-114 suppressed the proliferation of allo-reactive T cells in mixed leukocyte cultures, yet preserved CMV-specific, CD8 + memory T cells. Together, these results support the use of IMMU-114 as a novel conditioning regimen for maximally preventing aGVHD without altering preexisting anti-viral immunity.
- the immunoconjugates and compositions described herein may include monoclonal antibodies.
- Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art. (See, e.g., Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1- 2.6.7 (John Wiley & Sons 1991)).
- This publication also provides the nucleotide sequences of the LL2 light and heavy chain variable regions, V k and VH, respectively.
- Techniques for producing humanized antibodies are disclosed, for example, by Jones et al., Nature 321 : 522 (1986), Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993).
- a chimeric antibody is a recombinant protein that contains the variable domains including the CDRs derived from one species of animal, such as a rodent antibody, while the remainder of the antibody molecule; i.e., the constant domains, is derived from a human antibody. Accordingly, a chimeric monoclonal antibody can also be humanized by replacing the sequences of the murine FR in the variable domains of the chimeric antibody with one or more different human FR. Specifically, mouse CDRs are transferred from heavy and light variable chains of the mouse immunoglobulin into the corresponding variable domains of a human antibody.
- a fully human antibody can be obtained from a transgenic non-human animal.
- a transgenic non-human animal See, e.g., Mendez et al., Nature Genetics, 15: 146-156, 1997; U.S. Pat. No. 5,633,425.
- Methods for producing fully human antibodies using either combinatorial approaches or transgenic animals transformed with human immunoglobulin loci are known in the art (e.g., Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb. Chem. High
- the phage display technique may be used to generate human antibodies ⁇ e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res. 4:126-40, incorporated herein by reference).
- Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as an immune dysfunction disease (Dantas- Barbosa et al., 2005).
- the advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
- RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97). Library construction was performed according to Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual, Barbas et al.
- Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to make the phage display library.
- libraries may be screened by standard phage display methods. The skilled artisan will realize that this technique is exemplary only and any known method for making and screening human antibodies or antibody fragments by phage display may be utilized.
- transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols as discussed above.
- Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).
- a non-limiting example of such a system is the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods 231 :11-23, incorporated herein by reference) from Abgenix (Fremont, CA).
- the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
- the XENOMOUSE® was transformed with germline-configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Ig kappa loci, including the majority of the variable region sequences, along accessory genes and regulatory sequences.
- the human variable region repertoire may be used to generate antibody producing B cells, which may be processed into hybridomas by known techniques.
- XENOMOUSE® immunized with a target antigen will produce human antibodies by the normal immune response, which may be harvested and/or produced by standard techniques discussed above.
- a variety of strains of XENOMOUSE® are available, each of which is capable of producing a different class of antibody.
- Transgenically produced human antibodies have been shown to have therapeutic potential, while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999).
- the skilled artisan will realize that the claimed compositions and methods are not limited to use of the XENOMOUSE® system but may utilize any transgenic animal that has been genetically engineered to produce human antibodies.
- the claimed methods and compositions may utilize any of a variety of antibodies known in the art.
- Antibodies of use may be commercially obtained from a number of known sources.
- a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA).
- ATCC American Type Culture Collection
- VA Manassas
- a large number of antibodies against various disease targets have been deposited at the ATCC and/or have published variable region sequences and are available for use in the claimed methods and compositions. See, e.g., U.S. Patent Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403;
- Exemplary known antibodies include, but are not limited to, hPAM4 (U.S. Patent No. 7,282,567), hA20 (U.S. Patent No. 7,251,164), hA19 (U.S. Patent No. 7,109,304), hIMMU31 (U.S. Patent No. 7,300,655), hLLl (U.S. Patent No. 7,312,318, ), hLL2 (U.S. Patent No. 7,074,403), hMu-9 (U.S. Patent No. 7,387,773), hL243 (U.S. Patent No. 7,612,180), hMN-14 (U.S. Patent No. 6,676,924), hMN-15 (U.S.
- Patent No. 7,541,440 discloses hRl (U.S. Provisional Patent Application 61/145,896), hRS7 (U.S. Patent No. 7,238,785), hMN-3 (U.S. Patent No. 7,541,440), AB-PG1-XG1-026 (U.S. Patent Application 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO 2009/130575).
- Other known antibodies are disclosed, for example, in U.S. Patent Nos.
- Antibody fragments which recognize specific epitopes can be generated by known techniques.
- the antibody fragments are antigen binding portions of an antibody, such as F(ab) 2 , Fab', Fab, Fv, scFv and the like.
- Other antibody fragments include, but are not limited to, F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and Fab' fragments which can be generated by reducing disulfide bridges of the F(ab') 2 fragments.
- Fab' expression libraries can be constructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity.
- a single chain Fv molecule comprises a VL domain and a VH domain.
- the VL and VH domains associate to form a target binding site. These two domains are further covalently linked by a peptide linker (L).
- L peptide linker
- An antibody fragment can be prepared by known methods, for example, as disclosed by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647 and references contained therein. Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL.1, page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
- a single complementarity-determining region is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region. Accordingly, a CDR is sometimes referred to as hypervariable region.
- a variable region comprises three CDRs.
- CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells.
- Another form of an antibody fragment is a single-domain antibody (dAb), sometimes referred to as a single chain antibody.
- dAb single-domain antibody
- Techniques for producing single-domain antibodies are well known in the art (see, e.g., Cossins et al., Protein Expression and Purification, 2007, 51 :253-59; Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol. Chem. 2001, 276:24774-780).
- the sequences of antibodies may be varied to optimize the physiological characteristics of the conjugates, such as the half-life in serum.
- Methods of substituting amino acid sequences in proteins are widely known in the art, such as by site-directed mutagenesis (e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed, 1989).
- the variation may involve the addition or removal of one or more glycosylation sites in the Fc sequence (e.g., U.S. Patent No. 6,254,868, the Examples section of which is incorporated herein by reference).
- specific amino acid substitutions in the Fc sequence may be made (e.g., Hornick et al., 2000, J Nucl Med 41 :355-62; Hinton et al., 2006, J Immunol 176:346-56; Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Patent No.
- an anti-CD74 antibody or fragment thereof and an anti-HLA-DR antibody or fragment thereof may be joined together by means such as the dock-and-lock technique described below.
- Other combinations of antibodies or fragments thereof may be utilized.
- the anti-CD74 or anti-HLA-DR antibody could be combined with another antibody against a different epitope of the same antigen, or alternatively with an antibody against another antigen expressed by the APC or DC cell, such as CD209 (DC-SIGN), CD34, CD74, CD205, TLR 2 (toll-like receptor 2), TLR 4, TLR 7, TLR 9, BDCA-2, BDCA-3, BDCA-4 or HLA-DR.
- Methods for producing bispecific antibodies include engineered recombinant antibodies which have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. (See, e.g., FitzGerald et al, Protein Eng
- bispecific antibodies can be produced using molecular engineering.
- the bispecific antibody may consist of, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen.
- the bispecific antibody may consist of, for example, an IgG with two binding sites for one antigen and two scFv with two binding sites for a second antigen.
- compositions disclosed herein may also include functional bispecific single-chain antibodies (bscAb), also called diabodies.
- bscAb are produced by joining two single-chain Fv fragments via a glycine-serine linker using recombinant methods.
- the V light-chain (VL) and V heavy-chain (VH) domains of two antibodies of interest are isolated using standard PCR methods.
- the VL and VH CDNAS obtained from each hybridoma are then joined to form a single-chain fragment in a two-step fusion PCR.
- the first PCR step introduces the linker, and the second step joins the VL and VH amplicons.
- Each single chain molecule is then cloned into a bacterial expression vector.
- one of the single-chain molecules is excised and sub-cloned into the other vector, containing the second single-chain molecule of interest.
- the resulting bscAb fragment is subcloned into a eukaryotic expression vector.
- Functional protein expression can be obtained by transfecting the vector into Chinese
- a humanized, chimeric or human anti-CD74 and/or anti-HLA-DR monoclonal antibody can be used to produce antigen specific diabodies, triabodies, and tetrabodies.
- the monospecific diabodies, triabodies, and tetrabodies bind selectively to targeted antigens and as the number of binding sites on the molecule increases, the affinity for the target cell increases and a longer residence time is observed at the desired location.
- the two chains comprising the V H polypeptide of the humanized CD74 or HLA-DR antibody connected to the V polypeptide of the humanized CD74 or HLA-DR antibody by a five amino acid residue linker may be utilized. Each chain forms one half of the diabody.
- the three chains comprising VH polypeptide of the humanized CD74 or HLA-DR antibody connected to the V polypeptide of the humanized CD74 or HLA-DR antibody by no linker may be utilized.
- Each chain forms one third of the triabody.
- tandab tetravalent tandem diabody with dual specificity
- the bispecific tandab is a dimer of two identical polypeptides, each containing four variable domains of two different antibodies (V H j, V L i, VH2, V ⁇ ) linked in an orientation to facilitate the formation of two potential binding sites for each of the two different specificities upon self-association.
- bispecific or multispecific antibodies may be produced using the dock-and-lock (DNL) technology (see, e.g., U.S. Patent Nos. 7,521,056; 7,550,143; 7,534,866; 7,527,787 and 7,666,400; the Examples section of each of which is incorporated herein by reference).
- DNL dock-and-lock
- the DNL method exploits specific protein/protein interactions that occur between the regulatory (R) subunits of cAMP -dependent protein kinase (PKA) and the anchoring domain (AD) of A-kinase anchoring proteins (AKAPs) (Baillie et al, FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol.
- PKA which plays a central role in one of the best studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R subunits
- the structure of the holoenzyme consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, J. Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RI and RII), and each type has a and ⁇ isoforms (Scott, Pharmacol.
- PKA regulatory subunits there are four types of PKA regulatory subunits - RI , Rip, Rlla and RIi .
- the R subunits have been isolated only as stable dimers and the dimerization domain has been shown to consist of the first 44 amino-terminal residues (Newlon et al, Nat. Struct. Biol. 1999; 6:222). Binding of cAMP to the R subunits leads to the release of active catalytic subunits for a broad spectrum of serine/threonine kinase activities, which are oriented toward selected substrates through the compartmentalization of PKA via its docking with AKAPs (Scott et al, J. Biol. Chem. 1990;265;21561).
- AKAP microtubule-associated protein-2
- the amino acid sequences of the AD are quite varied among individual AKAPs, with the binding affinities reported for RII dimers ranging from 2 to 90 nM (Alto et al, Proc. Natl. Acad. Sci. USA. 2003; 100:4445). AKAPs will only bind to dimeric R subunits.
- the AD binds to a hydrophobic surface formed by the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999; 6:216).
- the dimerization domain and AKAP binding domain of human Rlla are both located within the same N-terminal 44 amino acid sequence (Newlon et al, Nat. Struct. Biol. 1999;6:222; Newlon et al, EMBO J. 2001 ;20: 1651), which is termed the DDD herein.
- Entity B is constructed by linking an AD sequence to a precursor of B, resulting in a second component hereafter referred to as b.
- the dimeric motif of DDD contained in a 2 will create a docking site for binding to the AD sequence contained in b, thus facilitating a ready association of a 2 and b to form a binary, trimeric complex composed of a 2 b.
- This binding event is made irreversible with a subsequent reaction to covalently secure the two entities via disulfide bridges, which occurs very efficiently based on the principle of effective local concentration because the initial binding interactions should bring the reactive thiol groups placed onto both the DDD and AD into proximity (Chmura et al, Proc. Natl. Acad. Sci. USA.
- DNL constructs of different stoichiometry may be produced and used, including but not limited to dimeric, trimeric, tetrameric, pentameric and hexameric DNL constructs (see, e.g., U.S. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and 7,666,400.)
- fusion proteins A variety of methods are known for making fusion proteins, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest.
- double-stranded nucleic acids may be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2 nd Ed, 1989).
- the AD and/or DDD moiety may be attached to either the N- terminal or C-terminal end of an effector protein or peptide.
- site of attachment of an AD or DDD moiety to an effector moiety may vary, depending on the chemical nature of the effector moiety and the part(s) of the effector moiety involved in its physiological activity.
- Site-specific attachment of a variety of effector moieties may be performed using techniques known in the art, such as the use of bivalent cross-linking reagents and/or other chemical conjugation techniques.
- the DNL technique may be utilized to produce complexes comprising multiple copies of the same anti-CD74 or anti-HLA-DR antibody, or to attach one or more anti-CD74 antibodies to one or more anti-HLA-DR antibodies, or to attach an anti-HLA-DR or anti-CD74 antibody to an antibody that binds to a different antigen expressed by APCs and/or DCs.
- the DNL technique may be used to attach antibodies to different effector moieties, such as toxins, cytokines, carrier proteins for siRNA and other known effectors.
- the disclosed methods and compositions may involve production and use of proteins or peptides with one or more substituted amino acid residues.
- the DDD and/or AD sequences used to make DNL constructs may be modified as discussed below.
- amino acid substitutions typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions).
- conservative amino acid substitutions The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.
- the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132).
- the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules.
- Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
- the use of amino acids whose hydropathic indices are within ⁇ 2 is preferred, within ⁇ 1 are more preferred, and within ⁇ 0.5 are even more preferred.
- Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 .+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.
- amino acid side chain For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine.
- a compact side chain such as glycine or serine
- an amino acid with a bulky side chain e.g., tryptophan or tyrosine.
- tryptophan or tyrosine The effect of various amino acid residues on protein secondary structure is also a
- arginine and lysine glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
- amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed.
- conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp.
- conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr.
- therapeutic agents may be administered by a pretargeting method, utilizing bispecific or multispecific antibodies.
- the bispecific or multispecific antibody comprises at least one binding arm that binds to an antigen exhibited by a targeted cell or tissue, such as CD74 or HLA-DR, while at least one other binding arm binds to a hapten on a targetable construct.
- the targetable construct comprises one or more haptens and one or more therapeutic and/or diagnostic agents.
- Pre-targeting is a multistep process originally developed to resolve the slow blood clearance of directly targeting antibodies, which contributes to undesirable toxicity to normal tissues such as bone marrow.
- a radionuclide or other diagnostic or therapeutic agent is attached to a small delivery molecule (targetable construct) that is cleared within minutes from the blood.
- a pre-targeting method of treating or diagnosing a disease or disorder in a subject may be provided by: (1) administering to the subject a bispecific antibody or antibody fragment; (2) optionally administering to the subject a clearing composition, and allowing the composition to clear the antibody from circulation; and (3) administering to the subject the targetable construct, containing one or more chelated or chemically bound therapeutic or diagnostic agents.
- an antibody or antibody fragment may be directly attached to one or more therapeutic agents to form an immunoconjugate.
- Therapeutic agents may be attached, for example to reduced SH groups and/or to carbohydrate side chains.
- a therapeutic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation.
- such agents can be attached using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et ai, Int. J. Cancer 56: 244 (1994). General techniques for such conjugation are well-known in the art.
- the therapeutic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody.
- the Fc region may be absent if the antibody component of the immunoconjugate is an antibody fragment. However, it is possible to introduce a carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment. See, for example, Leung et al, J. Immunol. 154: 5919 (1995); U.S. Patent Nos. 5,443,953 and 6,254,868, the
- the engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
- An alternative method for attaching therapeutic agents to a targeting molecule involves use of click chemistry reactions.
- the click chemistry approach was originally conceived as a method to rapidly generate complex substances by joining small subunits together in a modular fashion.
- Various forms of click chemistry reaction are known in the art, such as the Huisgen 1,3 -dipolar cycloaddition copper catalyzed reaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), which is often referred to as the "click reaction.”
- Other alternatives include cycloaddition reactions such as the Diels- Alder, nucleophilic substitution reactions (especially to small strained rings like epoxy and aziridine compounds), carbonyl chemistry formation of urea compounds and reactions involving carbon-carbon double bonds, such as alkynes in thi
- the azide alkyne Huisgen cycloaddition reaction uses a copper catalyst in the presence of a reducing agent to catalyze the reaction of a terminal alkyne group attached to a first molecule.
- a second molecule comprising an azide moiety
- the azide reacts with the activated alkyne to form a 1 ,4-disubstituted 1,2,3-triazole.
- the copper catalyzed reaction occurs at room temperature and is sufficiently specific that purification of the reaction product is often not required.
- a copper-free click reaction has been proposed for covalent modification of biomolecules.
- the copper- free reaction uses ring strain in place of the copper catalyst to promote a [3 + 2] azide-alkyne cycloaddition reaction (Id.)
- cyclooctyne is an 8-carbon ring structure comprising an internal alkyne bond.
- the closed ring structure induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole.
- cyclooctyne derivatives may be used for copper- free click reactions (Id.)
- the specificity of the click chemistry reaction may be used as a substitute for the antibody-hapten binding interaction used in pretargeting with bispecific antibodies.
- the specific reactivity of e.g., cyclooctyne moieties for azide moieties or alkyne moieties for nitrone moieties may be used in an in vivo cycloaddition reaction.
- An antibody or other targeting molecule is activated by incorporation of a substituted cyclooctyne, an azide or a nitrone moiety.
- a targetable construct is labeled with one or more diagnostic or therapeutic agents and a complementary reactive moiety.
- the targeting molecule comprises a cyclooctyne
- the targetable construct will comprise an azide
- the targeting molecule comprises a nitrone
- the targetable construct will comprise an alkyne, etc.
- the activated targeting molecule is administered to a subject and allowed to localize to a targeted cell, tissue or pathogen, as disclosed for pretargeting protocols.
- the reactive labeled targetable construct is then administered. Because the cyclooctyne, nitrone or azide on the targetable construct is unreactive with endogenous biomolecules and highly reactive with the complementary moiety on the targeting molecule, the specificity of the binding interaction results in the highly specific binding of the targetable construct to the tissue-localized targeting molecule.
- a wide variety of therapeutic reagents can be administered concurrently or sequentially with the anti-CD74 and/or anti-HLA-DR antibodies.
- the therapeutic agents recited here are those agents that also are useful for administration separately with an antibody or fragment thereof as described above.
- Therapeutic agents include, for example, cytotoxic agents such as vinca alkaloids, anthracyclines, gemcitabine, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2 inhibitors, antimitotics, anti-angjogenic and pro-apoptotic agents, particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans, proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, tyrosine kinase inhibitors, and others.
- cytotoxic agents such as vinca alkaloids, anthracyclines, gemcitabine, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2 inhibitors, antimitotics, anti-angjogenic and pro-apoptotic agents, particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans,
- cytotoxic agents include nitrogen mustards, alkyl sulfonates,
- Suitable cytotoxic agents are described in REMINGTON'S PHARMACEUTICAL
- conjugates of camptothecins and related compounds may be conjugated to an anti-CD74 or anti-HLA-DR antibody, for example as disclosed in U.S. Patent No. 7,591,994, the Examples section of which is incorporated herein by reference.
- a toxin can be of animal, plant or microbial origin.
- a toxin, such as Pseudomonas exotoxin, may also be complexed to or form the therapeutic agent portion of an
- toxins include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, onconase, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
- RNase ribonuclease
- DNase I DNase I
- Staphylococcal enterotoxin-A Staphylococcal enterotoxin-A
- pokeweed antiviral protein pokeweed antiviral protein
- onconase gelonin
- diphtheria toxin diphtheria toxin
- Pseudomonas exotoxin Pseudomonas endotoxin.
- Additional toxins suitable for use are known to those of skill in the art and are disclosed in U.S. Pat. No.
- the term "immunomodulator” includes cytokines, lymphokines, monokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSF), interferons (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, transforming growth factor (TGF), TGF-a, TGF- ⁇ , insulin-like growth factor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), TNF- a, TNF- ⁇ , mullerian-inhibiting substance, mouse gonadotropin- associated peptide, inhibin, activin, vascular endothelial
- the antibody or fragment thereof may be administered as an immunoconjugate comprising one or more radioactive isotopes useful for treating diseased tissue.
- Particularly useful therapeutic radionuclides include, but are not limited to 1 1 1 In, 177 Lu, 212 Bi, 213 Bi, 21 1 At, 62 Cu, "Cu, 67 Cu, 90 Y, 125 1, 131 1, 32 P, 33 P, 47 Sc, n i Ag, 67 Ga, 142 Pr, 153 Sm, 161 Tb, 166 Dy, 166 Ho, l86 Re, 188 Re, l89 Re, 212 Pb, 223 Ra, 225 Ac, 59 Fe, 75 Se, 77 As, 89 Sr, 99 Mo, 105 Rh, 109 Pd, 143 Pr, 149 Pm, 169 Er, 194 Ir, 198 Au, 199 Au, and 21 'Pb.
- the therapeutic radionuclide preferably has a decay energy in the range of 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter and 4,000-6,000 keV for an alpha emitter.
- Maximum decay energies of useful beta-particle-emitting nuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-I l l, Sb-119, 1-125, Ho- 161, Os-189m and Ir- 192. Decay energies of useful beta-particle-emitting nuclides are preferably ⁇ 1,000 keV, more preferably ⁇ 100 keV, and most preferably ⁇ 70 keV.
- radionuclides that substantially decay with generation of alpha-particles.
- Such radionuclides include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr- 221, At-217, Bi-213 and Fm-255. Decay energies of useful alpha-particle-emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.
- Additional potential therapeutic radioisotopes include n C, 13 N, 15 0, 75 Br, 198 Au, 224 Ac, 126 1, 133 1, 77 Br, 113m In, 95 Ru, 97 Ru, 103 Ru, 105 Ru, ,07 Hg, 203 Hg, 121m Te, 122m Te, 125m Te, 1 65 Tm, 167 Tm, 168 Tm, 197 Pt, 109 Pd, 105 Rh, l42 Pr, 143 Pr, 161 Tb, 16 1Ho, 199 Au, 57 Co, 58 Co, 51 Cr, 5 9 Fe, 75 Se, 201 T1, 225 Ac, 76 Br, 169 Yb, and the like.
- Interference RNA Interference RNA
- the therapeutic agent may be a siRNA or interference RNA species.
- the siRNA, interference RNA or therapeutic gene may be attached to a carrier moiety that is conjugated to an antibody or fragment thereof.
- a variety of carrier moieties for siRNA have been reported and any such known carrier may be incorporated into a therapeutic antibody for use.
- Non-limiting examples of carriers include protamine (Rossi, 2005, Nat Biotech 23:682-84; Song et al., 2005, Nat Biotech 23:709-17); dendrimers such as PAMAM dendrimers (Pan et al., 2007, Cancer Res. 67:8156-8163);
- siRNA carriers can also be used to carry other oligonucleotide or nucleic acid species, such as anti-sense oligonucleotides or short DNA genes.
- siRNA species of potential use include those specific for IKK-gamma (U.S. Patent 7,022,828); VEGF, Flt-1 and Flk-l/KDR (U.S. Patent 7,148,342); Bcl2 and EGFR (U.S. Patent 7,541,453); CDC20 (U.S. Patent 7,550,572); transducin (beta)-like 3 (U.S.
- Patent 7,576,196 K-ras (U.S. Patent 7,576,197); carbonic anhydrase II (U.S. Patent
- amyloid beta precursor protein U.S. Patent 7,635,771
- IGF-1R U.S. Patent 7,638,621
- ICAM1 U.S. Patent 7,642,349
- complement factor B U.S. Patent 7,696,344
- p53 7,781,575)
- apolipoprotein B 7,795,421
- siRNA species are available from known commercial sources, such as Sigma- Aldrich (St Louis, MO), Invitrogen (Carlsbad, CA), Santa Cruz Biotechnology (Santa Cruz, CA), Ambion (Austin, TX), Dharmacon (Thermo Scientific, Lafayette, CO), Promega (Madison, WI), Mirus Bio (Madison, WI) and Qiagen (Valencia, CA), among many others.
- Other publicly available sources of siRNA species include the siRNAdb database at the Swedish Bioinformatics Centre, the MIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the Broad Institute, and the Probe database at NCBI. For example, there are 30,852 siRNA species in the NCBI Probe database. The skilled artisan will realize that for any gene of interest, either a siRNA species has already been designed, or one may readily be designed using publicly available software tools. Any such siRNA species may be delivered using the subject DNL complexes.
- siRNA species known in the art are listed in Table 1. Although siRNA is delivered as a double-stranded molecule, for simplicity only the sense strand sequences are shown in Table 1.
- CEACAM1 AACCTTCTGGAACCCGCCCAC SEQ ID NO:38
- Table 1 represents a very small sampling of the total number of siRNA species known in the art, and that any such known siRNA may be utilized in the claimed methods and compositions.
- the claimed methods and compositions are of use for treating disease states, such as autoimmune disease or immune system dysfunction (e.g., aGVHD).
- the methods may comprise administering a therapeutically effective amount of a therapeutic antibody or fragment thereof or an immunoconjugate, either alone or in conjunction with one or more other therapeutic agents, administered either concurrently or sequentially.
- Multimodal therapies may include therapy with other antibodies, such as anti-CD209 (DC-SIGN), anti-CD34, anti-CD74, anti-CD205, anti-TLR-2, anti-TLR-4, anti- TLR-7, anti- TLR-9, anti-BDCA-2, anti- BDCA-3, anti- BDCA-4 or anti-HLA-DR (including the invariant chain) antibodies in the form of naked antibodies, fusion proteins, or as
- cytotoxic drugs are co-administered with a therapeutic antibody.
- the cytokines, cytotoxic drugs and therapeutic antibody can be administered in any order, or together.
- Therapeutic antibodies or fragments thereof can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic antibody is combined in a mixture with a pharmaceutically suitable excipient.
- a pharmaceutically suitable excipient Sterile phosphate- buffered saline is one example of a pharmaceutically suitable excipient.
- Other suitable excipients are well-known to those in the art. See, for example, Ansel et al,
- the therapeutic antibody can be formulated for intravenous administration via, for example, bolus injection or continuous infusion.
- the therapeutic antibody is infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours.
- the first 25-50 mg could be infused within 30 minutes, preferably even 15 min, and the remainder infused over the next 2-3 hrs.
- Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
- the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- the therapeutic antibody may also be administered to a mammal subcutaneously or even by other parenteral routes. Moreover, the administration may be by continuous infusion or by single or multiple boluses. Preferably, the therapeutic antibody is infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours.
- the dosage of an administered therapeutic antibody for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. It may be desirable to provide the recipient with a dosage of therapeutic antibody that is in the range of from about 1 mg/kg to 25 mg/kg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate.
- the dosage may be repeated as needed, for example, once per week for 4-10 weeks, once per week for 8 weeks, or once per week for 4 weeks. It may also be given less frequently, such as every other week for several months, or monthly or quarterly for many months, as needed in a maintenance therapy.
- a therapeutic antibody may be administered as one dosage every 2 or 3 weeks, repeated for a total of at least 3 dosages.
- the therapeutic antibody may be administered twice per week for 4-6 weeks. If the dosage is lowered to approximately 200- 300 mg/m 2 (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it may be administered once or even twice weekly for 4 to 10 weeks.
- the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3 months. It has been determined, however, that even higher doses, such as 20 mg/kg once weekly or once every 2- 3 weeks can be administered by slow i.v. infusion, for repeated dosing cycles.
- the dosing schedule can optionally be repeated at other intervals and dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule.
- Control release preparations can be prepared through the use of polymers to complex or adsorb the immunoconjugate or naked antibody.
- biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release of an immunoconjugate or antibody from such a matrix depends upon the molecular weight of the immunoconjugate or antibody, the amount of immunoconjugate or antibody within the matrix, and the size of dispersed particles.
- Anti-CD74 and/or anti-HLA-DR antibodies or immunoconjugates can be used to treat immune dysregulation disease and related autoimmune diseases.
- Immune diseases may include acute idiopathic thrombocytopenic purpura, Addison's disease, adult respiratory distress syndrome (ARDS), agranulocytosis, allergic conditions, allergic encephalomyelitis, allergic neuritis, amyotrophic lateral sclerosis (ALS), ankylosing spondylitis, antigen- antibody complex mediated diseases, anti-glomerular basement membrane disease, anti- phospholipid antibody syndrome, aplastic anemia, arthritis, asthma, atherosclerosis, autoimmune disease of the testis and ovary , autoimmune endocrine diseases, autoimmune myocarditis, autoimmune neutropenia, autoimmune polyendocrinopathies, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), autoimmune thrombocytopenia, Bechet disease, Berger's disease (IgA
- dermatomyositis colitis, conditions involving infiltration of T cells and chronic inflammatory responses, coronary artery disease, Crohn's disease, cryoglobulinemia, dermatitis, dermatomyositis, diabetes mellitus, diseases involving leukocyte diapedesis, eczema, encephalitis, erythema multiforme, erythema nodosum, Factor VIII deficiency, fibrosing alveolitis , giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, graft versus host disease (GVHD), granulomatosis, Grave's disease, Guillain-Barre Syndrome, Hashimoto's thyroiditis, hemophilia A, Henoch-Schonlein purpura, idiopathic hypothyroidism, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgA nephropathy
- thrombocytopenias juvenile onset diabetes, juvenile rheumatoid arthritis, Lambert-Eaton Myasthenic Syndrome, large vessel vasculitis , leukocyte adhesion deficiency, leukopenia, lupus nephritis, lymphoid interstitial pneumonitis (HIV), medium vessel vasculitis , membranous nephropathy, meningitis, multiple organ injury syndrome, multiple sclerosis, myasthenia gravis, osteoarthritis, pancytopenia, pemphigoid bullous, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia, polymyositis, post-streptococcal nephritis, primary biliary cirrhosis, primary hypothyroidism, psoriasis, psoriatic arthritis, pure red cell a
- CD74 (invariant chain, Ii) is a type-II transmembrane glycoprotein that associates with the major histocompatibility class (MHC) II a and ⁇ chains and directs the transport of the ⁇ complexes to endosomes and lysosomes.
- MHC major histocompatibility class
- MIF macrophage migration-inhibitory factor
- CD74 is expressed by certain normal HLA class II-positive cells, including B cells, monocytes, macrophages, Langerhans cells, dendritic cells, subsets of activated T cells, and thymic epithelium.
- CD74 is also expressed on a variety of malignant cells, including the vast majority of B-cell cancers (NHL, CLL, MM).
- the LLl monoclonal antibody was generated by hybridoma technology after immunization of BALB/c mice with Raji human Burkitt lymphoma cells.
- the LLl antibody reacts with an epitope in the extracellular domain of CD74.
- CD74-positive cell lines have been shown to very rapidly internalize LLl (nearly 10 molecules per cell per day). This rapid internalization enables LLl to be an extremely effective agent for delivery of cytotoxic agents, such as chemotherapeutics or toxins.
- milatuzumab humanized anti-CD74 LLl antibody
- IgG Fc antibody multiple myeloma
- milatuzumab is under clinical evaluation as a therapeutic antibody for relapsed or refractory B-cell malignancies (Berkova et al, 2010, Expert Opin Investig Drugs 19:141-149).
- CD74 is also present in normal APCs, such as B cells, monocytes, macrophages, Langerhans cells, and follicular and blood DCs (Stein et al, 2007, Clin Cancer Res. 13:5556s-5563s; Freudenthal & Steinman, 1990, Proc Natl Acad Sci USA 87:7698-7702).
- B cells normal APCs
- monocytes monocytes
- macrophages e.g., monocytes
- macrophages e.g., macrophages
- Langerhans cells follicular and blood DCs
- milatuzumab has any effects on the viability of mDCl, pDCs, mDC2, and monocytes.
- the present Example assessed the binding profile and cytotoxicity of milatuzumab on all APC subsets of human PBMCs, including mDCl, pDCs, mDC2, B cells, T cells, and monocytes. As shown below, exposure of PBMCs to milatuzumab caused potent depletion of mDCl and mDC2, mild depletion of B cells, and no effect on pDCs, monocytes, and T cells, which could be correlated with CD74 expression levels on these cells. These results distinguish milatuzumab from T-cell antibodies and support use of milatuzumab for preventing and treating GVHD.
- Antibodies and reagents - Milatuzumab (hLLl, U.S. Patent Nos. 7,312,318), labetuzumab (hMN-14, U.S. Patent No. 6,676,924), epratuzumab (hLL2, U.S. Patent No. 7,074,403), and hLLl -Fab- A3B3 (U.S. Patent No. 7,354,587), the Examples section of each cited patent incorporated herein by reference, were obtained as disclosed.
- Rituximab was purchased from IDEC Pharmaceuticals Corp. (San Diego, CA).
- Anti-CD86 2331[FUN-1]
- FITC-conjugated anti-CD74 M-B741
- PerCP-conjugated anti-HLA-DR L243 [G46-6]
- CD3 CD3
- Miltenyi Biotec Auburn, CA
- APC allophycocyanin
- Milatuzumab and anti-CD86 were labeled with the ZENON® ALEXA FLUOR® 488 human IgG labeling kit (Invitrogen, Carlsbad, CA) following the manufacturer's instructions.
- PBMCs - PBMCs were isolated from the buffy coats of healthy donors by standard density-gradient centrifugation over FICOLL-PAQUETM (Lonza, Walkersville, MD).
- mDCl were purified from PBMCs by depleting CD19 + B cells, followed by positive enrichment of BDCA-1 + cells.
- pDCs were purified by depleting all the cells that do not express BDCA-4 antigen.
- mDC2 were purified by enriching BDCA-3 + cells.
- the BDCA-3 " cells that contained no mDC2 were used for isolation of NK cells by depleting all the cells that do not express CD56. Those depleted cells that contained neither NK cells nor mDC2 were used as non-NK cells. All the purification procedures were performed according to the manual of MACS® kits (Miltenyi Biotec).
- mDCl were identified as CD14 19 " BDCA-1 + cell populations (Morel et al, 2002, Immunology 106:229-236).
- the lymphocyte population was analyzed for B cells (CD19 + SSC low ), non-B lymphocytes (primarily T cells) (CD19 " 14 " SSC low ), and monocytes (CD14 + SSC medium ).
- the live cell fraction of each cell population was quantitated as the percentage of 7-AAD " cells.
- PBMCs were stained with PE-labeled anti- CD 14 and anti-CD 19, in combination with FITC-labeled anti-BDCA-2 and APC-labeled anti-BDCA-3.
- mDC2 were identified as CD14T9 " BDCA-3 ⁇ cell population, whereas pDCs were identified as CD14 " 19 " BDCA-2 + cell population.
- Flow cytometry was performed using a FACSCALIBUR® (BD Bioscience) and analyzed with FlowJo software (Tree Star, Inc., Ashland, OR).
- Binding of anti-CD74 antibodies with human PBMC subsets - Human PBMCs isolated from buffy coats of healthy donors were treated with FcR-blocking reagent (Miltenyi Biotec), then co-stained with PE-conjugated antibodies to CD 19 and CD 14, FITC-labeled mouse anti-human CD74 antibody (M-B741), or its isotype control; or Alexa 488-conjugated milatuzumab, or human IgG control, and APC-conjugated antibody to BDCA-1, BDCA-2, or BDCA-3. The cells were washed and analyzed by flow cytometry.
- B cells and monocytes were gated according to the same FL2 signal (PE-labeled anti-CD 14 and anti-CD 19) combined with their differential SSC signals.
- the CD 14 " 19 " cell populations were used to gate the BDCA-1 + , BDCA-2 + , or BDCA-3 + cell populations for mDCl , pDCs, and mDC2, respectively (Dzionek et al., 2000, J Immunol 165: 6037-6046).
- the binding efficiency of milatuzumab or M-B741 with these cell populations was assessed by FL1 mean fluorescence intensity (MFI).
- T-cell proliferation in allogeneic mixed leukocyte reaction - PBMCs from different donors were labeled with 1 ⁇ carboxyfluorescein succinimidyl ester (CFSE) following the manufacturer's instructions (Invitrogen, CA). After extensive washings, the cells from two different donors were mixed and incubated for 11 days. The cells were then harvested and analyzed by flow cytometry. The proliferating cells were quantitated by measuring the CFSE l0W cell frequencies (Han et al, 2008, Mol Ther. 16:269-279).
- CFSE carboxyfluorescein succinimidyl ester
- the cells were then harvested and stained with PE-labeled HLA- A*0201 CMV pentamer (Prolmmune, Bradenton, FL) (Wills et al, 1996, J Virol 70:7569- 7579; Tabi et al, 2001, J Immunol 166:5695-5703), followed by washing and staining with PerCp-CD8 (BD Pharmingen).
- PerCp-CD8 BD Pharmingen
- Milatuzumab selectively depletes myeloid DCs in human PBMCs - Milatuzumab is an antagonist antibody against CD74, which has been shown to have potent cytotoxicity against CD74-expressing B-cell lymphomas and multiple myeloma (Stein et al, 2004, Blood 104:3705-3711 ; Burton et al, 2004, Clin Cancer Res. 10:6606-6611 ; Stein et al, 2009, Clin Cancer Res. 15:2808-2817). Since most normal APCs or DCs express CD74 (Stein et al, 2007, Clin Cancer Res. 13:5556s-5563s; Freudenthal et al.
- milatuzumab may also be cytotoxic to these normal cells.
- hMN-14 humanized anti-CEACAM5
- rituximab chimeric anti-CD20
- hLL2 humanized anti-CD22, epratuzumab
- Fc-lacking hLLl- Fab-A3B3 the Fab fragment of milatuzumab fused to the A3B3 domain of CEACAM5 (Hefta et al., 1992, Cancer Res.
- milatuzumab significantly reduced the counts of live mDCl and mDC2 in PBMCs.
- the cytotoxicity of milatuzumab on mDCl was greater in the presence of non- NK than NK cells (not shown). Because of the small number of mDC2 cells, restoration of milatuzumab toxicity on this cell population was only tested in the presence of added NK cells.
- milatuzumab acts through an Fc-mediated mechanism to deplete mDCl and mDC2 in PBMCs, which may preferentially involve non-NK cell components for the killing.
- Milatuzumab does not affect CD86 expression and IL-12 production by human PBMCs - . Because costimulatory molecules, including CD40, CD80 and CD86, are critical for donor APC function in intestinal and skin chronic GVHD (Anderson et al, 2005, Blood 105:2227-2234), we next investigated if milatuzumab had any effect on the expression of CD86 in mDCl, monocytes, B cells, and non-B lymphocytes.
- INF- ⁇ and lipopolysaccharide (LPS) stimulate maturation of APCs and were included in this study to evaluate the effect of milatuzumab on both immature (without IFN- ⁇ and LPS) and mature (with IFN- ⁇ and LPS) cells. As shown in FIG. 2A, milatuzumab had little or no effect on CD86 expression in either mature or immature APCs.
- IL-12 the "decisive" cytokine that drives type I immune response, may play a crucial role in the development of acute GVHD (Williamson et al, 1996, J Immunol 157:689-699; Yabe et al., 1999, Bone Marrow Transplant. 24:29-34).
- milatuzumab may not affect either "signal 2" (costimulatory molecules) or “signal 3” (cytokines) of APCs, suggesting that the antigen-presenting function of APCs is not affected by this antibody.
- Milatuzumab reduces T-cell proliferation in allo-MLR -
- milatuzumab or control antibodies The proliferated allo-reactive T cells were identified based on the CFSE dilution. As shown in FIG. 3A, the allo-MLR treated with the isotype control antibody, hMN-14, underwent robust T-cell proliferation characterized by 21.5% of T cells with CFSE dilution. In contrast, T-cell proliferation was only detected in 3.6% of cells in the MLR treated with milatuzumab. Statistical analysis of a total of 10
- milatuzumab causes a potent depletion of mDCls and mDC2s, but not non-B lymphocytes that are composed of mainly T cells. This is not unexpected, because the majority of T cells are resting cells, which lack the expression of CD74 (Stein et al, 2007, Clin Cancer Res 13:5556s-5563s). This result led us to reason that milatuzumab, while suppressing the proliferation of allo-reactive T cells, may preserve the preexisting pathogen-specific memory T cells.
- milatuzumab even at a 10-fold higher concentration than was used for depletion of mDCl and mDC2 (50 ⁇ g/ml), did not affect the CMV-specific EFN- ⁇ response in CD8 + T cells stimulated in vitro by a CMV pp65 peptide pool or CMV pp65 protein (data not shown).
- a 6-day allo-MLR was then performed, in which the responder PBMCs were from this CMV-positive, HLA-A*0201 donor, and the stimulator PBMCs were from another donor, irrespective of CMV status.
- CMV-specific CD8 + T cells were determined by staining the cells with HLA*A0201 CMV pentamer (NLVPMVATV) (SEQ ID NO: 100). As expected, CMV-specific CD8 + T cells were not altered by milatuzumab treatment (not shown). This result is important, because CMV is one of the most prevalent pathogens that cause severe infections after allo-HSCT.
- the current standard immunosuppressive agents such as high-dose steroids, effectively control GVHD but critically impair host immunity against pathogens. It is thus highly desired that any novel strategy against GVHD spare pathogen-specific immunity while suppressing the allo-specific response. Our results suggest that the third-party responses, such as pathogen-specific memory T-cell immunity, are not compromised by milatuzumab treatment.
- DC depletion of DCs can be achieved by a number of antibodies.
- One example is the anti-CD52 antibody, alemtuzumab (Klangsinsirikul et al, 2002, Blood 99: 2586-2591; Ratzinger et al, 2003, Blood 101: 1422-1429), which has been used clinically for prevention of acute GVHD and is currently in clinical trials for the treatment of chronic GVHD. It can efficiently deplete host DCs and suppress the proliferation of allo-reactive T cells, but it also impairs anti-viral responses.
- RA83 a rabbit anti-human CD83 polyclonal antibody
- RA83 is another DC-depleting agent, which targets activated DCs, leading to the suppression of allo-proliferation but without reducing CMV- or influenza- specific T cells (Munster et al, 2004, Int Immunol 16:33-42; Wilson et al, 2009, J Exp Med 206:387-398).
- use of rabbit polyclonal antibody for human therapy is likely to produce other undesirable side effects, such as immune response to the rabbit antibody.
- milatuzumab a humanized anti-CD74 antibody
- anti-CD74 antibodies in general and milatuzumab in particular are novel DC-depleting antibodies for the control of GVHD. This can be used prophylactically to prevent acute GVHD, or therapeutically for chronic GVHD. In both cases, milatuzumab could offer the advantage of life-saving third- party immune functions being spared. This differs from current immunosuppressive therapies that suppress the overall immune functions without discrimination.
- the mechanism of milatuzumab on DCs may differ from that on malignant B cells, in which the cytotoxicity of milatuzumab is not through either ADCC or CDC, as revealed by a 4-h cytotoxicity assay, but through a direct inhibition of the NF- ⁇ signaling pathway via blocking CD74 (Stein et al, 2009, Clin Cancer Res. 15:2808-2817; Stein et al, 2004, Blood 104:3705-3711; Binsky et a/., 2007 Proc Natl Acad Sci USA 104:13408-13413). It may also differ from the CDC-dependent mechanism by which anti-CD52 antibody, alemtuzumab, depletes DCs (Klangsinsirikul et al, 2002, Blood 99:2586-2591).
- rituximab The suppression of the allogeneic T-cell response by rituximab may be through both depletion and functional modification of B cells (Shimabukuro-Vornhagen et al, 2009, Blood 114:4919-4927). In the case of epratuzumab, it may regulate B-cell function to suppress the allo-response.
- Rituximab has been used clinically to effectively prevent acute GVHD and to treat chronic GVHD in allo-HSCT patients (Okamoto et al, 2006, Leukemia 20:172-173; Cutler et al, 2006, Blood 108:756-762).
- epratuzumab Although there is no report about the therapeutic effect on GVHD, epratuzumab has been shown to be effective in treating systemic lupus erythematosus patients Dorner & Goldenberg, 2007, Ther Clin RiskManag 3:953-959; Jacobi et al, 2008, Ann Rheum Dis 67:450-457). It would be worthwhile to investigate the potential efficacy of epratuzumab in managing GVHD, as proposed by Shimabukuro-Vornhagen, et al. (2009, Blood 114:4919-4927).
- Milatuzumab however, efficiently depletes myeloid DCs, the major and critical initiator of GVHD, and mildly but significantly depletes B cells, as well as downregulates CD 19 expression on B cells (data not shown). It is thus expected that milatuzumab might be more potent in controlling GVHD than rituximab or epratuzumab.
- milatuzumab can selectively deplete myeloid DCs, the critical initiator of GVHD after allo-HSCT.
- this antibody does not impair the anti-viral immune responses studied, while suppressing the allo-specific responses.
- the outcome following allo-HSCT is expected to be improved by the control of GVHD by using this novel antibody to deplete host and donor myeloid dendritic cells.
- IMMU-114 is a humanized IgG4 anti-HLA-DR antibody derived from the murine anti-human HLA-DR antibody, L243. It recognizes a conformational epitope in the a-chain of HLA-DR (Stein et al, 2006, Blood 108:2736-2744).
- the engineered IgG4 isotype (hL243y4P) of this humanized antibody abrogates its ADCC and CDC effector functions, but retains its antigen-binding properties and direct cytotoxicity against a variety of tumors (Stein et al, 2006, Blood 108:2736-2744), which is mediated through hyper-activation of ERK and J K MAP kinase signaling pathways (Stein et al., 2010, Blood 115:5180-90).
- B cells and monocytes are the two other major subsets of circulating APCs.
- B cells are involved in the pathogenesis of acute and chronic GVHD (Shimabukuro-Vornhagen et al, 2009, Blood 114:4919-4927) and that B-cell depleting therapy is effective in prevention and treatment of GVHD (Alousi et al, 2010, Leuk Lymphoma 51 :376-389).
- the anti-CD20 antibody, rituximab when included in the conditioning regimen, reduces the incidence of aGVHD (Christopeit et al, 2009, Blood 113:3130-3131).
- Monocytes may also be involved in the pathogenesis of GVHD, since higher numbers of blood monocytes before conditioning are associated with greater risk of aGVHD (Arpinati et al, 2007, Biol Blood Marrow Transplant 13:228-234).
- the proteosome inhibitor, bortezomib which efficiently depletes monocytes (Arpinati et al., 2009, Bone Marrow Transplant 43:253-259), is active in controlling acute and chronic GVHD (Sun et al, 2004, Proc Natl. Acad Sci USA 101 :8120-8125). Because each subset of APCs is involved in the pathogenesis of aGVHD, it is desirable to deplete all APC subsets during the preparative conditioning for allo-HSCT. This goal has not been attained by current regimens.
- IMMU-114 or hL243y4P can deplete all subsets of APCs, but not T cells, from human peripheral blood mononuclear cells (PBMCs), including myeloid DCs (mDCs), plasmacytoid DCs (pDCs), B cells and monocytes.
- PBMCs peripheral blood mononuclear cells
- mDCs myeloid DCs
- pDCs plasmacytoid DCs
- B cells monocytes.
- purified mDCs or pDCs were still killed efficiently by IMMU-114, suggesting that IMMU-114 depletes these APCs independently of antibody-dependent cellular cytotoxicity (ADCC) or complement- dependent cytotoxicity (CDC).
- ADCC antibody-dependent cellular cytotoxicity
- CDC complement- dependent cytotoxicity
- IMMU-114 suppressed the proliferation of allo- reactive T cells in mixed leukocyte cultures, yet preserved CMV-specific, CD8 + memory T cells. Together, these results demonstrate the potential of IMMU-114 as a novel conditioning regimen for maximally preventing aGVHD without alteration of preexisting anti-viral immunity.
- Antibodies - IMMU-114 (hL243y4p, U.S. Patent No. 7,612,180) and labetuzumab (hMN-14, U.S. Patent No. 6,676,924) were prepared as described.
- Rituximab was purchased from IDEC Pharmaceuticals Corp. (San Diego, CA).
- Commercially available antibodies were obtained from Miltenyi Biotec (Auburn, CA):FITC-conjugated antibody to BDCA-2
- allophycocyanin ylPQ-conjugated antibodies to BDCA-1 (AD5-8E7), BDCA-2 (AC 144), and BDCA-3 (AD5-14H12).
- PBMCs - PBMCs Purification of myeloid and plasmacytoid DCs from PBMCs - PBMCs were isolated from the buffy coats of healthy donors by standard density-gradient centrifugation over FICOLL-PAQUETM (Lonza, Walkersville, MD). MACS® kits (Miltenyi Biotec) were used to purify DC subsets from PBMCs.
- mDCl cells were purified from PBMCs by depleting CD19 + B cells, followed by positive enrichment of BDCA-1 + cells.
- pDCs were purified by depleting all the cells that do not express BDCA-4 antigen.
- mDC2 cells were purified by enriching BDCA-3 + cells.
- Flow cytometric analysis of APC subsets in human PBMCs - PBMCs from normal donors were treated with IMMU-114 or other antibodies at 37°C, 5% C0 2 , for 3 days.
- the live PBMCs were gated based on the forward scatter (FSC) and side scatter (SSC) signals.
- FSC forward scatter
- SSC side scatter
- mDCl cells were identified as CD14 " 19 " BDCA-1 + cell populations (Dzionek et al, 2000, J Immunol 165:6037-6046).
- the lymphocyte population was analyzed for B cells
- PBMCs were stained with PE-labeled anti-CD 14 and anti-CD 19, in combination with FITC-labeled anti- BDCA-2 and APC-labeled anti-BDCA-3.
- mDC2 cells were identified as the CDXA WROCA-l ⁇ cell population, whereas pDCs were identified as the CD14 " 19 " BDCA-2 + cell population.
- Flow cytometry was performed using a
- T-cell proliferation in allogeneic mixed leukocyte reaction - PBMCs from different donors were labeled with 1 ⁇ carboxyfluorescein succinimidyl ester (CFSE) following the manufacturer's instructions (Invitrogen, CA). After extensive washings, the cells from two different donors were mixed and incubated for 11 days. The cells were then harvested and analyzed by flow cytometry. The proliferating cells were quantitated by measuring the CFSE l0W cell frequencies.
- CFSE carboxyfluorescein succinimidyl ester
- IMMU-114 efficiently depletes B cells and monocytes, but not T cells or NK cells from human whole blood in vitro (Stein et al., 2010, Blood 115:5180-90). Since both mDCs and pDCs express HLA-DR, IMMU-114 may also deplete these two major subsets of blood DCs. To investigate this, we treated human PBMCs with IMMU-114 or a control antibody (hMN-14 or labetuzumab, humanized anti-CEACAM5 antibody) (Sharkey et al., 1995, Cancer Res.
- IMMU-114 but not hMN-14, depleted B cells and monocytes, but not non-B lymphocytes (the majority are T cells) (data not shown), which is consistent with our previous findings in whole blood samples (Stein et al., 2010, Blood 115:5180-90).
- mDC type 1 mDCl
- pDCs pDCs
- mDC2 the minor subset of mDCs, Déek et al., 2000, J Immunol 165:6037-6046
- Statistical analysis of a total of 10 stimulator/responder combinations showed a significant reduction (P ⁇ 0.01) in T-cell proliferation in IMMU-1 14-treated allo-MLR (FIG. 6).
- Alemtuzumab has been used extensively as a component of the conditioning regimen in patients undergoing allo-HSCT and has been demonstrated to significantly reduce GVHD (Kottaridis et al, 2000, Blood 96:2419-2425). However, alemtuzumab depletes both DCs and T cells, accounting for the increased reactivation of CMV in allo-HSCT patients (Perez- Simon et al, 2002, Blood 100:3121-3127; Chakrabarti et al, 2002, Blood 99:4357-4363). IMMU-114, however, does not affect T cells while depleting all subsets of APCs (FIG. 4).
- IMMU-114 does not affect CMV-specific memory T cells.
- CMV-specific CD8 + T cells were determined by staining the cells with HLA*A0201 CMV pentamer.
- CMV-specific CD8 + T cells were not altered by IMMU-1 14 treatment (not shown). This result shows that pathogen-specific memory T-cell immunity, such as CMV-specific memory T cells, is not compromised by IMMU-114 treatment.
- IMMU-114 a humanized anti-HLA-DR IgG4 antibody, can deplete all subsets of APCs efficiently, including mDCl, pDC, mDC2, B cells, and monocytes, leading to potent suppression of allo-reactive T cell proliferation, yet preserves CMV-specifie, CD8 + memory T cells.
- IMMU-114 does not affect T cells, leading to the preservation of pathogen-specific memory T cells, whereas alemtuzumab depletes T cells, leading to reactivation of CMV in allo-HSCT patients.
- IMMU-114 depletes APC subsets through direct action without the requirement of intact host immunity, whereas alemtuzumab depletes DCs through CDC- and ADCC-mediated mechanisms, which require intact host immune effector functions.
- IMMU-114 is rapidly cleared from the blood within several hours, followed by the clearance of remaining antibody with a half-life of ⁇ 2 days (not shown), from which the half-life of IMMU-1 14 in humans is predicted to be 2-3 days according to the allometric scaling of an immunoglobulin fusion protein described by Richter et al. (Drug Metab Dispos 27:21-25, 1999).
- IMMU-1 14 has the potential to be a novel component of the allograft conditioning regimen, with more efficiency, higher safety, and wider applicability, especially in patients with compromised immunity, compared to currently available agents.
- hL243y4P humanized anti-HLA-DR antibody hL243y4P (IMMU-114) on a panel of leukemia cell lines.
- hL243y4P bound to the cell surface of 2/3 AML, 2/2 mantle cell, 4/4 ALL, 1/1 hairy cell leukemia, and 2/2 CLL cell lines, but not on the 1 CML cell line tested (not shown).
- Cytotoxicity assays demonstrated that ⁇ 243 ⁇ 4 ⁇ was toxic to 2/2 mantle cell, 2/2 CLL, 3/4 ALL, and 1/1 hairy cell leukemia cell lines, but did not kill 3/3 AML cell lines despite positive staining (not shown).
- the CML cell line was also not killed by hL243y4P (not shown).
- hL243y4P cytotoxicity correlates with activation of ERK and JNK signaling and differentiates the mechanism of action of hL243y4P cytotoxicity from that of anti-CD20 MAbs (not shown).
- hL243y4P also changes mitochondrial membrane potential and generates ROS in Raji cells (not shown). Inhibition of ERK, JNK, or ROS by specific inhibitors partially abrogates the apoptosis. Inhibition of 2 or more pathways abolishes the apoptosis.
- IMMU-114-sensitive and -resistant HLA-DR- expressing cell lines were used as examples of HLA-DR + cell lines resistant to IMMU-114 cytotoxicity.
- IMMU-1 14-sensitive cells included NHL (Raji), MCL (Jeko-1 and Granta-519), CLL (WAC and MEC-1), and ALL (REH and MN60).
- IMMU-114 induces phosphorylation and activation of ERK and JNK mitogen activated protein (MAP) kinases in all the cells defined as IMMU- 11—sensitive by the cytotoxicity assays, but not the IMMU-114-resistant cell lines, Kasumi- 3 and GDM-1 (data not shown).
- MAP mitogen activated protein
- ERK, JNK, and ROS inhibitors used were: NAC (5 mM) blocks ROS, U0126 (10 ⁇ ) blocks MEK phosphorylation and the ERKl/2 pathway, and SP600125 (10 ⁇ ) blocks the JNK pathway. Inhibition of ERK, JNK, or ROS by their respective inhibitors decreased apoptosis in Raji cells, although the inhibition was not complete when any single inhibitor was used (not shown).
- IMMU-114 mitochondrial membrane potential and generation of ROS also were not observed on treatment of these AML cell lines with IMMU-114 (not shown).
- Activation of ERKl/2 and JNK signaling pathways was also assessed in CLL patient samples (not shown). Patient cells were incubated with EVIMU-114 for 4 hours because the cells in these samples were much smaller than those of the established cell lines. Moreover, the shorter incubation time avoids the risk of higher apoptosis and cell death. Similar to our observations in the IMMU-11—sensitive cell lines, activation and phosphorylation of the ERKl/2 and JNK pathways were observed in the CLL patient cells, indicating the generation of stress in these samples (not shown). Almost 4- to 5-fold activation of ERK and JNK pathways was observed on incubation with IMMU-114 over untreated controls, although no such activation was seen on treatment with rituximab or milatuzumab (not shown).
- IMMU-114 induces cell death.
- Treatment with IMMU-114 induced a time-dependent mitochondrial membrane depolarization that could be detected in Raji cells as well as in other sensitive lines (not shown).
- a time-course analysis in Raji cells indicated a change in mitochondrial membrane depolarization of 46% in as little as 30 minutes of treatment, and a further increase to 66% in 24 hours (not shown). Similar changes in ROS levels were observed (not shown).
- a thirty minute incubation with IMMU-114 induced a 24% change in ROS levels that increased to 33% to 44% on overnight incubation (not shown).
- IMMU-114 cytotoxicity correlates with activation of ERK and JNK signaling.
- the results of these studies differentiate the mechanism of action of IMMU-114 cytotoxicity from that of the anti-CD74 (milatuzumab) and anti- CD20 MAbs.
- the DNL technique can be used to make dimers, trimers, tetramers, hexamers, etc. comprising virtually any antibody, antibody fragment, cytokine or other effector moiety.
- antibodies, cytokines, toxins or other protein or peptide effectors may be produced as fusion proteins comprising either a dimerization and docking domain (DDD) or anchoring domain (AD) sequence.
- DDD and AD moieties may be joined to antibodies, antibody fragments, cytokines or other effectors as fusion proteins, the skilled artisan will realize that other methods of conjugation exist, such as chemical cross-linking, click chemistry reaction, etc.
- the technique is not limiting and any protein or peptide of use may be produced as an AD or DDD fusion protein for incorporation into a DNL construct.
- the AD and DDD conjugates may comprise any molecule that may be cross-linked to an AD or DDD sequence using any cross-linking technique known in the art.
- a dendrimer or other polymeric moiety such as polyethyleneimine or polyethylene glycol (PEG), may be incorporated into a DNL construct, as described in further detail below.
- AD or DDD sequences may be utilized. Exemplary DDD and AD sequences are provided below.
- DDDl SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:45)
- DDD2 CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:46)
- AD2 CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:48)
- DDDl and DDD2 comprise the DDD sequence of the human RIIcc form of protein kinase A.
- the DDD and AD moieties may be based on the DDD sequence of the human RIa form of protein kinase A and a corresponding AKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.
- the plasmid vector pdHL2 has been used to produce a number of antibodies and antibody-based constructs. See Gillies et al., J Immunol Methods (1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.
- the di-cistronic mammalian expression vector directs the synthesis of the heavy and light chains of IgG.
- the vector sequences are mostly identical for many different IgG-pdHL2 constructs, with the only differences existing in the variable domain (VH and VL) sequences. Using molecular biology tools known to those skilled in the art, these IgG expression vectors can be converted into Fab-DDD or Fab- AD expression vectors.
- Fab-DDD expression vectors To generate Fab-DDD expression vectors, the coding sequences for the hinge, CH2 and CH3 domains of the heavy chain are replaced with a sequence encoding the first 4 residues of the hinge, a 14 residue Gly-Ser linker and the first 44 residues of human Rlla (referred to as DDD1).
- DDD1 human Rlla
- AD1 AKAP- S
- Two shuttle vectors were designed to facilitate the conversion of IgG-pdHL2 vectors to either Fab-DDD 1 or Fab- AD 1 expression vectors, as described below.
- the CHI domain was amplified by PCR using the pdHL2 plasmid vector as a template.
- the left PCR primer consisted of the upstream (5') end of the CHI domain and a Sacll restriction endonuclease site, which is 5' of the CHI coding sequence.
- the right primer consisted of the sequence coding for the first 4 residues of the hinge (P SC, SEQ ID NO:98) followed by four glycines and a serine, with the final two codons (GS) comprising a Bam HI restriction site.
- the 410 bp PCR amplimer was cloned into the PGEMT® PCR cloning vector (PROMEGA®, Inc.) and clones were screened for inserts in the T7 (5') orientation.
- a duplex oligonucleotide was synthesized to code for the amino acid sequence of DDD1 preceded by 11 residues of the linker peptide, with the first two codons comprising a BamHI restriction site. A stop codon and an Eagl restriction site are appended to the 3 'end.
- the encoded polypeptide sequence is shown below.
- oligonucleotides designated RIIAl-44 top and RIIAl-44 bottom, which overlap by 30 base pairs on their 3' ends, were synthesized and combined to comprise the central 154 base pairs of the 174 bp DDD1 sequence.
- the oligonucleotides were annealed and subjected to a primer extension reaction with Taq polymerase. Following primer extension, the duplex was amplified by PCR. The amplimer was cloned into PGEMT® and screened for inserts in the T7 (5') orientation.
- a duplex oligonucleotide was synthesized to code for the amino acid sequence of AD1 preceded by 1 1 residues of the linker peptide with the first two codons comprising a BamHI restriction site. A stop codon and an Eagl restriction site are appended to the 3 'end. The encoded polypeptide sequence is shown below.
- a 190 bp fragment encoding the DDDl sequence was excised from PGEMT® with BamHI and Notl restriction enzymes and then li gated into the same sites in CHI -PGEMT® to generate the shuttle vector CHI -DDDl -PGEMT®.
- a 110 bp fragment containing the AD1 sequence was excised from PGEMT® with BamHI and Notl and then ligated into the same sites in CHI -PGEMT® to generate the shuttle vector CHI -AD 1 -PGEMT®.
- CHI -DDDl or CHl-ADl can be incorporated into any IgG construct in the pdHL2 vector.
- the entire heavy chain constant domain is replaced with one of the above constructs by removing the SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacing it with the SacII/EagI fragment of CHI -DDDl or CHl-ADl, which is excised from the respective pGemT shuttle vector.
- h679-Fd-ADl-pdHL2 is an expression vector for production of h679 Fab with AD1 coupled to the carboxyl terminal end of the CHI domain of the Fd via a flexible Gly/Ser peptide spacer composed of 14 amino acid residues.
- a pdHL2 -based vector containing the variable domains of h679 was converted to h679-Fd-ADl-pdHL2 by replacement of the SacII/EagI fragment with the CHl-ADl fragment, which was excised from the CH1-AD1- SV3 shuttle vector with SacII and Eagl.
- C-DDDl-Fd-hMN-14-pdHL2 is an expression vector for production of a stable dimer that comprises two copies of a fusion protein C-DDDl-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxyl terminus of CHI via a flexible peptide spacer.
- the plasmid vector hMN-14(I)-pdHL2 which has been used to produce hMN-14 IgG, was converted to C-DDDl-Fd-hMN-14-pdHL2 by digestion with SacII and Eagl restriction endonucleases to remove the CH1-CH3 domains and insertion of the CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttle vector with SacII and Eagl.
- AD- and DDD-fusion proteins comprising a Fab fragment of any of such antibodies may be combined, in an approximate ratio of two DDD-fusion proteins per one AD-fusion protein, to generate a trimeric DNL construct comprising two Fab fragments of a first antibody and one Fab fragment of a second antibody.
- N-DDDl-Fd-hMN-14-pdHL2 is an expression vector for production of a stable dimer that comprises two copies of a fusion protein N-DDDl-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the amino terminus of VH via a flexible peptide spacer.
- the expression vector was engineered as follows. The DDD1 domain was amplified by PCR.
- the hMN-14 Fd sequence was amplified by PCR. As a result of the PCR, a BamHI restriction site and the coding sequence for part of the linker were appended to the 5' end of the amplimer. A stop codon and Eagl restriction site was appended to the 3' end. The 1043 bp amplimer was cloned into pGemT. The hMN-14-Fd insert was excised from pGemT with BamHI and Eagl restriction enzymes and then ligated with DDD1-SV3 vector, which was prepared by digestion with those same enzymes, to generate the construct N-DDDl-hMN- 14Fd-SV3.
- the N-DDDl-hMN-14 Fd sequence was excised with Xhol and Eagl restriction enzymes and the 1.28 kb insert fragment was ligated with a vector fragment that was prepared by digestion of C-hMN-14-pdHL2 with those same enzymes.
- the final expression vector was N-DDDl-Fd-hMN-14-pDHL2.
- the N-linked Fab fragment exhibited similar DNL complex formation and antigen binding characteristics as the C-linked Fab fragment (not shown).
- C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production of C-DDD2-Fab- hMN-14, which possesses a dimerization and docking domain sequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14 via a 14 amino acid residue Gly/Ser peptide linker.
- the fusion protein secreted is composed of two identical copies of hMN-14 Fab held together by non-covalent interaction of the DDD2 domains.
- the expression vector was engineered as follows. Two overlapping, complimentary oligonucleotides, which comprise the coding sequence for part of the linker peptide and residues 1-13 of DDD2, were made synthetically. The oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and Pstl, respectively.
- the duplex DNA was ligated with the shuttle vector CH1-DDD1 -PGEMT®, which was prepared by digestion with BamHI and Pstl, to generate the shuttle vector CH1-DDD2- PGEMT®.
- a 507 bp fragment was excised from CH 1 -DDD2-PGEMT® with SacII and Eagl and ligated with the IgG expression vector hMN-14(I)-pdHL2, which was prepared by digestion with SacII and Eagl.
- the final expression construct was designated C-DDD2-Fd- hMN-14-pdHL2. Similar techniques have been utilized to generated DDD2-fusion proteins of the Fab fragments of a number of different humanized antibodies.
- h679-Fab-AD2 was designed to pair as B to C-DDD2-Fab-hMN- 14 as A.
- h679-Fd- AD2-pdHL2 is an expression vector for the production of h679-Fab-AD2, which possesses an anchoring domain sequence of AD2 appended to the carboxyl terminal end of the CHI domain via a 14 amino acid residue Gly/Ser peptide linker.
- AD2 has one cysteine residue preceding and another one following the anchor domain sequence of AD1.
- the expression vector was engineered as follows. Two overlapping, complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprise the coding sequence for AD2 and part of the linker sequence, were made synthetically.
- oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and Spel, respectively.
- duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, which was prepared by digestion with BamHI and Spel, to generate the shuttle vector CH1-AD2- PGEMT®.
- a 429 base pair fragment containing CHI and AD2 coding sequences was excised from the shuttle vector with SacII and Eagl restriction enzymes and ligated into h679-pdHL2 vector that prepared by digestion with those same enzymes.
- the final expression vector is h679-Fd-AD2-pdHL2.
- TF1 A large scale preparation of a DNL construct, referred to as TF1, was carried out as follows. N-DDD2-Fab-hMN- 14 (Protein L-purified) and h679-Fab-AD2 (IMP-291 -purified) were first mixed in roughly stoichiometric concentrations in ImM EDTA, PBS, pH 7.4. Before the addition of TCEP, SE-HPLC did not show any evidence of a 2 b formation (not shown). Instead there were peaks representing a4 (7.97 min; 200 kDa), a 2 (8.91 min; 100 kDa) and B (10.01 min; 50 kDa).
- TF1 is a highly stable complex.
- HSG IMP- 239
- TF1 is a highly stable complex.
- IMP- 239 HSG
- C- DDDl-Fab-hMN-14 and h679-Fab-ADl was tested under similar conditions, the observed increase in response units was accompanied by a detectable drop during and immediately after sample injection, indicating that the initially formed a 2 b structure was unstable.
- a trimeric DNL construct designated TF2 was obtained by reacting C-DDD2-Fab- hMN-14 with h679-Fab-AD2.
- a pilot batch of TF2 was generated with >90% yield as follows.
- Protein L-purified C-DDD2-Fab-hMN- 14 200 mg was mixed with h679-Fab-AD2 (60 mg) at a 1.4: 1 molar ratio.
- the total protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.
- Subsequent steps involved TCEP reduction, HIC chromatography, DMSO oxidation, and IMP 291 affinity chromatography. Before the addition of TCEP, SE- HPLC did not show any evidence of a 2 b formation.
- TF2 was purified to near homogeneity by IMP 291 affinity chromatography (not shown).
- IMP 291 is a synthetic peptide containing the HSG hapten to which the 679 Fab binds (Rossi et al., 2005, Clin Cancer Res 1 1 :7122s-29s).
- SE-HPLC analysis of the IMP 291 unbound fraction demonstrated the removal of a4, a 2 and free kappa chains from the product (not shown).
- TF2 The functionality of TF2 was determined by BIACORE® assay.
- TF2, C-DDD1- hM -14+h679-ADl (used as a control sample of noncovalent a 2 b complex), or C-DDD2- hMN-14+h679-AD2 (used as a control sample of unreduced a 2 and b components) were diluted to 1 ⁇ ⁇ (total protein) and passed over a sensorchip immobilized with HSG.
- the response for TF2 was approximately two-fold that of the two control samples, indicating that only the h679-Fab-AD component in the control samples would bind to and remain on the sensorchip.
- the IgG and Fab fusion proteins shown in Table 2 were constructed and incorporated into DNL constructs.
- the fusion proteins retained the antigen-binding characteristics of the parent antibodies and the DNL constructs exhibited the antigen-binding activities of the incorporated antibodies or antibody fragments.
- DDDl, DDD2, DDD3, DDD3C, ADl, AD2 and AD3 sequence variants of AD and/or DDD moieties may be utilized in construction of the DNL complexes.
- Rlla DDD sequence is the basis of DDDl and DDD2 disclosed above.
- the four human PKA DDD sequences are shown below.
- the DDD sequence represents residues 1-44 of Rlla, 1-44 of RIIp, 12-61 of RIa and 13-66 of Rip. (Note that the sequence of DDD1 is modified slightly from the human PKA Rlla DDD moiety.)
- AD moiety binding may also be readily determined by standard binding assays, for example as disclosed in Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50).
- Alto et al. performed a bioinformatic analysis of the AD sequence of various AKAP proteins to design an RII selective AD sequence called AKAP-IS (SEQ ID NO:47), with a binding constant for DDD of 0.4 nM.
- the AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA. Residues in the AKAP-IS sequence where substitutions tended to decrease binding to DDD are underlined in SEQ ID NO:47 below.
- the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moiety sequence to prepare DNL constructs.
- Other alternative sequences that might be substituted for the AKAP-IS AD sequence are shown in SEQ ID NO:61-63. Substitutions relative to the AKAP-IS sequence are underlined. It is anticipated that, as with the AD2 sequence shown in SEQ ID NO:48, the AD moiety may also include the additional N-terminal residues cysteine and glycine and C-terminal residues glycine and cysteine.
- Figure 2 of Gold et al. disclosed additional DDD-binding sequences from a variety of AKAP proteins, shown below.
- LAWKIAKMIVSDVMQQ (SEQ ID NO:73)
- AKAP 1 -pep EEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO:86)
- AKAP2-pep LVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 87)
- AKAP 10-pep NTDEAQEELAWKIAKMlVSDIMQQA (SEQ ID NO:90)
- AKAP12-pep NGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO:92)
- Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree of sequence homology between different AKAP -binding DDD sequences from human and non-human proteins and identified residues in the DDD sequences that appeared to be the most highly conserved among different DDD moieties. These are indicated below by underlining with reference to the human PKA RJIa DDD sequence of SEQ ID NO:45. Residues that were particularly conserved are further indicated by italics. The residues overlap with, but are not identical to those suggested by Kinderman et al. (2006) to be important for binding to AKAP proteins.
- Cationic polymers such as polylysine, polyethylenimine, or polyamidoamine (PAMAM)-based dendrimers, form complexes with nucleic acids.
- PAMAM polyamidoamine
- One approach to improve selectivity and potency of a dendrimeric nanoparticle may be achieved by conjugation with an antibody that internalizes upon binding to target cells.
- E1-G5/2 We synthesized and characterized a novel immunoconjugate, designated E1-G5/2, which was made by the DNL method to comprise half of a generation 5 (G5) PAMAM dendrimer (G5/2) site-specifically linked to a stabilized dimer of Fab derived from hRS7, a humanized antibody that is rapidly internalized upon binding to the Trop-2 antigen expressed on various solid cancers.
- G5/2 generation 5
- Fab derived from hRS7 a humanized antibody that is rapidly internalized upon binding to the Trop-2 antigen expressed on various solid cancers.
- E1-G5/2 was prepared by combining two self-assembling modules, AD2-G5/2 and hRS7-Fab-DDD2, under mild redox conditions, followed by purification on a Protein L column.
- AD2-G5/2 we derivatized the AD2 peptide with a maleimide group to react with the single thiol generated from reducing a G5 PAMAM with a cystamine core and used reversed-phase HPLC to isolate AD2-G5/2.
- hRS7-Fab-DDD2 as a fusion protein in myeloma cells, as described in the Examples above.
- E1-G5/2 The molecular size, purity and composition of E1-G5/2 were analyzed by size- exclusion HPLC, SDS-PAGE, and Western blotting. The biological functions of E1-G5/2 were assessed by binding to an anti-idiotype antibody against hRS7, a gel retardation assay, and a DNase protection assay.
- E1-G5/2 was shown by size-exclusion HPLC to consist of a major peak (>90%) flanked by several minor peaks.
- the three constituents of E1-G5/2 (Fd-DDD2, the light chain, and AD2-G5/2) were detected by reducing SDS-PAGE and confirmed by Western blotting.
- Anti-idiotype binding analysis revealed E1-G5/2 contained a population of antibody-dendrimer conjugates of different size, all of which were capable of recognizing the anti-idiotype antibody, thus suggesting structural variability in the size of the purchased G5 dendrimer.
- the DNL technique can be used to build dendrimer-based nanoparticles that are targetable with antibodies. Such agents have improved properties as carriers of drugs, plasmids or siRNAs for applications in vitro and in vivo.
- anti- APC and/or anti-DC antibodies such as anti-CD74 and/or anti-HLA-DR, may be utilized to deliver cytotoxic or cytostatic siRNA species to targeted DCs and/or APCs for therapy of GVHD and other immune dysfunctions.
- the peptide IMP 498 up to and including the PEG moiety was synthesized on a Protein Technologies PS3 peptide synthesizer by the Fmoc method on Sieber Amide resin (0.1 mmol scale).
- the maleimide was added manually by mixing the ⁇ -maleimidopropionic acid NHS ester with diisopropylethylamine and DMF with the resin for 4 hr.
- the peptide was cleaved from the resin with 15 mL TFA, 0.5 mL H 2 0, 0.5 mL triisopropylsilane, and 0.5 mL thioanisole for 3 hr at room temperature.
- the peptide was purified by reverse phase HPLC using H 2 0/CH 3 CN TFA buffers to obtain about 90 mg of purified product after
- RNA interference has been shown to down-regulate the expression of various proteins such as HER2, VEGF, Raf-1, bcl-2, EGFR and numerous others in preclinical studies. Despite the potential of RNAi to silence specific genes, the full therapeutic potential of RNAi remains to be realized due to the lack of an effective delivery system to target cells in vivo.
- a DDD2-L-thPl module comprising truncated human protamine (thPl, residues 8 to 29 of human protamine 1) was produced, in which the sequences of DDD2 and thPl were fused respectively to the N- and C -terminal ends of a humanized antibody light chain (not shown).
- the sequence of the truncated hPl (thPl) is shown below.
- El-L-thPl The purity and molecular integrity of El-L-thPl following Protein A purification were determined by size-exclusion HPLC and SDS-PAGE (not shown). In addition, the ability of El-L-thPl to bind plasmid DNA or siRNA was demonstrated by the gel shift assay (not shown). El-L-thPl was effective at binding short double-stranded oligonucleotides (not shown) and in protecting bound DNA from digestion by nucleases added to the sample or present in serum (not shown).
- the DNL technique was employed to generate El-L-thPl .
- the hRS7 IgG-AD module constructed as described in the Examples above, was expressed in myeloma cells and purified from the culture supernatant using Protein A affinity chromatography.
- the DDD2-L-thPl module was expressed as a fusion protein in myeloma cells and was purified by Protein L affinity chromatography. Since the CH3-AD2-IgG module possesses two AD2 peptides and each can bind to a DDD2 dimer, with each DDD2 monomer attached to a protamine moiety, the resulting El-L-thPl conjugate comprises four protamine groups.
- El-L- thpl was formed in nearly quantitative yield from the constituent modules and was purified to near homogeneity (not shown) with Protein A.
- DDD2-L-thPl was purified using Protein L affinity chromatography and assessed by size exclusion HPLC analysis and SDS-PAGE under reducing and nonreducing conditions (data not shown). A major peak was observed at 9.6 min (not shown). SDS-PAGE showed a major band between 30 and 40 kDa in reducing gel and a major band about 60 kDa
- DDD2-L-thPl retarded the mobility of 500 ng of a linear form of 3-kb DNA fragment in 1% agarose at a molar ratio of 6 or higher (not shown).
- El-L- thPl retarded the mobility of 250 ng of a linear 200-bp DNA duplex in 2% agarose at a molar ratio of 4 or higher (not shown), whereas no such effect was observed for hRS7-IgG-AD2 alone (not shown).
- the ability of El-L-thPl to protect bound DNA from degradation by exogenous DNase and serum nucleases was also demonstrated (not shown).
- El-L-thPl (10 ⁇ g) was mixed with FITC-siRNA (300 nM) and allowed to form El-L-thPl -siRNA complexes which were then added to Trop-2-expressing Calu-3 cells. After incubation for 4 h at 37°C the cells were checked for internalization of siRNA by fluorescence microscopy (not shown).
- El-L-thPl The ability of El-L-thPl to induce apoptosis by internalization of siRNA was examined.
- El-L-thPl (10 ⁇ g) was mixed with varying amounts of siRNA (AllStars Cell Death siRNA, Qiagen, Valencia, CA).
- the El-L-thPl -siRNA complex was added to ME- 180 cells. After 72 h of incubation, cells were trypsinized and annexin V staining was performed to evaluate apoptosis.
- the DNL technology provides a modular approach to efficiently tether multiple protamine molecules to the anti-Trop-2 hRS7 antibody resulting in the novel molecule El-L- thP 1.
- SDS-P AGE demonstrated the homogeneity and purity of E 1 -L-thP 1.
- DNase protection and gel shift assays showed the DNA binding activity of El-L-thPl.
- El-L-thPl internalized in the cells like the parental hRS7 antibody and was able to effectively internalize siRNA molecules into Trop-2-expressing cells, such as ME- 180 and Calu-3.
- DNL technique is not limited to any specific antibody or siRNA species. Rather, the same methods and compositions demonstrated herein can be used to make targeted delivery complexes comprising any antibody, any siRNA carrier and any siRNA species.
- the use of a bivalent IgG in targeted delivery complexes would result in prolonged circulating half-life and higher binding avidity to target cells, resulting in increased uptake and improved efficacy.
- HIDS hexavalent IgG-based DNL structures
- modules Two types of modules, which were produced as recombinant fusion proteins, were combined to generate a variety of HIDS.
- Fab- DDD2 modules were as described for use in generating trivalent Fab structures (Rossi et al. Proc Natl Acad Sci USA.2006; 103(18): 6841-6).
- the Fab-DDD2 modules form stable homodimers that bind to AD2-containing modules.
- IgG-AD2 modules were created to pair with the Fab-DDD2 modules: C-H-AD2-IgG and N-L-AD2- IgG.
- C-H-AD2-IgG modules have an AD2 peptide fused to the carboxyl terminus (C) of the heavy (H) chain of IgG via a 9 amino acid residue peptide linker.
- the DNA coding sequences for the linker peptide followed by the AD2 peptide are coupled to the 3 ' end of the CH3 (heavy chain constant domain 3) coding sequence by standard recombinant DNA methodologies, resulting in a contiguous open reading frame.
- the C-H- AD2-IgG module can be combined with any Fab-DDD2 module to generate a wide variety of hexavalent structures composed of an Fc fragment and six Fab fragments. If the C-H-AD2- IgG module and the Fab-DDD2 module are derived from the same parental monoclonal antibody (MAb) the resulting HIDS is monospecific with 6 binding arms to the same antigen. If the modules are instead derived from two different MAbs then the resulting HIDS are bispecific, with two binding arms for the specificity of the C-H-AD2-IgG module and 4 binding arms for the specificity of the Fab-DDD2 module.
- MAb parental monoclonal antibody
- N-L-AD2-IgG is an alternative type of IgG-AD2 module in which an AD2 peptide is fused to the amino terminus (N) of the light (L) chain of IgG via a peptide linker.
- the L chain can be either Kappa (K) or Lambda ( ⁇ ) and will also be represented as K.
- the DNA coding sequences for the AD2 peptide followed by the linker peptide are coupled to the 5' end of the coding sequence for the variable domain of the L chain (VL), resulting in a contiguous open reading frame.
- the N-L-AD2-IgG module can be combined with any Fab- DDD2 module to generate a wide variety of hexavalent structures composed of an Fc fragment and six Fab fragments.
- DNL complexes comprising an IgG moiety attached to four effector moieties, such as cytokines.
- an IgG moiety was attached to four copies of interferon-a2b.
- the antibody-cytokine DNL construct exhibited superior pharmacokinetic properties and/or efficacy compared to
- Hex-hA20 a monospecific anti-CD20 HIDS, by combining C-H-AD2-hA20 IgG with hA20-Fab-DDD2.
- the Hex-hA20 structure contains six anti-CD20 Fab fragments and an Fc fragment, arranged as four Fab fragments and one IgG antibody.
- Hex-hA20 was made in four steps.
- the molecular weights of C-H-AD2-hA20 IgG and (hA20-Fab-DDD2) 2 are 168 kDa and 107 kDa, respectively.
- 134 mg of hA20-Fab-DDD2 would be mixed with 100 mg of C-H-AD2-hA20 IgG to achieve a 210% molar equivalent of the former.
- the mixture is typically made in phosphate buffered saline, pH 7.4 (PBS) with 1 mM EDTA.
- Step 2 Mild Reduction: Reduced glutathione (GSH) was added to a final
- Step 3 Mild Oxidation: Following reduction, oxidized glutathione (GSSH) was added directly to the reaction mixture to a final concentration of 2 mM and the solution was held at room temperature for 1 -24 hours.
- GSSH oxidized glutathione
- Step 4 Isolation of the DNL product: Following oxidation, the reaction mixture was loaded directly onto a Protein-A affinity chromatography column. The column was washed with PBS and the Hex-hA20 was eluted with 0.1 M glycine, pH 2.5. Since excess hA20-Fab- DDD2 was used in the reaction, there was no unconjugated C-H-AD2-hA20 IgG, or incomplete DNL structures containing only one (hA20-Fab-DDD2) 2 moiety. The
- the Protein A-purified material contains only the desired product.
- the calculated molecular weight from the deduced amino acid sequences of the constituent polypeptides is 386 kDa.
- Size exclusion HPLC analysis showed a single protein peak with a retention time consistent with a protein structure of 375 - 400 kDa (not shown).
- SDS-PAGE analysis under non-reducing conditions showed a cluster of high molecular weight bands indicating a large covalent structure (not shown).
- SDS-PAGE under reducing conditions showed the presence of only the three expected polypeptide chains: the AD2-fused heavy chain (HC-AD2), the DDD2-fused Fd chain (Fd-DDD2), and the kappa chains (not shown).
- the DNL method was used to create a monospecific anti-CD22 HIDS (Hex-hLL2) by combining C-H-AD2-hLL2 IgG with hLL2-Fab-DDD2.
- the DNL reaction was accomplished as described above for Hex-hA20.
- the calculated molecular weight from the deduced amino acid sequences of the constituent polypeptides is 386 kDa.
- Size exclusion HPLC analysis showed a single protein peak with a retention time consistent with a protein structure of 375 - 400 kDa (not shown).
- SDS-PAGE analysis under non-reducing conditions showed a cluster of high molecular weight bands, which were eliminated under reducing conditions to leave only the three expected polypeptide chains: HC-AD2, Fd-DDD2, and the kappa chain (not shown).
- the DNL method was used to create bispecific HIDS by combining C-H-AD2-hLL2 IgG with either hA20-Fab-DDD2 to obtain DNL1 or hMN-14-DDD2 to obtain DNL1C.
- DNL1 has four binding arms for CD20 and two for CD22.
- hMN-14 is a humanized MAb to carcinoembryonic antigen (CEACAM5)
- CEACAM5 humanized MAb to carcinoembryonic antigen
- the DNL reactions were accomplished as described for Hex-hA20 above.
- the calculated molecular weights from the deduced amino acid sequences of the constituent polypeptides are ⁇ 386 kDa.
- Size exclusion HPLC analysis showed a single protein peak with a retention time consistent with a protein structure of 375 - 400 kDa for each structure (not shown).
- SDS-PAGE analysis under non-reducing conditions showed a cluster of high molecular weight bands, which were eliminated under reducing conditions to leave only the three expected polypeptides: HC-AD2, Fd-DDD2, and the kappa chain (not shown).
- the DNL method was used to create bispecific HIDS by combining C-H-AD2-hA20 IgG with either hLL2-Fab-DDD2 to obtain DNL2 or hMN-14-DDD2 to obtain DNL2C.
- DNL2 has four binding arms for CD22 and two for CD20.
- DNL2C has four binding arms for CEACAM5 and two for CD20. The DNL reactions were accomplished as described for Hex- hA20.
- the calculated molecular weights from the deduced amino acid sequences of the constituent polypeptides are -386 kDa.
- Size exclusion HPLC analysis showed a single protein peak with a retention time consistent with a protein structure of 375 - 400 kDa for each structure (not shown).
- SDS-PAGE analysis under non-reducing conditions showed high molecular weight bands, but under reducing conditions consisted solely of the three expected polypeptides: HC-AD2, Fd-DDD2, and the kappa chain (not shown).
- the DNL method was used to create a monospecific anti-CD20 HIDS ( -Hex-hA20) by combining N-L-AD2-hA20 IgG with hA20-Fab-DDD2. The DNL reaction was accomplished as described above for Hex-hA20.
- the calculated molecular weight from the deduced amino acid sequences of the constituent polypeptides is 386 kDa.
- SDS-PAGE analysis under non-reducing conditions showed a cluster of high molecular weight bands, which under reducing conditions were composed solely of the four expected polypeptides: Fd-DDD2, H-chain, kappa chain, and AD2-kappa (not shown).
- a bispecific HIDS was generated by combining N-L-AD2-hA20 IgG with hLL2-Fab- DDD2.
- the DNL reaction was accomplished as described above for Hex-hA20.
- the calculated molecular weight from the deduced amino acid sequences of the constituent polypeptides is 386 kDa.
- Size exclusion HPLC analysis showed a single protein peak with a retention time consistent with a protein structure of 375 - 400 kDa (not shown).
- SDS-PAGE analysis under non-reducing conditions showed a cluster of high molecular weight bands that under reducing conditions showed only the four expected polypeptides: Fd-DDD2, H-chain, kappa chain, and AD2-kappa (not shown).
- DNL 1 and DNL2 were highly stable in serum and maintained complete bispecific binding activity.
- HIDS generated as described above retained the binding properties of their parental Fab/IgGs.
- Competitive ELISAs were used to investigate the binding avidities of the various HIDS using either a rat anti-idiotype MAb to hA20 (WR2) to assess the binding activity of the hA20 components or a rat anti-idiotype MAb to hLL2 (WN) to assess the binding activity of the hLL2 components.
- WR2 rat anti-idiotype MAb to hA20
- WN rat anti-idiotype MAb to hLL2
- hLL2 binding plates were coated with hLL2 IgG and the HIDS were allowed to compete with the immobilized IgG for WN binding.
- the relative amount of anti- Id bound to the immobilized IgG was detected using peroxidase-conjugated anti-Rat IgG.
- DNL2 and DNL3 contain two hA20 Fabs and four hLL2 Fabs, they showed similar strength in binding to the same anti-id antibody (not shown).
- HIDS Some of the HIDS were observed to have potent anti-proliferative activity on lymphoma cell lines. DNLl, DNL2 and Hex-hA20 inhibited cell growth of Daudi Burkitt Lymphoma cells in vitro (not shown). Treatment of the cells with 10 nM concentrations was substantially more effective for the HIDS compared to rituximab (not shown). Using a cell counting assay, the potency of DNLl and DNL2 was estimated to be more than 100-fold greater than that of rituximab, while the Hex-hA20 was shown to be even more potent (not shown).
- Rap-DDD Rap-DDD
- humanized IgG-AD modules which were produced in myeloma cells and targeted B-cell lymphomas and leukemias via binding to CD20 (hA20, veltuzumab), CD22 (hLL2, epratuzumab) or HLA-DR (hL243, IMMU-114), to generate 20-Rap, 22-Rap and C2-Rap, respectively.
- a dimer of Rap was covalently tethered to the C-terminus of each heavy chain of the respective IgG.
- a control construct, 14-Rap was made similarly, using labetuzumab (hMN-14), that binds to an antigen (CEACAM5) not expressed on B-cell
- lymphomas/leukemias are lymphomas/leukemias.
- Rap-DDD2 The deduced amino acid sequence of secreted Rap-DDD2 is shown above (SEQ ID NO:99). Rap, underlined; linker, italics; DDD2, bold; pjQ, amino-terminal glutamine converted to pyroglutamate. Rap-DDD2 was produced in E. coli as inclusion bodies, which were purified by IMAC under denaturing conditions, refolded and then dialyzed into PBS before purification by Q-Sepharose anion exchange chromatography. SDS-PAGE under reducing conditions resolved a protein band with a Mr appropriate for Rap-DDD2 (18.6 kDa) (not shown). The final yield of purified Rap-DDD2 was 10 mg/L of culture.
- the DNL method was employed to rapidly generate a panel of IgG-Rap conjugates.
- the IgG-AD modules were expressed in myeloma cells and purified from the culture supernatant using Protein A affinity chromatography.
- the Rap-DDD2 module was produced and mixed with IgG-AD2 to form a DNL complex. Since the CH3-AD2-IgG modules possess two AD2 peptides and each can tether a Rap dimer, the resulting IgG-Rap DNL construct comprises four Rap groups and one IgG. IgG-Rap is formed nearly quantitatively from the constituent modules and purified to near homogeneity with Protein A.
- the CH3-AD2-IgG exists as both a monomer, and a disulfide-linked dimer (not shown).
- the IgG-Rap resolves as a cluster of high molecular weight bands of the expected size between those for monomelic and dimeric CH3-AD2-IgG (not shown).
- Reducing conditions, which reduces the conjugates to their constituent polypeptides shows the purity of the IgG-Rap and the consistency of the DNL method, as only bands representing heavy-chain- AD2 (HC-AD2), kappa light chain and Rap-DDD2 were visualized (not shown).
- Rap-DDD2 Reversed phase HPLC analysis of 22-Rap (not shown) resolved a single protein peak at 9.10 min eluting between the two peaks of CH3-AD2-IgG-hLL2, representing the monomelic (7.55 min) and the dimeric (8.00 min) forms.
- the Rap-DDD2 module was isolated as a mixture of dimer and tetramer (reduced to dimer during DNL), which were eluted at 9.30 and 9.55 min, respectively (not shown).
- LC/MS analysis of 22-Rap was accomplished by coupling reversed phase HPLC using a C8 column with ESI-TOF mass spectrometry (not shown).
- the spectrum of unmodified 22-Rap identifies two major species, having either two G0F (G0F/G0F) or one G0F plus one GIF (G0F/G1F) N-linked glycans, in addition to some minor glycoforms (not shown). Enzymatic deglycosylation resulted in a single deconvoluted mass consistent with the calculated mass of 22-Rap (not shown).
- the resulting spectrum following reduction with TCEP identified the heavy chain-AD2 polypeptide modified with an N-linked glycan of the G0F or GIF structure as well as additional minor forms (not shown).
- Each of the three subunit polypeptides comprising 22-Rap were identified in the deconvoluted spectrum of the reduced and deglycosylated sample (not shown).
- the results confirm that both the Rap- DDD2 and HC-AD2 polypeptides have an amino terminal glutamine that is converted to pyroglutamate (pQ); therefore, 22-Rap has 6 of its 8 constituent polypeptides modified by pQ.
- hLL2 internalization rate for hLL2 (anti-CD22) is much faster than hA20 (anti-CD20).
- 14-Rap shares the same structure as 22-Rap and 20-Rap, but its antigen (CEACAM5) is not expressed by the NHL cells.
- Cells were treated continuously with IgG-Rap as single agents or with combinations of the parental MAbs plus rRap.
- Both 20-Rap and 22-Rap killed each cell line at concentrations above 1 nM, indicating that their action is cytotoxic as opposed to merely cytostatic (not shown).
- 20-Rap was the most potent IgG-Rap, suggesting that antigen density may be more important than internalization rate.
- the DNL method provides a modular approach to efficiently tether multiple cytotoxins onto a targeting antibody, resulting in novel immunotoxins that are expected to show higher in vivo potency due to improved pharmacokinetics and targeting specificity.
- LC/MS, RP-HPLC and SDS-PAGE demonstrated the homogeneity and purity of IgG-Rap.
- Targeting Rap with a MAb to a cell surface antigen enhanced its tumor-specific cytotoxicity.
- Antigen density and internalization rate are both critical factors for the observed in vitro potency of IgG-Rap.
- In vitro results show that CD20-, CD22-, or HLA-DR-targeted IgG-Rap have potent biologic activity for therapy of B-cell lymphomas and leukemias.
- the trimeric DNL constructs may comprise three different effector moieties, for example two different antibody moieties and a cytokine moiety.
- the generation and characterization of the first bispecific MAb-IFNa designated 20-C2-2b, which comprises two copies of IFN-a2b and a stabilized F(ab) 2 of hL243
- the 20-C2-2b displayed greater cytotoxicity against KMS12-BM (CD20 + /HLA- DR + myeloma) than monospecific MAb-IFNa that targets only HLA-DR or CD20, indicating that all three components in 20-C2-2b can contribute to toxicity.
- Our findings indicate that a given cell's responsiveness to MAb-IFNa depends on its sensitivity to IFNa and the specific antibodies, as well as the expression and density of the targeted antigens.
- 20-C2-2b has antibody-dependent cellular cytotoxicity (ADCC), but not CDC, and can target both CD20 and HLA-DR, it is useful for therapy of a broad range of hematopoietic disorders that express either or both antigens.
- ADCC antibody-dependent cellular cytotoxicity
- HLA-DR hematopoietic disorders that express either or both antigens.
- DCC antibody-dependent cellular cytotoxicity
- veltuzumab or v-mab anti-CD20 Igd
- hL243y4p Immu-114, anti-HLA-DR IgG 4
- a murine anti-IFNa MAb and rat anti-idiotype MAbs to v-mab (WR2) and hL243 (WT).
- DNL constructs were provided by Immunomedics, Inc.: veltuzumab or v-mab (anti-CD20 Igd), hL243y4p (Immu-114, anti-HLA-DR IgG 4 ), a murine anti-IFNa MAb, and rat anti-idiotype MAbs to v-mab (WR2) and hL243 (WT).
- DNL constructs DNL constructs
- Monospecific MAb-IFNa (20-2b-2b, 734-2b-2b and C2-2b-2b) and the bispecific HexAb (20-C2-C2) were generated by combination of an IgG-AD2 -module with DDD2- modules using the DNL method, as described in the preceding Examples.
- the construction of the mammalian expression vector as well as the subsequent generation of the production clones and the purification of C H 3-AD2-IgG-v-mab are disclosed in the preceding Examples.
- the expressed recombinant fusion protein has the AD2 peptide linked to the carboxyl terminus of the CH3 domain of v-mab via a 15 amino acid long flexible linker peptide.
- Co-expression of the heavy chain- AD2 and light chain polypeptides results in the formation of an IgG structure equipped with two AD2 peptides.
- the expression vector was transfected into Sp/ESF cells (an engineered cell line of Sp2/0) by electroporation.
- the pdHL2 vector contains the gene for dihydrofolate reductase, thus allowing clonal selection, as well as gene amplification with methotrexate (MTX).
- Stable clones were isolated from 96-well plates selected with media containing 0.2 ⁇ MTX. Clones were screened for C H 3-AD2-IgG-vmab productivity via a sandwich ELISA. The module was produced in roller bottle culture with serum- free media.
- the DDD-module, IFNa2b-DDD2 was generated as discussed above by recombinant fusion of the DDD2 peptide to the carboxyl terminus of human IFNa2b via an 18 amino acid long flexible linker peptide. As is the case for all DDD-modules, the expressed fusion protein spontaneously forms a stable homodimer.
- the C H l-DDD2-Fab-hL243 expression vector was generated from hL243-IgG-pdHL2 vector by excising the sequence for the 3 ⁇ 41- ⁇ 3 ⁇ 4 ⁇ -3 ⁇ 42-3 ⁇ 43 domains with SacII and Eagl restriction enzymes and replacing it with a 507 bp sequence encoding CH1-DDD2, which was excised from the C-DDD2-hMN-14-pdHL2 expression vector with the same enzymes.
- the culture broth containing the C H l-DDD2-Fab-hL243 module was applied directly to KAPPASELECT® affinity gel (GE-Healthcare), which was washed to baseline with PBS and eluted with 0.1 M Glycine, pH 2.5.
- the DNL mixture was purified with Protein A (MABSELECTTM), which binds the C H 3-AD2-IgG-v-MAb group and eliminates un-reacted IFNa2b-DDD2 or C H l-DDD2-Fab-hL243.
- the Protein A-bound material was further purified by IMAC using HIS-SELECT® HF Nickel Affinity Gel, which binds specifically to the IFNa2b-DDD2 moiety and eliminates any constructs lacking this group.
- the final process step, using an hL243-anti-idiotype affinity gel removed any molecules lacking C H l-DDD2-Fab-hL243.
- affinity chromatography may be used to purify DNL complexes comprising any combination of effector moieties, so long as ligands for each of the three effector moieties can be obtained and attached to the column material.
- the selected DNL construct is the one that binds to each of three columns containing the ligand for each of the three effector moieties and can be eluted after washing to remove unbound complexes.
- the eluate which contained ⁇ 20 mg protein, was neutralized with 3 M Tris-HCl, pH 8.6 and dialyzed into HIS-SELECT® binding buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH 2 P0 4 , pH 8.0) prior to application to a 5-mL HIS-SELECT® IMAC column.
- HIS-SELECT® binding buffer 10 mM imidazole, 300 mM NaCl, 50 mM NaH 2 P0 4 , pH 8.0
- the column was washed to baseline with binding buffer and eluted with 250 mM imidazole, 150 mM NaCl, 50 mM NaH 2 P0 4 , pH 8.0.
- the IMAC eluate which contained ⁇ 11.5 mg of protein, was applied directly to a WP (anti-hL243) affinity column, which was washed to baseline with PBS and eluted with 0.1 M glycine, pH 2.5.
- the process resulted in 7 mg of highly purified 20-C2-2b. This was approximately 44% of the theoretical yield of 20-C2-2b, which is 50% of the total starting material (16 mg in this example) with 25% each of 20-2b-2b and 20-C2-C2 produced as side products.
- the bispecific MAb-IFNa was generated by combining the IgG-AD2 module, CH3- AD2-IgG-v-mab, with two different dimeric DDD-modules, C H l-DDD2-Fab-hL243 and IFN 2b-DDD2. Due to the random association of either DDD-module with the two AD2 groups, two side-products, 20-C2-C2 and 20-2b-2b are expected to form, in addition to 20- C2-2b.
- Non-reducing SDS-PAGE resolved 20-C2-2b (-305 kDa) as a cluster of bands positioned between those of 20-C2-C2 (-365 kDa) and 20-2b-2b (255 kDa).
- Reducing SDS-PAGE resolved the five polypeptides (v-mab HC-AD2, hL243 Fd-DDD2, IFNa2b- DDD2 and co-migrating v-mab and hL243 kappa light chains) comprising 20-C2-2b (not shown).
- IFNa2b-DDD2 and hL243 Fd-DDD2 are absent in 20-C2-C2 and 20-2b-2b.
- MABSELECTTM binds to all three of the major species produced in the DNL reaction, but removes any excess IFNa2b-DDD2 and C H l-DDD2-Fab-hL243.
- the HIS-SELECT® unbound fraction contained mostly 20-C2-C2 (not shown).
- the unbound fraction from WT affinity chromatography comprised 20-2b-2b (not shown).
- Each of the samples was subjected to SE-HPLC and immunoreactivity analyses, which corroborated the results and conclusions of the SDS-PAGE analysis.
- LC/MS analysis of 20-C2-2b identified both the O-glycosylated and non-glycosylated species of IFNa2b-DDD2 with mass accuracies of 15 ppm and 2 ppm, respectively (not shown).
- the observed mass of the O-glycosylated form indicates an O-linked glycan having the structure NeuGc-NeuGc-Gal-GalNAc, which was also predicted ( ⁇ 1 ppm) for 20-2b-2b (not shown).
- LC/MS identified both v-mab and hL243 kappa chains as well as hL243-Fd-DDD2 (not shown) as single, unmodified species, with observed masses matching the calculated ones ( ⁇ 35 ppm).
- v-mab HC-AD2 Two major glycoforms of v-mab HC-AD2 were identified as having masses of 53,714.73 (70%) and 53,877.33 (30%), indicating G0F and GIF N-glycans, respectively, which are typically associated with IgG (not shown). The analysis also confirmed that the amino terminus of the HC-AD2 is modified to pyroglutamate, as predicted for polypeptides having an amino terminal glutamine.
- Immunoreactivity assays demonstrated the homogeneity of 20-C2-2b with each molecule containing the three functional groups (not shown). Incubation of 20-C2-2b with an excess of antibodies to any of the three constituent modules resulted in quantitative formation of high molecular weight immune complexes and the disappearance of the 20-C2-2b peak.
- the HIS-SELECT® and WT affinity unbound fractions were not immunoreactive with WT and anti-IFNa, respectively (not shown).
- the MAb-IFNa showed similar binding avidity to their parental MAbs (not shown).
- the 20-C2-2b DNL construct depleted lymphoma cells more effectively than normal B cells and had no effect on T cells (not shown). However, it did efficiently eliminate monocytes (not shown). Where v-mab had no effect on monocytes, depletion was observed following treatment with hL243a4p and MAb-IFNa, with 20-2b-2b and 734-2b-2b exhibiting similar toxicity (not shown). Therefore, the predictably higher potency of 20-C2-2b is attributed to the combined actions of anti-HLA-DR and IFNa, which may be augmented by HLA-DR targeting.
- monocyte depletion may be a pharmacodynamic effect associated anti-HLA-DR as well as IFNa therapy; however, this side affect would likely be transient because the monocyte population should be repopulated from hematopoietic stem cells.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Oncology (AREA)
- Inorganic Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Transplantation (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Endocrinology (AREA)
- Mycology (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2794499A CA2794499A1 (en) | 2010-04-01 | 2011-03-29 | Antibody-based depletion of antigen-presenting cells and dendritic cells |
AU2011235328A AU2011235328A1 (en) | 2010-04-01 | 2011-03-29 | Antibody-based depletion of antigen-presenting cells and dendritic cells |
EP11763313.1A EP2552483A4 (en) | 2010-04-01 | 2011-03-29 | Antibody-based depletion of antigen-presenting cells and dendritic cells |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US31990210P | 2010-04-01 | 2010-04-01 | |
US61/319,902 | 2010-04-01 | ||
US32928210P | 2010-04-29 | 2010-04-29 | |
US61/329,282 | 2010-04-29 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011123428A1 true WO2011123428A1 (en) | 2011-10-06 |
Family
ID=44709922
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2011/030294 WO2011123428A1 (en) | 2010-04-01 | 2011-03-29 | Antibody-based depletion of antigen-presenting cells and dendritic cells |
Country Status (5)
Country | Link |
---|---|
US (3) | US20110243841A1 (en) |
EP (1) | EP2552483A4 (en) |
AU (1) | AU2011235328A1 (en) |
CA (1) | CA2794499A1 (en) |
WO (1) | WO2011123428A1 (en) |
Families Citing this family (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1355666B1 (en) | 2000-12-22 | 2012-06-13 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Use of repulsive guidance molecule (RGM) and its modulators |
US8883160B2 (en) * | 2004-02-13 | 2014-11-11 | Ibc Pharmaceuticals, Inc. | Dock-and-lock (DNL) complexes for therapeutic and diagnostic use |
US9550838B2 (en) | 2004-02-13 | 2017-01-24 | Ibc Pharmaceuticals, Inc. | Dock-and-lock (DNL) complexes for therapeutic and diagnostic use |
US8158129B2 (en) * | 2005-04-06 | 2012-04-17 | Ibc Pharmaceuticals, Inc. | Dimeric alpha interferon PEGylated site-specifically shows enhanced and prolonged efficacy in vivo |
CA2624562A1 (en) | 2005-09-30 | 2007-04-12 | Abbott Gmbh & Co. Kg | Binding domains of proteins of the repulsive guidance molecule (rgm) protein family and functional fragments thereof, and their use |
US8962803B2 (en) | 2008-02-29 | 2015-02-24 | AbbVie Deutschland GmbH & Co. KG | Antibodies against the RGM A protein and uses thereof |
US9175075B2 (en) | 2009-12-08 | 2015-11-03 | AbbVie Deutschland GmbH & Co. KG | Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein |
US11214610B2 (en) | 2010-12-01 | 2022-01-04 | H. Lundbeck A/S | High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris |
US9884909B2 (en) | 2010-12-01 | 2018-02-06 | Alderbio Holdings Llc | Anti-NGF compositions and use thereof |
CN103619879A (en) | 2010-12-01 | 2014-03-05 | 奥尔德生物控股有限责任公司 | Anti-ngf compositions and use thereof |
US9067988B2 (en) | 2010-12-01 | 2015-06-30 | Alderbio Holdings Llc | Methods of preventing or treating pain using anti-NGF antibodies |
US9078878B2 (en) | 2010-12-01 | 2015-07-14 | Alderbio Holdings Llc | Anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75 |
US9539324B2 (en) | 2010-12-01 | 2017-01-10 | Alderbio Holdings, Llc | Methods of preventing inflammation and treating pain using anti-NGF compositions |
SG10202006762PA (en) | 2012-01-27 | 2020-08-28 | Abbvie Deutschland | Composition and method for diagnosis and treatment of diseases associated with neurite degeneration |
WO2014028502A1 (en) * | 2012-08-13 | 2014-02-20 | ImmunGene Inc. | Engineered antibody-interferon fusion molecules for treatment of autoimmune diseases |
KR20230078823A (en) * | 2012-12-13 | 2023-06-02 | 이뮤노메딕스, 인코오포레이티드 | Dosages of immunoconjugates of antibodies and sn-38 for improved efficacy and decreased toxicity |
IL300029A (en) | 2014-05-16 | 2023-03-01 | Baylor Res Institute | Methods and compositions for treating autoimmune and inflammatory conditions |
EP4249916A3 (en) * | 2015-04-06 | 2023-11-01 | President and Fellows of Harvard College | Compositions and methods for non-myeloablative conditioning |
IL255664A0 (en) | 2017-11-14 | 2017-12-31 | Shachar Idit | Hematopoietic stem cells with improved properties |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070086942A1 (en) * | 2005-10-19 | 2007-04-19 | Ibc Pharmaceuticals, Inc. | Methods and compositions for generating bioactive assemblies of increased complexity and uses |
US20080210475A1 (en) * | 2003-08-21 | 2008-09-04 | Microsoft Corporation | Ink Editing Architecture |
US20100015048A1 (en) * | 1999-06-09 | 2010-01-21 | Immunomedics, Inc. | Internalizing Anti-CD74 Antibodies and Methods of Use |
US20100068137A1 (en) * | 2005-10-19 | 2010-03-18 | Ibc Pharmaceuticals, Inc. | Dock-and-Lock (DNL) Vaccines for Cancer Therapy |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
HUT77342A (en) * | 1994-12-07 | 1998-03-30 | F.Hoffmann-La Roche Ag. | Monoclonal antibody fragments having immunosuppressant activity |
US8119101B2 (en) * | 1999-05-10 | 2012-02-21 | The Ohio State University | Anti-CD74 immunoconjugates and methods of use |
US20060222633A1 (en) * | 2000-05-11 | 2006-10-05 | Yale University | Prevention, decrease, and/or treatment of immunoreactivity by depleting and/or inactivating antigen presenting cells in the host |
EP1667717A2 (en) * | 2003-09-09 | 2006-06-14 | GPC Biotech AG | Therapeutic human anti-mhc class ii antibodies and their uses |
US20060280738A1 (en) * | 2005-06-08 | 2006-12-14 | Tedder Thomas F | Anti-CD19 antibody therapy for transplantation |
JP5011277B2 (en) * | 2005-04-06 | 2012-08-29 | アイビーシー・ファーマシューティカルズ・インコーポレーテッド | Methods and uses for generating stably linked complexes consisting of homodimers, homotetramers or dimeric dimers |
WO2007075270A2 (en) * | 2005-12-16 | 2007-07-05 | Ibc Pharmaceuticals, Inc. | Multivalent immunoglobulin-based bioactive assemblies |
JP5740772B2 (en) * | 2009-08-31 | 2015-07-01 | アイビーシー ファーマスーティカルズ,インコーポレイテッド | Bispecific immune cytokine dock-and-lock (DNL) complex and therapeutic use thereof |
EP2523680A4 (en) * | 2010-01-11 | 2013-06-19 | Ct Molecular Med & Immunology | Enhanced cytotoxicity of anti-cd74 and anti-hla-dr antibodies with interferon-gamma |
-
2011
- 2011-03-29 CA CA2794499A patent/CA2794499A1/en not_active Abandoned
- 2011-03-29 US US13/074,351 patent/US20110243841A1/en not_active Abandoned
- 2011-03-29 AU AU2011235328A patent/AU2011235328A1/en not_active Abandoned
- 2011-03-29 EP EP11763313.1A patent/EP2552483A4/en not_active Withdrawn
- 2011-03-29 WO PCT/US2011/030294 patent/WO2011123428A1/en active Application Filing
-
2012
- 2012-10-19 US US13/656,159 patent/US20130164214A1/en not_active Abandoned
-
2016
- 2016-12-14 US US15/378,972 patent/US20170088619A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100015048A1 (en) * | 1999-06-09 | 2010-01-21 | Immunomedics, Inc. | Internalizing Anti-CD74 Antibodies and Methods of Use |
US20080210475A1 (en) * | 2003-08-21 | 2008-09-04 | Microsoft Corporation | Ink Editing Architecture |
US20070086942A1 (en) * | 2005-10-19 | 2007-04-19 | Ibc Pharmaceuticals, Inc. | Methods and compositions for generating bioactive assemblies of increased complexity and uses |
US20100068137A1 (en) * | 2005-10-19 | 2010-03-18 | Ibc Pharmaceuticals, Inc. | Dock-and-Lock (DNL) Vaccines for Cancer Therapy |
Non-Patent Citations (5)
Title |
---|
"HLA-DR.", WIKIPEDIA, 26 February 2010 (2010-02-26), XP008161685, Retrieved from the Internet <URL:http://en.wiklpedia.org/w/index.php?title=HLA-DR&oidid=346491680> [retrieved on 20100802] * |
BARRERA ET AL.: "Polarized expression of CD74 by gastric epithellal cells.", J HISTOCHEM CYTOCHEM, vol. 53, no. 12, 2005, pages 1481 - 1489, XP008161721 * |
CHEN ET AL.: "Differential Effects of Milatuzumab On Human Antigen-Pressenting Cells in Comparison to Malignant B Cells.", 51ST ASH ANNUAL MEETING, 2009, XP008161688, Retrieved from the Internet <URL:http://ash.corifex.com/ash/2009/webprogram/Paper23675.html> [retrieved on 20110802] * |
LOKSHIN ET AL.: "Differential regulation of maturation and apoptosis of humen monocyte-derived dendritic celle mediated by MHC dass II", INT IMMUNOL, vol. 14, no. 9, 2002, pages 1027 - 1037, XP008161720 * |
STEIN ET AL.: "Combining milatuzumab with bortezomib, doxorubicin, or dexamethasone improves responses in multiple myeloma cell lines.", CLIN CANCER RES, vol. 15, no. 8, 2009, pages 2808 - 2817, XP008161722 * |
Also Published As
Publication number | Publication date |
---|---|
US20170088619A1 (en) | 2017-03-30 |
EP2552483A1 (en) | 2013-02-06 |
AU2011235328A1 (en) | 2012-09-27 |
EP2552483A4 (en) | 2013-09-25 |
CA2794499A1 (en) | 2011-10-06 |
US20110243841A1 (en) | 2011-10-06 |
US20130164214A1 (en) | 2013-06-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20170088619A1 (en) | Antibody-Based Depletion of Antigen-Presenting Cells and Dendritic Cells | |
US9737617B2 (en) | Multiple signaling pathways induced by hexavalent, monospecific and bispecific antibodies for enhanced toxicity to B-cell lymphomas and other diseases | |
US9359443B2 (en) | Combination therapy with anti-CD74 and anti-CD20 antibodies provides enhanced toxicity to B-cell diseases | |
AU2011203890B2 (en) | Enhanced cytotoxicity of anti-CD74 and anti-HLA-DR antibodies with interferon-gamma | |
US8883160B2 (en) | Dock-and-lock (DNL) complexes for therapeutic and diagnostic use | |
WO2012162583A1 (en) | Design and construction of novel multivalent antibodies | |
CA2781717C (en) | Dock-and-lock (dnl) complexes for delivery of interference rna | |
US9550838B2 (en) | Dock-and-lock (DNL) complexes for therapeutic and diagnostic use | |
US20130078263A1 (en) | Anti-HLA-DR Antibodies Suppress Allogeneic and Xenogeneic Immune Responses to Organ Transplants | |
AU2011292178B8 (en) | Compositions and methods of use comprising combinations of anti-CD74 antibodies with a therapeutic agent. the therapeutic agent may be attached to the anti-CD74 antibody or may be separately administered, either before, simultaneously with or after the anti-CD74 antibody | |
US20170088635A1 (en) | Dock-and-Lock (DNL) Complexes for Therapeutic and Diagnostic Use | |
CHANG et al. | Patent 2794499 Summary | |
AU2013203542B2 (en) | Enhanced cytotoxicity of anti-CD74 and anti-HLA-DR antibodies with interferon-gamma | |
AU2013203542B8 (en) | Enhanced cytotoxicity of anti-CD74 and anti-HLA-DR antibodies with interferon-gamma |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11763313 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011235328 Country of ref document: AU |
|
ENP | Entry into the national phase |
Ref document number: 2794499 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2011235328 Country of ref document: AU Date of ref document: 20110329 Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011763313 Country of ref document: EP |