EP1850872A2 - Procede d'utilisation d'un anticorps anti-cd137 en tant qu'agent de radio-immunotherapie ou radio-immunodetection - Google Patents

Procede d'utilisation d'un anticorps anti-cd137 en tant qu'agent de radio-immunotherapie ou radio-immunodetection

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Publication number
EP1850872A2
EP1850872A2 EP05723386A EP05723386A EP1850872A2 EP 1850872 A2 EP1850872 A2 EP 1850872A2 EP 05723386 A EP05723386 A EP 05723386A EP 05723386 A EP05723386 A EP 05723386A EP 1850872 A2 EP1850872 A2 EP 1850872A2
Authority
EP
European Patent Office
Prior art keywords
antibody
human
antibodies
cell
transgenic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05723386A
Other languages
German (de)
English (en)
Other versions
EP1850872A4 (fr
Inventor
Scott E. Strome
Daniel Schindler
Lieping Chen
Harry Meade
Yann Echelard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
rEVO Biologics Inc
Original Assignee
GTC Biotherapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/058,458 external-priority patent/US20060182744A1/en
Priority claimed from PCT/US2005/004739 external-priority patent/WO2006088447A1/fr
Application filed by GTC Biotherapeutics Inc filed Critical GTC Biotherapeutics Inc
Publication of EP1850872A2 publication Critical patent/EP1850872A2/fr
Publication of EP1850872A4 publication Critical patent/EP1850872A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • A61K47/6817—Toxins
    • 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
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • 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
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/12—Immunoglobulins specific features characterized by their source of isolation or production isolated from milk
    • 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
    • C07K2317/74—Inducing 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
    • C07K2317/75—Agonist effect on antigen

Definitions

  • the present invention relates to the recombinant production of an agonistic anti-CD 137 antibody and variants thereof.
  • the current invention provides for the transgenic production of anti-CD137 antibodies, in which the glycosylation profiles of the antibodies are altered to enhance their use in the treatment of specific types of cancers or other disease states.
  • the present invention relates generally to the field of the recombinant production of therapeutic antibodies and the modification of their glycosylation profile. More particularly, it concerns improved methods for generating transgenic agonistic anti-CD 137 antibodies optimized for the treatment of various types of cancer and/or autoimmune disorders.
  • glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins.
  • transgenic animals As will be seen the glycosylation profile of recombinant anti-CD 137 antibody and other proteins of interest produced in several transgenic goat lines, from cloned animals and at different stages of lactation including induced lactations was evaluated.
  • Recombinant proteins provide effective therapies for many life- threatening diseases.
  • the use of high expression level systems such as bacterial, yeast and insect cells for production of therapeutic protein is limited to small proteins without extensive post-translational modifications.
  • Mammalian cell systems, while producing many of the needed post-translational modifications, are more expensive due to the complex, and therefore sophisticated culture systems are required.
  • reduced protein expression levels are often seen.
  • Proteins have been produced in mammary glands of various transgenic animals with expression levels suitable for cost effective production at the scale of hundreds of kilograms of protein per year. Although the post-translational modification of proteins produced using transgenic technology has been published (Edmunds T 1998; James DC 1995), the effect of expression level and genetic polymorphisms on these post- translational modifications, especially glycosylation, has not been reported and is highly variable.
  • CD137 (also called 4-1BB) is a membrane glycoprotein that is inducibly expressed on activated T cells, B cells, dendritic cells and natural killer (NK) cells.
  • Anti- human CD 137 antibodies are potential biotherapeutic agents to shrink solid tumors in vivo and prevent their recurrence.
  • CD 137 is a member of the tumor necrosis factor receptor (TNFR) superfamily of costimulatory molecules. This molecule is inducibly expressed on activated T-, B-, dendritic and natural killer (NK) cells. Stimulation of CD137 by its natural ligand, CD137L, or by agonistic antibody induces vigorous T-cell proliferation and prevents activation-induced cell death.
  • TNFR tumor necrosis factor receptor
  • CD 137 signaling The intracellular biochemical pathway for CD 137 signaling is not fully understood, but TNFR associated factors (TRAF) 1 and 2 are believed to play a role.
  • TNFR associated factors (TRAF) 1 and 2 are believed to play a role.
  • the development of therapeutic anti-CD 137 would fill a critical unmet need for an effective immunomodulatory treatment of solid tumors. Despite significant advances in cancer therapy in recent decades, the majority of solid tumors in advanced stages have remained remarkably resistant to effective treatment. These include melanoma and carcinomas of the breast, colon, ovaries, kidney, prostate and lung.
  • Agonistic anti- CD 137 antibody has induced complete or partial regression in murine tumor models with diverse histological origin, either alone or in combination with other modalities. The development of a novel immuno-modulatory therapy would substantially reduce suffering and improve the quality of life for patients with these types of cancers. Moreover, and according to the current invention, an anti-CD 137 also appears to ameliorate experimental autoimmune encephalo-myelitis and systemic lupus erythematosis in mouse models.
  • CD 137 antibody preparations that are optimized for use as human biotherapeutics: 1) a first preferred embodiment would entail constructing a fully glycosylated and humanized antibody containing, which should reduce or prevent inactivation of the therapeutic protein by Human Anti-Mouse Antibody (HAMA) response, while retaining activity against solid tumors and usefulness in conjunction in bone marrow transplant operations ("BMT"); and 2) a second preferred embodiment would entail constructing an aglycosylated form of an agonistic anti CD 137 antibody, which would offer simpler manufacture and separate indications of specific utility such as leukemia and lymphoma, as well as utility against autoimmune disease states.
  • HAMA Human Anti-Mouse Antibody
  • BMT bone marrow transplant operations
  • Other objects of the current invention include the production of a humanized version of the agonistic antibody anti-human CD 137, an immune modulator that is effective in shrinking solid tumors and preventing their recurrence.
  • Specific indications against which the antibody variants of the current invention would provide beneficial therapeutic effects include: an effective immunomodulatory treatment of solid tumors; melanomas; as well as carcinomas of the breast, colon, ovaries, kidney, prostate and lung.
  • the anti-CD 137 antibodies of the invention are effective in the treatment of autoimmune derived encephalo-myelitis and systemic lupus erythematosis.
  • compositions which comprise an amount of a transgenic protein of interest, a prodrug thereof, or a pharmaceutically acceptable salt of said compound or of said prodrug and a pharmaceutically acceptable vehicle, diluent or carrier.
  • This invention is also directed to pharmaceutical compositions for the treatment of disease conditions which may be optimally treated with biologically active protein molecules that have had their glycosylation profile changed or modified.
  • the present invention also provides at least one recombinant 41-BB antibody that may be glycosylated or aglycosylated conjugated to a radionuclide or toxin to enhance radioimmunodetection, radioimmunotherapy or toxin delivery to a specific tissue or cell type.
  • the recombinant agonistic 41-BB antibody of the invention can be chimeric, humanized or fully human as well.
  • An antibody according to the present invention can include any protein or peptide containing molecule that comprises at least a portion of an 41-BB immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) (also termed the hypervariable region or HV) of a heavy or light chain variable region, or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, wherein the antibody can be incorporated into an antibody of the present invention.
  • CDR complementarity determining region
  • an isolated nucleic acid encoding at least one isolated recombinant 41-BB antibody; an isolated nucleic acid vector comprising the isolated nucleic acid, and/or a prokaryotic or eukaryotic host cell comprising the isolated nucleic acid.
  • the host cell can optionally be at least one selected from prokaryotic or eukaryotic cells, or fusion cells thereof, e.g., but not limited to, mammalian, plant or insect, such as but not limited to, CHO, myeloma, or lymphoma cells, bacterial cells, yeast cells, silk worm cells, or any derivative, immortalized or transformed cell thereof.
  • Also provided is a method for producing at least one 41-BB antibody comprising translating the protein encoding nucleic acid under conditions in vitro, in vivo or in situ, such that the recombinant 41-BB antibody is expressed in detectable or recoverable amounts.
  • an article of manufacture for human pharmaceutical or diagnostic use comprising packaging material and a container comprising a solution or a lyophilized form of at least one isolated recombinant 41-BB antibody of the present invention.
  • the article of manufacture can optionally comprise having the container as a component of a parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal,
  • FIG. 1 Shows a graph of the spleen size of sacrificed mice after treatment with the antibodies of the current invention.
  • FIG. 2 Shows a graph of the survival time of animals treated with antibodies of the current invention according to different treatment regimens.
  • FIG. 3. Shows the antibodies of that invention biotinylated. Goat anti-human
  • H&L-AP detect bound to both biotinylated and non-biotinylated antibody. Strep- AP only bound to the biotinylated antibody. Both glycosylated and aglycosylated were bound equally.
  • FIG. 4 Shows a graph of the survival time of animals treated with antibodies of the current invention according to different treatment regimens.
  • FIG. 5 Shows Anti-human CD 137 mAb binds to CHO/CD137 cells (left) and to CD3-activated human T cells (right). Flow cytometric analysis of GW, a mouse anti-human CD 137 mAb (bottom) is compared to binding by commercial anti- human CD 137 and anti-human CD3 (top). FITC-labeled antibodies were used for either direct (anti-CD3) or indirect immunofluorescence. X axis, fluorescence intensity; Y axis, no. of cells. Histogram at left in each panel is isotype-matched control IgG.
  • FIG. 6 Shows the Co-stimulation of human T cell growth by anti-CD3 and anti- CD 137 mAb of the invention. T cell proliferation was measured by incorporation of radioactive thymidine.
  • FIG. 7 Shows a silver-stained SDS-PAGE gel of transgenic goat milk samples at different stages of antibody purification. Lane 1: milk sample containing human IgGl. Lane 2: Protein A eluate. Lane 3: CM HyperD column eluate; Lane 4: Methyl HyperD column eluate.
  • FIG. 8 Shows a histological representation of the tissues after various treatment regimens according to the antibodies of the invention.
  • FIG. 9 Shows flow cytometry data related to the bioactivity of antibodies produced by transgenic animals versus CHO cell produced 4-1BB at various doses.
  • FIG. 10 Shows flow cytometry data related to the bioactivity of antibodies produced by transgenic animals, glycosylated versus non-glycosylated.
  • FIG. 11 Shows flow cytometry data related to the bioactivity of antibodies produced by transgenic animals as measured by various bioactivity markers.
  • FIG. 12 Shows the activity of the antibodies of the invention in treatment regimen with 100 IU of IL-2.
  • NK cells were separated from fresh Buffy Coat blood via Ficoll-Paque separation followed by positive selection CD56 PE and anti-PE magnetic beads.
  • NK cells were cultured for four days in 100 IU/ml of IL-2 before being transferred to another plate coated with lOug/ml of the appropriate protein. 24 hours later the supernatants were harvested for IFN gamma ELISA and the cells were triple stained with CD3 PerCP, CD56 PE, and CD 137 FITC. The NK cells were analyzed by flow cytometry
  • FIG. 13 Shows the activity of the antibodies of the invention in treatment regimen with 200 IU of IL-2.
  • NK cells were separated from fresh Buffy Coat blood via Ficoll-Paque separation followed by positive selection CD56 PE and anti-PE magnetic beads. NK cells were cultured for four days in 200 IU/ml of IL-2 before being transferred to another plate coated with 10ug/ml of the appropriate protein. 24 hours later the supernatants were harvested for IFN gamma ELISA and the cells were triple stained with CD3 PerCP, CD56 PE, and CD 137 FITC. The NK cells were analyzed by flow cytometry.
  • FIG. 14 Shows a graph of 4-1BB activity versus an IGgI.
  • FIG. 15 Shows a general schematic describing the general production of transgenic mammals.
  • FIG. 16 Shows a graph of spleen size after treatment with the antibodies of the invention.
  • FIG. 17 Shows a graph of spleen size in whole animal models after time being treated with the antibodies of the invention.
  • FIG. 18 Shows activity of the antibodies of the invention in a cellular assay over various times after stimulation.
  • FIG. 19 Shows a graph of treatment with the antibodies of the current invention in the presence of IL-2 and/or ⁇ interferon.
  • FIG. 20 Shows a general schematic of transgene constructs for milk expression of antibodies.
  • the gene of interest replaces the coding region of caprine beta-casein, a milk specific gene.
  • the 6.2 kb promoter region is linked to the coding regions of either the H or L IgG chains, followed by untranslated caprine beta casein 3' sequences and downstream elements.
  • Fig. 21 Shows a comparison of the carbohydrates in anti CD137 antibodies from transgenic animal and human 293 cell line.
  • the antibodies including both glycosylated and non-glycosylated forms from transgenic animals were expressed and purified, while the same antibody from human 293 cell line was expressed and purified.
  • the antibodies in 5 ug were applied to a 4-20% SDS-PAGE in reducing condition and stained with Coomassie blue.
  • Fig. 22 Shows a comparison of the carbohydrates in anti CD 137 antibodies from transgenic animal and human 293 cell line when applied to a 4-20% SDS-PAGE and transferred to a PVDF membrane. A western blot was performed using a goat anti human IgG (Fc specific) antibody.
  • Fig. 23 (a) - (c) Shows a MALDI-TOF analysis of the carbohydrates. The carbohydrates were released using PNGase F in the presence of 1% ⁇ - mercaptoethanol from glycosylated antibodies.
  • FIG. 24(a)-(b) Shows chromatographs of glycosylated and non-glycosylated transgenic antibodies on Con A column.
  • Fig. 25(a)-(b) Shows the use of a Lentil lectin column used to determine the presence of core fucose. Both glycosylated and non-glycosylated transgenic antibodies were applied to a Lentil lectin column, respectively. The bound protein was eluted by ⁇ -methylmannoside.
  • FIG. 26 Shows response curves differences of the Antibodies of the invention over time versus controls.
  • FIG. 27 Shows a graph of NK cell ELISA for ⁇ interferon.
  • NK cells were separated from fresh Buffy Coat blood via Ficoll-Paque separation followed by positive selection CD56 PE and anti-PE magnetic beads.
  • NK cells were cultured for four days in 100 IU/ml of IL-2 before being transferred to another plate coated with lOug/ml of the appropriate protein. 24 hours later the supernatants were harvested for IFN gamma ELISA and the cells were triple stained with CD3 PerCP, CD56 PE, and CD 137 FITC. The NK cells were analyzed by FACS.
  • FIG. 28 Shows the activity of the antibodies of the invention in treatment regimen with 100 IU of IL-2.
  • NK cells were separated from fresh Buffy Coat blood via Ficoll-Paque separation followed by positive selection CD56 PE and anti-PE magnetic beads. NK cells were cultured for four days in 100 IU/ml of IL-2 before being transferred to another plate coated with 10ug/ml of the appropriate protein. 24 hours later the supernatants were harvested for IFN gamma ELISA and the cells were triple stained with CD3 PerCP, CD56 PE, and CD137 FITC. The NK cells were analyzed by The NK cells were analyzed by flow cytometry.
  • FIG. 29 Shows a western blot of anti-CD 137 production levels in the milk of various lines of transgenic mice.
  • FIG. 30 Shows a graph of the survival time of animals treated with antibodies of the current invention according to different treatment regimens.
  • FIG. 31 Shows a graph of the survival time of animals treated with antibodies of the current invention according to different treatment regimens with regard to PBMC.
  • FIG. 32 Shows a graph of the survival statistics of animals treated with antibodies of the current invention.
  • FIG. 33 Shows a figure of the survival statistics of animals in graphic form.
  • FIG. 34 Shows an ELISA assay of ⁇ -interferon production from cell cultures exposed to antibodies of the invention.
  • the ELISA measures supernatant ⁇ - interferon, which is selectively stimulated by anti-CD 137.
  • FIG. 35 Shows a bar graph of average spleen size.
  • FIG. 36 Shows a bar graph of mice with or without lymphoma.
  • FIG. 37 Shows the spleen sizes of the animals treated with the antibody variants of the invention. It appears that mice given PBMC and GW or glycosylated antibody die with massive splenomegaly. The B cell depleted animals treated with GW also die. Animals with antibody and no cells seem appear to be in good health. Likewise, animals with aglycosylated antibody and cells seem in good health. DETAILED DESCRIPTION
  • the aglycosylated antibody is used in an immunotherapy against cancer and the development of cancerous tumors.
  • the physiological pathway involved works through 4- IBB, stimulating its activity, to prolong survival.
  • the following abbreviations have designated meanings in the specification and are provided for convenience:
  • PAD pulsed amperometric detection
  • MALDI matrix-assisted laser desorption/ionization
  • TOF time of flight
  • Endo H endo- ⁇ -N-acetylglucosaminidase H
  • EHS Engelbreth-holm-swarm
  • CHO cells Chinese hamster ovary cells
  • sDHB 2,5-dihydroxybenzoic acid matrix
  • NeuAc N-acetylneuraminic acid
  • NeuGc N-glycolylneuraminic acid
  • NT Nuclear Transfer
  • GIcNAc N-acetylglucosamine
  • GaINAc N-acetylgalactosamine
  • Gal galactose
  • Man mannose
  • Chimeric Antibody - A genetically engineered fusion of parts of a mouse antibody with parts of a human antibody.
  • chimeric antibodies contain approximately 33% mouse protein and 67% human protein.
  • they combine the specificity of the murine antibody with the efficient human immune system interaction of a human antibody.
  • Expression Vector - A genetically engineered plasmid or virus, derived from, for example, a bacteriophage, adenovirus, retrovirus, poxvirus, herpes virus, or artificial chromosome, that is used to transfer an biologically active transgenic protein coding sequence, operably linked to a promoter, into a host cell, such that the encoded recombinant transgenic protein is expressed within the host cell.
  • Fully Human Antibody Recently the term “fully human” and “human” antibody has been used to label those antibodies derived from transgenic mice carrying human antibody genes or from human cells. To the human immune system, however, the difference between "fully human”,
  • Homologous Sequences refers to genetic sequences that, when compared, exhibit similarity.
  • the standards for homology in nucleic acids are either measures for homology generally used in the art or hybridization conditions.
  • Substantial homology in the nucleic acid context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 60% of the residues, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95 to 98% of the nucleotides.
  • substantial homology exists when the segments did hybridize under selective hybridization conditions, to a strand, or its complement.
  • Humanized Antibody - A genetically engineered antibody in which the minimum mouse part from a murine antibody is transplanted onto a human antibody; generally humanized antibodies are 5-10% mouse and 90-95% human. Humanized antibodies were developed to counter the HAMA and HACA responses seen with murine and chimeric antibodies. Data from marketed humanized antibodies and those in clinical trials show that humanized antibodies exhibit minimal or no response of the human immune system against them.
  • Leader sequence or a "signal sequence” a nucleic acid sequence that encodes a protein secretory signal, and, when operably linked to a downstream nucleic acid molecule encoding a transgenic protein and directs secretion.
  • the leader sequence may be the native human leader sequence, an artificially-derived leader, or may obtained from the same gene as the promoter used to direct transcription of the transgene coding sequence, or from another protein that is normally secreted from a cell.
  • Milk-producing cell - A cell e.g., a mammary epithelial cell that secretes a protein into milk.
  • Milk-specific promoter - A promoter that naturally directs expression of a gene in a cell that secretes a protein into milk e.g., a mammary epithelial cell
  • the casein promoters e.g., ⁇ -casein promoter (e.g., alpha S-I casein promoter and alpha S2-casein promoter), ⁇ -casein promoter (e.g., the goat beta casein gene promoter (DiTullio, BIOTECHNOLOGY 10:74-77, 1992), ⁇ -casein promoter, and ⁇ -casein promoter; the whey acidic protein (WAP) promoter (Gorton et al., BIOTECHNOLOGY 5: 1183-1187, 1987); the ⁇ -lactoglobulin promoter (Clark et al., BIOTECHNOLOGY 7: 487-492, 1989); and the ⁇ -lactalbumin promoter (Soulier et al., FEBS
  • Nuclear Transfer - This refers to a method of cloning wherein the nucleus from a donor cell is transplanted into an enucleated oocyte.
  • Operably Linked - A gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
  • Parthenogenic The development of an embryo from an oocyte without the penetration of sperm.
  • compositions suitable for unequivocal biological testing as well as for appropriate administration to effect treatment of a human patient are suitable for unequivocal biological testing as well as for appropriate administration to effect treatment of a human patient.
  • substantially pharmaceutically pure means at least about 90% pure.
  • Porcine - of or resembling pigs or swine Porcine - of or resembling pigs or swine.
  • Promoter - A minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific, temporal-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' or intron sequence regions of the native gene.
  • Recombinant - refers to a nucleic acid sequence which is not naturally occurring, or is made by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site.
  • it is performed to join together nucleic acid segments of desired functional polypeptide sequences to generate a single genetic entity comprising a desired combination of functions not found in the common natural forms.
  • Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • a similar concept is intended for a recombinant, e.g., a non- glycosylated or glycan-modified transgenic protein according to the instant invention.
  • Therapeutically-effective amount An amount of a therapeutic molecule or a fragment thereof that, when administered to a patient, inhibits or stimulates a biological activity modulated by that molecule.
  • Transformed cell or Transfected cell - A cell (or a descendent of a cell) into which a nucleic acid molecule encoding desired protein of the invention related has been introduced by means of recombinant DNA techniques.
  • the nucleic acid molecule may be stably incorporated into the host chromosome, or may be maintained episomally.
  • Transgene Any piece of a nucleic acid molecule that is inserted by artifice into a cell, or an ancestor thereof, and becomes part of the genome of the animal which develops from that cell.
  • a transgene may include a gene which is partly or entirely exogenous (i.e., foreign) to the transgenic animal, or may represent a gene having identity to an endogenous gene of the animal.
  • Transgenic Organism An organism into which genetic material from another organism has been experimentally transferred, so that the host acquires the genetic information of the transferred genes in its chromosomes in addition to that already in its genetic complement.
  • Vector - As used herein means a plasmid, a phage DNA, or other DNA sequence that (1) is able to replicate in a host cell, (2) is able to transform a host cell, and (3) contains a marker suitable for identifying transformed cells.
  • a method for the production of a transgenic antibody of interest comprising expressing in the milk of a transgenic non-human placental mammal a transgenic antibody construct that has a modified sugar profile and is amenable to the modification of its glycosylation pattern to improve certain parameters of its performance as a therapeutic agent or as a treatment for a variety of disease conditions.
  • the term "treating”, “treat” or “treatment” as used herein includes preventative (e.g., prophylactic) and palliative treatment.
  • two general forms of agonistic anti- CD 137 antibody are contemplated a form expressed as a aglycosylated form and a second glycosylated form both produced by recombinant caprines or other mammalian "bioreactor.”
  • the primary difference between the two forms of the antibody of interest is their glycosylation state, though according to the current invention both are bioactive there are observed differences in their effectiveness profile for specific therapeutic applications.
  • the aglycosylated form of the 4-1BB antibody stimulates the EMH through FC cross-linking causing a secondary cytokine cascade causing a prolongation of life of animals carrying life-threatening cancers.
  • Therapeutic mouse mAbs that require repeated administration for a full clinical effect are unsuitable for human use because the HAMA response neutralizes the antibody, clears it quickly from the circulation and, in the worst case, induces serious allergic hypersensitivity.
  • Several strategies have been developed to replace most of the murine Ig sequences with human sequences, resulting in fewer side effects while retaining efficacy.
  • the HAMA response may not be a serious problem with anti-CD 137 because of the potential inhibitory effects of anti-CD 137 on antibody production.
  • the most cost effective strategy for developing a human therapeutic mAb is to replace the murine heavy chain (H) and light chain (L) constant regions (CH and CL, respectively) with human regions so that the resulting chimeric antibody is comprised mostly of human IgG protein sequence except for the antigen-binding domains.
  • This is the strategy used for Rituxan® (Rituximab anti-human CD20, Genentech), the first monoclonal antibody approved in the U.S. to treat non-Hodgkin lymphoma.
  • providing therapeutic mAbs with human CH and CL sequences should eliminate approximately 90% of the immunogenicity of murine antibody proteins.
  • An alternative strategy for developing a clinical mAb product is to produce antibody in transgenic mice in which the entire native Ig repertoire has been replaced with human Ig genes. Such mice produce fully human antibody proteins.
  • a chimeric, humanized or fully human antibody is produced as one of several preferred embodiments of the current invention.
  • both this antibody and a chimeric one would retain their effector function and would be useful in the treatment of cancer and cancerous lesions.
  • the proposed chimeric antibody embodiment of the current invention retains the original murine variable (antigen-binding) sequences and hence should retain its binding and functional properties.
  • Glycosylation is a post-translational modification that can produce a variety of final protein forms in the natural state.
  • IgG molecules are glycosylated at the ASN 2 97 residue of the CH2 domain, within the Fc region.
  • One important aspect of purifying recombinant proteins from any expression system is demonstrating that the final product has a glycosylation pattern that is comparable to the native protein, but this is difficult given the natural micro-heterogeneity in carbohydrate structures. Failure to achieve comparable glycosylation during protein expression could lead to the addition of specific carbohydrate processing steps during purification, which would add complexity and cost.
  • glycosylated and aglycosylated IgG' s have comparable binding to Fc receptors and Protein A in vitro and comparable circulating half-lives in vivo.
  • the aglycosylated form is more successful in protecting against physiological conditions such as leukemia and lymphoma as opposed to a glycosylated embodiment of the current invention that demonstrates effectiveness against solid tumors, cancerous BMT conditions and cancerous lesions.
  • An agonistic anti -human CD 137 mAb has been developed for testing as a potential immunotherapeutic treatment for cancer.
  • Antibodies against murine CDl 37 were raised in rats that were immunized with a fusion protein consisting of the extracellular domain of murine CD 137 and human Ig constant (C) region.
  • the leading candidate reagent, clone 2A is an IgG 2a protein that has been well characterized in vitro and in vivo, as described in the background section above.
  • the antibody so produced was a murine anti-human CD 137 mAb that specifically recognizes human CD 137 and does not cross-react with murine CD 137.
  • the leading candidate reagent, designated Clone GW binds specifically to transfected Chinese hamster ovary (CHO) cells expressing human antibody. ⁇
  • Antibodies are covalent heterotetramers comprised of two identical Ig H chains and two identical L chains that are encoded by different genes. Formation of a mature functional antibody molecule requires that the two proteins must be expressed in the same cell at the same time in stoichiometric quantities and must self-assemble with the proper configuration. According to the current invention the mice and goats expressing mature functional antibodies by co-transfecting separate constructs containing the H and L chains. It is important that both transgenes integrate into the same chromosomal site so that the genes are transmitted together to progeny and protein expression is jointly regulated in individual mammary duct epithelial cells that produce milk proteins. In practice, these requirements have been met in transgenic mice and goats.
  • Transgenic animals capable of recombinant antibody expression, are made by co-transfecting separate constructs containing the heavy and light chains. Glycosylated and aglycosylated versions were made by site-directed mutagenesis.
  • the anti- human CD 137 antibodies of the invention were developed and tested to determine their anti-tumor activity.
  • Two xenograft human tumor models were used: ovarian carcinoma in NOD-SCID mice and EBV-induced B lymphoma in SCID mice.
  • the inventors have constructed a variety of transgene expression vectors containing human constant region sequences for the four major IgG subclasses. These vectors also carry the goat beta-casein promoter and other 5' and 3' regulatory sequences that are used to ensure mammary-specific transgene expression.
  • the chimeric antibody variant of the current invention is constructed by inserting the variable region sequences of the mouse anti- human CD 137 into the constructs developed for the current invention. The first step is to clone and sequence the amino termini of the anti-CD 137 H and L chains to identify the murine sequences corresponding to the antibody variable regions.
  • the inventors have also assembled a collection of oligonucleotides that represent sequences from the 5' coding region of various families of murine immunoglobulins. These sequences were used individually as 5' primers for polymerase chain reaction (PCR) to amplify cDNA prepared from hybridoma RNA, and the resulting PCR products were cloned and sequenced.
  • the 3' PCR primers were prepared from the known sequences of the constant regions. These PCR primers did include appropriate restriction endonuclease sites so that the resulting amplified sequences were inserted into our expression vectors. These sequences were inserted into the constructs to produce genes encoding chimeric proteins.
  • the methods used for the genetic engineering of antibody proteins are known.
  • the methods used to clone and sequencing the anti-CD 137 antibody gene variable regions included the following steps:
  • RNA were prepared from the hybridoma by standard methods and cDNA were prepared by reverse transcription with a commercially available kit of reagents (Reverse Transcription System, Promega, Madison, WI).
  • variable region sequences by inserting the PCR- generated sequences into cloning vectors with the neomycin resistance (neo R ) selectable marker and isolating neo R colonies.
  • neo R neomycin resistance
  • Sequence H and L chain cDNA prepared from approximately 6 colonies to determine the consensus sequence for each variable region. It were important to ensure that no mutations have been introduced into the sequences from PCR artifacts. DNA sequencing were performed on a fee-for-service basis by SequeGen, Co. (Worcester, MA).
  • the murine anti-CD 137 variable region sequences obtained according to the methods provided above were used to replace human variable region sequences in existing human IgGi expression vectors to produce chimeric transgene constructs, as illustrated in Figure 5.
  • the antibody expression vectors utilized contained the necessary IgGi H gene in its native glycosylated form.
  • the IgGi glycosylation site is an Asn residue at position 297 in the CH2 domain.
  • Also produced was an aglycosylated form of the IgGi H chain by altering Asn 297 to GIn 2 C 17 by site specific mutagenesis. This did give us three constructs: L chain, glycosylated H chain and aglycosylated H chain.
  • constructs Two forms of each construct were prepared for testing and for the generation of transgenic animals. The constructs were used in transient transfection studies to test bioactivity of the genetically engineered chimeric protein.
  • the constructs used for transgenic animal development contained the goat ⁇ -casein promoter and other 5' and 3' regulatory sequences that are used to ensure high level mammary-specific transgene expression. Because of the cross-species recognition of the promoter and other regulatory elements, the same construct was used to generate transgenic mice and goats.
  • constructs were evaluated by restriction mapping via Southern blot analysis after cleavage with specific restriction endonucleases to confirm that the transgenes are regulatory elements remain structurally intact.
  • the constructs completed were used to make transiently transfected cells and transgenic animals according to the current invention.
  • the chimeric anti-human CD 137 antibody were expressed in a transient transfection system so that it could be confirmed that its binding affinity and specificity are comparable to the original murine monoclonal antibody. It was important to test the chimeric mAb to confirm that it retains the binding and functional properties of the original mAb.
  • Myeloma cells can express "irrelevant" Ig proteins that are unrelated to the designated mAb, and mutations were introduced by the PCR amplification step. As a result, the cloning process can produce sequences for antibodies that lack the desired binding and functional characteristics.
  • Binding affinity were evaluated by measuring the ability of chimeric CD 137 to inhibit binding of the original monoclonal antibody in a semi-quantitative competitive binding assay.
  • Chimeric L chain constructs were co-transfected with either glycosylated or aglycosylated H chain constructs into 293T cells, a human renal epithelial cell line that has been transformed by the adenovirus ElA gene product.
  • the 293T subline also express SV40 large T antigen, which allows episomal replication of plasmids containing the SV40 origin and early promoter region.
  • Transfections were carried out by the standard calcium phosphate precipitation method. After transfection, cells were washed free of calcium phosphate and cultured for 4 days. Supernatant did collected and either tested directly or separated over a Protein A column to isolate IgG.
  • CHO/CD137 and activated human T-cells Freshly isolated human peripheral blood T- cells were activated for 24 lir in the plates coated with anti-CD3 and anti-CD28 monoclonal antibodies (PharMingen, San Diego, CA). Cells were harvested and stained with anti-CD 137 or an isotype-matched control mAb, in the presence or absence of purified human CD 137Ig fusion protein, and then with FITC-conjugated goat anti-human IgGl antibody. Stained cells were fixed in 1% paraformaldehyde and analyzed by flow cytometry.
  • Binding affinity were measured semi-quantitatively by the dose range over which chimeric anti-CD 137 inhibits binding by the original GW mAb, compared to control IgG. The glycosylated and aglycosylated chimeric preparations were compared to the original GW mAb. Dose-dependent Co-stimulation of T-ceII growth and cytokine production by immobilized anti-CD137.
  • a co-stimulation assay for anti-CD 137 were performed. Briefly, fresh human T-cells that have been purified on a nylon-wool column were stimulated with plate- bound anti-CD3 and various concentrations of chimeric anti-CD137. Typical concentrations used to test the original GW mAb ranged from about 1 to 25 ⁇ g/ml. 3 H- thymidine was added during the last 15 hr of the 3-day culture. Radioactivity in harvested cells were measured with a MicroBeta TriLux liquid scintillation counter (Wallac).
  • glycosylated and aglycosylated chimeric preparations were compared to the original GW mAb with plate-bound isotype-matched IgG as a control.
  • the supernatants from these cultures with ELISA were assayed to measure supernatant gamma-interferon, which is selectively stimulated by anti-CD 137.
  • the ELISA was performed with Human IFN-r ELISA kit (eBioscience) following the instruction. Capture antibodies were coated to the plate with incubation under 37°C, 4hrs. After wash with TPBSx4, blocking solution was applied and incubated 30min under RT. After wash with TPBSx4, standard were add to the plate with the starting concentration of 500pg/ml. Serum were diluted 1/5 with blocking solution and add to the plate, then stayed 4°C overnight. Detect antibodies were add after plate wash and incubated 1 hour under RT. Then developed with TMB and stopped by 2 N H 2 SO 4 . The plate was read by MRX revelation plate reader.
  • Transgene constructs for the chimeric antibodies were used to generate transgenic mice and gosts to test secretion and bioactivity of the chimeric anti-CD 137 preparations.
  • Transgenic animals produce mature antibodies by introducing a 1:1 mixture of H chain and separately L chain constructs.
  • the L chain construct were combined with either the glycosylated or aglycosylated H chain construct.
  • the relative and absolute levels of bioactive product in milk was measured by Western blot analysis and measure antibody binding in vitro.
  • the most practical strategy for testing the feasibility of the inducible systems in transgenic mice was to evaluate transgenic protein expression in the milk of first-generation (F 1 ) mice.
  • transgenic founder animals are identified by PCR analysis of tail tissue DNA and relative copy number were determined using Southern blot analysis. The goal was to produce 10 transgenic first-generation transgene-bearing "founder" (F 0 ) females from each construct (glycosylated and aglycosylated). This allowed for variations in expression due to possible chromosomal rearrangements and position-dependent variegation that were generated by transgene integration. These F 0 mice were mated at maturity to initiate lactation.
  • Protein A-purified IgG fractions isolated from pooled milk samples from each line were analyzed in vitro to characterize antibody binding specificity and affinity and dose-dependent enhancement of T-cell proliferation.
  • a second major hurdle in the clinical transition of co-stimulatory approaches to cancer immunotherapy is the demonstration of effectiveness of the antibodies in an appropriate model system in vivo.
  • two xenograft mouse models for testing the effects of ability of immune modulators to amplify T-cell-mediated immune responses were used:
  • NOD-SCID non-obese diabetic / severe combined immune deficiency
  • Ascites samples from patients with ovarian cancer are fractionated to recover tumor cells, which are injected into the NOD- SCBD mice either subcutaneously or intraperitoneally to induce tumors.
  • a lymphocyte-enriched cell fraction from the same patients is injected into the mice after the tumors have become established. These lymphocytes alone are not sufficient to cause significant tumor regression, but treatment with immune modulators can augment the immunological response to tumor.
  • NOD-SCID mice have multiple immune defects, which allow reconstitution with human cancer cells and hematopoietic cells
  • LPD LPD due to transformation of B cells by EBV virus.
  • human NK cells and T-cells survive for a long period of time and were activated by IL-2 and GM-CSF to prevent the development of EBV-LPD.
  • Both of these systems provide clinically relevant models for evaluating the bioactivity of chimeric anti-human CD 137 antibody in activating T-cells and antitumor immunity.
  • treatment success were evaluated by the increase in survival and (in one variant of NOD-SCID model) a decrease in solid tumor volume.
  • NOD-SCID ovarian carcinoma model has been used to evaluate antitumor effects of various co-stimulatory molecules and mAb.
  • both models can be used in parallel to test the chimeric anti-CD 137 preparations.
  • mice Female NOD-SCID mice (Strain NOD.CB 17-SCID, Jackson Laboratory, Bar Harbor, ME) were sublethally irradiated to kill residual non-thymic-derived NK cells and used as described by Dr. Chen. [9] with small modifications. Briefly, ascites fluid from patients with primary ovarian cancer were collected and centrifuged over Ficoll/Hypaque to separate two fractions: tumor cells and a lymphocyte-enriched fraction. A portion of the tumor cells and all of the lympho-cytes were cryopreserved.
  • Washed suspensions of tumor cells were injected at doses of 2 x 10 7 cells in 200 microliters buffered saline into one of two sites on different mice: dorsal subcutaneous tissue, to establish solid tumors, or intraperitoneally (i.p.), to establish an ascites tumor.
  • An ascites aspirate from one patient usually provides enough cells to reconstitute approximately 20 mice.
  • Solid tumor size were measured twice weekly with calipers fitted with a Vernier scale and calculated on the basis of 3 perpendicular measurements.
  • the lymphocyte fraction were thawed and resuspended with an expected recovery of about 80% viable cells.
  • the cell suspension were injected either intravenously (iv) into mice with solid subcutaneous tumors (2 x 10 7 cells) or i.p (5 x 10 6 cells) into mice with ascites tumors. Mice received an i.p. injection of 100 ⁇ g to 300 ⁇ g of chimeric anti-human CD137 and the same treatment did repeat weekly for three more times. Control mice did receive isotype-matched mAb.
  • mice with ascites tumors were sacrificed for humane reasons, in accordance with IACUC guidelines. All mice were sacrificed at the end of the experiment. In addition, some mice with either solid or ascites tumors were assayed to measure the cytolytic activity of their tumor-specific cytotoxic T lymphocytes (CTLs). Briefly, 7 to 10 days after the second antibody treatment, animals were sacrificed and lymphocytes were harvested from tumor-draining lymph nodes. The lymphocytes were restimulated in vitro with irradiated carcinoma cells from the original donor.
  • CTLs tumor-specific cytotoxic T lymphocytes
  • mice After 4-6 days in culture, the stimulated cells were used as effectors in a standard 4-hour 51 Cr release assay against tumor target cells. T-cells whose responsiveness was augmented in vivo by anti-CD 137 should kill the target cells more effectively than T-cells treated with isotype-matched control antibody. The survival of the mice were analyzed by the log rank test.
  • This model assay system was used essentially as provided in the prior art. Briefly, normal healthy donors who are EBV seropositive and HIV seronegative, and provide informed consent under a then-current IRB protocol at the Mayo Clinic, did undergo leukophoresis. PBMCs were separated on a Ficoll/Hypaque gradient (Sigma), washed and injected i.p. into SCID mice at a dose of 5 x 10 7 cells/mouse in 0.5 ml PBS. SCID mice were treated with weekly injections of anti-AsialoGM-1 antiserum to deplete their natural killer (NK) cells and increase the rate of engrafting.
  • NK natural killer
  • mice Within 6 weeks of PBMC injection, untreated mice usually develop B-cell lymphomas and begin to die. In previous studies, approximately 81% of mice that received human PBMC were successfully engrafted, as established by detection of circulating human Ig by ELISA. Only successfully engrafted mice were used for these studies.
  • mice received weekly 3 i.p. injections of 100-300 ⁇ g of either anti-CD137 test preparation or an isotype-matched negative control (as described above). Mice that receive low-dose GM-CSF plus IL-2 served as positive controls. Outcomes were measured as survival and expansion of human T-cells, detected by flow cytometry analysis using anti-human MHC class I from blood PBMC. The survival of the mice were analyzed by the log rank test.
  • the chimeric anti-CD137 produced in the milk of transgenic animals was bioactive. Pro-longed survival and increased immune responses in mice after chimeric anti-CD 137 treatment also established this recombinant antibody as a potential cancer therapeutic.
  • NK-cell-depleted SCID mice NK-cell-depleted SCID mice
  • these components probably are sufficient to elicit a successful response to anti-CD137.
  • NOD-SCID ovarian carcinoma model it is sometimes difficult to harvest enough TDLN T-cells to measure their cytolytic activity by 10 days after antibody treatment. If we this is a problem, then we did sacrifice additional mice 14 to 21 days after treatment and recover T-cells from their spleens, which generally provide a higher yield.
  • transgenic goats were produced and characterizing cells lines for use as donors in the nuclear transfer procedure.
  • the production of transgenic goats was facilitated by utilizing the same constructs utilized in the development of transgenic mice.
  • the inventors used nuclear transfer techniques to generate transgenic goats with pre-defined genetics.
  • the transgene construct was introduced into primary cell lines by a standard transfection method, examples of such techniques include lipofection or electroporation.
  • the recombinant primary cell lines are screened in vitro for important characteristics such as transgene copy-number, integrity and integration site before they are used to produce transgenic animals.
  • Nuclear transfer eliminates the problem with transgene mosaicism in the first few generations because all of the animals derived from a transgenic cell line should be fully transgenic.
  • Fibroblasts from fresh goat skin biopsy samples were maintained in primary culture in vitro. Briefly, skin samples were minced in Ca ++ -free and Mg ++ -free phosphate buffered saline (PBS), harvested with dilute trypsin in EDTA to recover single cell suspensions and cultured at 37°C. When the cells become confluent they were trypsinized and sub-cultured. Aliquots of cells were cryopreserved in liquid nitrogen.
  • PBS Ca ++ -free and Mg ++ -free phosphate buffered saline
  • Each cell line were characterized by Southern blot analysis with probes specific for the transgene such as beta-casein, chimeric anti-CD 137 H and L chain cDNAs to establish the transgene copy number and to look for gross rearrangements.
  • Each cell line also was analyzed by FISH to confirm that there was a single integration site and to determine its chromosomal location, and by cytogenetic analysis to confirm that it has a normal karyotypes. Only primary cultures that are subsequently found to exhibit transgene structural integrity, uniform integration characteristics and normal karyotypes were analyzed further.
  • Interphase FISH a few hundred cells from each expanded colony were immobilized on filters and hybridized to amplified transgene-specific digoxigenin- labeled probes.
  • metaphase FISH cells were cultured on Lab Tek Chamber slides and pulsed with 5-bromo-2'deoxyuridine (BrdU) to allow for replication banding. Probe binding were detected with FITC-conjugated anti-digoxigenin, and the chromosomes were counterstained with 4' ,6-Diamidino-2-phenylindole (DAPI). Images were captured using a Zeiss Axioskop microscope, a Hamamatsu digital camera, and Image Pro-Plus software.
  • Some probes are relatively large and easy to detect by FISH but probes for individual IgG H and L chains, which are encoded by relatively short cDNA sequences, are too small to give good resolution by themselves. These small probes were mixed with sequences from the milk-specific promoter for goat beta-casein.
  • the goat beta casein probe also detects the single copy endogenous goat beta casein gene on chromosome 4, this is a known binding site that does not interfere with interpretation of the results.
  • Cell lines that are used to generate first- generation transgenic goats must be karyo-typically normal and must carry structurally intact chimeric anti-CD 137 H and L chain genes along with the beta-casein promoter and other essential regulatory elements.
  • test diluent is CETS diluted 1/10 with plate wash solution
  • the antibody was biotinylated.
  • the Goat anti- human H&L-AP detect bound to both biotinylated and non-biotinylated antibody.
  • Strep- AP only bound to the biotinylated antibody.
  • the 4-1BB antibody CD137 produced according to the current invention was cloned and expressed in the milk of several lines of transgenic mice and goats as a genomic "mini-gene.” The expression of this gene is under the control of the goat ⁇ -casein regulatory elements. Substantial expression of the antibody variants according to the current invention in both mice and goats has been established.
  • One of the initial targets for immuno therapeutic use of the current agonistic anti-CD 137 antibody is for use with patients suffering from squamous cell carcinoma of the head and neck.
  • One of the objectives of the current invention is to establish the production of bioactive anti-human CD 137 antibody, an immune modulator that may be effective against solid tumors, in the milk of transgenic animals.
  • CD137 also called A- IBB
  • A- IBB is a membrane glycoprotein that were induced in several types of lymphoid cells.
  • An agonistic monoclonal antibody (mAb) against murine CD 137 shrank mouse tumors in vivo and prevented their recurrence, suggesting that anti-CD 137 may be effective against human tumors.
  • the next technical hurdle to clinical translation is to develop a genetically engineered form of the anti-human CD 137 that is suitable for clinical use, and to demonstrate that it is effective against human tumors in an appropriate mouse model.
  • the present invention also includes a method of cloning a genetically engineered or transgenic mammal, by which a desired gene is inserted, removed or modified in the differentiated mammalian cell or cell nucleus prior to insertion of the differentiated mammalian cell or cell nucleus into the enucleated oocyte.
  • Suitable mammalian sources for oocytes include goats, sheep, cows, pigs, rabbits, guinea pigs, mice, hamsters, rats, primates, etc.
  • the oocytes were obtained from ungulates, and most preferably goats or cattle. Methods for isolation of oocytes are well known in the art.
  • oocytes may preferably be matured in vivo before these cells may be used as recipient cells for nuclear transfer, and before they were fertilized by the sperm cell to develop into an embryo.
  • Metaphase II stage oocytes which have been matured in vivo, have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes are collected surgically from either non-super ovulated or super ovulated animals several hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone.
  • hCG human chorionic gonadotropin
  • the current invention enables the use of transgenic production of biopharmaceuticals, transgenic proteins, plasma proteins, and other molecules of interest in the milk or other bodily fluid (i.e., urine or blood) of transgenic animals homozygous for a desired gene that then optimizes the glycosylation profile of those molecules.
  • a desired gene that then optimizes the glycosylation profile of those molecules.
  • the current invention when multiple or successive rounds of transgenic selection are utilized to generate a cell or cell line homozygous for more than one trait such a cell or cell line were treated with compositions to lengthen the number of passes a given cell line can withstand in in vitro culture. Telomerase would be among such compounds that could be so utilized.
  • the use of living organisms as the production process means that all of the material produced were chemically identical to the natural product.
  • non-glycosylated related transgenic proteins are produced in the milk of transgenic animals.
  • the human recombinant protein of interest coding sequences were obtained by screening libraries of genomic material or reverse-translated messenger RNA derived from the animal of choice (such as cattle or mice), or through appropriate sequence databases such as NCBI, genbank, etc. These sequences along with the desired polypeptide sequence of the transgenic partner protein are then cloned into an appropriate plasmid vector and amplified in a suitable host organism, usually E. coll
  • the DNA sequence encoding the peptide of choice can then be constructed, for example, by polymerase chain reaction amplification of a mixture of overlapping annealed oligonucleotides.
  • the DNA construct After amplification of the vector, the DNA construct would be excised with the appropriate 5' and 3' control sequences, purified away from the remains of the vector and used to produce transgenic animals that have integrated into their genome the desired non-glycosylated related transgenic protein.
  • some vectors such as yeast artificial chromosomes (YACs)
  • YACs yeast artificial chromosomes
  • non-glycosylated related refers to the presence of a first polypeptide encoded by enough of a protein sequence nucleic acid sequence to retain its biological activity, this first polypeptide is then joined to a the coding sequence for a second polypeptide also containing enough of a polypeptide sequence of a protein to retain its physiological activity.
  • the coding sequence being operatively linked to a control sequence which enables the coding sequence to be expressed in the milk of a transgenic non-human placental mammal.
  • a DNA sequence which is suitable for directing production to the milk of transgenic animals carries a 5 '-promoter region derived from a naturally-derived milk protein and is consequently under the control of hormonal and tissue-specific factors. Such a promoter should therefore be most active in lactating mammary tissue. According to the current invention the promoter so utilized were followed by a DNA sequence directing the production of a protein leader sequence which would direct the secretion of the transgenic protein across the mammary epithelium into the milk.
  • a suitable 3'-sequence preferably also derived from a naturally secreted milk protein, and may be added to improve stability of mRNA.
  • suitable control sequences for the production of proteins in the milk of transgenic animals are those from the caprine beta casein promoter.
  • transgenic animals can now be performed using a variety of methods.
  • the method preferred by the current invention is nuclear transfer.
  • the antibody preparations provided herein is preferably employed for in vivo applications.
  • the compositions used may be in the dosage form of solid, semi-solid or liquid such as, e.g., tablets, pills, powders, capsules, gels, ointments, liquids, suspensions, or the like.
  • the antibody compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts.
  • the compositions may also include, depending on the formulation desired, pharmaceutically acceptable carriers or diluents, which are defined as aqueous-based vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the human recombinant protein of interest.
  • diluents examples include distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank's solution. The same diluents may be used to reconstitute lyophilized a human recombinant protein of interest.
  • the pharmaceutical composition may also include other medicinal agents, pharmaceutical agents, carriers, adjuvants, nontoxic, non- therapeutic, non-immunogenic stabilizers, etc. Effective amounts of such diluent or carrier were amounts which are effective to obtain a pharmaceutically acceptable formulation in terms of solubility of components, biological activity, etc.
  • compositions herein may be administered to human patients via oral, parenteral or topical administrations and otherwise systemic forms for anti-melanoma, anti-lymphoma, anti-leukemia and anti-breast cancer treatment.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they maybe presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • the composition may take the form of tablets or lozenges formulated in conventional manner.
  • Therapeutic methods involve administering to a subject in need of treatment a therapeutically effective amount of a transgenic antibody.
  • “Therapeutically effective” is employed here to denote the amount of transgenic antibodies that are of sufficient quantity to inhibit or reverse a disease condition (e.g., reduce or inhibit cancer growth).
  • Some methods contemplate combination therapy with known cancer medicaments or therapies, for example, chemotherapy (preferably using compounds of the sort listed above) or radiation.
  • the patient may be a human or non-human animal.
  • a patient typically were in need of treatment when suffering from a cancer characterized by increased levels of receptors that promote cancer maintenance or proliferation.
  • Administration during in vivo treatment may be by any number of routes, including parenteral and oral, but preferably parenteral.
  • Intracapsular, intravenous, intrathecal, and intraperitoneal routes of administration may be employed, generally intravenous is preferred. The skilled artisan did recognize that the route of administration did vary depending on the disorder to be treated.
  • Toxicity may be determined using methods well known in the art and found in the foregoing references. Efficacy may be determined utilizing the same guidance in conjunction with the methods described below in the Examples. A pharmaceutically effective amount, therefore, is an amount that is deemed by the clinician to be toxicologically tolerable, yet efficacious. Efficacy, for example, were measured by the induction or substantial induction of T lymphocyte cytotoxicity at the targeted tissue or a decrease in mass of the targeted tissue. Suitable dosages were from about 1 mg/kg to 10 mg/kg.
  • plOO BC2083 mayo heavy (BC2197) 6) BC1060 7) p85 pCR-blunt-1060 kappa constant rev
  • plO4 BC1060 mayo LC chim (BC2198)
  • pH2 pCEP4-Xho-mayo-IgGl-aglycos BC2206
  • a murine anti-human CD 137 mAb that specifically recognizes human CD137 and does not cross-react with murine CD137 The leading candidate reagent, designated Clone GW, binds specifically to transfected Chinese hamster ovary (CHO) cells expressing human CD 137 (CHO/CD137) at levels comparable with commercially available anti-human CD 137.
  • CHO/CD137 Chinese hamster ovary
  • Well established methods for the genetic engineering of antibody proteins were used to clone and sequence the anti-CD 137 antibody gene variable regions (Maynard and Georgiou 2000; Sacchi, Federico et al. 2001). To identify the family of the antibody, it was sequenced chemically from the amino terminus. In this manner we would be able to use family specific primers for PCR.
  • a purified anti-human 4- IBB antibody was developed from hybridoma GW.
  • the antibody concentration was 670 ng/ ⁇ l.
  • SDS sample buffer After reduction in SDS sample buffer, 4.7 ⁇ g was run on a 10% Bis-Tris gel in MOPS buffer. After running, the gel was equilibrated in 10 mM CAPS, pH 11.0-10% Methanol buffer and blotted at 100V for lhr onto Invitrogen PVDF membrane, 0.2 ⁇ m pore size
  • the amino terminal sequencing results for the light chain was DIVLTQSPASLAVSL. This matches MUSIGKM195 or Swissprot KV3B_MOUSE, a member of family 21, a family which is used in about 7% of all antibodies.
  • the amino terminal sequence of the heavy chain is: KVQLQQS GAGLVKPG. This matches MUSIGAPCJ in the Genbank database, a member of family 1, the J558 family which contains the bulk of the germline genes and is used about 30% of the time.
  • HAMA human anti-mouse antibodies
  • MAbs are partly 'humanized' through genetic engineering.
  • the most common method of antibody humanization involves replacement of the constant region of the mouse MAb with a human constant region, resulting in a mouse:human chimera.
  • Chimeric antibodies are created by cloning the murine gene that codes for the antibody variable region and the human gene that codes for the antibody constant region.
  • This type of genetic engineering enables scientists to produce antibodies with a murine variable region combined with a human constant region.
  • Potential advantages for chimeric antibodies include less immunogenicity and longer circulation of the antibody (LoBuglio, Wheeler et al. 1989; Knight, Wagner et al. 1995).
  • An antibody which stimulates 4- IBB has been reported to suppress antigen-induced humoral immune response (Hong, Lee et al. 2000).
  • the BC2083 expression vector containing the ⁇ mmunogen human antibody sequences with a mouse leader sequence was used (Plasmid 1). This gene has a splice donor site eliminated by a G to A silent mutation which did not change the coding for glycine near the C terminus. Unique sites were put into the BC2083 expression vector surrounding the variable region. DraIII and PmII were put into the N terminus and Apal exists in the amino portion of the heavy constant region.
  • the human IgGl constant portion was put back into the unique Apal and Xhol sites by cutting it out of BC2083 and cloning it into p80 to give p83 BC2083 DraIII IgGl (Plasmid 3).
  • This plasmid has unique Dralll/Pmll and Apal sites flanking the heavy variable region so that any heavy variable region were attached to the human IgGl constant region coding sequences.
  • the heavy chain variable region of the anti-4-lBB antibody was prepared for insertion by putting Drain and PmII sites on the amino terminus and an Apa site on the C terminus by PCR.
  • the Apal site is naturally occurring near the amino terminus of the human IgGl constant region.
  • PCR was performed with primers MHE and MHECusing PfuTurbo (Stratagene Cat No. 600153-81) and cDNA.
  • the PCR fragment was cloned into pCR-Bluntll-TOPO (Invitrogen Cat. No.: K28602) and sequenced with primers pcr2.1f and pcr2.1b (List 3). This give p96, containing the heavy chain variable region flanked by Dralll-Pmll and Apal (Plasmid 4).
  • the beta-casein expression vector, plOO BC2083 mayo heavy (BC2197)(Plasmid 5), was constructed by isolating the p96 pCR-Bluntll-mayo-heavy Dralll-Apal fragment and ligating it to cut DraIII- Apal cut p83 BC2083 DraIII IgGl (Plasmid 3).
  • the expression vector used for the light chain was BC1060 (Plasmid 6).
  • a Kpnl site was introduced by changing the codon for a glycine from GGG or GGC to GGT.
  • the coding sequence for a leucine was changed to CTT from CTG to create a HindIII site (plasmid 8).
  • PfuTurbo (Stratagene) the coding region of the human constant region of the kappa chain was isolated from BC 1060 with Kpnl and HindIII sites at the beginning and a naturally occurring Sad site near the end of the coding region using primers from Table 9.
  • variable region was isolated from cDNA by PCR with primers from Table 10 and cloned into pCR2.1-Blunt-TOPO to make p92 pCR2.1 -Blunt- Mayo kappa variable (Plasmid 9) where the variable region is flanked by a Xhol site at nucleotide 340 and Kpnl and HindlM sites around nucleotide 731. These plasmids were sequenced and the resulting sequences are listed in List 5.
  • the light chain chimera was first constructed in pCR-Blunt using 3 pieces of DNA.
  • the backbone from Xhol to Sad was contributed by p86 pcr-blunt-1060 kappa constant .
  • the kappa constant region was the Hindlll-Sacl piece from p85 pcr-blunt-1060 kappa constant rev.
  • the variable region was supplied by p92 pCR-Blunt- Mayo kappa variable rev using the Xhol-Hindlll piece.
  • Colonies were checked by PCR with primers, pcr2.1f and pcr2.1b, looking for production of a 863 bp fragment. This gives p94 pCR- Bluntll-mayo-kap-chim (Plasmid 10).
  • the plasmid was checked by cutting with Xhol and Sad to give a 684 bp fragment.
  • the light chain chimera was put into the beta-casein expression vector BC 1060 containing the Immunogen human light chain with the mouse heavy leader sequence.
  • p94 was cut with Xhol-Sacl and the small piece isolated.
  • BC1060 was cut with Kpnl-Sacl and the 5206 bp piece isolated.
  • BC 1060 was cut with Kpnl, Xhol, and Pad to isolate the large backbone. These three pieces were ligated and colonies were screened with the needed primers.
  • the positive plasmid was checked with BgIII and the PCR product sequenced. This plasmid is plO4 BC1060 mayo LC chim (BC2198)(Plasmid 11).
  • the recent large-scale transient transfection technology is now generating great interest because of its demonstrated ability to produce large amounts of recombinant proteins within a few days.
  • the human embryonic kidney 293 cell line (293) is suitable for transient transfection technology as it were efficiently transfected.
  • a 293 genetic variant stably expressing the EBV EBNAl protein (293E) has been shown to provide significantly higher protein expression when EBVs oriP is present in the vector backbone.
  • the increased expression obtained by the use of oriP/EBNAl systems appears to be independent of episomal replication when performing transient transfection.
  • the utility of the pCEP4 vector has been found to be limited to the human 293 EBNA cell line (Parham et al., 2001).
  • the 293EBNA/ebv vector host system represents a significant improvement over COS7/SV40ori based systems.
  • An important issue for high level recombinant protein expression is to use vectors with promoters that are highly active in the host cell line, such as the CMV promoter, which is particularly powerful in 293 cells where it has been shown to be strongly trans activated by the constitutively expressed adenovirus El a protein. (Durocher et al., 2002).
  • the Xhol fragment from pi 04 BC 1060 Mayo LC chim was ligated into the Xhol site of pCEP4 to give plO6 and pl07 pCEP4- Mayo-LC (#2203) (Plasmid 12). Positive colonies were detected by PCR with oligos CEPF & KVC.
  • Antibodies are glycosylated at Asn297 of the heavy chain constant region (Wright and Morrison, 1998).
  • the carbohydrate is sequestered between the heavy chains and has a complex biantennary structure composed of a core saccharide structure consisting of two mannosyl residues attached to a mannosyl-di-N-acetylchitobiose unit (Rademacher et al. 1985).
  • the outer arms arise from the terminal processing of the oligosaccharide in the Golgi; although the overall structure of the carbohydrate is conserved, considerable heterogeneity is seen in the identity of the terminal sugar residues.
  • Analysis of carbohydrates isolated from normal human serum IgG has yielded up to 30 different structures.
  • Antibodies lacking glycosylation lack effector functions like antibody mediated cell dependent cytotoxicity (ADCC) since they can not bind Fc gammaRl receptor and complement activation by their failure to bind CIq (Nose and Wigzell 1983; Leatherbarrow et al. 1985; Tao 1989; Jefferis et al. 1998; Mimura et al. 2000; Mimura et al. 2001; Dorai et al. 1991).. They can still bind the neonatal receptor. (Simmons et al. 2002) Since the Mayo anti-4-lBB antibody is an agonist antibody and, like 4- IBB ligand, activates the 4- IBB receptor loss of effector functions is not detrimental and would possibly be beneficial.
  • ADCC antibody mediated cell dependent cytotoxicity
  • oligosaccharides from all transgenic animals were a mixture of high mannose, hybrid and complex type oligosaccharides with or without fucose.
  • Sialic acid was present as 2, 6-linked sialic acid and no ⁇ 1,3-linked galactose was observed in the transgenic glycoprotein.
  • the heavy chain coding sequence was prepared by PCR with PfuTurbo from BC2083 using primers heavy constant N and heavy constant C subcloned into pCR-Zero - Blunt. This gave p76 and p77 pCR2.1-Blunt-IgGl-heavy-constant . These plasmids were sequenced giving the sequences in List 6 to ensure no mutations were introduced into the constant region during the PCR.
  • the 4- IBB antibodies produced by transgenic mice and goats augment the initial graft versus host disease and stimulates the EMH. This then requires Fc cross-linking which may explain differences between "g” and "gw”. Animals that die in the experiments provided in the development of the current invention likely die of GVH secondary to a cytokine cascade. Also according to the current invention, the aglycosylated antibodies developed stimulate 4-1BB and result in prolonged survival in the whole animal lymphoma model. Therefore, according to the current invention the aglycosylated antibodies (chimeric humanized and human) have beneficial attributes for the treatment of cancer and autoimmune disorders. This while the glycosylated version has treatment potential for BMT conditions and those of similar cause.
  • the chimera with the heavy chain variable region of the anti-CD 137 antibody was prepared by ligating the small Kpnl-Agel piece of #110 pCEP4-BamHI-HC (#2202) containing the variable region into Kpnl-Agel cut #88 pCR2.1-Blunt-IgGl-heavy- mut. This gives plasmid pll l pCR2.1-Mayo-IgGl-heavy-mut (Plasmid 14). This plasmid was checked with BsaAI-Pstl.
  • the small Xhol fragment from pi l l containing the chimeric antibody coding region was inserted into the Xhol site of pCEP4. Colonies were checked by PCR with HVC C09 and CEPF. This gave pi 12 pCEP4-Xho-mayo-IgGl-aglycos (BC2206). Expected fragments were obtained with EcoRV- Hindi ⁇ digestion (2479 bp) and BamHI digestion (1454 bp).
  • the small Xhol fragment from pil l containing the chimeric antibody coding region was inserted into the Xhol site of BC2083. Colonies were checked by PCR with oligos HVC 09 and CA5. Digestion with MluI-Eco47III-NotI gave the expected 2479 bp fragment, while digestion with BamHI gave the expected 1454 bp fragment.
  • Antibody Humanization It has been established that stimulation of CD 137 through its natural ligand or agonistic antibodies potentiates the antitumor immune response in vivo through stimulation of tumor-reactive effector T-cells and enhanced regulatory NK activity.
  • mAbs anti-murine CD137 monoclonal antibodies
  • mice such as the poorly immunogenic AGF104A sarcoma and the highly tumorigenic P815 mastocytoma, as well as EL4 thymoma, K1735 melanoma, B 10.2 and 87 sarcoma, RENCA renal carcinoma, J558 plasmacytoma, MCA205 sarcoma, JC breast cancer, MCA26 colon cancer and GL261 glioma, alone or in combination with other therapeutic modalities.
  • CD 137 agonistic antibodies elicit potent T-cell responses but their role in humoral immune responses is inhibitory.
  • Systemic administration of anti-mouse CD 137 mAb suppresses antigen-specific antibody production by energizing T-helper cells and inhibits autoantibody production by deleting autoreactive B cells.
  • This unique feature of CD137 signaling has important clinical implications because it may minimize the Human Anti- Mouse Antibody (HAMA) response, which inactivates murine antibody proteins in the circulation.
  • HAMA Human Anti- Mouse Antibody
  • Anti-CD 137 offers great promise as a potential therapeutic agent against certain solid tumors.
  • the difficulties not addressed by the prior art include the development of an anti-CD 137 for human cancer therapeutic suitable for clinical applications, that is, to demonstrate its effect against human tumors and to establish a reliable and cost-effective production source.
  • a chimeric or humanized agonistic anti-CD 137 antibody that: 1) contains human constant region sequences on the heavy and light chains of the immunoglobulin molecule (IgG) to minimize neutralization by Human Anti-Mouse Antibody (HAMA) responses in vivo; and 2) lacks the glycosyl group found on native IgG to simplify antibody purification from the milk of transgenic goats.
  • IgG immunoglobulin molecule
  • HAMA Human Anti-Mouse Antibody
  • Humanization also called Reshaping or CDR-grafting
  • mAbs monoclonal antibodies
  • a mouse-human chimeric monoclonal antibody agonist anti-CD137 was developed. Humanization of the anti-CD137 antibody is expected to enhance its use for patients undergoing immunotherapy or for other indications. On the basis of the observed amino acid sequence identity, complementary determining regions (CDRs) of the VL and VH regions were grafted onto the human anti- DNA-associated idiotype immunoglobulin clone. It was observed by competitive ELISA that the recombinant chimeric antibody of the invention exhibited a similar bioactivity profile when compared with the murine monoclonal antibody. The anti-CD 137 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement- mediated cytotoxicity when assayed. Humanization of the antibody sequences of the current invention are expected to eliminate any undesired human anti-mouse antibody response, allowing for repeated i.v. administration into humans.
  • G2F is also present as minor species.
  • the binding of transgenic glycosylated and non-glycosylated anti CD 137 to lectin columns was also investigated. It was found that the majority of transgenic glycosylated antibody bound to Con A, a lectin specific for high mannose type carbohydrates. The interaction between antibody and Con A confirms the presence of high mannose type oligosaccharides present on transgenic glycosylated antibody. See Figures 21-25. It is also possible that increasing the ADCC levels could enhance the effectiveness of anti-CD- 137 antibodies. This could be done by any number of methods. [00172] Turning to Figure 21, the results indicates that glycosylated anti CD137 antibodies from either transgenic animal or human 293 cell line migrated in similar pattern.
  • the carbohydrate profiles are identified in the transgenic antibody vis-a-vis.
  • the major carbohydrate in transgenic antibody is non- fucosylated Man5.
  • GIF and G2F core fucose containing oligosaccharides
  • Gl and Man6 non-fucosylated oligosaccharides
  • the carbohydrates identified in the same antibody expressed from human 293 cell line are mostly fucosylated oligosaccharides.
  • the major structures in these oligosaccharides are GOF and GIF.
  • G2F is also G2F as minor species.
  • Figures 24(a)-(b) the lectins were also used to confirm the presence of specific carbohydrates in transgenic antibody.
  • the following figures show the chromatographes of glycosylated and non-glycosylated transgenic antibodies on Con A column.
  • the results from Figures 24(a)-(b) show that the majority of glycosylated transgenic antibody bound to Con A column and eluted by ⁇ -methylmannoside starting from fraction 11.
  • most of non-glycosylated anti CD 137 antibody and an antibody without any high mannose oligosaccharides were not bound.
  • the data is consistent with the MALDI-TOF analysis and indicate that the presence of high mannose type oligosaccharides in transgenic glycosylated antibody.
  • the Lentil lectin column was also used to determine the presence of core fucose because the lectin is known to interact with core fucosylated oligosaccharides.
  • Both glycosylated and non-glycosylated transgenic antibodies were applied to a Lentil lectin column, respectively.
  • the bound protein was eluted by ⁇ -methylmannoside. It was found that neither of these antibodies bound to the lectin column.
  • an antibody which contains mainly core fucosylated oligosaccharides, also didn't bind to the column (data not shown).
  • Majority of a control glycoprotein bound to the column (data not shown). The result suggests that the core fucose in some of the antibody may not expose or be accessible to the lectin column. Therefore, the binding of antibody to Lentil lectin column cannot be used as tool to determine the presence of core fucose in the antibody studied.
  • the Mayo anti-CD137 antibody was previously expressed in mouse milk.
  • the construction of BC2197 (plOO BC2083 mayo heavy) and (BC2198) plO4 BC1060 mayo LC chim were described in the quarterly report of September 2003.
  • the parental plasmids were those of the Immunogen antibody expression vectors, BC2083 for the heavy chain and BC 1060 for the light chain.
  • the variable regions including the leader sequences in those parental plasmids were exchanged with the cDNA sequence from the variable region of the heavy and light chains of the Mayo anti-CD 137 antibody cDNA.
  • the goat expression vector we replaced the constant regions, IgGl of the heavy chain and kappa of the light chain with sequences which were cloned at GTC.
  • PCR was done from brain cDNA as above to give plasmids, pi 16, pi 17, pi 18, and pi 19. None of these plasmids had the correct sequence. For example, most of the plasmids were missing the Apal and/or the Xhol sites at the end of the sequence, which should have been provided by the PCR primers. The PCR was done again using pi 16 as template or brain cDNA again.
  • PCR of pi 16 yielded 121, 122, and 123.
  • PCR of brain cDNA yielded 124.
  • the insert from plasmid 121 was used to make pl33, pl34, pl35, and pl36 which are BC2083 mayo heavy Glm(17) by cutting 100c BC2083 mayo heavy with Apal & Xhol and pl21 ZeroBlunt-IgGl Glm(17) with Apal & Xhol, ligating and selecting on kanamycin. pi 33 was used.
  • variable regions were changed in plasmid BC2083, an expression vector containing the human antibody sequences with a mouse leader sequence was used. This gene has a splice donor site at the end of the IgGl constant region eliminated by a G to A silent mutation which did not change the coding for glycine.
  • the constant region was changed to an IgGl constant region that was cloned. The cloned constant region from pi 14 was used to create pl37 and 138.
  • plOO BC2083 mayo heavy (BC2197) was cut with Apal -Xhol and 114 ZeroBlunt-IgGl Glm(3) cut with Apal & Xhol and ligated to give pl38 (BC2228).
  • the cloned constant region from pl21 (Glm(17)) was used to create pl33, 134, 135, and 136 BC2083 mayo heavy Glm(17).
  • the kappa constant region was also replaced with one cloned at GTC. (May 11, 2004)
  • primer 1 (diluted 4 ul + 4 ul H2O) 5' AGGGTACCAAGCTTGAAATCAAACGAAC Kappa Constant Human HOl
  • primer 2 (diluted 1 ul + 7 ul H2O) 5 '
  • This PCR product was rePCRed with the same primers and cloned into the Invitrogen plasmid ZeroBlunt to give plasmids 127, 128, 129, and 130.
  • Monoclonal antibodies have the inherent property of specificity for a certain target antigen. With the 4-1BB antibody of the current invention this property of reactive specificity to tumor-associated antigens is also useful for additional therapeutic potential when conjugated to radionuclides, cytotoxic drugs, or toxins. The same specificity is exploited in radioimmunodetection, wherein the antibody is labeled with a suitable radionuclide that can be detected using available camera/sensor imaging technology.
  • Radiolabeled antibodies are known in the prior art and have often been used as an immunoconjugate.
  • the specific glycosylation state of the 41-BB antibody utilized for a particular radioimmunodetection or radioimmunotherapy task is determined according to the utility of the combination for a target cancer, neoplasm, or cell type.
  • 41-BB mAbs conjugated to a radionuclide or toxin can be administered to a patient in a therapeutically effective dose for a specific disease indication.
  • the toxicity of immunoconjugates is limited to the toxicity of the nuclide or toxin attached to the antibody.
  • the critical organ is the bone marrow, with thrombocytopenia being the dose-limiting toxicity.
  • the utilization of relatively nonimmunogenic antibody forms that are chimeric, humanized or fully human, that will permit multiple administrations of a specific immunoconjugate to a specific patient. This is also true regardless of the glycosylation state of the 41-BB antibody.
  • Indium-111, Technetium-99, may be used in radioimmunodetection
  • Iodine-131, rhenium- 186 may be used for radioimmunotherapy
  • radioimmunotherapy especially for solid tumors, may require either large doses of immunoconjugated antibody with marrow support or carefully planned fractionations.
  • Beta particle emitters such as yttrium-90, iodine-131, and rhenium-186 are nuclides of choice for established disease as their major toxicity is hematopoietic. Radioimmunodetection studies have been carried out not only with intact immunoglobulin but with mAb fragments as well. These have included F(ab')2 and, more frequently, Fab' fragments. According to the current invention, fragments such as these of the 41-BB antibodies may be used in conjunction with desirable radionuclides.
  • the radionuclide with energy emission characteristics most suitable for use with current nuclear medicine cameras is technetium-99, which has gamma emissions of 140 KeV.
  • This transitional element has no particulate emission; its short half-life further reduces radiation dose to the patient, permitting use of relatively large amounts of radioactivity.
  • Rhenium is another transitional element with chemical characteristics comparable to those of technetium-99. Rhenium-186 and -188 have been used in therapy. They emit beta-minus radiation and concurrent gamma emissions that thereby permit imaging. Stability of binding of rhenium to antibody is an important consideration in the development of suitable rhenium-labeled antibodies in immunotherapy.
  • Radioiodines [00195] Radioiodination of proteins, including mAbs, is a known process, and thus, the greatest number of radioimmunodetection trials have been carried out with radioiodinated mAbs. Iodine- 123 has a short half-life (13 hours) and ideal emission characteristics (no particulate emission, 159-KeV photon emission).
  • Iodine-131 has a long half-life of 8 days and a complex decay scheme that includes beta-minus emission, precluding use of large amounts of radioactivity. This constraint, in addition to its high energy (364-Ke V) gamma emission makes it less than optimal for gamma camera imaging, necessitating special collimation protocols for many gamma cameras.
  • the benefits for Iodine-131 include:
  • Iodine-131 is easily available and relatively inexpensive; 2) Protein radioiodination is relatively easy to carry out;
  • Radioiodinated MAbs are relatively stable in vivo; and,
  • Persistence of free radioiodine in the body can be obviated by saturation of physiologic iodine stores with nonradioactive iodine, permitting prompt clearance of free radioiodine by the kidneys without significant thyroid or stomach uptake; thus, nonspecific uptake of iodine-131-labeled MAbs is not a problem.
  • Technetium-99 and other transitional elements have been labeled to intact immunoglobulin and fragments by direct labeling, which involves reduction of the disulfide bonds to sulfhydryl groups, or by indirect labeling via attachment of a technetium-99-avid ligand. The latter method is usually more cumbersome but may produce a more stable label in vivo. Radiometals are often attached to antibodies via a chelating agent, that is, by indirect labeling. Other methods include the use of the modification of the diethylenetriamine-pentaacetic molecule or use of macrocyclic chelates.
  • Radioimmunodetection allows a survey of the entire body to be made for evidence of recurrent or metastatic disease with far lower radiation burdens and costs than CT. Thus, radioimmunodetection appears to be of greatest utility in the follow-up of patients at high risk for recurrent or metastatic cancer. Many studies agree that radioimmunodetection is not as sensitive as CT scanning for the detection of hepatic metastatic disease but is more specific. Especially when indium-I l l -labeled antibodies have been used, considerable "nonspecific" hepatic uptake has precluded visualization of hepatic metastases as areas of increased (“hot”) tracer uptake.
  • radioimmunodetection may be the procedure of choice. Because radioimmunodetection appears to be more sensitive than CT for recurrent disease and through the avoidance of HAMA through the use of a chimeric, humanized or fully human recombinant 41-BB may make the antibodies of the current invention available for screening procedures.
  • Tamada K. et al. LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response, J. IMMUNOL.

Abstract

L'invention concerne le développement et des procédés d'utilisation d'un anticorps agoniste recombiné anti-humain CD137, et de variantes de glycosylation correspondantes. Ces anticorps agissent en tant qu'agents anticancéreux et/ou modulateurs du système immunitaire qui sont efficaces pour réduire des tumeurs solides et/ou d'autres manifestations cancéreuses, ainsi que pour empêcher la récurrence de celles-ci. Parmi les types de cancer efficacement traités au moyen de l'anticorps selon l'invention figurent en outre les leucémies et les lymphomes. Dans un mode de réalisation préféré de cette invention, les anticorps recombinés sont produits dans le lait d'animaux transgéniques, puis extraits de ce lait par purification. Dans un autre mode de réalisation préféré de la présente invention, les anticorps agonistes anti-CD137 peuvent être conjugués à des radionucléides à des fins de radio-immunodétection ou de radio-immunothérapie, ou être conjugués à une toxine pour mieux traiter divers cancers.
EP05723386A 2005-02-15 2005-02-18 Procede d'utilisation d'un anticorps anti-cd137 en tant qu'agent de radio-immunotherapie ou radio-immunodetection Withdrawn EP1850872A4 (fr)

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PCT/US2005/005405 WO2006088464A2 (fr) 2005-02-15 2005-02-18 Procede d'utilisation d'un anticorps anti-cd137 en tant qu'agent de radio-immunotherapie ou radio-immunodetection

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