EP2150261A2 - Kit de parties pour le traitement du cancer ou de maladies infectueuses - Google Patents

Kit de parties pour le traitement du cancer ou de maladies infectueuses

Info

Publication number
EP2150261A2
EP2150261A2 EP08789068A EP08789068A EP2150261A2 EP 2150261 A2 EP2150261 A2 EP 2150261A2 EP 08789068 A EP08789068 A EP 08789068A EP 08789068 A EP08789068 A EP 08789068A EP 2150261 A2 EP2150261 A2 EP 2150261A2
Authority
EP
European Patent Office
Prior art keywords
kit
antibody
monoclonal antibody
cells
parts
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Withdrawn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
EP08789068A
Other languages
German (de)
English (en)
Inventor
Nadine Fernandez
Christophe De Romeuf
Rémi Urbain
Jacques Bartholeyns
Aurélie Boyer
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.)
IDM Immuno Designed Molecules
LFB Biotechnologies SAS
Original Assignee
IDM Immuno Designed Molecules
LFB Biotechnologies SAS
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
Application filed by IDM Immuno Designed Molecules, LFB Biotechnologies SAS filed Critical IDM Immuno Designed Molecules
Priority to EP08789068A priority Critical patent/EP2150261A2/fr
Publication of EP2150261A2 publication Critical patent/EP2150261A2/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Withdrawn legal-status Critical Current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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the invention relates generally to the treatment of hematopoietic tumor or autoimmune diseases, in particular through the use of activated macrophages and a monoclonal antibody.
  • ADCC antibody-dependent cell cytotoxicity
  • the Fc receptors are surface glycoproteins that can bind the Fc portion of immunoglobulin (Ig) molecules. Fc receptors are defined by their specificity for immunoglobulin subtypes. Fc receptors for IgG are referred to as FcyR, for IgE as FccR, and for IgA as Fc ⁇ R. Different accessory cells bear Fc receptors for antibodies of different isotype, and the isotype of the antibody determines which accessory cells will be engaged in a given response (reviewed by Gerber J. S. et al . 2001 Microbes and Infection, 3: 131-139; Billadeau D. D. et al. 2002, The Journal of Clinical Investigation, 2(109): 161- 1681; Ravetch J.
  • Fc ⁇ Rs There are three known Fc ⁇ Rs , designated Fc ⁇ RI(CD64), FcyRII (CD32) , and Fc ⁇ RIII (CD16) .
  • autoimmune and/or inflammatory disorders the immune system triggers an inflammatory response when there are no foreign substances to fight and the body's normally protective immune system causes damage to its own tissues by mistakenly attacking self.
  • autoimmune disorders which affect the body in different ways.
  • the brain is affected in individuals with multiple sclerosis
  • the gut is affected in individuals with Crohn's disease
  • the synovium, bone and cartilage of various joints are affected in individuals with rheumatoid arthritis.
  • destruction of one or more types of body tissues, abnormal growth of an organ, or changes in organ function may result.
  • the autoimmune disorder may affect only one organ or tissue type or may affect multiple organs and tissues.
  • autoimmune disorders Organs and tissues commonly affected by autoimmune disorders include hematopoietic cells, blood vessels, connective tissues, endocrine glands (e.g., the thyroid or pancreas), muscles, joints, and skin.
  • Auto-immune diseases are often associated with an inflammatory disease.
  • Inflammatory disease such as rheumatoid arthritis (RA) and juvenile rheumatoid arthritis are types of inflammatory arthritis.
  • Arthritis is a general term that describes inflammation in joints. Besides rheumatoid arthritis, other types of arthritis associated with inflammation include the following: psoriatic arthritis, Reiter's syndrome, ankylosing spondylitis arthritis, and gouty arthritis .
  • a tumor is a neoplastic mass resulting from abnormal uncontrolled cell growth which can be benign or malignant. Benign tumors generally remain localized. Malignant tumors are collectively termed cancers.
  • malignant generally means that the tumor can invade and destroy neighboring body structures and spread to distant sites to cause death (for review, see Robbins and Angel1, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-122). Cancer can arise in many sites of the body and behave differently depending upon its origin. Cancerous cells destroy the part of the body in which they originate and then spread to other part(s) of the body where they start new growth and cause more destruction.
  • cancer therapy may involve surgery, chemotherapy, hormonal therapy and/or radiation treatment to eradicate neoplastic cells in a patient (Stockdale, 1998, "Principles of Cancer Patient Management", in Scientific American: Medicine, vol. 3, Rubenstein and Federman, eds . , Chapter 12, Section IV) .
  • chemotherapy has many drawbacks. Almost all chemotherapeutic agents are toxic, and chemotherapy causes significant, and often dangerous, side effects, including severe nausea, bone marrow depression, immunosuppression, etc.
  • the beneficial effects of chemotherapy can be compromised by cellular mechanisms that allow neoplastic tissue to evade the toxicity of drugs.
  • pleiotropic resistance to a variety of unrelated drugs has been observed, and this phenomenon has been called multidrug resistance.
  • Development of multidrug resistance is frequently observed in second or third intention cytotoxic treatment of cancer.
  • Resistance to chemotherapy, whether it is intrinsic or acquired, is a major cause of failure in the curative treatment of neoplastic malignancies.
  • a promising alternative is immunotherapy, in which cancer cells are specifically targeted by cancer antigen-specific antibodies.
  • B cells are the immune system's "arms factories," producing antibodies that target invading microbes for destruction. Abnormal B cell proliferation causes such leukemias as multiple myeloma and acute lymphoblastic leukemia. They also have a critical role in antibody mediated autoimmune diseases such as rheumatoid arthritis and lupus. Preliminary clinical studies suggest therapeutic benefits in patients with classic autoantibody-mediated syndromes, such as autoimmune cytopenias . B-cell-targeted therapy promises to rapidly secure an important place in the treatment of autoantibody-associated conditions .
  • Rituximab (marketed as Rituxan® by Genentech and MabThera® by Roche) was the first FDA-approved monoclonal antibody and was developed at IDEC Pharmaceuticals (see U.S. Pat. Nos . 5,843,439; 5,776,456 and 5,736, 137) for treatment of human B- cell lymphoma, an haematological cancer (Reff et al . , Blood, 83: 435-445 (1994)).
  • Rituxan is a chimeric, anti-CD20 monoclonal antibody (mab) .
  • CD20 is a tetraspanning transmembrane phospho-protein that is expressed predominantly in pre-B cells and in mature peripheral B cells in humans and mice.
  • CD20 is also strongly and homogeneously expressed on most mature B-cell malignancies, except for chronic lymphocytic leukemia (CLL) cells, where expression is more variable.
  • Rituxan ® has been first approved for the treatment of relapsed or refractory, CD20+, B-cell low-grade Non-Hodgkin' s Lymphoma (NHL). In 2006, it has been approved in combination with chemotherapy for three others types of NHL.
  • Rituxan is also approved in combination with methotrexate in adult patients with moderately-to-severely active rheumatoid arthritis who have had an inadequate response to one or more tumor necrosis factor antagonist therapies.
  • follicular center cell lymphoma FCC
  • MCL mantle cell lymphoma
  • DLCL diffuse large cell lymphoma
  • SLL/CLL small lymphocytic lymphoma/chronic lymphocytic leukemia
  • auto-immune diseases such as systemic lupus or idiopathic thrombocytopenic purpura.
  • Macrophages play a major role in the antitumoral response, and they are able to be activated by immunological activators against cancer cells (Adams D. and Hamilton T.: Activation of macrophages for tumor cell kill: effector mechanism and regulation. In Heppner & Fulton (eds), Macrophages and cancer. CRC Press, 1988, p. 27; Fidler I.: Macrophages and metastases. A biological approach to cancer therapy. Cancer Res. 45: 4714, 1985) .
  • macrophages or other cells derived from monocytes or from their precursors, with their strong capacity for endocytosis, digestion, and surface antigen presentation, are capable of inducing a specific immune response.
  • Macrophages presenting cytotoxic activity which is particularly significant also designated as activated macrophages or MAK (registered trade mark)
  • MAK registered trade mark
  • kits of parts for treating haematopoietic tumors including abnormal B-cell proliferation such as chronic lymphocytic leukaemia (CLL) , or autoimmune disease, comprising an activated macrophage and a monoclonal antibody directed against an antigen expressed by tumor cells or involved in auto-immune diseases.
  • CLL chronic lymphocytic leukaemia
  • autoimmune disease comprising an activated macrophage and a monoclonal antibody directed against an antigen expressed by tumor cells or involved in auto-immune diseases.
  • the present invention concerns the combined use of activated macrophage and antibody in a subject suffering from a hematopoietic tunnor or autoimmune disease. More specifically, the present invention provides a kit of parts for the treatment of hematopoietic tumor or autoimmune disease comprising an activated macrophage and a monoclonal antibody wherein the affinity of the Fc region of said antibody to Fc gamma receptor is higher than the affinity of IgG polyclonal antibodies to said Fc gamma receptor.
  • the invention concerns a use of such kit of parts as a medicament for the treatment of a patient suffering from a blood cancer or autoimmune disease.
  • a first object of the invention is a kit of parts for the treatment of cancer or infectious diseases, preferably hematologic cancer or auto-immune disease, comprising activated macrophages and a monoclonal antibody, wherein the affinity of the Fc region of said antibody to Fcyreceptor III (CD16) is higher than the affinity of IgG polyclonal antibodies to said CDl6.
  • the said affinity of the Fc region of said antibody is at least of 2.10 6 M "1 .
  • said monoclonal antibody is not displaced by IgG polyclonal antibodies, especially by IgG present in blood serum.
  • said monoclonal antibody binds to CD16 of said macrophage with an affinity of at least 2.10 6 M "1 , and more advantageously of at least 1.10 7 M “1 , or preferably of 1.10 8 M “1 or even of 1.10 9 M "1 , as determined by Scatchard analysis or BIAcore technology (Label-free surface plasmon resonance based technology) .
  • the monoclonal antibody is produced in the form of a composition of monoclonal antibodies, each of said monoclonal antibody having N-glycoside-linked sugar chains bound on the Fc ⁇ glycosylation site (Asn 297, EU numbering), and wherein among said N-glycoside-linked sugar chains of all the antibodies of the composition, the fucose content is less than 65%.
  • said activated macrophage is a MAK® (IDM, Paris, France).
  • said antibody has a variable region directed against an antigen expressed by tumor cells or involved in auto-immune diseases.
  • said antigen is CD20.
  • said monoclonal antibody is EMAB6 , produced by the clone R509, deposited to the CNCM under the accession number 1-3314.
  • said monoclonal antibody is EMAB603, produced by the clone R603, deposited to the CNCM under the accession number 1-3529.
  • said cancer is chronic lymphoid leukaemia (CLL) .
  • CLL chronic lymphoid leukaemia
  • Another object of the invention is the use of a kit of parts for the preparation of a medicament.
  • Another object of the invention is the use of the kit of parts for the preparation of a medicament for the treatment of hematopoietic tumor or autoimmune disease.
  • the hematopoietic tumor comprises leukemia, lymphoma and myeloma. More particularly, the hematopoietic tumor comprising B-cell malignancy is selected from the group consisting of indolent forms of B-cell lymphomas, aggressive forms of B-cell lymphomas, chronic lymphatic leukemias, acute lymphatic leukemias, Waldenstrom's rriacroglobulinemia and multiple myeloma.
  • the autoimmune disease comprises immune dysregulation disease, inflammatory disorders, organ graft rejection or graft versus host disease, septicemia and septic shock, Hashimoto's thyroiditis, pernicious anemia, sarcoidosis, primary biliary cirrhosis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis /dermatomyositis , polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis obliterans, ulcerative colitis, erythema multiform
  • kit of parts » designates a combined preparation containing, as active substance, activated macrophages and a monoclonal antibody, for the simultaneous, separate or sequential use, for the treatment of hematologic diseases or autoimmune diseases.
  • macrophages and antibodies are associated to form a single active unit before injection to the patient.
  • the two active ingredients form a functional unit, i.e. a functional true combination through a purpose-directed application. Due to their use in the kit of parts of the invention, the two active ingredients (activated macrophages and monoclonal antibody) show a joint effect.
  • activated macrophages » as used herein is intended to include macrophages which have at least one of the following properties : their cytotoxic activity with IFN- ⁇ is increased by about 20 to about 40% with respect to standard macrophages, and is preferably of about 93%; the extension of deactivation of cytotoxic activity in reply to an activation of IFN- ⁇ is in a ratio such that after 6Oh of activation with IFN- ⁇ , cytotoxic activity is higher than or equal to 30%, preferably of about 55%, compared to the maximum cytotoxic activity displayed by the macrophages due to IFN- ⁇ activation, with said cytotoxic activity being measured as the percentage of inhibition of 3-H thymidine (tritiated thymidine) incorporation by target tumoral cells, particularly U
  • standard macrophages corresponds to those obtained by the culture of monocytes in a standard medium such as RPMI 1640, AIMV or I ⁇ cove modified media.
  • Cytotoxic activity corresponds to that measured as a percentage of inhibition of 3-H thymidine incorporation by target tumoral cells, particularly U937 cells (human histiocytic cell line, ATCC CRL 1593.2), and a test for this measure is hereafter given: differentiated macrophages were seeded into 96-well flat microtiter plates at 104 macrophages /well in 0.2 ml.
  • cytolysis cytostasis and phagocytosis.
  • the macrophages originate from human subjects, who can be either healthy subjects in case of allogeneic therapy or more preferably from the patient himself, suffering from various diseases .
  • the macrophages originate from the patient to be treated by adoptive immunotherapy.
  • Activated macrophages as well as a culture medium and process for obtaining said macrophages are detailed in the international patent application WO 94/26875 or in the European patent EP 0806959, which are included herein for references .
  • the process for preparing macrophages defined in the above mentioned patent application WO 94/26875 is carried out with a composition rich in blood cells obtained by apheresis from a healthy individual or from a patient, and includes a stage of monocyte culture in a culture medium containing GM-CSF (Granulocyte-Macrophage colony stimulating factor) and 2% of autologous serum.
  • GM-CSF Gramulocyte-Macrophage colony stimulating factor
  • the monocytes are cultivated in the presence of lymphocytes for about 6 to 7 days.
  • the differentiated macrophages thus obtained are activated in the culture medium by the addition of IFN-y.
  • the macrophages can be enriched by elutriation.
  • the culture step may be preceded by a separation step of mononuclear cells on the one hand and red cells, granulocytes and part of the platelets contained in the composition derived from blood obtained by apheresis on the other hand; and then by a purification step, by washing part of the blood platelets and anticoagulants remaining after the preceding separation step.
  • the activated macrophage may be prepared according to the method comprising the following steps: 1) recovery of blood derived mononuclear cells directly from blood apheresis or from blood bag collection, followed by centrifugation, to eliminate a substantial part of red blood cells, granulocytes and platelets, and collection of peripheral blood leukocytes; 2) washing peripheral blood leukocytes obtained from the preceeding steps for instance by centrifugation (to remove 90% of platelets, red blood cells and debris) to obtain mononuclear cells;
  • said activated macrophage is a macrophage activated killer, MAK® (IDM, Paris, France), described in the international patent application WO 94/26875 or in the European patent EP 0806959 or in the document Immunobiology, 210, (2005), 267-277: Antitumor properties of human-activated macrophages produced in large scale for clinical application; Veronique Baron-Bodo, Paula Doceur, Marie-Laure Lefebvre, Karine Labroquere, Catherine Defaye, Christophe Cambouris, Didier Prigent, Margarita Salcedo, Aurelie Boyer, Alessandra Nardin, which are incorporated herein by reference.
  • MAK® macrophage activated killer
  • antibody or “immunoglobulin” (used interchangeably herein) refers to an antigen-binding protein having a basic four-polypeptide chain structure consisting of two heavy and two light chains, said chains being stabilized, for example, by interchain disulfide bonds, which has the ability to specifically bind antigen.
  • Each chain is divided into regions or domains consisting of around 110 amino acid residues.
  • the light chain has two domains and the heavy has four.
  • the N-terminal domain at the tip of the arms of the "Y" on both the heavy and light chain are known to be variable in amino acid sequence composition and are thus called “variable region” (VL and VH) .
  • the other domains are called constant for a similar reason (CL, CHl, CH2 , CH3 ) .
  • variable domains show three regions of hypervariability in sequence called the complementarity determining regions (CDRs). They differ in length and sequence between different antibodies and are mainly responsible for the specificity (recognition) and affinity (binding) of the antibodies to their target markers. Proteolytic digestion of antibodies releases different fragments termed Fv (Fragment variable), Fab (Fragment antigen binding) and Fc (Fragment crystallisation) . Note that antibody engineering can join the separate segments of the heavy and light chains in the Fv with a flexible peptide linker to form a single-chain Fv (scFv). The Fc region mediates the effector function of the antibodies, which are induced as a result of interaction of the antibody with its antigen via the variable moiety.
  • the antibody of the present invention comprises a variable region and a constant region (Fc) .
  • the constant region (Fc) of the monoclonal antibody is human.
  • Human constant region DNA sequences can be isolated m accordance with well known procedures from a variety of human cells, for example immortalized B cells (see Kabat et al . , supra, and Liu et al , WO 87/02671) (each of which is incorporated by reference m its entirety for all purposes) .
  • the antibody will contain both light chain and heavy chain constant regions
  • the heavy chain constant region usually includes CHl, hinge, CH2 , CH3 , and CH4 regions.
  • the antibodies described herein include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA and IgE, and any lsotype, including IgGl, IgG2 , IgG3 and IgG4.
  • constant region depends, in part, whether antibody- dependent complement and/or cellular mediated toxicity is desired. For example, isotopes IgGl and IgG3 have complement activity and isotypes IgG2 and IgG4 do not
  • the term "monoclonal antibody” is intended to include a preparation of antibody whose specificity is unique and identical These antibodies have an identical ammo-acid sequence because they are produced by one type of cell line and are all clones of a single parent cell. It is to be noted that post-translational modifications, like glycosylation, depend on the nature of the cell line, and that as such the monoclonal antibodies may not be identical with respect to these post- translational modifications.
  • the monoclonal antibody of the invention may be prepared by conventional methods, such as the production of hybridoma ⁇ as described by Koehler and Milstein (1975), immortalization of human B lymphocytes by Epstem-Barr ' s virus (EBV), or more recent ones, such as phage display technology, use of a combinatorial library of human or transgenic animal antibodies, notably from the mouse, and of course monoclonal antibodies may also be prepared by molecular engineering.
  • conventional methods such as the production of hybridoma ⁇ as described by Koehler and Milstein (1975), immortalization of human B lymphocytes by Epstem-Barr ' s virus (EBV), or more recent ones, such as phage display technology, use of a combinatorial library of human or transgenic animal antibodies, notably from the mouse, and of course monoclonal antibodies may also be prepared by molecular engineering.
  • the monoclonal antibody of the invention is prepared in genetically modified cells by introducing at least one vector allowing antibodies to be expressed, these cells being eukaryotic or prokaryotic cells, notably cells from non-human transgenic mammals, insects, plants, bacteria or yeasts.
  • these cells are selected from rat myeloma cell lines, notably YB2/0 and
  • IR983F human myeloma lines such as Namalwa or any other cell of human origin such as PERC6 , CHO lines, notably CHO-K, CHO-
  • LeclO CHO-Lecl, CHO Pro-5, CHO dhfr-, CHO Lecl3 , or other lines selected from Wil-2, Jurkat, Vero, Molt-4, COS-7, 293- HEK, BHK 1 K6H6, NSO, SP2/0-Ag 14 and P3X63Ag8. 653.
  • the monoclonal antibody of the invention is monospecific, e.g. its Fab are directed to the same antigen.
  • the monoclonal antibody of the present invention may be a recombinant, chimeric, humanized or human antibody.
  • chimeric antibody » as used herein is intended to include all immunoglobulin molecules comprising a human and non-human portion. More specifically, the antigen combining region (or variable region) of a chimeric antibody is derived from a non-human source (e.g., murine) and the constant region of the chimeric antibody (which confers biological effector function to the immunoglobulin) is derived from a human source.
  • the chimeric antibody should have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule.
  • the variable region may be murine, from rats, rabbits, non- human primate, goat, and the like, without limitation.
  • the monoclonal antibody comprises a murine variable region, and a human constant region.
  • humanized antibody refers to an antibody that includes at least one humanized antibody chain (i.e., at least one humanized light or heavy chain) .
  • humanized antibody chain refers to an antibody chain (i.e., a light or heavy chain, respectively) having a variable region that includes a variable framework region substantially from an human antibody, and complementarity determining regions (CDRs)
  • human antibody is intended to include an antibody which, when aligned to a human antibody amino acid sequence for comparison purposes, the region shares at least 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e., local sequence identity) with the human framework or constant region sequence, allowing, for example, for conservative substitutions, consensus sequence substitutions, germline substitutions, backmutations , and the like.
  • conservative substitutions, consensus sequence substitutions, germline substitutions, backmutations, and the like is often referred to as "optimization" of a humanized antibody or chain.
  • human antibody is also intended to include a human antibody which naturally exists in the human body, and antibodies obtained from a human antibody phage library and from a human antibody-producing transgenic animal or a human antibody-producing transgenic plant, which are prepared based on recent advances in genetic engineering, cell engineering and developmental engineering techniques.
  • chimeric, humanized and human antibodies are produced by recombinant expression.
  • Nucleic acids encoding chimeric, humanized or human light and heavy chain variable regions, optionally linked to constant regions, are inserted into expression vectors.
  • the light and heavy chains can be cloned in the same or different expression vectors.
  • heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.
  • the whole antibodies, their dimers , individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, HPLC purification, gel electrophoresis and the like (see generally Scopes, Protein Purification (Springer-Verlag, N. Y., (1982)).
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
  • the DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector (s) that ensure the expression of immunoglobulin polypeptides.
  • Expression control sequences include, but are not limited to, promoters (e.g., naturally-associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the crossreacting antibodies .
  • expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DMA.
  • expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin- resistance, tetracycline resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences (see, e.g., Itakura et al . , U.S. Pat. No. 4,704,362) .
  • Prokaryotic hosts particularly useful for cloning the polynucleotides (e.g., DNA sequences) of the present invention may be E. coli, bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various
  • MAK®e expression vectors which will typically contain expression control sequences compatible with the host cell
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribo ⁇ ome binding site sequences and the like, for initiating and completing transcription and translation.
  • yeast Other microbes, such as yeast, are also useful for expression.
  • Saccharomyces is a preferred yeast host, with suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired.
  • Typical promoters include 3- phosphoglycerate kinase and other glycolytic enzymes .
  • Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
  • mammalian tissue cell culture may also be used to express and produce the polypeptides of the present invention (e.g., polynucleotides encoding immunoglobulins or fragments thereof) .
  • Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art, and include CHO cell lines, YB2/0 rat cell lines, various Cos cell lines, HeLa cells, preferably, myeloma cell lines, or transformed B-cells or hybridomas .
  • the cells are nonhuman .
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al . , Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, KNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149 (1992).
  • antibody-coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., Deboer et al . , U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No 5,304,489, and Meade et al . , U.S. Pat. No. 5,849,992).
  • Suitable transgenes include coding sequences for light and/or heavy chains in operable linkage with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulm.
  • the vectors containing the polynucleotide sequences of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection may be used for other cellular hosts. (See generally Sambrook et al , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
  • transgenic animals can be itiicromjected into fertilized oocytes, or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes .
  • the monoclonal antibodies according to the invention have reduced immunogenicity , and long serum half-life.
  • Fcgamma receptors Fcgamma receptors
  • Activated macrophages of the invention express on their cell membrane three types of Fcgamma receptors: FcgammaRl (CD64), FcgammaRII (CD32) and FcgammaRlII (CD16).
  • CD16 is also called “low affinity receptor", and is constitutively expressed on polymorphonuclear neutrophils (PMNs), monocytes and NK-cells.
  • PMNs polymorphonuclear neutrophils
  • monocytes monocytes
  • NK-cells NK-cells.
  • CD16 participates in various effector functions, notably phagocytosis of opsonized particles or of immune complexes, and in antibody-dependent cellular cytotoxicity (ADCC) .
  • ADCC antibody-dependent cellular cytotoxicity
  • the monoclonal antibody of the kit of parts of the invention has a high affinity of its Fc region toward Fcgamma receptors present on activated macrophages, especially on CD16. More particularly, the invention describes the synergic interaction of particular monoclonal antibodies and activated macrophages. Contrary to the association of the state of the art of a conventional monoclonal antibody and an activated macrophage, in which the addition of blood serum in the medium inhibit the ADCC activity of the association, the blood serum has no influence on the ADCC of the association, i.e. of the kit of parts of the invention.
  • Polyvalent IgG Polyclonal IgG
  • CD16 and CD64 Polyclonal IgG
  • polyvalent IgG can inhibit the mechanism of lysis of the effector cells via CD64, the CD64 being saturated by the addition of polyvalent IgG (see patent application WO 01/77181) .
  • the applicant has shown that, surprisingly, the association in a kit of parts of monoclonal antibody whose affinity of the Fc region of said monoclonal antibody to CDl6 is higher than the affinity of IgG present in the blood serum to the CD16 induces an ADCC activity that is not inhibited by the addition of blood serum in vitro.
  • the monoclonal antibody is therefore not displaced by IgG polyclonal antibodies in case of presence or addition of blood serum to the medium.
  • the Fc region of the monoclonal antibody binds the CD16 of the activated macrophages, and this binding is not displaced by the polyclonal IgG, even present at high concentrations in the blood serum.
  • the kit of parts of the invention achieves optimal killing of the target cells even at low concentration of monoclonal antibody.
  • concentration of monoclonal antiboby of the kit of parts is inferior to the concentration of a conventional antibody of the same specificity, traditionally used alone to treat an autoimmune or a blood cancer.
  • the kit of parts achieves target cell death even if the E:T ratio (effector cells to target cells ratio) is not necessarily high, which can be for example as low as 1:1.
  • the E:T ratio is comprised between 1:1 and 50:1.
  • the Fc region of the monoclonal antibody of the invention has an association constant of at least 1.10 7 M "1 preferentially 1.10 8 M "1 or even 1.10 9 M '1 for CDl6.
  • association constant of the antibody of the invention is measured according to the method described in the document Maenaka et al . (Katsu ⁇ ii Maenaka, P.Anton van der
  • the human low affinity Fey receptors Ha, lib and III bind IgG with fast kinetics and distinct thermodynamic properties.
  • the concentration of monoclonal antibody in the kit of parts is inferior to 50 mg/billion MAK, preferably, 2,5 mg/100 million MAK.
  • the monoclonal antibody binds to CD16 of said activated macrophages with an affinity of at least 1.10 7 M "1 preferably of 1.10 8 M “1 or even of 1.10 9 M "1 measured by Scatchard analysis or BlAcore technology.
  • the monoclonal antibody of the invention can be prepared by means of the process described in patent application WO 01/77181.
  • This process of preparing a monoclonal antibody capable of activating effector cells expressing CDl6, and preferentially activated macrophages comprises the following steps: a) purifying several different monoclonal antibodies obtained from various cell lines or clones originating from cell lines selected from hybridomas , in particular heterohybridomas , and animal or human cell lines transfected with a vector comprising the gene encoding the antibody, b) adding each monoclonal antibody obtained in step a) to a reaction mixture comprising: i. the target cells of the monoclonal antibody, ii.
  • the monoclonal antibody of the invention is in fact a composition containing antibodies, which are qualified as "monoclonal” because they originate from the same cell clone. All the antibodies of a composition of monoclonal antibody have the same amino acid sequence, and the same specificity. However, because of the post- translational modifications of eukaryote cells, all of the antibodies of one composition of monoclonal antibodies may not have the same glycan structure.
  • the antibodies of all human and animal subclasses have a N- oligosaccharide attached to the CH 2 domain of each heavy chain, at residue asparagine 297 (EU numbering) for human IgG.
  • This asparagine (Asn) residue is also called “Fey glycosylation site”, or “N-glycoside-linked sugar chain biding site”, and the M-oligosaccharide is also called “N-glycoside-linked sugar chain” .
  • the sugar chain terminus which binds to Asn297 is called a reducing end, and the opposite side is called a non-reducing end.
  • N-glycoside- linked sugar chains have various structures, depending on the glycosylation specifics of the producing cell line, but it is known that they have a basic common core structure shown by the following structural formula:
  • This core structure may comprise the following additional sugars: N-acetylglucosamine (GIcNAc), fucose (fuc), galactose (gal) .
  • GIcNAc N-acetylglucosamine
  • fucose fucose
  • gal galactose
  • N-glycoside-linked sugar chain which binds to an antibody includes any sugar chain possibly containing one or several of these additional sugars, there are a number of combinations of sugar chains for the two N-glycoside sugar chains which bind to the antibody.
  • compositions of monoclonal antibody in which fucose content is less than 65%, or advantagesouly comprised between 15% and 45%, and preferably betv/een 20% and 40% exhibit a strong affinity of the Fc region for CD16. More particularly, this kind of composition of monoclonal antibody has an affinity of the Fc region of said antibody to CDl6 higher than the affinity of IgG polyclonal antibodies to CD16. Moreover, the antibodies of such monoclonal antibody composition are not easily displaced by IgG present in blood serum.
  • composition of monoclonal antibody is produced in a cell which has a low fucose-adding enzyme activity to N-acetylglucosamine of the reducing terminal, wherein such enzyme may be fucosyltransferase .
  • suitable enzymes such as endoglycosidases , including fucosidases.
  • composition of monoclonal antibody of the invention is produced in YB2/0 (ATCC CRL-1662) .
  • the monoclonal antibody of the kit of parts of the invention is directed against an antigen expressed by tumor cells or cells involved in auto-immune diseases.
  • antigen » designates an entity (e.g., a proteinaceous entity or peptide) to which an antibody specifically binds.
  • Specific binding of an antibody means that the variable region of the antibody exhibits appreciable affinity for antigen or a preferred epitope and, preferably, does not exhibit significant crossreactivity.
  • Appreciable or preferred binding include binding with an affinity of at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M "1 . Affinities greater 10 7 M "1 , preferably greater than 10 8 M "1 are more preferred.
  • an antibody that "does not exhibit significant crossreactivity" is one that will not appreciably bind to an undesirable entity (e.g., an undesirable proteinaceous entity) .
  • an undesirable entity e.g., an undesirable proteinaceous entity
  • specific binding is determined according to Scatchard analysis and/or competitive binding assays.
  • the monoclonal antibody of the invention has specificity for the CD20 antigen, or in specific therefor.
  • CD20 is a cell surface antigen expressed on more than 90% of B-cell lymphomas, which does not shed or modulate in neoplastic cells (McLaughlin et al . , J. Clin. Oncol. 16: 2825- 2833 (1998b)).
  • the CD20 antigen is a non-glycosylated, 35 kDa B-cell membrane protein involved in intracellular signaling, B-cell differentiation and calcium channel mobilization (Clark et al., Adv. Cancer Res. 52: 81-149 (1989); Tedder et al . , Immunology Today 15: 450-454 (1994)).
  • the antigen appears as an early marker of the human B-cell lineage, and is ubiquitously expressed at various antigen densities on both normal and malignant B-cell populations. However, the antigen is absent on fully, mature B-cells (e.g., plasma cells), early B-cell populations and stem cells, making it a suitable target for antibody mediated therapy.
  • mature B-cells e.g., plasma cells
  • stem cells e.g., stem cells
  • the heavy chain amino acid sequence of this monoclonal antibody is the sequence SEQ ID NO: 1.
  • the light chain amino acid sequence of this monoclonal antibody is the sequence SEQ ID NO: 2 or the sequence SEQ ID NO : 3.
  • this antibody may be obtained, as described in the patent application, WO2006/064121 which is incorporated herein by reference, by means of the transfection of an appropriate cell, preferably YB2/0, by vectors permitting the expression of the heavy and the light chains described above.
  • the composition of monoclonal anti-CD20 has a fucose content of less than 65%, and preferentially comprised between 20 and 40%.
  • the monoclonal antibody of the kit of parts of the invention is produced by the clone R509 which is deposited at the CNCM under the accession number CNCM 1-3314 (Collection Nationale de Cultures de Microorganismes , Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15 - France) .
  • the monoclonal antibody of the kit of parts of the invention is produced by the clone R603 which is deposited at the CNCM under the accession number CNCM 1-3529.
  • the kit of parts of the invention contains the monoclonal antibody produced by the clone R603 or by the clone R509, and the activated macrophages MAK® (IDM, Paris, France).
  • the Applicant has shown that the kit of parts of the invention efficiently killed ex vivo Chronic Lymphoid Leukaemia cells isolated from cancer patients, even at 1 to 1 effector to tumor ratio, at low monoclonal antibody concentration (0.025 mg/million MAK), and in the presence of 50% blood serum.
  • kit of parts of the invention so achieves an optimal destruction of the target cells recognised by the variable 002119
  • the concentration of the monoclonal antibody in the kit of parts of the invention for the treatment of B-CLL can be less than 375 mg/m 2 , and advantageously less than 20 mg/m 2 .
  • the kit of parts of the invention may be used for the treatment of any of the following diseases or disorders, where the disease or disorder is selected from the group consisting of an immune dysregulation disease, organ graft rejection and graft versus host disease.
  • the malignant disease or disorder is selected from the group consisting of a hematopoietic tumor
  • the B-cell malignancy is selected from the group consisting of indolent forms of B-cell lymphomas, aggressive forms of B-cell lymphomas, chronic lymphatic leukemias, acute lymphatic leukemias, Waldenstrom's macroglobulinemia, and multiple myeloma.
  • the kit of parts of the invention may be used for the treatment of non-malignant B-cell disorders and related diseases, such as many autoimmune and immune dysregulatory diseases, including septicemia and septic shock among immune dysregulatory diseases.
  • Auto-immune and/or inflammatory diseases can be systemic or organ specific and associated or not with pathogenic auto-antibodies.
  • autoimmune and/or inflammatory disorders include, but are not limited to, Hashimoto's thyroiditis, pernicious anemia, sarcoidosis, primary biliary cirrhosis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis , polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis obliterans, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, Henoch
  • the diseases that can be treated with the kit of parts of the invention are those where the level of effector cells is diminished or where the functionality of effector cells is diminished.
  • the kit of parts of the invention is used as an autologous therapy.
  • the present treatment consists in the local or systemic injection of autologous activated macrophages (preferentially MAK® killer cells) which have access to injured areas, and in particular to hypoxic areas, where they tend to concentrate and of monoclonal antibody according to the invention, linked to the MAK before the injection.
  • autologous activated macrophages preferentially MAK® killer cells
  • the activated macrophages and the monoclonal antibody are in the form of injectable solutions.
  • the injectable solutions are m the form of locally injectable solutions.
  • the injectable solutions are in the form of systemically injectable solutions.
  • the kit of parts formed of antibodies associated to MAK cells are kept frozen m separate aliquoted vials in the presence of 4% human serum albumin and of 10% DMSO, at -8O 0 C until use.
  • 6 administrations are carried out on the patient (3 to 10 administrations over a two year period).
  • the activated macrophages are to be administered at a dose of about 10 8 activated macrophages per injection.
  • the monoclonal antibody is to be administered at a dose of about 2.5 mg antibody per injection if linked to the MAK® prior to injection.
  • the activated macrophages are to be administered in a repeated way up to ten times, the interval between each administration being between three days to three months
  • the monoclonal antibody and the activated macrophages are to be injected simultaneously.
  • they may be administered in the same solution, or in a different one, but at the same time.
  • the activated macrophages may be administered in a right arm, whilst at the same time the monoclonal antibody may be administered in the left arm.
  • the monoclonal antibody and the activated macrophages are to be administered in a sequential manner, the monoclonal antibody being administered before the activated macrophages.
  • the monoclonal antibody is administered in one arm, for example the left one, before the macrophage, which is administered in the other arm.
  • the activated macrophages and the monoclonal antibody are to be administered sequentially, the activated macrophages being administered before the monoclonal antibody.
  • monoclonal antibody is administered in one arm, for example the left one, before the macrophage, which is administered in the other arm.
  • the time interval between the administration of activated macrophages and administration of monoclonal antibody is of one day to two weeks. More preferably, activated macrophages and antibodies are associated in vitro at room temperature and then kept frozen at -80 0 C until injection.
  • kit of parts of the invention can be administered after the treatment of the patient by a conventional therapy, e.g. by chemotherapeutic agents.
  • This treatment can be conducted after first failure and relapse following conventional chemotherapies, or before chemotherapy, to prevent chemoresistance .
  • Local treatment with chemotherapy drugs causes cell necrosis and release of chemokines which call and activelylaste macrophages and monocyte derived cells. Therefore, combining conventional chemotherapy with the immunotherapy based on the kit of parts of the invention can synergistically increase cytotoxicity and increase immune response at the same time as preventing the establishment of resistance.
  • kit of parts adoptive therapy can be proposed after failure and relapse.
  • the active ingredients which are administered either at the same time, or separately, or sequentially, according to the invention do not represent a mere aggregate of known agents, but a new combination with the surprising valuable property described above.
  • kits-of-parts means that the components of the combined preparation are not necessarily present as a union e.g. in composition, in order to be available for separate or sequential application.
  • kits of parts are combined in vitro, to freeze the resulting cell drug until use, allowing all quality controls before batch release.
  • Another object of the invention is a pharmaceutical composition comprising the kit of parts of the invention.
  • kits of parts of the invention for the preparation of a medicament for the treatment of a hematopoietic tumor or an autoimmune disease.
  • the medicament may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the compositions may be sterilised by conventional, well-known sterilisation techniques.
  • the formulation is in the form of 3 to 15 flasks of 10 ml each, containing 200 million MAK and 2.5 mg of antibodies, resuspended in 4% of HSA (human serum albumin) and 10% of DMSO (dimethylsulfoxide) and frozen at -80 0 C. Following safety controls, the product is released, and each dose can be thawed and diluted in HSA 4% prior to injection. DESCRIPTION OF THE FIGURES
  • Figure 1 Fc expression by MAK® cells from HD497 (one representative healthy donor) . Isotype controls are shown as dotted lines and specific labelling as solid lines.
  • PBMC Peripheral blood mononuclear cells
  • MAK-cell technology (Immunobiology 210 (2005) 267-277 :Anti-tumor properties of human-activated macrophages produced in large scale for clinical application; Veronique Baron-Bodo, Paula Doceur, Marie-Laure Lefebvre, Karine Labroquere, Catherine Defaye, Christophe Cambouris, Didier Prigent, Margarita Salcedo, Aure ' lie Boyer, Alessandra Nardin) IDM, Paris, France) , and also Thiounn N, Pages F, Mejean A, et al : Adoptive immunotherapy for superficial bladder cancer with autologous macrophage activated killer cells.
  • the MAK-cells are prepared according to GMP procedures by culture at a concentration of approximately 5 x 10 6 /mL in Iscove modified Dulbecco medium devoid of any animal or human proteins, and supplemented with granulocyte- macrophage colony stimulating factor (GM-CSF) (500 IU/mL, Novartis, France) and 2% of autologous serum.
  • GM-CSF granulocyte- macrophage colony stimulating factor
  • Cell culture was carried out at 37 0 C for six days in a humidified incubator under 5% of CO 2 , in hydrophobic EVA bags. After six days of culture, iFN- ⁇ (Boehringer Ingelheim, France) was added to the culture containers at the dose of 250 U/mL. On the seventh day of culture, macrophages were purified by an elutriation in physiological solution. The number of macrophages collected was at least 10 9 per culture. They were eventually combined with monoclonal antibodies by 1 h contact at room temperature and aliquoted at 200 million cells per vial and frozen at - 8O 0 C in the presence of 4% Human Serum Albumin and 10% DMSO.
  • iFN- ⁇ Boehringer Ingelheim, France
  • MAK cell preparations were incubated in presence of various monoclonal antibodies and resuspended in TO-PRO-3 (Molecular Probes) solution. Stained cells were further acquired using a FACSCalibur flow cytometer. TO-PRO-3 dye allows exclusion of dead cells during acquisition. Samples were analyzed by flow cytometry by gating on the viable (TO-PRO-3) cell population of interest (FSC/SSC) .
  • TO-PRO-3 Molecular Probes
  • Figure 1 shows the molecule cell surface expression by MAK.
  • Day 7 activated and elutriated macrophages from healthy donors were stained with antibodies specific for CD64, CD32 and CD16. Cells were analyzed by flow cytometry. lsotype controls are shown as dotted lines and specific labelling as solid lines.
  • One representative donor HD497 is presented here.
  • Example 2 Manufacture of anti-CD2Q antibody for inclusion in the kit of parts of the invention.
  • EMAB6 produced by the cell line R509, deposited at the CNCM under the accession number 1-331
  • EMAB603 produced by the cell line R603, deposited at the CNCM under the accession number 1-3529 were prepared as described in patent WO 2006/064121, whose content is incorporated herein by reference, in particular pages 26-33.
  • EMAB6 and EMAB603 are also named "LFB-antibodies” or "EMABling antibodies”.
  • variable regions of the murine antibody CAT-13.6E12 were determined.
  • the expression vectors of the heavy and of the light chain of the monoclonal antibodies EMAB6 and EMAB603 were constructed.
  • the light chain variable sequence was chimerized with a constant light chain sequence and cloned in an expression vector. The same protocol was applied for the chimerization of the heavy chain.
  • cell line YB2/0 is transfected with the two vectors expressing the heavy and the light chains of the antibody EMAB6 and EMAB603.
  • N-glycan of the heavy chain of EMAB6 and EMAB603 antibodies was analysed by HPCE-LIF (high performance capillary electrophoresis with detection of laser induced fluorescence) .
  • the anti-CD20 monoclonal antibody was desalted on a Sephadex G-25 column (HiTrap Desalting, Amersham Biosciences ) , dried by evaporation and put in suspension in a hydrolysis buffer of PNGase F (Glyko) in presence of 50 mM de ⁇ - mercaptoethanol . After 16 h of incubation at 37 0 C, the protein fraction was precipitated by addition of absolute ethanol and the supernatant, which contain the N-glycans, was dried by evaporation.
  • the oligosaccharides thus obtained were labelled directly by a fluorochrome : the APTS (l-amino-pyrene-3 , 6 , 8- trisulfonate) , or submitted to the action of specific exoglycosida ⁇ es before being labelled by the APTS.
  • the oligosaccharides thus labelled were injected on a cappilary JV- CHO, separated and quantified by HPCE-LIF.
  • fucose content was carried out either by addition of isolated fucosylated forms, or more specifically after simultaneous action of neuraminidase, ⁇ -galactosidase and iv-acetyHexosaminidase, leading to 2 peaks on the electrophoresis which correspond to the fucosylated or unfucosylated pentasaccharide [GlcNac2-Man3 ] .
  • the content of galactose was calculated by adding the percentages of the oligosaccharide forms containing galactose, obtained after the action of neuraminidase and fucosidase.
  • the formula used was the following :
  • Example 3 Antitumoral efficacy of the kit of parts; in vitro experintents
  • MAK cells are armed with increasing doses of rituximab or with the anti-CD20 antibody according to the invention and cultured for up to 2Oh with CLL tumor cells or Raji Burkitt Lymphoma cell line (ATCC-CLL-86 ) in absence or in presence of up to 50% blood serum. Following incubation, a flow cytometry assay allows quantification of the remaining alive tumor cells. Briefly, macrophages and tumor cells are stained with specific antibodies and acquired on the flow cytometer in presence of a dead cell exclusion dye together with an internal beads control allowing cell quantification. Cells are analyzed on the flow cyto ⁇ ieter and the percentage of cell kill was calculated.
  • % cell kill [ (number of alive CLL in the absence of MAK + Ab) - (number of alive CLL in the presence of MAK + Ab) / (number of alive CLL in the absence of MAK + Ab) ] x 100
  • Example 4 Experimental murine model (SCID mice grafted with tumors expressing CD20) showing anti-tumor efficacy of the new binary complex MAK-Ab (according to the invention)
  • SCID mice were administered cells from B-CLL patients labelled ex vivo with a fluorescent dye (CFSE) .
  • the percent of tumor cells is monitored in different organs such as the spleen, the liver, the kidney.
  • the absence of effect of injection of human serum on the antitumor efficacy is also shown. Different E/T ratios and administration route of the therapeutic product.
  • Example 5 Infusion of MAK combined to anti-CD20 antibodies to patients with chronic lymphoid leukaemia.
  • CLL chronic lymphocytic leukemia
  • MRD persistent minimal residual disease
  • MAK® autologous macrophage-activated killer cells
  • Heavily pretreated MRD-positive CLL patients were enrolled in a phase II trial to receive infusions of MAK®/anti-CD20.
  • Mononuclear cells were isolated from peripheral blood by cytapheresis .
  • Activated macrophages were obtained after 7 days of differentiation and activation with gamma interferon before purification by apheresis (Immuno-Designed Molecules, Paris, France) .
  • MAKs were then incubated for 1 h at room temperature with rituximab or with the EMAB6 antib-CD20 antibody according to the invention.
  • the six injections were completed in 80% of the patients enrolled with MAK-Rituximab without significant adverse events. Several patients achieved a durable immunophenotypic, and molecular remission. After a median-follow-up of 60 months, some patients were still in complete phenotypic plus molecular remission, and in complete clinical remission. The median progression-free survival was more significantly increased in patients receiving MAK plus EMEAB6 than in patients receiving MAK-Rituximab.

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Abstract

Trousse pour le traitement du cancer ou de maladies infectieuses, comprenant des macrophages activés et un anticorps monoclonal. L'affinité de la région Fc dudit anticorps pour le récepteur Fc gamma III (CDI 6) est plus forte que l'affinité d'anticorps polyclonaux IgG pour ledit récepteur Fc gamma III.
EP08789068A 2007-04-26 2008-04-25 Kit de parties pour le traitement du cancer ou de maladies infectueuses Withdrawn EP2150261A2 (fr)

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EP07290522A EP1985633A1 (fr) 2007-04-26 2007-04-26 Kit de parties pour le traitement du cancer ou de maladies infectueuses
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FR2940616A1 (fr) * 2008-12-30 2010-07-02 Lfb Biotechnologies Utilisation d'un anticorps anti-cd20 pour le traitement du lymphome primaire intraoculaire.
FR2966043A1 (fr) * 2010-10-14 2012-04-20 Lfb Biotechnologies Utilisation d'un anticorps anti-cd20 pour le traitement du lymphome cerebral primitif
FR2976811A1 (fr) * 2011-06-22 2012-12-28 Lfb Biotechnologies Utilisation d'un anticorps anti-cd20 a haute adcc pour le traitement de la maladie de waldenstrom
JP6433786B2 (ja) * 2011-08-10 2018-12-05 ラボラトワール フランセ デュ フラクショヌマン エ デ ビオテクノロジーＬａｂｏｒａｔｏｉｒｅ Ｆｒａｎｃａｉｓ ｄｕ Ｆｒａｃｔｉｏｎｎｅｍｅｎｔ ｅｔ ｄｅｓ Ｂｉｏｔｅｃｈｎｏｌｏｇｉｅｓ 高度ガラクトシル化抗体
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WO2008146165A3 (fr) 2009-02-19
TW200918555A (en) 2009-05-01
EP1985633A1 (fr) 2008-10-29
WO2008146165A2 (fr) 2008-12-04

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