EP1487487A2 - Verfahren und zusammensetzungen zur prävention, diagnose und behandlung von krebs mit bispezifischen molekülen - Google Patents

Verfahren und zusammensetzungen zur prävention, diagnose und behandlung von krebs mit bispezifischen molekülen

Info

Publication number
EP1487487A2
EP1487487A2 EP02778405A EP02778405A EP1487487A2 EP 1487487 A2 EP1487487 A2 EP 1487487A2 EP 02778405 A EP02778405 A EP 02778405A EP 02778405 A EP02778405 A EP 02778405A EP 1487487 A2 EP1487487 A2 EP 1487487A2
Authority
EP
European Patent Office
Prior art keywords
antigen
tumor
binds
mammal
tumor associated
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Withdrawn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
EP02778405A
Other languages
English (en)
French (fr)
Inventor
David Jackson
Linda Nardone
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.)
Elusys Therapeutics Inc
Original Assignee
Elusys Therapeutics 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
Application filed by Elusys Therapeutics Inc filed Critical Elusys Therapeutics Inc
Publication of EP1487487A2 publication Critical patent/EP1487487A2/de
Withdrawn legal-status Critical Current

Links

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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

  • the present invention relates to bispecific molecules having a first antigen recognition portion which binds a C3b-like receptor (known as complement receptor 1 (CRl) or CD35 in primates) and a second antigen recognition portion which binds an epitope of a tumor associated antigen.
  • the present invention also relates to methods of using the bispecific molecules in cancer prevention, diagnosis, and treatment.
  • Curing a cancer is a difficult task despite current available treatments and the body's own defense system again cancer cells. Surgery can rarely ferret out every metastasis, and treatments that kill cancer cells are generally toxic to normal cells as well. If even a few cancerous cells remain, they can proliferate to produce a resurgence of the disease. Unlike the normal cells, cancer cells may also quickly evolve resistance to the poisonous drugs that are used against them. Because of the special targeting ability towards the tumor cells that antibodies and certain lymphocytes possess, immunological cancer therapy have generated great interest.
  • Antibody responses involve the production of antibodies, which are proteins called immunoglobulins.
  • the antibodies circulate in the bloodstream and permeate the other body fluids, where they bind specifically to the foreign antigen that elicited them. Binding by antibody inactivates viruses and bacteria by blocking their ability to bind to receptors on target cells. Antibody binding also marks invading microorganisms for destruction, either by making it easier for a phagocytic cell to ingest them or by activating a system of blood proteins, collectively called complement, that kills the invaders. (MOLECULAR BIOLOGY OF THE CELL, Albert B.
  • T lymphocytes which develop in the thymus, are responsible for cell-mediated immunity.
  • helper T cells and suppressor T cells play a regulatory role in immunity, acting either
  • T lymphocytes called cytotoxic T cells, kill virus-infected cells, parasites, and cancer cells.
  • cytotoxic T cells kill virus-infected cells, parasites, and cancer cells.
  • the surface of T cells contains transmembrane proteins called T cell receptors that recognize foreign molecules on the surface of other cells. T cell receptors are antibody-like proteins.
  • MHC major histocompatibility complex
  • helper T cells recognize foreign antigens in association with class II MHC glycoproteins on the surface of an antigen-presenting cell.
  • a cytotoxic T lymphocyte will kill a virus-infected cell when it recognize fragments of viral protein bound to class I MHC molecules on the surface of the infected cell.
  • the cell membrane-bound tumor antigens have been shown to represent targets or potential targets for antibody-mediated or cytotoxic T lymphocyte (CTL)-mediated therapy directed against the tumor cells, while the shed antigens have been used clinically in evaluating cancer risk, diagnosis, prognosis, or response to treatment. (See Stearns, et al., 1998, Breast Cancer Research and Treatment, 52:239-259).
  • CTL cytotoxic T lymphocyte
  • Antibodies can be used in several ways in cancer therapy. First, as the first line of the body's defenses against foreign intruders, each antibody will bind to a specific antigen, such as a tumor specific antigen or a tumor associated antigen, and triggers the rest of the immune system to destroy the tumor. (MOLECULAR BIOLOGY OF THE GENE, Watson J. D. et al., 4 th ed., The Benjamin/Cumming Publishing Company, Inc., 1987). Second,
  • antibodies can be used in chemotherapy to improve the antitumor efficacy and to reduce the toxicities of the drug.
  • Chemotherapeutic drugs spread throughout the body, reaching not only the tumor but also healthy organs such as the intestines and bone marrow, where they kill off normal dividing cells.
  • An antibody conjugated drug is able to selectively target the tumor cells and thus increase the sensitivity and specificity of the drug.
  • antibodies can also be used in some other tumor therapies as a targeting tool. For example,
  • Panorex targets a protein which helps cells stick
  • HERCEPTIN containing antibody Trastuzumab
  • HER-2/neu a growth factor receptor that is present in larger than normal amounts on some breast cancer cells.
  • Numerous studies have shown that the unlucky 25% to 30% of breast cancer patients whose tumors produce more HER-2/neu have worse prognoses and shorter life expectancies.
  • Clinical trials of HERCEPTIN showed that it can slow the progression of breast cancer in women whose
  • U.S. Pat. 6,241,985 is another example of cancer immunotherapy.
  • the invention employs a binding agent, which binds to a pre-determined epitope of a multi-epitopic tumor associated antigen. Such binding will alter the antigen in a manner so that the host immune system can recognize and initiate an immune response to the previous unrecognized tumor
  • U.S. Pat. 6,086,873 describes an invention using UN-exposed antibodies to a cancer patient, with the specific purpose of generating an immune response to the UN-exposed antibody. This response may provide a therapeutic advantage via enhanced humoral and cellular consequences directed to the cancer cells.
  • cytotoxic T lymphocytes also defend the body against
  • non-T, and non-B cells mediate antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Immune complexes can also cause blocking in vitro. Immune complexes in cancer sera may be composed of cell surface material of varying antigenic specificities, e.g., tumor antigen, fetal antigen, or viral antigen.
  • the tumor antigen levels are a function of expression of the protein by malignant cells, the number of
  • Shed tumor antigens can be but are not limited to: (1) carcinoembryonic antigen (CEA), an antigen exist in extracts of fetal gut and varies types of cancer, especially carcinomas of entodermal origin (colorectal, pulmonary, pancreatic, gastric) (Hansen H.J. et al., 1974, Human Path.
  • CEA carcinoembryonic antigen
  • MUC-1 gene which is a cell-associated mucin-like protein implicated in breast cancer (assays detect circulating MUC-1 products include CA15-3, CA27-29, CA 549, breast cancer mucin (BCM, mammary serum antigen (MSA), and mucin-like carcinoma-associated antigen (MCA)) (Stearns V. et al., 1998, Breast Cancer Research and Treatment 52: 239-
  • tumor antigens may create various problems in cancer immunotherapy or diagnosis. For instance, tumor antigens may affect the pharmacokinetics of an antibody regime, so that large dosage and more frequent administration of the antibodies are required.
  • HERCEPTIN it was observed that presence of shed antigen from
  • HER2/neu receptor increases the clearance of HERCEPTIN. Since most antibody drugs or antibody conjugated drugs have severe side effects, e.g., administration of HERCEPTIN can result in severe hypersensitivity reactions (including anaphylaxis), infusion reactions and pulmonary events (see HERCEPTP PI boxed warning), an increased dosage or frequent administration of the drug due to the shed antigen will also exacerbate the side effects of the treatment. Moreover, Davies Q. et al showed that radiolabeled hCTMOl
  • the present invention relates to bispecific molecules having a first antigen recognition portion which binds a C3b-like receptor or its functional equivalent and a second antigen recognition portion which binds an epitope of a shed tumor antigen.
  • the first antigen recognition portion of a bispecific molecule can be any polypeptide that contains a C3b-like receptor binding domain and an effector domain.
  • the first antigen recognition portion is an anti-CRl mAb.
  • the first antigen recognition portion is an anti-CRl polypeptide antibody, including but is not limited to, a single-chain variable region fragment (scFv) with specificity for a C3b-like receptor fused to the N-terminus of an immunoglobulin Fc domain.
  • the second antigen recognition portion of a bispecific molecule can be any molecular moiety, including but is not limited to any antibody or antigen binding fragment thereof, that recognizes and binds an epitope of a shed tumor antigen.
  • the tumor associated antigen that the second antigen recognition portion binds can be a antigen selected from the group consisting of carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM), HER2-/neu protein, CA125, tumor-associated glycoprotein - 72 (TAG-72), prostate-specific antigen (PSA), carbohydrate antigen 19-9 (CA19-9), tissue polypeptide specific antigen (TPS), and product of MUC-1 gene.
  • CEA carcinoembryonic antigen
  • PEM polymorphic epithelial mucin
  • HER2-/neu protein CA125
  • TAG-72 tumor-associated glycoprotein - 72
  • PSA prostate-specific antigen
  • CA19-9 carbohydrate antigen 19-9
  • TPS tissue polypeptide specific antigen
  • the second antigen recognition portion of the bispecific molecule can recognize and bind any epitope of a shed tumor associated antigen.
  • the bispecific molecule can recognize and bind the same epitope as a therapeutic agent, e.g., a therapeutic antibody or a therapeutic cell, e.g., a lymphocyte.
  • the bispecific molecule can also recognize and bind a different epitope as a therapeutic agent, e.g., a therapeutic antibody or a therapeutic cell, e.g., lymphocyte.
  • the epitope that is recognized and bound by the second antigen recognition portion of the bispecific molecule can be an epitope that is not exposed by the cell-surface bound tumor associated antigen.
  • the bispecific molecule used in the present invention can be cross-linked antibodies, wherein the first antibody is specific to a primate C3b-like receptor and the second antibody is specific to a shed tumor antigen.
  • the bispecific molecule comprises an anti-CRl monoclonal antibody cross-linked to a monoclonal antibody that recognizes and binds an epitope of said shed tumor associated antigen.
  • the bispecific molecule can also be antibodies that are produced recombinantly and have one domain recognizing a primate C3b-like receptor and a second domain recognizing a shed tumor antigen.
  • the bispecific molecule can as well be produced using the method of protein trans- splicing and has the first antigen recognition portion recognizing a primate C3b-like receptor and the second antigen recognizing portion recognizing a shed tumor antigen.
  • the present invention also relates to polyclonal populations of bispecific molecules which comprise a plurality of different bispecific molecules, each of the different bispecific molecules comprising a first antigen recognition portion that binds a C3b-like receptor and a different second antigen recognition portion that binds an epitope of a tumor associated antigenic molecule.
  • the polyclonal population of the invention comprises a plurality of different bispecific molecules having different second antigen recognition portions that have specificities directed to, e.g., a plurality of recognition sites on a shed tumor antigen.
  • the population of bispecific molecules can have a plurality of different second antigen recognition portions that recognize and bind different epitopes on a shed tumor antigen.
  • the population of bispecific molecules can also have a plurality of different second antigen recognition portions that recognize and bind the same epitope on a shed tumor antigen.
  • each bispecific molecule in the polyclonal population of bispecific molecules comprises an anti-CRl monoclonal antibody cross-linked to a monoclonal antibody that recognizes and binds an epitope of a shed tumor associated antigen.
  • kits comprising: (a) a bispecific molecule, and (b) an agent, wherein said bispecific molecule comprises a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds an epitope of a tumor associated antigen shed by cells of a tumor, and wherein said agent binds said tumor associated antigen on cells of said tumor in order to achieve a therapeutic effect in treating said mammal.
  • the first antigen recognition portion of the bispecific molecule is an anti-CRl mAb.
  • the bispecific molecule comprises an anti-CRl mAb cross-linked to a monoclonal antibody that recognizes and binds an epitope of a tumor associated antigen.
  • the tumor associated antigen that the second antigen recognition portion binds can be a antigen selected from the group consisting of carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM), HER2-/neu protein, CA125, tumor-associated glycoprotein - 72 (TAG-72), prostate-specific antigen (PSA), carbohydrate antigen 19-9 (CA19-9), tissue polypeptide specific antigen (TPS), and product of MUC-1 gene.
  • CEA carcinoembryonic antigen
  • PEM polymorphic epithelial mucin
  • HER2-/neu protein CA125
  • TAG-72 tumor-associated glycoprotein - 72
  • PSA prostate-specific antigen
  • CA19-9 tissue polypeptide specific antigen
  • TPS tissue polypeptide specific antigen
  • kits comprising: (a) a polyclonal population of bispecific molecules, and (b) an agent, wherein said polyclonal population of bispecific molecules comprises a plurality of different bispecific molecules, each bispecific molecule in said plurality comprising a first antigen recognition portion that binds a C3b-like receptor and a different second antigen recognition portion that binds an epitope of a tumor associated antigen shed by cells of a tumor, and wherein said agent binds said tumor associated antigen on cells of said tumor in order to achieve a therapeutic effect in treating said mammal.
  • the first antigen recognition portion of the bispecific molecules is an anti-CRl mAb.
  • each of the bispecific molecules comprises an anti-CRl mAb cross-linked to a monoclonal antibody that recognizes and binds an epitope of a tumor associated antigen.
  • the tumor associated antigen that the second antigen recognition portion binds can be a antigen selected from the group consisting of carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM), HER2-/neu protein, CA125, tumor-associated glycoprotein - 72 (TAG-72), prostate-specific antigen (PSA), carbohydrate antigen 19-9 (CA19-9), tissue polypeptide specific antigen (TPS), and product of MUC-1 gene.
  • CEA carcinoembryonic antigen
  • PEM polymorphic epithelial mucin
  • HER2-/neu protein CA125
  • TAG-72 tumor-associated glycoprotein - 72
  • PSA prostate-specific antigen
  • CA19-9 tissue polypeptide specific antigen
  • TPS tissue polypeptide specific antigen
  • the present invention also provides a population of modified hematopoietic cells that comprise a plurality of hematopoietic cells each bound to one or more bispecific molecules, wherein each of said bispecific molecules comprises a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds an epitope of a tumor associated antigenic molecule, wherein said bispecific molecules bound to said population of modified hematopoietic cells forms a population of bispecific molecules comprising different second antigen recognition portions.
  • the present invention provides methods for treating a mammal, e.g., a human, having a tumor, cells of said tumor shedding a tumor associated antigen into the circulation of said mammal, said method comprising administering to said mammal a therapeutically sufficient amount of a bispecific molecule, said bispecific molecule comprising a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds an epitope of said shed tumor associated antigen, wherein said mammal is subject to a cancer therapy, said cancer therapy comprising treating said mammal with an agent that binds said tumor associated antigen on cells of said tumor in order to achieve a therapeutic effect in treating said mammal.
  • a mammal e.g., a human, having a tumor, cells of said tumor shedding a tumor associated antigen into the circulation of said mammal
  • said method comprising administering to said mammal a therapeutically sufficient amount of a bispecific molecule, said bispecific molecule
  • the invention provides a method for treating a mammal having a tumor, cells of said tumor shedding a tumor associated antigen into the circulation of said mammal, said method comprising (a) administering to said mammal a bispecific molecule, wherein said bispecific molecule comprises a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds an epitope of said shed tumor associated antigen; and (b) administering to said mammal an agent that binds said tumor associated antigen on cells of said tumor in order to achieve a therapeutic effect in treating said mammal.
  • the first antigen recognition portion of the bispecific molecule is an anti-CRl mAb.
  • the bispecific molecule comprises an anti-CRl mAb cross-linked to a monoclonal antibody that recognizes and binds an epitope of a tumor associated antigen.
  • the tumor associated antigen that the second antigen recognition portion binds can be a antigen selected from the group consisting of carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM), HER2-/neu protein, CA125, tumor-associated glycoprotein - 72 (TAG-72), prostate-specific antigen (PSA), carbohydrate antigen 19-9 (CA19-9), tissue polypeptide specific antigen (TPS), and product of MUC-1 gene.
  • CEA carcinoembryonic antigen
  • PEM polymorphic epithelial mucin
  • HER2-/neu protein CA125
  • TAG-72 tumor-associated glycoprotein - 72
  • PSA prostate-specific antigen
  • CA19-9 tissue polypeptide specific antigen
  • TPS tissue polypeptide specific antigen
  • the agent is a therapeutic antibody that binds the tumor associated antigen on cells of the tumor.
  • the agent is a chemotherapeutic drug conjugated to an antibody that binds the tumor associated antigen on cells of the tumor.
  • the agent is a radiolabeled antibody that binds the tumor associated antigen on cells of the tumor.
  • the agent is a stimulated or cultured lymphocyte that binds the tumor associated antigen on cells of the tumor.
  • Bispecific molecules can be administered before, at the same time, or after the administration of the therapeutic antibody or drugs. In one embodiment, the bispecific molecule is administered concurrently with the agent. In another bispecific molecule is administered for a period of time before the agent is administered. In still another embodiment, the bispecific molecule is administered for a period of time after the agent is administered.
  • the present invention also provides a method for treating a mammal having a tumor, cells of said tumor shedding a tumor associated antigen into the circulation of said mammal, said method comprising administering to said mammal a therapeutically sufficient amount of a bispecific molecule, said bispecific molecule comprising a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds an epitope of said shed tumor associated antigen, wherein said mammal is subject to a cancer therapy, said cancer therapy comprising treating said mammal with an agent that stimulates production of cells that bind said tumor associated antigen on cells of said tumor in order to achieve a therapeutic effect in treating said mammal.
  • the invention provides a method for treating a mammal having a tumor, cells of said tumor shedding a tumor associated antigen into the circulation of said mammal, said method comprising (a) administering to said mammal a bispecific molecule, wherein said bispecific molecule comprises a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds an epitope of said shed tumor associated antigen; and (b) administering to said mammal an agent that binds said tumor associated antigen on cells of said tumor in order to achieve a therapeutic effect in treating said mammal.
  • the first antigen recognition portion of the bispecific molecule is an anti-CRl mAb.
  • the bispecific molecule comprises an anti-CRl mAb cross-linked to a monoclonal antibody that recognizes and binds an epitope of a tumor associated antigen.
  • the tumor associated antigen that the second antigen recognition portion binds can be a antigen selected from the group consisting of carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM), HER2-/neu protein, CA125, tumor-associated glycoprotein - 72 (TAG-72), prostate-specific antigen (PSA), carbohydrate antigen 19-9 (CA19-9), tissue polypeptide specific antigen (TPS), and product of MUC-1 gene.
  • CEA carcinoembryonic antigen
  • PEM polymorphic epithelial mucin
  • HER2-/neu protein CA125
  • TAG-72 tumor-associated glycoprotein - 72
  • PSA prostate-specific antigen
  • CA19-9 tissue polypeptide specific antigen
  • TPS tissue polypeptide specific antigen
  • the agent is a cytokine that stimulates production of an immune response against the tumor associated antigen.
  • Bispecific molecules can be administered before, at the same time, or after the administration of the therapeutic agent. In one embodiment, the bispecific molecule is administered concurrently with the agent. In another bispecific molecule is administered for a period of time before the agent is administered. In still another embodiment, the bispecific molecule is administered for a period of time after the agent is administered.
  • the present invention also provides a method for treating a mammal having a tumor, wherein cells of said tumor shed a tumor associated antigen into the circulation of said mammal, and wherein said tumor associated antigen elicits in said mammal an immune response against said tumor, said method comprising administering to said mammal a o therapeutically sufficient amount of a bispecific molecule, said bispecific molecule comprising a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds an epitope of said shed tumor associated antigen.
  • the first antigen recognition portion of the bispecific molecule is an anti-CRl mAb.
  • the bispecific molecule comprises an 5 anti-CRl mAb cross-linked to a monoclonal antibody that recognizes and binds an epitope of a tumor associated antigen.
  • the tumor associated antigen that the second antigen recognition portion binds can be a antigen selected from the group consisting of carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM), HER2-/neu protein, CA125, tumor-associated glycoprotein - 72 (TAG-72), prostate-specific antigen (PSA), 0 carbohydrate antigen 19-9 (CA19-9), tissue polypeptide specific antigen (TPS), and product of MUC-1 gene.
  • CEA carcinoembryonic antigen
  • PEM polymorphic epithelial mucin
  • HER2-/neu protein CA125
  • TAG-72 tumor-associated glycoprotein - 72
  • PSA prostate-specific antigen
  • TPS tissue polypeptide specific antigen
  • the present invention also provides a method for detecting tumor in a mammal having a tumor, cells of said tumor shedding a tumor associated antigen into the circulation of said mammal, said method comprising (a) administering to said mammal a 5 therapeutically sufficient amount of a bispecific molecule, said bispecific molecule comprising a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds an epitope of said shed tumor associated antigen; (b) administering to said mammal an agent conjugated with a label, said agent recognizes and binds said tumor associated antigen; and (c) detecting said label.
  • the 0 agent is a radiolabeled antibody that binds said tumor associated antigen on cells of said tumor.
  • the first antigen recognition portion of the bispecific molecule is an anti-CRl mAb.
  • the bispecific molecule comprises an anti-CRl mAb cross-linked to a monoclonal antibody that recognizes and binds an epitope of a tumor associated antigen.
  • the tumor associated antigen that the 5 second antigen recognition portion binds can be a antigen selected from the group consisting of carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM), HER2-/neu protein, CA125, tumor-associated glycoprotein - 72 (TAG-72), prostate-specific antigen (PSA), carbohydrate antigen 19-9 (CA19-9), tissue polypeptide specific antigen (TPS), and product of MUC-1 gene.
  • CEA carcinoembryonic antigen
  • PEM polymorphic epithelial mucin
  • HER2-/neu protein CA125
  • TAG-72 tumor-associated glycoprotein - 72
  • PSA prostate-specific antigen
  • TPS tissue polypeptide specific antigen
  • product of MUC-1 gene a antigen selected from the group consisting of carcinoembryonic antigen (CEA), polymorphic epithelial mucin (PEM), HER2-/neu protein, CA125, tumor-associated glycoprotein - 72 (TA
  • Figure 1 depicts a bispecific molecule comprising cross-linked monoclonal antibodies. The wavy line indicates cross-linker.
  • Figure 2 depicts a bispecific molecule of the invention, that is a bispecific immunoglobulin created by the fusion of the hybridomas producing the antibodies; the left arm of the antibody as depicted binds C3b-like receptor; the right arm binds Ag2, which is a shed tumor antigen.
  • Figures 3 A-F illustrate some bispecific molecule embodiments as illustrated in Figure 2.
  • Left to right depicts amino- to carboxy-terminal order.
  • Panel A depicts a bispecific molecule which is a single polypeptide consisting essentially of a first binding domain (BD1), fused to the amino terminus of a CH2 and CH3 portion of an immunoglobulin heavy chain fused to a second binding domain (BD2) at its carboxy terminus.
  • BD1 first binding domain
  • BD2 second binding domain
  • Panel B depicts a dimer consisting of a first polypeptide consisting essentially of a BD1 fused to the amino terminus of a Fc domain of an antibody(a hinge region, a CH2 domain and a CH3 domain) and a second polypeptide consisting essentially of a Fc domain with a BD2 domain fused to the Fc domain's carboxy terminus.
  • Panel C depicts the structure, in a specific embodiment, of one or both of the polypeptides of the dimer of Panel B.
  • Panel C depicts a polypeptide that consists essentially of a variable light chain domain (NL) and constant light chain domain (CL) fused via a linker molecule to the amino terminus of a NH domain followed by a CHI domain, a hinge region, a CH2 domain and a CH3 domain.
  • Panel D depicts the structure, in a specific embodiment, of one or both of the polypeptides of the dimer of Panel B.
  • Panel D depicts a polypeptide containing a scFv fused to the amino terminus of a CHI domain, followed by a hinge region, a CH2 domain and a CH3 domain.
  • Panel E depicts a polypeptide comprising two separate scFv with specificity for two separate antigens, the polypeptide consisting essentially of a first scFv domain fused to a CH2 domain, followed by a CH3 domain, and a second scFv domain. " " indicates "binds to.”
  • Panel F depicts a polypeptide comprising two variable regions with specificity for two separate antigens, the polypeptide consisting essentially of a first variable heavy chain fused to a variable light chain, a CH2 domain, a CH3 domain, a variable heavy chain and variable light chain.
  • Figures 4 A-B illustrate bispecific molecules of the present invention that are 5 produced by trans-splicing methods.
  • Fig. 4A schematic illustration of some configurations of bispecific molecules;
  • Fig. 4B schematic illustration of some configurations of bispecific molecules including a streptavidin-biotin linker.
  • the present invention provides methods and compositions for the prevention, diagnosis, and treatment of tumor.
  • the invention is based on the observation that a variety of tumor cells produce antigens, e.g., tumor associated antigens or tumor specific antigens, on their surfaces. Some of these antigens are shed or secreted into the circulation. Removal of the shed tumor antigens from the circulation may improve the specificity and efficacy of
  • the bispecific molecules of the present invention utilize the unique properties of the C3b-like receptor, expressed on the surface of hematopoietic cells (for example, CRl on erythrocytes in humans), to rapidly and efficiently clear shed tumor antigens from the circulation.
  • the C3b receptor is known as the complement receptor 1 (CRl) in primates or
  • C3b-like receptor is understood to mean any mammalian circulatory molecule which has an analogous function to a primate C3b receptor, for example, CRl.
  • tumor or cancer, which is used interchangeably
  • tumor is understood to refer to an abnormal benign or malignant mass of tissue or
  • tumor associated antigen refers to an antigenic molecule that can be but need not to be specifically associate to tumor cells, e.g., antigens that are expressed on both cancer cells and certain
  • tumor associated antigens include but are not limited to: carcinoembryonic antigen (CEA), an antigen existing in extracts of fetal gut and varies types of cancer, especially carcinomas of entodermal origin (colorectal, pulmonary, pancreatic, gastric) (Hansen H.J. et al., 1974, Human Path. 5:139-147; Hagan P.L. et al.,
  • CEA carcinoembryonic antigen
  • tumor specific antigen refers to an antigen that is specifically associated to tumor cells, i.e., normal cells do not express such antigen.
  • Tumor associated antigen is expressed on the surface of a tumor cell, which is also referred to by the term "surface-bound tumor associated antigen.” Tumor associated antigen can also be shed or secreted by the tumor cell into circulation. The shed form of a tumor associated antigen is referred to by the term “shed tumor antigen”, which is understood to include both circulating tumor specific antigens and the tumor associated antigens that are ejected or secreted by cancer cells.
  • the present invention provides methods and compositions which can be used in antibody-mediated therapy.
  • bispecific molecules are used in conjunction with a therapeutic antibody or antibodies.
  • bispecific molecules are used in conjunction with an antibody-mediated chemotherapeutic drug or drugs.
  • a therapeutic antibody can refer to antibodies that are used to target cancer cells and have an anti-cancer growth or anti-metastasis effects, e.g., therapeutic antibodies may activate a host's immune system that leads to the killing of the targeted cancer cells, or they may prevent cancer cells from sticking together so that the metastasis rate can be lowered.
  • therapeutic antibody can be but are not limited to Panorex and Trastuzumab (HERCEPTIN). (See Dickman S., 1998, Science, 280: 1196-1197).
  • the antibodies that are used in antibody-mediated therapies target cancer cells more specifically so that the treatments have better efficacies and less side effects.
  • the present invention also provides methods and compositions which can be used in cell-mediated therapy.
  • the bispecific molecules are used in conjunction with cytokines that stimulate production of cells that target and kill cancer cells.
  • cytokines include, but are not limited to, interleukin-1 (IL-la and b), interleukin-2 (IL-2), interleukin-4 (IL-4), tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN-a, b), macrophage cology stimulating factor, and granulocyte macrophage colony stimulating factor.
  • the bispecific molecules are used in conjunction with stimulated and cultured lymphocytes that specifically target and kill tumor cells.
  • the bispecific molecules are used in conjunction with cytokine activated tumor-specific tumor infiltrating lymphocytes (TIL) that specifically target tumor cells.
  • TIL tumor-specific tumor infiltrating lymphocytes
  • bispecific molecules are used in conjunction with cytotoxic T lymphocytes (CTL) in CTL-mediated cancer therapy.
  • CTL cytotoxic T lymphocytes
  • bispecific molecules are used in conjunction with K cells in antibody- dependent cellular cytotoxicity (ADCC).
  • K cells mediate antibody-dependent cellular cytotoxicity (ADCC) wherein said K cells are able to kill cells that express foreign antigens.
  • the bispecific molecules are used to remove shed tumor antigens so that upon such removal, the therapeutic agents, e.g., lymphocytes, will be more efficiently targeting tumor cells because of the removal of the blocking or suppressing effects conveyed by the shed tumor antigens.
  • the present invention provides methods and compositions that enhances a host's immune responses to tumor.
  • the bispecific molecules are administered to remove the shed tumor antigens so that upon the removal of shed tumor antigens from circulation, the host's own immune components, such as antibodies and cytotoxic T cells that are elicited by the tumor antigens, are not blocked by the circulating tumor antigens, and therefore, target the tumor cells more specifically and more efficiently. As a result, less tumor cells will escape the attacks by a host's own immune system.
  • the bispecific molecules are administered at an early stage of the tumor development so that the host's immune response will be more specifically targeting the tumor cells to slow tumor growth and lower the metastasis rate.
  • the bispecific molecules are administered to patient after surgical removal of the tumor to enhance the patient's immune system's ability to kill residue tumor cells, thereby increasing survival rates.
  • Bispecific molecules can also be used to assist therapies which enhance the antigenic abilities of a cell expressed tumor associated antigens, which elicit stronger immune response in a host.
  • bispecific molecules are used in conjunction with a binding agent, which binds to a pre-determined epitope of a multi-epitopic tumor associated antigen, and such binding will alter the antigen in a manner so that the host immune system can recognize and initiate an immune response to the previous unrecognized tumor associated antigen (U.S. Pat. Nos. 6,241,985 and 6,086,873).
  • epitope refers to an antigenic determinant, i.e., a region of a molecule that provokes an immunological response in a host or is bound by an antibody. This region can but need not comprise consecutive amino acids.
  • the term epitope is also known in the art as "antigenic determinant.”
  • An epitope may comprise as few as three amino acids in a spatial conformation which is unique to the immune system of the host. Generally, an epitope consists of at least five such amino acids, and more usually consists of at least 8-10 such amino acids. Methods for determining the spatial conformation of such amino acids are known in the art.
  • the present invention also provides methods and compositions that can be used in conjunction with radiolabeled antibodies, which are used in diagnosis of tumor or in radiotherapy of cancer.
  • the bispecific molecules are used in conjunction with radiolabeled antibodies in detecting a tumor, e.g., radiolabeled antibodies can be injected to a host and then visualized by any imaging methods that detects specifically the radiation site(s) known in the art.
  • radiolabeled antibodies can be injected to a host and then visualized by any imaging methods that detects specifically the radiation site(s) known in the art.
  • the term "radiolabeled antibody” refers to antibodies that are linked with radioactive markers, such as indium- 111 ( In). (See Hagan P.L. et al., 1985, J. Nucl. Med. 26:1418-1423).
  • radiolabeled antibodies used in the radiotherapy can be, but are not limited to, radiolabeled anti-CEA monoclonal antibodies (mABs), radiolabeled anti-PEM mABs, radiolabeled anti-HER2/neu mABs, radiolabled anti-CA-125 mABs, radiolabeled anti-TAG-72 mABs, radiolabeled anti-PSA mABs, radiolabeled anti-CA19-9 mABs, radiolabeled anti-TPS mABs, and radiolabeled anti-MUC-1 mABs.
  • the bispecific molecules are used in conjunction with radiotherapy.
  • radiotherapy radiation is employed to deposit energy to injure or destroy cells in the area being treated by damaging their genetic material, making it impossible for the cells to grow.
  • radiotherapy can be but are not limited to, radiolabeled antibodies that deliver doses of radiation, e.g., X-rays, Gamma rays, directly to the cancer sites. Radiotherapy may be used alone or in combination with other cancer treatments, such as chemotherapy or surgery.
  • compositions of the present invention can be used in any cancer therapy, wherein the removal of shed tumor antigens would be beneficial to a patient.
  • the methods and the compositions of the present invention can be used to any mammal, including but not limited to human and non-human animals (e.g., horses, cows, pigs, dogs, cats, sheep, goats, mice, rats, etc.).
  • methods and compositions are used to treat cancer in a human or non-human primates.
  • Preferred characteristics of a mammal treated with the methods and compositions of the present invention include sufficient volume of blood flow to the liver to provide rapid and efficient clearance of the shed tumor antigens, and also the presence of fixed tissue macrophages in the liver and spleen (e.g., Kupffer cells). Shed tumor antigen clearance is relatively independent of the animal species, rather, the clearance depends on the animal size, total macrophage cell numbers, and the dose of the bispecific molecules.
  • the present invention relates to bispecific molecules, e.g., bispecific antibodies that are characterized by having a first antigen recognition portion which binds a C3b-like receptor or its functional equivalent and a second antigen recognition portion which binds an epitope of a shed tumor antigen to be cleared from the circulation of a subject.
  • the complement component, C3b is the ligand for the C3b receptor and is activated to bind cells or immune complexes (IC), which are targeted for clearance by the immune system.
  • the C3b component after binding the targeted cell or IC, subsequently binds the C3b receptor, thereby tethering the antigen, e.g., a cell or an IC, to the circulating red blood cell (RBC) in a complex.
  • RBC red blood cell
  • This red blood cell-antigen complex then passes through the circulation to the liver or spleen where the complex is then thought to be recognized and eliminated by the reticuloendothelial system.
  • the antigen is then phagocytosed by macrophages in the reticuloendothelial system, and the red blood cell is released back into the circulation (Cornacoff, J., et al., 1983, J. Clin. Invest, 71:236-47).
  • the first antigen recognition portion of a bispecific molecule can be any polypeptide that contains a CRl binding domain and an effector domain.
  • the first antigen recognition portion is an anti-CRl mAb.
  • the first antigen recognition portion is an anti-CRl polypeptide antibody, including but is not limited to, a single-chain variable region fragment (scFv) with specificity for a C3b-like receptor fused to the N-terminus of an immunoglobulin Fc domain.
  • scFv single-chain variable region fragment
  • the first antigen binding portion can also be a chimeric antibody, such as but is not limited to a humanized monoclonal antibody wherein the complementarity determining regions are mouse, and the framework regions are human thereby decreasing the likelihood of an immune response in human patients treated with the antibody (United States Patent Nos. 4,816,567, 4,816,397, 5,693,762; 5,585,089; 5,565,332 and 5,821,337 which are incorporated herein by reference in their entirety).
  • the Fc domain of the chimeric antibody can be recognized by the Fc receptors on phagocytic cells, thereby facilitating the transfer and subsequent proteolysis of the RBC-immune complex.
  • the second antigen recognition portion of a bispecific molecule can be any molecular moiety, including but is not limited to any antibody or antigen binding fragment thereof, that recognizes and binds a shed tumor antigen.
  • tumor associated antigens that are shed by tumor cells into the circulation and can be bound by the second antigen recognition portion include, but are not limited to, carcinoembryonic antigen (CEA), an antigen existing in extracts of fetal gut and varies types of cancer, especially carcinomas of entodermal origin (colorectal, pulmonary, pancreatic, gastric) (Hansen H.J. et al, 1974, Human Path. 5:139-147; Hagan P.L. et al., 1985, J. Nucl. Med.
  • CEA carcinoembryonic antigen
  • PEM polymorphic epithelial mucin
  • HER2-/neu protein which is elevate in breast cancer
  • SBA HERCEPTIN Prescribing Information - Genentech
  • SBA HERCEPTIN Summary Basis for Approval
  • CA125 which associates with ovarian carcinomas
  • TAG-72 tumor-associated glycoprotein - 72
  • MUC-1 gene which is a cell-associated mucin-like protein implicated in breast cancer (assays detect circulating MUC-1 products include CA15-3, CA27-29, CA 549, breast cancer mucin (BCM, mammary serum antigen (MSA), and mucin-like carcinoma-associated antigen (MCA)) (Stearns N. et al., 1998, Breast Cancer Research and Treatment 52: 239-259).
  • the epitope of a shed tumor antigen that can be recognized and bound by the second antigen recognition portion of the bispecific molecule can be any epitope of a shed tumor antigen.
  • the epitope that is recognized and bound by the second antigen recognition portion of the bispecific molecule can be but do not need to be the same epitope that is recognized and bound by the therapeutic antibodies or lymphocytes that target the tumor cells.
  • the epitope that is recognized and bound by the second antigen recognition portion of the bispecific molecule can be an epitope that is not exposed by the cell-surface bound tumor associated antigen.
  • the second antigen recognition portion of the bispecific molecule can also be a non-proteinaceous moiety.
  • the second antigen recognition portion is a nucleic acid.
  • the second antigen recognition portion is an organic small molecule.
  • the second antigen binding portion is an oligosaccharide.
  • a cocktail of bispecific molecules of the present invention refers to a mixture of purified bispecific molecules for targeting one or a mixture of shed tumor antigens.
  • the cocktail of bispecific molecules refers to a mixture of purified bispecific molecules having a plurality of second antigen binding domains that target different or same antigenic molecules and that are of mixed types.
  • the mixture of the second antigen binding domains can be a mixture of peptides, nucleic acids, and/or organic small molecules.
  • a cocktail of bispecific molecules is generally prepared by mixing various purified bispecific molecules. Such bispecific molecule cocktails are useful, inter alia, as personalized medicine tailored according to the need of individual patients.
  • the bispecific molecule used in the present invention can be cross-linked antibodies, wherein the first antibody is specific to a primate C3b-like receptor and the second antibody is specific to a shed tumor antigen.
  • the bispecific molecule can also be antibodies that are produced recombinantly and have one domain recognizes a primate C3b-like receptor and o the second domain recognize a shed tumor antigen.
  • the bispecific molecule can as well be produced using the method of protein trans-splicing and has the first antigen recognition portion recognizing a primate C3b-like receptor and the second antigen recognizing portion recognizing a shed tumor antigen. See Figures 1-4, see also section 5.5, infra.
  • the second antigen recognition portion of the bispecific molecule can be any molecular moiety, including but are not limited to any antibody or antigen binding fragment.
  • antibody that recognizes and binds a shed tumor antigen as used herein refers to immunoglobulin molecules or fragments thereof.
  • the present invention also envisions the 0 use of antibody fragments that contain an antigen binding site which specifically binds a shed tumor antigen.
  • immunologically active fragments of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin or papain. Examples of methods of generating and expressing immunologically active fragments of antibodies can be found in U.S. Patent No. 5 5,648,237 which is inco ⁇ orated herein by reference in its entirety.
  • the immunoglobulin molecules are encoded by genes which include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant regions, as well as a myriad of immunoglobulin variable regions.
  • Light chains are classified as either kappa or lambda.
  • Light chains comprise a variable light (VL) and a constant light (CL) domain.
  • Heavy chains 0 are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.
  • Heavy chains comprise variable heavy (VH), constant heavy 1 (CHI), hinge, constant heavy 2 (CH2), and constant heavy 3 (CH3) domains.
  • the IgG heavy chains are further sub-classified based on their sequence variation, and the subclasses are designated IgGl, IgG2, IgG3 and IgG4.
  • 5 Antibodies can be further broken down into two pairs of a light and heavy domain.
  • the paired NL and NH domains each comprise a series of seven subdomains: framework region 1 (FR1), complementarity determining region 1 (CDR1), framework region 2 (FR2), complementarity determining region 2 (CDR2), framework region 3 (FR3), complementarity determining region 3 (CDR3), framework region 4 (FR4) which constitute the antibody-antigen recognition domain.
  • a chimeric antibody may be made by splicing the genes from a monoclonal antibody of appropriate antigen specificity together with genes from a second human antibody of appropriate biologic activity. More particularly, the chimeric antibody may be made by splicing the genes encoding the variable regions of an antibody together with the constant region genes from a second antibody molecule.
  • This method is used in generating a humanized monoclonal antibody wherein the complementarity determining regions are mouse, and the framework regions are human thereby decreasing the likelihood of an immune response in human patients treated with the antibody (United States Patent Nos. 4,816,567, 4,816,397, 5,693,762; 5,585,089; 5,565,332 and 5,821,337 which are inco ⁇ orated herein by reference in their entirety).
  • An antibody suitable for use in the present invention may be obtained from natural sources or produced by hybridoma, recombinant or chemical synthetic methods, including modification of constant region functions by genetic engineering techniques (United States Patent No. 5,624,821).
  • the bispecific antibody of the present invention may be of any isotype, but is preferably human IgGl.
  • Antibodies exist for example, as intact immunoglobulins or can be cleaved into a number of well-characterized fragments produced by digestion with various peptidases, such as papain or pepsin.
  • Pepsin digests an antibody below the disulfide linkages in the hinge region to produce a F(ab)'2 fragment of the antibody which is a dimer of the Fab composed of a light chain joined to a NH-CHl by a disulfide bond.
  • the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the F(ab)'2 dimer to a Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region.
  • antibody fragments includes antibody fragments produced by the modification of whole antibodies or those synthesized de novo.
  • an antibody can also be a single-chain antibody (scFv), which generally comprises a fusion polypeptide consisting of a variable domain of a light chain fused via a polypeptide linker to the variable domain of a heavy chain.
  • scFv single-chain antibody
  • the present invention provides polyclonal populations of bispecific molecules which comprises a plurality of different bispecific molecules, each of the different bispecific molecules comprising a first antigen recognition portion that binds a C3b-like receptor and a
  • the population thus comprises a plurality of different antigen recognition specificities, e.g., directed to different epitopes or different variants of a tumor associated antigenic molecule, and/or different shed antigens.
  • the polyclonal population of bispecific molecules are produced by cross-linking a first antigen
  • polyclonal population of bispecific molecules are produced by using the method of protein trans- splicing. (See U.S. Provisional Patent Application No. 60/244,811, filed November 1, 2000).
  • the first antigen recognition portion in a bispecific molecule in the plurality of 0 different bispecific molecules in the polyclonal population of the present invention can be any molecule or fragment thereof comprising a C3b-like receptor binding domain and preferably an effector domain.
  • the first antigen recognition portion comprises an anti-CRl monoclonal antibody.
  • the first antigen recognition portion can also be a single chain Fv fragment fused to an Fc domain or a chimeric antibody 5 comprising a C3b-like receptor binding domain and an effector domain.
  • the second antigen recognition portion of a bispecific molecule in the plurality of bispecific molecules in the polyclonal population of the present invention can be any molecular moiety that recognizes and binds a tumor associated antigenic molecule, including but is not limited to any antibody or antigen binding fragment thereof.
  • the o polyclonal population of the invention comprises a plurality of different bispecific molecules having different second antigen recognition portions that have specificities directed to, e.g., a plurality of recognition sites on a shed tumor antigen.
  • the population of bispecific molecules can have a plurality of different second antigen recognition portions that recognize and bind different epitopes on a shed tumor 5 antigen.
  • the population of bispecific molecules can also have a plurality of different second antigen recognition portions that recognize and bind the same epitope on a shed tumor antigen.
  • each bispecific molecule in the plurality of bispecific molecules in the polyclonal population can be known or unknown. The exact
  • each bispecific molecule in the plurality of bispecific molecules in the polyclonal population can also be known or unknown.
  • the characteristics and the proportions of at least some bispecific molecules in the plurality of bispecific molecules in the polyclonal population are known so that if desired, the exact proportions of such bispecific molecules can be adjusted for optimal therapeutic and/or prophylactic efficacy.
  • the polyclonal population of bispecific molecules can comprise bispecific molecules that do not bind the target tumor antigen.
  • the population of bispecific molecules can be prepared from a hyperimmune serum that contains antibodies that bind antigenic molecules other than those on the target tumor antigens.
  • the plurality of bispecific molecules in the polyclonal population constitutes at least 10%, 20%, 50% or
  • the plurality of bispecific molecules in the polyclonal population constitutes at least 90% of the population.
  • the plurality of bispecific molecules in the polyclonal population of bispecific molecules preferably does not comprise any single bispecific molecule which has a proportion exceeding 90%, 80%, or 50% of the plurality. More preferably, the plurality of bispecific molecules in the polyclonal population
  • the plurality of bispecific molecules in the polyclonal population comprises at least 2 different bispecific molecules with different antigen recognition specificities.
  • the plurality of bispecific molecules in the polyclonal population comprises at least 10 different bispecific molecules with different 5 antigen recognition specificities.
  • the plurality of bispecific molecules in the polyclonal population comprises at least 100 different bispecific molecules with different antigen recognition specificities.
  • the polyclonal population can be generated from a suitable polyclonal population of antigen recognition portions, such as but not limited to a polyclonal immunoglobulin preparation.
  • polyclonal population does not inhibit or impair other bispecific molecule's activity. More preferably, one or more bispecific molecules in the polyclonal population enhance the effectiveness of one or more other bispecific molecule(s).
  • the invention provides a population of modified hematopoietic cells that comprise a plurality of hematopoietic cells each bound to one or 5 more bispecific molecules, wherein each of said bispecific molecules comprises a first antigen recognition portion that binds a C3b-like receptor cross-linked to a different second antigen recognition portion that binds a tumor associated antigenic molecule, wherein said bispecific molecules bound to said population of modified hematopoietic cells forms a population of bispecific molecules comprising different second antigen recognition portions.
  • the present invention provides methods and compositions that can be used in cancer diagnosis and treatments.
  • Bispecific molecules can be used to augment antibody- mediated cancer therapy, cell-mediated cancer therapy, radiotherapy, and/or to enhance a host's own immune responses to cancer, or any combinations thereof.
  • Bispecific molecules can also be used in antibody-mediated tumor imaging technology to enhance visualization of the tumor.
  • the present invention provides methods of using bispecific molecules in augmenting antibody-mediated cancer therapy.
  • one or more bispecific molecules are used in combination with therapeutic antibody, antibodies, or chemotherapeutic agent conjugated with monoclonal or polyclonal antibodies for cancer treatment.
  • bispecific molecules After administration, bispecific molecules bind to shed tumor antigens and remove them from the circulation. The removal of shed tumor antigens by the bispecific molecules enhances the specificity and efficacy as well as lowing the side effects of the therapeutic antibody, antibodies, or chemotherapeutic agent conjugated with monoclonal or polyclonal antibodies in cancer treatments. Any bispecific molecules described in section 5.1, supra, may be used for this pu ⁇ ose.
  • the bispecific molecules are used in conjunction with therapeutic monoclonal antibody or polyclonal antibody drugs.
  • Antigens such therapeutic monoclonal or polyclonal antibody drugs bind can be any tumor associated antigen, e.g., tumor specific antigens or a growth factor receptors overexpressed on tumor cells.
  • the bispecific molecules are used to remove the shed forms of such tumor associated antigens that are targeted by the therapeutic antibody drugs from the circulation. The removal of the shed antigens improves binding of the therapeutic antibodies to the surface-bound tumor antigens and enhances the localization of the therapeutic antibodies to cancer cells, and thus the specificity and efficacy of the therapeutic antibodies.
  • the bispecific molecules are used in conjunction with chemotherapeutic agent(s) conjugated with monoclonal or polyclonal antibodies.
  • the chemotherapeutic agent targets the tumor cells through the guidance of the conjugated monoclonal or polyclonal antibodies.
  • Antigens that the conjugated monoclonal or polyclonal antibodies bind can be any tumor associated antigens, e.g., tumor specific antigens or growth factor receptors overexpressed on tumor cells.
  • the bispecific molecules are used to remove the shed forms of such tumor associated antigens that are targeted by the antibodies that are conjugated to the chemotherapeutic agents from the circulation.
  • the removal of such shed antigens improves binding of the antibodies to the surface-bound tumor antigens and enhances the localization of the chemotherapeutic agents to the cancer cells, and thus the specificity and efficacy of chemotherapeutic agents.
  • the increased efficacy of the chemotherapeutic agent may lower the dosage requirement of such agent, and thus lower the side effects of chemotherapy of cancer.
  • bispecific molecules When used in combination with a therapeutic antibody or a chemotherapeutic agent conjugated to an antibody, bispecific molecules can target the same or different antigenic site as the therapeutic antibody or the chemotherapeutic antibody drug. Bispecific molecules can also target the same or different shed tumor antigens as the therapeutic antibody or the chemotherapeutic antibody drug. For example, a shed tumor antigen can complex to other molecules of the cell membrane and form different macromolecular aggregates. (Black, P.H., Advances in Cancer Research, 1980, 32:75-199). Such aggregates may possess epitopes that are not exposed on surface-bound antigens.
  • the bispecific molecules target the same epitope of the antigen as the therapeutic antibody or the chemotherapeutic drug, e.g., both the bispecific molecules and the therapeutic antibody target HER2/neu in breast cancer treatment.
  • the bispecific molecules target a different epitope as the therapeutic antibody or the antibodies conjugated to a chemotherapeutic agent.
  • a therapeutic antibody targets a particular epitope of a tumor associated antigen expressed on the cancer cell
  • the bispecific molecule targets a different epitope of the shed form of the antigen.
  • the epitope targeted by the bispecific molecule can be an epitope that is not exposed when the antigen is cell surface bound.
  • a cocktail of bispecific molecules or a polyclonal population of bispecific molecules can also be used in conjunction with a therapeutic antibody or a chemotherapeutic agent conjugated to an antibody.
  • Bispecific molecules can be administered before, at the same time, or after the administration of the therapeutic antibody or drugs that are used in antibody-mediated cancer therapy. In one embodiment, bispecific molecules are administered before the administration of therapeutic antibodies or chemotherapeutic drugs.
  • the time intervals between the administration of the bispecific molecules and the therapeutic antibodies or drugs can be determined by routine experiments that are familiar to one skilled in the art.
  • the therapeutic antibody or drug is given after the shed tumor antigen level in the circulation falls below a desirable threshold.
  • the level of the shed tumor antigen can be determined by using any techniques known in the art, e.g., an enzyme linked immunosorbert assay (ELISA). (Leitzel et al., 1992, J. of Clinical Oncology, 10:1436-1443).
  • ELISA enzyme linked immunosorbert assay
  • a tumor may shed a large quantities of antigen in a rather o short time period, e.g., shedding of approximately half of cell surface human melanoma tumor antigen occurs in three hours.
  • bispecific molecules are given after the initial treatment with one or more bispecific molecules, which lowered the shed antigen level in the circulation to a satisfactory threshold.
  • bispecific molecules are given at at least 1, 2, 4, or 5 days before every administration of the therapeutic antibodies or chemotherapeutic drugs. This is especially beneficial when a tumor is shedding antigens at a small but constant rate. 0 h another embodiment, the bispecific molecules are administered at the same time with the therapeutic antibodies or the chemotherapeutic drugs.
  • the bispecific molecules are administered at at least 1, 2, 4, or 5 days after the administration of the therapeutic antibodies or chemotherapeutic drugs.
  • Such administration can be beneficial especially when the therapeutic antibody or 5 drug has a longer half life than that of the bispecific molecules used in the treatment.
  • bispecific molecules any combination of different timing of the administration of the bispecific molecules can be used.
  • the therapeutic antibody has a longer half life than that of the bispecific molecules used, it is preferable to administer the bispecific molecules before and after the administration of the 0 therapeutic antibody.
  • the frequency or intervals of administration of bispecific molecules depends on the serum concentration of the shed tumor antigens, which can be determined by any techniques known in the art, e.g., ELISA. (Leitzel et al, 1992, J. of Clinical Oncology, 10:1436-1443).
  • the administration frequency of bispecific molecules can be increased or decreased when 5 the shed tumor antigen level changes in the circulation either higher or lower than previously determined level.
  • the dosage of bispecific molecules can be determined by routine experiments that are familiar to one skilled in the art. It can be determined based on the shed tumor antigen level in the circulation, the half life of the bispecific molecule, as well as the number of RBCs and the number of CRl sites on each RBC.
  • the shed tumor antigen level in the circulation can be determined by any technology known in the art, e.g., ELISA.
  • the half life of the bispecific molecule can also be determined by different experiments, e.g., using ELISA to measure serum concentration of the bispecific molecules at different time points.
  • the half life of a bispecific molecule depends both on the bispecific molecule itself and the particular shed tumor antigen and level the shed tumor antigen the bispecific molecule complexes to.
  • bispecific molecules can be evaluated by any methods known in the art, e.g., by methods that based on measuring the survival rate, side effects, dosage requirement of the therapeutic antibody or chemotherapeutic drugs, clearance rate of the therapeutic drug, or any combinations thereof. If the administration of a bispecific molecule achieves any one or more of the benefits in a patient, such as increasing the survival rate, decreasing side effects, lowing the dosage requirement for a therapeutic antibody or an antibody conjugated to a chemotherapeutic agent, decreasing the clearance rate of a therapeutic antibody or chemotherapeutic drug, the bispecific molecules are said to have augmented the antibody therapy, and the method is said to have efficacy.
  • the present invention provides methods of using bispecific molecules in augmenting cell-mediated cancer therapy.
  • the removal of one or more shed tumor antigens from the circulation leads to the elimination or redirection of the blocking or suppressing effects conveyed by the shed tumor antigens, thereby rendering the cell-mediated therapy more effective.
  • Any bispecific molecules described in section 5.1, supra, may be used for this pu ⁇ ose.
  • the bispecific molecules are used in conjunction with cytokines that stimulate the production of cell that target and kill tumor cells. In another embodiment of the invention, the bispecific molecules are used in conjunction with stimulated and cultured lymphocytes that specifically target and kill tumor cells (CTL-mediated therapy).
  • CTL-mediated therapy stimulated and cultured lymphocytes that specifically target and kill tumor cells
  • bispecific molecules are used in conjunction with K cells
  • the bispecific molecules are used in conjunction with cytokine gene-transfected tumor-specific tumor infiltrating lymphocytes (TIL) that specifically deliver the gene product at the site of the tumor tissue.
  • TIL tumor-specific tumor infiltrating lymphocytes
  • genes that encode a cytokine can be, but are not limited to, interleukin-1 (IL-l ⁇ and ⁇ ), interleukin-2 (IL-2), interleukin-4 (IL-4), tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN- ⁇ , ⁇ , ⁇ ), macrophage cology stimulating factor, and granulocyte macrophage colony stimulating factor.
  • IL-1 interleukin-1
  • IL-4 interleukin-4
  • TNF tumor necrosis factor
  • LT lymphotoxin
  • IFN- ⁇ , ⁇ , ⁇ interferon
  • macrophage cology stimulating factor granulocyte macrophage colony stimulating factor.
  • IL-2 has been shown to have multiple functions, e.g., stimulating lymphokine-activated killer (LAK) cells, proliferating the activated T lymphocytes specific for tumor cells, inducing the natural killer cells, and helping in the secretion of IFN- ⁇ , LT, and TNF.
  • LAK lymphokine-activated killer
  • Rosenberg et al. demonstrated that patients with melanoma treated with LAK cells plus high-dose IL-2 showed tumor regression. (Rosenberg S.A. et al., 1985, N Engl. J. Med. 313:1485).
  • Administration of IL-2 alone also showed promising results in patients with melanoma.
  • bispecific molecules When used in combination with a cell-mediated therapy, bispecific molecules can target the same or different antigenic site as the therapeutic cells. For example, a shed tumor antigen can complex to other molecules of the cell membrane and form different macromolecular aggregates. (Black, P.H., Advances in Cancer Research, 1980, 32:75-199).
  • the bispecific molecules target the same epitope or the same antigen as the therapeutic cells.
  • the bispecific molecules target a different epitope as the therapeutic cells. For example, a therapeutic cell targets a particular epitope of a tumor associated antigen expressed on the cancer cell, whereas the bispecific molecule targets a different epitope of the shed form of the antigen.
  • the epitope targeted by the bispecific molecule can be an epitope that is not exposed when the antigen is cell surface bound.
  • a cocktail of bispecific molecules or a polyclonal population of bispecific molecules can also be used in conjunction with a cell- mediated therapy.
  • Bispecific molecules can be administered before, at the same time, or after the administration of the agents, e.g., cytokines and/or therapeutic cells, that are used in cell-mediated cancer therapy. In one embodiment, bispecific molecules are administered before the administration of the agent. The time intervals between the administration of the bispecific molecules and the agent can be determined by routine experiments that are familiar to one skilled in the art.
  • the agent is given after the shed tumor antigen level in the circulation falls below a desirable threshold.
  • the level of the shed tumor antigen can be determined by using any techniques known in the art, e.g., an enzyme linked immunosorbert assay (ELISA). (Leitzel et al, 1992, J. of Clinical Oncology, 10:1436-1443).
  • ELISA enzyme linked immunosorbert assay
  • a tumor may shed a large quantities of antigen in a rather short time period, e.g., shedding of approximately half of cell surface human melanoma tumor antigen occurs in three hours. (Black, P.H., Advances in Cancer Research, 1980, 32:75-199).
  • bispecific molecules are given at at least 1, 2, 4, or 5 days before every administration of the agent. This is especially beneficial when a tumor is shedding antigens at a small but constant rate.
  • the bispecific molecules are administered at the same time with the agent.
  • the bispecific molecules are administered at at least 1, 2, 4, or 5 days after the administration of the agent.
  • Such administration can be beneficial especially when the agent used in the cell-mediated therapy has a long half life than that of the bispecific molecules used in the treatment.
  • bispecific molecules any combination of different timing of the administration of the bispecific molecules can be used.
  • the therapeutic antibody has a longer half life than that of the bispecific molecules used, it is preferable to administer the bispecific molecules before and after the administration of the therapeutic antibody.
  • the frequency or intervals of administration of bispecific molecules depends on the serum concentration of the shed tumor antigens, which can be determined by any techniques known in the art, e.g., ELISA. (Leitzel et al., 1992, J. of Clinical Oncology, 10:1436-1443).
  • the administration frequency of bispecific molecules can be increased or decreased when the shed tumor antigen level changes either higher or lower than previously determined level in the circulation.
  • the dosage of bispecific molecules can be determined by routine experiments that are familiar to one skilled in the art. It can be determined based on the shed tumor antigen level in the circulation, the half life of the bispecific molecule, as well as the number of RBCs and the number of CRl sites on each RBC.
  • the shed tumor antigen level in the circulation can be determined by any technology known in the art, e.g., ELISA.
  • the half life of the bispecific molecule can also be determined by different experiments, e.g., using ELISA to measure serum concentration of the bispecific molecules at different time points.
  • the half life of a bispecific molecule depends both on the bispecific molecule itself and the particular shed tumor antigen it complexes to.
  • the effects or benefits of administration of bispecific molecules can be evaluated by any methods known in the art, e.g., by methods that based on measuring the survival rate, side effects, dosage requirement of the agent used in cell-mediated therapy, clearance rate of the agent, or any combinations thereof.
  • the bispecific molecules achieves any one or more of the benefits in a patient, such as increasing the survival rate, decreasing side effects, lowing the dosage requirement for an agent used in cell-mediated therapy, decreasing the clearance rate of an agent, the bispecific molecules are said to have augmented the cell-mediated therapy, and the method is said to have efficacy.
  • the present invention provides methods and compositions to enhance the effects of a host's immune response to cancer.
  • the present invention is particularly useful in the treatment of a tumor that produces strong antigenic tumor associated antigens that elicit a strong immune response in the host.
  • Any bispecific molecules described in section 5.1, supra, may be used for this pu ⁇ ose.
  • the bispecific molecules are administered to remove shed tumor antigens, e.g., shed tumor specific antigens and shed tumor associated antigens that elicit immune response in the host.
  • the removal of shed tumor antigens from circulation by bispecific molecules eliminates or reduces the blocking or suppressing effect conveyed or induced by the shed tumor antigens to a host's own immune components, e.g., antibodies and/or cytotoxic T cells that are elicited by the tumor antigens.
  • the host's immune components therefore, target the tumor cells more specifically and more efficiently. As a result, less tumor cells will escape the host's own immune attack.
  • the bispecific molecules are administered at the early stage of the tumor development. This is particularly beneficial because at the early stage it is much easier for a host to eliminate a tumor using its own immune defense mechanisms.
  • the bispecific molecules are administered to a patient after surgical removal of the tumor. Since surgery often does not remove every tumor cells, the administration of bispecific molecules can be beneficial for enhancing the ability of a host's own immune system to attack the remaining tumor cells. Bispecific molecules can also be used to assist therapies which enhance the antigenic abilities of a cell expressed TAA, which elicit stronger immune response in a host.
  • bispecific molecules are used in conjunction with a binding agent, which binds to a pre-determined epitope of a multi-epitopic tumor associated antigen, and such binding alters the antigen in a manner so that the host immune system can recognize and initiate an immune response to the previously unrecognized tumor associated antigen (U.S. Pat. Nos. 6,241,985 and 6,086,873).
  • the frequency of the administration can be determined by routine experiments that are familiar to one skilled in the art.
  • the shed tumor antigen level in the circulation can be measured before the administration of bispecific molecules and at different time point, e.g., every hour, after the administration of bispecific molecules.
  • the time intervals between administration of bispecific molecules should not be longer than what is needed for shed tumor antigen level in the circulation to rise up again. It is preferred to administer the bispecific molecules at the rate that will keep lowing the concentration of the shed tumor antigen in the circulation.
  • bispecific molecules When used in combination with an agent used to increase the antigenicity of a tumor antigen, bispecific molecules can target the same or different antigenic site as the agent. Bispecific molecules can also target the same or different shed forms of the tumor antigens as the agent. For example, a shed tumor antigen can complex to other molecules of the cell membrane and form different macromolecular aggregates.
  • the bispecific molecules target the same epitope as the agent. In a preferred embodiment, the bispecific molecules target a different epitope as the agent. For example, an agent targets a particular epitope of a tumor associated antigen expressed on the cancer cell, whereas the bispecific molecule targets a different epitope of the shed form of the antigen.
  • the bispecific molecule targets an epitope that is different from the epitope targeted by the agent used to increase the antigenicity of a tumor antigen
  • the epitope targeted by the bispecific molecule can be an epitope that is not exposed when the antigen is cell surface bound.
  • a cocktail of bispecific molecules or a polyclonal population of bispecific molecules can also be used in conj unction with an agent.
  • bispecific molecules When bispecific molecules are administered to aid agent(s) that are used to enhance the antigenicity to stimulate a host's own immune system, bispecific molecules can be administered at the same time, or after the administration of the agents. In one embodiment, bispecific molecules are administered at the same time as the administration of the agent.
  • the ratio of doses of the bispecific molecules and the agent can be determined by routine experiments that are familiar to one skilled in the art, which depend at least in part on the level of the shed tumor antigen in the circulation. The ratio also depends on relative strength of binding of the bispecific molecules and the agents to the shed tumor antigens.
  • bispecific molecules remove shed tumor antigens at a rate that the level is enough to elicit immune response.
  • the dosage of bispecific molecules can be determined by routine experiments that are familiar to one skilled in the art. It can be determined based on the shed tumor antigen level in the circulation, the half life of the bispecific molecule, as well as the number of RBCs and the number of CRl sites on each RBC.
  • the shed tumor antigen level in the circulation can be determined by any technology known in the art, e.g., ELISA.
  • the half life of the bispecific molecule can also be determined by different experiments, e.g., using ELISA to measure serum concentration of the bispecific molecules at different time points.
  • the half life of a bispecific molecule depends both on the bispecific molecule itself and the particular shed tumor antigen it complexes to.
  • the level of the shed tumor antigen can be determined by using any techniques known in the art, e.g., an enzyme linked immunosorbert assay (ELISA). (Leitzel et al., 1992, J. of Clinical Oncology, 10:1436-1443).
  • the bispecific molecules are administered at at least 1, 2, 4, or 5 days after the administration of the agent. Such administration is especially beneficial when the bispecific molecules are administered after the immune response has been established.
  • the frequency or intervals of administration of bispecific molecules depends on the serum concentration of the shed tumor antigens, which can be determined by any techniques known in the art, e.g., ELISA. (Leitzel et al., 1992, J. of Clinical Oncology, 10:1436-1443).
  • the administration frequency of bispecific molecules can be increased or decreased when the shed tumor antigen level changes to either higher or lower level than previously determined level in the circulation.
  • the effects or benefits of administration of bispecific molecules can be evaluated by any methods known in the art, e.g., by methods that based on measuring the survival rate, side effects, dosage requirement of the agent used to increase the antigenicity of a tumor antigen, clearance rate of the agent, or any combinations thereof. If the administration of a bispecific molecule achieves any one or more of the benefits in a patient, such as increasing the survival rate, decreasing side effects, lowing the dosage requirement for an agent used to increase the antigenicity of a tumor antigen, decreasing the clearance rate of an agent, the bispecific molecules are said to have augmented the host's immune responses, and the method is said to have efficacy.
  • the present invention provides methods of using bispecific molecules in antibody- mediated tumor imaging or radiotherapy.
  • Bispecific molecules are administered before the antibody-mediated tumor imaging to remove shed tumor antigens from the circulation, thereby enhancing the localization of radiolabeled antibodies to tumor cells.
  • the methods are, therefore, useful for enhancing the quality of images of the tumor, and the sensitivity as well as the specificity of the diagnosis of a tumor.
  • Bispecific molecules can also be used to remove shed tumor antigens from the circulation in radiotherapy to enhance localization of radiolabeled antibodies to tumor cells. Upon such removal of shed tumor antigens, the 5 antibody-mediated radiotherapy is more efficient, more specifically targeting cancer cells, and having less side effects. Any bispecific molecules described in section 5.1, supra, may be used for these pu ⁇ oses.
  • the bispecific molecules are used in conjunction with radiolabeled antibodies in detecting tumor.
  • Any radioactive marker can be used to label the antibody, including but not limited to indium-111 ( In).
  • radiolabeled antibodies include, but are not limited to, radiolabeled anti-CEA monoclonal antibodies (mABs), radiolabeled anti-PEM mABs, radiolabeled anti-HER2/neu mABs, radiolabled anti-CA-125 mABs, radiolabeled anti-TAG-72 mABs, radiolabeled anti-PSA mABs, radiolabeled anti-CA19-9 mABs, radiolabeled anti-TPS mABs, and radiolabeled J anti-MUC-1 mABs.
  • Radiolabeled antibodies can be administered to a host and then visualized by using any imaging methods known in the art that detects specifically the radiation site(s).
  • the bispecific molecules are used in conjunction with radiotherapy.
  • Any agents for radiotherapy known in the art can be used.
  • Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for the cells to grow.
  • radiotherapy include, but are not limited to, radiolabeled antibodies that deliver doses of radiation, e.g., X-rays, Gamma rays, directly to the cancer sites. Radiotherapy may be used alone or in combination with chemotherapy or surgery.
  • radiolabeled antibodies 5 used in the radiotherapy can be, but are not limited to, radiolabeled anti-CEA monoclonal antibodies (mABs), radiolabeled anti-PEM mABs, radiolabeled anti-HER2/neu mABs, radiolabled anti-CA-125 mABs, radiolabeled anti-TAG-72 mABs, radiolabeled anti-PSA mABs, radiolabeled anti-CA19-9 mABs, radiolabeled anti-TPS mABs, and radiolabeled anti-MUC-1 mABs.
  • mABs radiolabeled anti-CEA monoclonal antibodies
  • mABs radiolabeled anti-PEM mABs
  • radiolabeled anti-HER2/neu mABs radiolabled anti-CA-125 mABs
  • radiolabeled anti-TAG-72 mABs radiolabeled anti-PSA mABs
  • the shed tumor antigen level in the circulation can be measured before the administration of bispecific molecules and at different time point, e.g., every hour, after the administration of bispecific molecules.
  • the time intervals between administration of bispecific molecules should not be longer than what is needed for shed tumor antigen level in the circulation to rise up again. It is preferred to administer the bispecific molecules at the rate that will keep the concentration of the shed tumor antigen in the circulation below a threshold level. Once the concentration of shed bispecific molecules in the circulation is at a satisfactory threshold, the radiolabeled antibodies can be administered for visualization of the tumor site(s).
  • bispecific molecules When used in combination with an agent used in radiotherapy, bispecific molecules can target the same or different antigenic site as the agent. Bispecific molecules can also target the same or different shed forms of a tumor antigen as the agent. For example, a shed tumor antigen can complex to other molecules of the cell membrane and form different macromolecular aggregates (Black, P.H., Advances in Cancer Research, 1980, 32:75-199). Such aggregates may possess epitopes that are not exposed on surface-bound antigens. In one embodiment, the bispecific molecules target the same epitope or the same antigen as the agent. In another embodiment, the bispecific molecules target a different epitope as the agent.
  • an agent targets a particular epitope of a tumor associated antigen expressed on the cancer cell
  • the bispecific molecule targets a different epitope of the shed form of the antigen.
  • the epitope targeted by the bispecific molecule can be an epitope that is not exposed when the antigen is cell surface bound.
  • a cocktail of bispecific molecules or a polyclonal population of bispecific molecules can also be used in conjunction with an agent.
  • bispecific molecules When bispecific molecules are used in radiotherapy, bispecific molecules can be administered before, at the same time, or after the administration of the agents that are used in radiotherapy, e.g., radiolabeled antibodies. In one embodiment, bispecific molecules are administered before the administration of the agent. The time intervals between the administration of the bispecific molecules and the agent can be determined by routine experiments that are familiar to one skilled in the art. In one embodiment, the agent is given after the shed tumor antigen level in the circulation falls below a desirable threshold. The level of the shed tumor antigen can be determined by using any technique known in the art, e.g., an enzyme linked immunosorbert assay (ELISA). (Leitzel et al., 1992, J. of Clinical Oncology, 10:1436-1443).
  • ELISA enzyme linked immunosorbert assay
  • a tumor may shed a large quantities of antigen in a rather short time period, e.g., shedding of approximately half of cell surface human melanoma tumor antigen occurs in three hours.
  • a large dosage or more frequent administration of bispecific molecules can be given in a short time period and then tapered down to a maintenance dosage.
  • an agent used in radiotherapy is given after the initial treatment with bispecific molecules, which lowered the shed antigen level in the circulation to a desired threshold.
  • bispecific molecules are given at at least 1, 2, 4, or 5 days before every administration of the agent. This is especially beneficial when a tumor is shedding antigens at a small but constant rate.
  • the bispecific molecules are administered at the same time with the agent.
  • the bispecific molecules are administered 1, 2, 4, or 5 days after the administration of the agent.
  • Such administration can be beneficial especially when the agent used in the radiotherapy has a long half life than that of the bispecific molecules used in the treatment. It wi l be apparent to one skilled in the art that any combination of different timing of the administration of the bispecific molecules can be used.
  • the agent used in the radiotherapy has a longer half life than that of the bispecific molecules used, it is preferable to administer the bispecific molecules before and after the administration of the agent.
  • the frequency or intervals of administration of bispecific molecules depends on the serum concentration of the shed tumor antigens, which can be determined by any technique known in the art, e.g., ELISA. (Leitzel et al, 1992, J. of Clinical Oncology, 10:1436-1443).
  • the administration frequency of bispecific molecules can be increased or decreased when the shed tumor antigen level changes to either higher or lower level than previously determined level in the circulation.
  • the dosage of bispecific molecules can be determined by routine experiments that are familiar to one skilled in the art. It can be determined based on the shed tumor antigen level in the circulation, the half life of the bispecific molecule, as well as the number of RBCs and the number of CRl sites on each RBC.
  • the shed tumor antigen level in the circulation can be determined by any technology known in the art, e.g., ELISA.
  • the half life of the bispecific molecule can also be determined by different experiments, e.g., using ELISA to measure serum concentration of the bispecific molecules at different time points.
  • the half life of a bispecific molecule depends both on the bispecific molecule itself and the particular shed tumor antigen it complexes to.
  • the effects or benefits of administration of bispecific molecules can be evaluated by any methods known in the art, e.g., by methods that based on measuring the survival rate, side effects, dosage requirement of the agent used in radiotherapy, clearance rate of the agent, or any combinations thereof. If the administration of a bispecific molecule achieves any one or more of the benefits in a patient, such as increasing the survival rate, decreasing side effects, lowing the dosage requirement for an agent used in radiotherapy, decreasing the clearance rate of an agent, the bispecific molecules are said to have augmented the antibody- mediated tumor imaging or radiotherapy, and the method is said to have efficacy.
  • the efficacy Prior to administration to humans, the efficacy is preferably shown in animal models. Any animal model for a circulatory disease known in the art can be used.
  • the dose of the bispecific antibody can be determined based on the hematopoietic cell concentration and the number of C3b-like receptor epitope sites bound by the anti-C3b-like receptor monoclonal antibodies per hematopoietic cell. If the bispecific antibody is added in excess, a fraction of the bispecific antibody will not bind to hematopoietic cells, and will inhibit the binding of pathogenic antigens to the hematopoietic cell. The reason is that when the free bispecific antibody is in solution, it will compete for available shed tumor antigens with bispecific antibody bound to hematopoietic cells. Thus, the bispecific antibody-mediated binding of the shed tumor antigens to hematopoietic cells follows a bell-shaped curve when binding is examined as a function of the concentration of the input bispecific antibody concentration.
  • the dose of therapeutic bispecific antibodies should preferably be, at a minimum, approximately 10 times the number of the targeted shed antigen in the blood.
  • the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.
  • partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration are often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake
  • a therapeutically effective amount of bispecific antibody ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to
  • 10 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a bispecific antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with a bispecific antibody in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between
  • the effective dosage of a bispecific antibody, used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • bispecific antibody agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the bispecific antibody will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect
  • bispecific antibodies depend upon the potency of the bispecific antibody with respect to the antigen to be cleared. Such appropriate doses may be determined using the assays described herein.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the bispecific antibody employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the concentration of antigen to be cleared.
  • the bispecific antibodies of the invention can be inco ⁇ orated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise bispecific antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the bispecific antibody, use thereof in the compositions is contemplated. Supplementary bispecific antibodies can also be inco ⁇ orated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • the preferred route of administration is intravenous.
  • Other examples of routes of administration include parenteral, intradermal, subcutaneous, transdermal (topical), and transmucosal.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediammetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF; Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that the viscosity is low and the bispecific antibody is injectable. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the bispecific antibody
  • bispecific antibodies e.g., one or more bispecific antibodies
  • dispersions are prepared by inco ⁇ orating the bispecific antibody into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the bispecific antibodies are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of bispecific antibody calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the bispecific antibody and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such a bispecific antibody for the treatment of individuals .
  • compositions can be included in a kit, in a container, pack, or dispenser together with instructions for administration.
  • Bispecific molecules used in the present invention can be produced in various ways. Examples of methods for production include, but are not limited to, cross-linking, recombinant technique, or protein trans-splicing. (See sections 5.5.1-5.5.4).
  • Antibodies can be prepared by immunizing a suitable subject with an antigen, e.g., tumor specific antigen or tumor associated antigen, as an immunogen.
  • an antigen e.g., tumor specific antigen or tumor associated antigen
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), the human B cell hybridoma technique by Kozbor et al. (1983, Immunol. Today 4:72), the EBN-hybridoma technique by Cole et al. (1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497), the human B cell hybridoma technique by Kozbor et al. (1983, Immunol. Today 4:72), the EBN-hybridoma technique by Cole et al. (1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques.
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., 1975, Nature, 256:495, or may be made
  • a mouse or other appropriate host animal such as a hamster, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • the culture medium for the hybridomas typically will include hypoxanthine,
  • HGPRT medium 20 aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine
  • 25 myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, 1984, J. Immunol.,
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as
  • radioimmunoassay RIA
  • enzyme-linked immuno-absorbent assay ELISA
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., 1980, Anal. Biochem. 107:220.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this pu ⁇ ose include, for example, D-MEM or RPMI- 1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • a monoclonal antibody directed against a pathogen or pathogenic antigenic molecule polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the antigen of interest.
  • Kits for generating and screening phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene antigen SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent Nos. 5,223,409 and 5,514,548; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No.
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison, et al., 1984, Proc. Natl. Acad. Sci., 81, 6851-6855; Neuberger, et al., 1984, Nature 312, 604-608; Takeda, et al., 1985, Nature, 314, 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule, (see e.g., U.S. Patent No. 5,585,089, which is inco ⁇ orated herein by reference in its entirety.)
  • CDRs complementarity determining regions
  • 5 humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567 and 5,225,539; European Patent Application 125,023; Better et al., 1988, Science
  • Complementarity determining region (CDR) grafting is another method of humanizing antibodies. It involves reshaping murine antibodies in order to transfer full antigen specificity and binding affinity to a human framework (Winter et al. U.S. Patent No.
  • CDR-grafted antibodies have been successfully constructed against various antigens, for example, antibodies against IL-2 receptor as described in Queen et al., 1989 (Proc. Natl. Acad. Sci. USA 86:10029); antibodies against cell surface receptors-CAMPATH as described in Riechmann et al. (1988, Nature, 332:323; antibodies against hepatitis B in Cole et al. (1991, Proc. Natl. Acad. Sci. USA 88:2869); as well as
  • CDR-grafted antibodies are generated in which the CDRs of the murine monoclonal antibody are grafted into a human antibody. Following grafting, most antibodies benefit from additional amino acid changes in the framework region to maintain affinity, presumably because framework residues are necessary to maintain CDR conformation, and
  • Fully human antibodies are particularly desirable for therapeutic treatment of 5 human patients.
  • Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of an immunogen.
  • Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93).
  • this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies see e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S.
  • Patent 5,661,016 and U.S. Patent 5,545,806.
  • companies such as Abgenix, Inc. (Freemont, CA (see, for example, U.S. Patent No. 5,985,615)) and Medarex, Inc. (Princeton, NJ), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
  • Completely human antibodies which recognize and bind a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • is used to guide the selection of a completely human antibody recognizing the same epitope Jespers et al. (1994) antigen Bio/technology 12:899-903).
  • a pre-existing antibody directed against a shed tumor antigen can be used diagnostically to monitor pathogen levels in tissue as part of a clinical testing procedure, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazmylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 1251, 1311, 35S or 3H.
  • Antibodies that are commercially available can be purchased and used to generate bispecific antibodies, e.g., from ATCC.
  • the antibody is produced by a commercially available hybridoma cell line.
  • the hybridoma secretes a human antibody.
  • bispecific molecules used in the present invention can be produced by chemical cross-linking antibodies, see e.g., U.S. Pat. Nos. 5,487,890, 5,470,570, 5,879,679, and U.S. Provisional Application No. 60/276,200, filed March 15, 2001, each of which is inco ⁇ orated herein by reference in its entirety.
  • bispecific molecules used in the invention are prepared by a method comprising cross-linking anti-CRl portions and antigen recognition portions by a chemical cross-linking agent.
  • Any standard chemical cross-linking methods can be used in the present invention.
  • cross-linking agents including but are not limited to, protein A, glutaraldehyde, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) can be used.
  • cross-linking agents N-succinimidyl S-acetylthioacetate (SATA) and sulfosuccinimidy 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC) are used to cross-link anti-CRl portions and antigen recognition portions.
  • SATA S-acetylthioacetate
  • sulfo-SMCC sulfosuccinimidy 4-(N-maleimidomethyl)cyclohexane-l-carboxylate
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • the bispecific molecules used in the present invention can also be produced recombinantly, whereby nucleotide sequences which encode antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to nucleotide sequences which encode immunoglobulin constant domain sequences, see e.g., U.S. Provisional Application Nos. 60/199,603, filed April 26, 2000, and 60/244,812, filed November 1, 2000, each of which is inco ⁇ orated herein by reference in its entirety.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm fused to the constant CH2 and CH3 domains, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm (see, e.g., WO 94/04690 published March 3, 1994).
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • the coding sequences for two or all three polypeptide chains are inserted in one expression vector.
  • the bispecific molecules comprising single polypeptides can also be produced recombinantly.
  • the nucleic acid encoding an antigen recognition portion that binds a shed tumor antigen is fused to the nucleic acid encoding an antigen recognition portion that binds a C3b-like receptor to obtain a fusion nucleic acids encoding a single polypeptide bispecific molecule.
  • the bispecific molecule is produced by a method comprising producing a bispecific immunoglobulin-secreting cell which has a first antigen recognition portion which binds a C3b-like receptor and a second antigen recognition portion which binds an epitope of a shed tumor associated antigen.
  • the method comprises the steps of fusing a first cell expressing an immunoglobulin which binds to the C3b-like receptor with a second cell expressing an immunoglobulin which binds to the shed tumor associated antigen, and selecting for cells that express the bispecific immunoglobulin.
  • a bispecific molecule comprising at least a first antigen recognition portion which binds a C3b-like receptor and a second antigen recognition portion which binds an epitope of a shed tumor associated antigen is produced by a method comprising the steps of transforming a cell with a first DNA sequence encoding at least the first antigen recognition portion and a second DNA sequence encoding at least the second antigen recognition portion, and independently expressing said first DNA sequence and said second DNA sequence so that said first and second antigen recognition portions are produced as separate molecules which assemble together in said transformed single cell, that is capable of binding to a C3b-like receptor with a first antigen recognition portion and also capable of binding an antigen to be cleared from the circulation with a second antigen recognition portion is formed.
  • the bispecific molecules used in the present invention can also be produced using the method of protein trans-splicing, see e.g., U.S. Provisional Application No. 60/244,811 , filed November 1, 2000, which is inco ⁇ orated herein by reference in its entirety.
  • the method can be used to directly or via a linker conjugate a first antigen recognition portion, e.g., an anti-CRl mAB, with a second antigen recognition portion that binds an epitope of a shed tumor associated antigen, e.g., a peptide or polypeptide, a nucleic acid, and an organic small molecules, to form a bispecific molecule.
  • the method can be used to conjugate a first antigen recognition portion with streptavidin to form a first antigen recognition portion-streptavidin fusion molecule which can be conjugated with a biotinylated second antigen recognition portion.
  • the first antigen recognition portion is conjugated to the N-terminus of an N-intein of a suitable split intein to produce an N-intein first antigen recognition portion fragment
  • the second antigen recognition portion is conjugated to the C-terminus of the C-intein of the split intein to produce a C-intein second antigen recognition portion fragment.
  • the N-intein first antigen recognition portion fragment and the C-intein second antigen recognition portion fragment are then brought together such that they reconstitute and undergo trans-splicing to produce the bispecific molecule.
  • the bispecific molecule produce by protein trans-splicing can contain a single second antigen recognition portion conjugated to the first antigen recognition portion.
  • the bispecific molecule of the invention can also contain two or more second antigen recognition portions conjugated to different regions of the first antigen recognition portion.
  • the bispecific molecule can contain two second antigen recognition portions conjugated to each of the heavy chains of a first antigen recognition monoclonal antibody.
  • such second antigen recognition portions can be the same or different antigen recognition portions.
  • the first and second antigen recognition portions can be different antigen recognition portions that target the same shed tumor associated antigen to be cleared.
  • the first and second second antigen recognition portions target an antigenic molecule to be cleared cooperatively.
  • one of the second antigen recognition portions may enhance the binding of the other second antigen recognition portion to a shed tumor associated antigen, thereby facilitating the removal of the shed tumor associated antigen.
  • the first and second second antigen recognition portions can also be different antigen recognition portions that target different shed tumor associated antigens to be cleared.
  • Various split inteins can be used for the production of the bispecific molecules of the present invention. In one aspect of the invention, naturally occurring split inteins are used for the production of bispecific molecules.
  • a split intein can be modified by adding, deleting, and/or mutating one or more amino acid residues to the N- intein and/or the C-intein such that the modification improves or enhances the intein' s proficiency in trans-splicing and/or permits control of trans-splicing processes.
  • a Cys residue can be included at the carboxy terminus of a C-intein so that the requirement that the molecular moiety conjugated to the C-intein must start with a Cys is alleviated.
  • one or more native proximal extein residues are added to the N- and/or C-intein to facilitate trans-splicing in a foreign extein content.
  • the trans-splicing system of the split intein encoded in the DnaE gene of Synechocystis sp. PCC6803 is used for the production of the bispecific molecules of the present invention.
  • an engineered split intein system based on the Mycobacterium tuberculosis RecA intein is used.
  • the production of bispecific molecules can be carried out in vitro wherein the intein antigen recognition portion fragments are expressed in separate hosts.
  • the production of bispecific molecules can also be carried out in vivo.
  • nucleic acids encoding the intein antigen recognition portion fragments are inserted into separate vectors which are then co-transfected into a host for in vivo production of the bispecific molecule.
  • nucleic acids encoding the intein fragments are inserted into the same vector which is then transfected into a host for in vivo production of the bispecific molecule.
  • the N-intein first antigen recognition portion fragment is preferably produced by fusing an appropriate antigen recognition moiety that binds a C3b-like receptor to the N-terminus of the N-intein of a suitable split intein.
  • the C- terminus of the heavy chain of an anti-CRl mAb is fused to the N-terminus of the N-intein of a split intein.
  • the C-intein second antigen recognition portion fragment is preferably produced by fusing an appropriate antigen recognition moiety that binds an epitope of a shed tumor associated antigen to be cleared to the C-terminus of the C-intein of a suitable split intein.
  • the amino acid residue immediately at the C-terminal side of the splice junction of the C-intein is a cysteine, serine, or threonine.
  • a C- intein streptavidin is produced by fusing a streptavidin to the C-terminus of a C-intein comprising a Cys, Ser, or Thr immediately downstream of the splice junction and is used in trans-splicing to produce a first antigen recognition portion-streptavidin fusion molecule which subsequently reacts with a biotinylated second antigen recognition portion to produce the bispecific molecule.
  • biotinylated second antigen recognition portion to produce the bispecific molecule.
  • the bispecific molecule is produced by mixing the N-intein first antigen recognition portion fragment and the C-intein second antigen recognition portion fragment in vitro so that the fragments reconstitute and undergo trans-splicing.
  • a first antigen recognition portion-streptavidin molecule is produced by mixing the N-intein first antigen recognition portion fragment and the C-intein streptavidin fragment in vitro to produce a first antigen recognition portion-streptavidin molecule.
  • the bispecific molecule is then produced by reaction of the first antigen recognition-streptavidin molecule with a biotinylated second antigen recognition portion.
  • a polyclonal population of bispecific molecules of the present invention can be produced using any method known in the art, e.g., by cross-linking, by recombination, or by protein trans-splicing, see e.g., U.S. Provisional Application Nos. 60/276,200, filed March 15, 2001; 60/199,603, filed April 26, 2000; 60/244,812, filed November 1, 2000; and 60/244,811, filed November 1, 2000; each of which is inco ⁇ orated herein by reference in its entirety.
  • the polyclonal population of bispecific molecules of the present invention is produced by cross-linking a polyclonal population of antigen recognition portions that bind a shed tumor antigen to a population of antigen recognition portions that bind a C3b-like receptor.
  • the entire polyclonal population of bispecific molecules can be produced in one reaction. Such can normally be done by first producing a polyclonal population of antigen recognition portions and cross-linking the entire population of such antigen recognition portions to a population of C3b-like receptor binding portions without isolation of individual members.
  • members and/or fractions of the polyclonal population can be produced separately and then combined to form the polyclonal population. Such embodiments are useful when polyclonal populations with specific compositions are to be produced.
  • the polyclonal population of bispecific molecules can be produced by transfecting a hybridoma cell line that expresses an immunoglobulin that binds a C3b-like receptor with a population of eukaryotic expression vectors containing nucleic acids encoding the heavy and light chain variable regions of a polyclonal population of immunoglobulins that bind different shed tumor antigens.
  • Cells that express bispecific immunoglobulins that comprise a first binding domain which binds to a C3b-like receptor and a second binding domain which binds to a shed tumor antigen are then selected using standard methods known in the art.
  • the polyclonal population of immunoglobulins can be obtained by any method known in the art, e.g., from a phage display library. If a phage display library is used, the number of specificities of such phage display library is preferably near the number of different specificities that are expressed at any one time by lymphocytes. More preferably the number of specificities of the phage display library is higher than the number of different specificities that are expressed at any one time by lymphocytes. Most preferably the phage display library comprises the complete set of specificities that can be expressed by lymphocytes. Kits for generating and screening phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No.
  • the polyclonal populations of bispecific antibodies are produced recombinantly, whereby the polyclonal population of nucleic acids which encode antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to nucleotides which encode immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
  • CHI first heavy-chain constant region
  • Polyclonal populations of bispecific molecules comprising single polypeptide bispecific molecules can be produced recombinantly.
  • a polyclonal population of nucleic acids encoding a polyclonal population of selected antigen recognition portions is fused to nucleic acids encoding the antigen recognition portion that binds a C3b-like receptor to obtain a population of fusion nucleic acids encoding a population of bispecific molecules.
  • the population of nucleic acids are then expressed in a suitable host to produce a polyclonal population of bispecific molecules.
  • Protein trans-splicing can also be used for producing populations of bispecific molecules comprising a plurality of bispecific molecules with different antigen recognition specificities.
  • polyclonal antibodies can be obtained by affinity screening of an antibody phage display library having a sufficiently large and diverse repertoire of specificities with a shed tumor antigen before recombinant fusion with a C-intein.
  • the nucleic acid encoding each member of the selected antibodies is then fused to a C-intein of a suitable trans-splicing system and expressed in a suitable host.
  • the C-intein antigen recognition portion fragments are allowed to reconstitute with the corresponding N-intein anti-CRl fragments and undergo trans-splicing reactions.
  • polyclonal populations wherein the plurality of antigen recognition portions has specificities for multiple epitopes of a targeted antigenic molecule and/or multiple variants of a targeted shed tumor antigen.
  • Such polyclonal libraries of bispecific molecules can be used for more efficient clearance of shed tumor antigens that have multiple epitopes and/or shed tumor antigens that have multiple variants or mutants, which normally cannot be effectively targeted and cleared by a monoclonal antibody having a single specificity.
  • the bispecific molecule such as a bispecific antibody
  • hematopoietic cells are collected from the individual to be treated (or alternatively hematopoietic cells from a non-autologous donor of the compatible blood type are collected) and incubated with an appropriate dose of the therapeutic bispecific antibody for a sufficient time so as to allow the antibody to bind the C3b-like receptor on the surface of the hematopoietic cells.
  • the hematopoietic cell bispecific antibody mixture is then administered to the subject to be treated in an appropriate dose (see, for example, Taylor et al., U.S. Patent No. 5,487,890).
  • the hematopoietic cells are preferably blood cells, most preferably red blood cells. Accordingly, in a specific embodiment, the invention provides a method of treating a cancer patient with the presence of undesirable shed tumor antigens, comprising the step of administering a hematopoietic cell/bispecific molecule complex to the patient in a therapeutically effective amount, said complex consisting essentially of a hematopoietic cell expressing a C3b-like receptor bound to one or more bispecific molecules, wherein said bispecific molecule (a) comprises a first binding domain which binds the C3b-like receptor on the hematopoietic cell, and (b) comprises a second binding domain which binds the shed tumor antigens.
  • the method alternatively comprises a method of treating a cancer patient with the presence of shed tumor antigens comprising the steps of (a) contacting a bispecific molecule with hematopoietic cells expressing a C3b-like receptor, to form a hematopoietic cell/bispecific molecule complex, wherein the bispecific molecule (i) comprises a first binding domain which binds the C3b-like receptor, and (ii) comprises a second binding domain which binds the shed tumor antigens; and (b) administering the hematopoietic cell/bispecific molecule complex to the patient in a therapeutically effective amount.
  • the invention also provides a method of making a hematopoietic cell/bispecific molecule complex comprising contacting a bispecific molecule with hematopoietic cells that express a C3b-like receptor under conditions conducive to binding, such that a complex forms, said complex consisting essentially of a hematopoietic cell bound to one or more bispecific molecules, wherein said bispecific molecule (a) comprises a first binding domain that binds the C3b-like receptor on the hematopoietic cells, (b) comprises a second binding domain that binds a shed tumor antigen.
  • bispecific antibodies which contain monoclonal antibodies that bind to different sites on a C3b-like receptor.
  • the monoclonal antibodies 7G9 and 1B4 bind to separate and non-competing sites on the primate C3b receptor. Therefore, a "cocktail" containing a mixture of two bispecific antibodies, each made with a different monoclonal antibody to the C3b-like receptor, may give rise to greater binding of antibodies to red blood cells.
  • the bispecific antibodies of the present invention can also be used in combination with certain fluids used for intravenous infusions.
  • the bispecific molecule such as a bispecific antibody
  • the bispecific molecule is prebound to red blood cells in vitro as described above, using a "cocktail" of at least two different bispecific antibodies.
  • the two different bispecific antibodies bind to the same antigen, but also bind to distinct and non-overlapping recognition sites on the C3b-like receptor.
  • the number of bispecific antibody-antigen complexes that can bind to a single red blood cell is increased.
  • antigen clearance is enhanced, particularly in cases where the antigen is in very high concentrations (see for example the '679 patent, column 6, lines 41-64).
  • HERCEPTIN trastuzumab
  • Trastuzumab is a recombinant DNA-derived humanized monoclonal antibody (mAB) that attacks a specific target: HER-2/neu, a growth factor receptor that is present in larger than normal amounts on some breast cancer cells. Numerous studies have shown that 25% to 30%» of breast cancer patients whose tumors produce more HER-2/neu have worse prognoses and shorter life expectancies. Clinical trials of HERCEPTIN showed that it can slow the progression of breast cancer in women whose cancer had already metastasized. It can also extend the median time to relapse from 4 months to as much as 11 months. (Dickman S., 1998, Science, 280:1196-1197; HERCEPTIN Summary Basis for Approval (SBA)-FDA).
  • SBA HERCEPTIN Summary Basis for Approval
  • HER2/neu patients with the highest levels of HER2/neu expression on the surface of breast cancer cells as measured by immunohistochemistry assay (IHC) have the best clinical response to HERCEPTIN. Specifically, those patients with the highest levels of expressed HER2/neu on tumor tissue (3+ for HER2/neu by the IHC assay) have statistically significant benefits in response rates, time to progression and short-term survival. Removal of shed HER2/neu further enhance the efficacy of treatment by HERCEPTIN. Those patient with lower levels of HER2/neu on tumor tissue (2+ for HER2/neu) have none of above benefits. if the 2+ cells produce no less HER2/neu protein than 3+ cells, but the 2+ cells shed a large proportion of what they produce in soluble form, the removal of shed antigen could also benefit therapy.
  • IHC immunohistochemistry assay
  • Anti-CRl monoclonal antibody 7G9 (see, U.S. Patent No. 5,879,679) is cross-linked to Trastuzumab to produce a bispecific molecule designated as 7G9 x Trastuzumab.
  • sulfo-SMCC 4-(N-maleimidomethyl)cyclohexane-l-carboxylate
  • 7G9 x Trastuzumab is administered at a lOmg/kg dose.
  • HERCEPTIN is administered at a
  • HERCEPTIN clearance (Clt) is measured and compared to the Clt without the use of the bispecific molecule.
  • the combination therapy using 7G9 x Trastuzumab and HERCEPTIN significantly improves the response rates, time to progression and short-term survival in both

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
EP02778405A 2001-09-28 2002-09-27 Verfahren und zusammensetzungen zur prävention, diagnose und behandlung von krebs mit bispezifischen molekülen Withdrawn EP1487487A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US32573201P 2001-09-28 2001-09-28
US325732P 2001-09-28
PCT/US2002/031020 WO2003026490A2 (en) 2001-09-28 2002-09-27 Methods and compositions for prevention, diagnosis, and treatment of cancer using bispecific molecules

Publications (1)

Publication Number Publication Date
EP1487487A2 true EP1487487A2 (de) 2004-12-22

Family

ID=23269190

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02778405A Withdrawn EP1487487A2 (de) 2001-09-28 2002-09-27 Verfahren und zusammensetzungen zur prävention, diagnose und behandlung von krebs mit bispezifischen molekülen

Country Status (4)

Country Link
EP (1) EP1487487A2 (de)
JP (1) JP2005510470A (de)
CA (1) CA2461631A1 (de)
WO (1) WO2003026490A2 (de)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
WO2003095626A2 (en) * 2002-05-13 2003-11-20 Elusys Therapeutics, Inc. Purified composition of bispecific molecules and methods of production
WO2005045058A2 (en) * 2003-10-27 2005-05-19 Monogram Biosciences, Inc. Detecting human anti-therapeutic antibodies
EP1814918A1 (de) * 2004-10-29 2007-08-08 Elusys Therapeutics, Inc. Verwendung von cr1-bindenden molekülen bei der clearance und der induktion von immunreaktionen
JP2008530123A (ja) * 2005-02-09 2008-08-07 ジェネンテック・インコーポレーテッド マトリックスメタロプロテアーゼアンタゴニストを用いたｈｅｒ２のシェディングの阻害
UA97469C2 (uk) 2005-07-25 2012-02-27 Емерджент Продакт Дівелопмент Сіетл, Елелсі Гуманізована специфічна до cd37 зв'язувальна молекула імуноглобуліну
NZ573646A (en) 2006-06-12 2012-04-27 Wyeth Llc Single-chain multivalent binding proteins with effector function
NZ603059A (en) 2008-04-11 2014-07-25 Emergent Product Dev Seattle Cd37 immunotherapeutic and combination with bifunctional chemotherapeutic thereof
TWI682941B (zh) 2013-02-01 2020-01-21 美商再生元醫藥公司 含嵌合恆定區之抗體
TWI701042B (zh) 2014-03-19 2020-08-11 美商再生元醫藥公司 用於腫瘤治療之方法及抗體組成物
PL3221359T3 (pl) 2014-11-17 2020-11-16 Regeneron Pharmaceuticals, Inc. Sposoby leczenia nowotworów przy użyciu dwuswoistego przeciwciała CD3XCD20
AU2016242866B2 (en) 2015-03-30 2021-06-03 Regeneron Pharmaceuticals, Inc. Heavy chain constant regions with reduced binding to FC gamma receptors
US11352426B2 (en) 2015-09-21 2022-06-07 Aptevo Research And Development Llc CD3 binding polypeptides
CN108290951B (zh) * 2015-09-23 2022-04-01 瑞泽恩制药公司 优化抗cd3双特异性抗体和其用途
CA3110513A1 (en) 2018-08-31 2020-03-05 Regeneron Pharmaceuticals, Inc. Dosing strategy that mitigates cytokine release syndrome for cd3/c20 bispecific antibodies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03026490A2 *

Also Published As

Publication number Publication date
JP2005510470A (ja) 2005-04-21
WO2003026490A2 (en) 2003-04-03
WO2003026490A3 (en) 2004-10-21
CA2461631A1 (en) 2003-04-03

Similar Documents

Publication Publication Date Title
US20210206858A1 (en) Anti-b7-h6 antibody, fusion proteins, and methods of using the same
JP5185815B2 (ja) 抗ｃｄ７１モノクローナル抗体および悪性腫瘍細胞を治療するためのその使用
ES2534288T3 (es) Composiciones terapéuticas contra el cáncer que seleccionan como objetivo 4Ig-B7-H3
CN109071642B (zh) 连接蛋白(Cx)43半通道结合抗体及其用途
US20230190796A1 (en) Engineered cells expressing prostate-specific membrane antigen (psma) or a modified form thereof and related methods
KR20120102714A (ko) 항-종양 항체 치료를 향상시키는 방법
EP1487487A2 (de) Verfahren und zusammensetzungen zur prävention, diagnose und behandlung von krebs mit bispezifischen molekülen
US10246516B2 (en) Anti-La antibodies and their use for immunotargeting
US20120027763A1 (en) Antibody for Targeted induction of Apoptosis, CDC and ADCC mediated killing of Cancer cells, TBL-CLN1
US20090053136A1 (en) Methods of Therapy and Diagnosis Using Immunotargeting of CD84Hy1-expressing Cells
AU2012277784B2 (en) Antibodies against prostate-specific stem cell antigen and use thereof
JP2022519377A (ja) がんのバイオマーカーとしてのｉｌ－４ｒ
CN112794911B (zh) 人源化抗叶酸受体1抗体及其应用
US20230365686A1 (en) Humanized antibody specific for cd22 and chimeric antigen receptor using the same
CN115175694A (zh) 抗baff受体抗体及其用途
EP4261225A1 (de) Anti-pd-1-antikörper und verwendungen davon
US20240084011A1 (en) Anti-CTLA4 monoclonal antibodies and chimeric antigen receptors
WO2012015662A1 (en) Il-18 receptor as a novel target of regulatory t cells in cancer
AU2002340069A1 (en) Methods and compositions for prevention, diagnosis, and treatment of cancer using bispecific molecules
JP2016013104A (ja) 抗イヌｃｄ２０モノクローナル抗体又は抗体フラグメント、キット、診断方法、治療用組成物、治療方法、核酸、ベクター、キメラ抗原受容体及びｔ細胞
CN116535509B (zh) 抗CD79b抗体、其制备方法及其应用
CN116444672B (zh) 人表皮生长因子3的抗体、其制备方法及其应用
WO2022251853A1 (en) C-x-c motif chemokine receptor 6 (cxcr6) binding molecules, and methods of using the same
US20040109863A1 (en) Methods of therapy and diagnosis using targeting of cells that express Ly-9
US20040109862A1 (en) Methods of therapy and diagnosis using targeting of cells that express Ly-9

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20040428

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060401