Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
  • A61K51/1072—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell being from the reproductive system, e.g. ovaria, uterus, testes or prostate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/10—Drugs for disorders of the urinary system of the bladder
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2896—Immunoglobulins [IG], 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 [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• Such candidate epitopes must be expressed at high levels on the cancer cells compared to normal tissue.
• this method requires the development of high affinity mAbs specific for these epitopes and these mAbs must show minimal cross-reactivity with self tissue.
• the biological mechanism of killing with mAbs will be variable, depending upon the epitopes identified on the cancer cells, and the effector functions ofthe specific mAb isotype.
• the cancer cells may reduce the available levels ofthe target epitope per cell, or eliminate it from their surface altogether. Thus, the use of mAbs in cancer diagnosis and treatment remains problematic.
• a more widely applicable approach to treatment of cancer with mAbs would be to identify a ubiquitous antigenic site, present on virtually all cancer cells, and then to develop a panel of mAbs specific for this antigen.
• a voluminous literature reveals that cancer cells share certain common characteristics. Many types of human cancer cells are characterized by substantial abnormalities in the glycosylation patterns of their cell-surface proteins and lipids (Hakomori et. al., 1996, Cane Res. 56:5309-18; Castronovo et al.J989, J Nat Cane Inst 81 :212-6; Springer et al.,1984, Science 224: 1198-206; and Springer et al., 1997, J Mol Med 75:594-602).
• C3b(i) complement activation products
• cancer patients have substantially lowered levels ofthe potentially protective IgM antibodies.
• the cancer cells cannot easily be killed by complement 5 activation because ofthe reduced levels of protective IgM antibody and the increased expression of human complement control proteins on their surface.
• C3b(i)-opsonized HIV can persist in the body bound to the surface of dendritic cells for very long periods of time, and this cell-bound HIV may represent one ofthe main obstacles to the permanent elimination of HIV from the body (Hiavacek et al., 1999, Proc. Nat!. Acad. Sci. 96:14681; and Schmitz, et al.,. 1994,. J. Immunol. 153:1352). Citation of a reference in this section or any section of this application shall not be construed as an admission that such reference is prior art to the present invention.
• the present invention also provides compositions comprising one or more nucleic acid molecules encoding one or more antibodies immunospecific for C3b(i) or C3b(i) linked to a second molecule, in an amount effective for the treatment or prevention of cancer in an animal.
• the present invention further provides a composition comprising one or more bispecific antibodies which are immunospecific for C3b(i) or C3b(i) linked to a second molecule and an effector cell receptor or antigen, in an amount effective for the treatment or prevention of cancer.
• C3b(i) refers to C3b and its fragments, including, but not limited to, C3b(i), C3b, and C3d. In certain embodiments,”C3b(i)" refers to C3b. In other embodiments, “C3b(i)” refers to C3b(i). In yet other embodiments, “C3b(i)” does not refer to C3dg or the fragment of C3b or C3b(i) which corresponds to C3dg.
• the "second molecule" that C3b(i) is linked to is IgG or IgM.
• the "second molecule” that C3b(i) is linked to is a cancer cell antigen such as, e.g., a. protein, glycoprotein, peptide, polypeptide, or glycolipid differentially or preferentially expressed by cancer cells.
• the "second molecule" that C3b(i) is linked to is a microbial or viral antigen.
• C3b(i) is covalently linked to a second molecule.
• antibodies immunospecific for one or more cancer antigens, viral antigens, or microbial antigens refer to antibodies thereof that immunospecifically bind to cancer antigens, viral antigens, or microbial antigens and do not non-specifically bind to other polypeptides.
• Antibodies that immunospecifically bind to cancer antigens, viral antigens, or microbial antigens may have cross-reactivity with other antigens.
• antibodies that immunospecifically bind to cancer antigens, viral antigens, or microbial antigens do not cross-react with other antigens.
• a peptide or polypeptide preferentially or differentially expressed on a cancer cell a viral peptide or polypeptide, a microbial peptide or polypeptide or an antibody that immunospecifically binds to C3b(i).
• the antibody or polypeptide When the antibody or polypeptide is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of he volume ofthe protein preparation.
• culture medium represents less than about 20%, 10%, or 5% of he volume ofthe protein preparation.
• the antibody or polypeptide When the antibody or polypeptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis ofthe protein. Accordingly such preparations ofthe antibody or polypeptide have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the antibody or polypeptide fragment of interest.
• antibodies ofthe invention are isolated or purified.
• nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid molecule.
• an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
• nucleic acid molecules encoding antibodies of the invention are isolated or purified.
• a "therapeutically effective amount” is the amount of a composition ofthe invention that results in a reduction ofthe growth or spread of cancer by at least 2.5 %, at least 5 %, at least 10 %, at least 15%, at least 25 %, at least 35 %, at least 45%, at least 50 %, at least 75%, at least 85 %, by at least 90 %, at least 95 %, or at least 99 % in an animal administered a composition ofthe invention relative to the growth or spread of cancer in an animal or group of animals (e.g. , two, three, five, ten or more animals) not administered a composition ofthe invention.
• FIG. 1 A-D. Representative flow cytometry data from a study with serum from a normal donor (A, B) and a cancer patient (C, D). Measurement of C3b(i) (A, C) and IgM (B, D) deposition on C4-2 human prostate cancer cells is shown. Abundant C3b(i) is deposited on C4-2 cancer cells in response to the addition of normal human serum; this opsonization appears to be facilitated by both the classical and alternative complement pathways. After opsonization with serum from a prostate cancer patient, significantly less C3b(i) and IgM are deposited on the tumor cells (C, D). C3b(i) deposition via the alternative pathway (serum with Mg-EGTA), however, is comparable for both the normal and cancer patient serum, suggesting that the alternative pathway ofthe complement system remains intact in prostate cancer patient serum.
• FIG. 4 Flow cytometry results from surveys of sera from normal donors and patients with prostate cancer. Binding of human immunoglobulin to LNCaP and C4-2 prostate cancer cells was measured. Significant differences were determined by t-tests.
• FIG. 5 (A-B). Immunohistochemical staining of normal (A) and neoplastic (B) human prostate tissue after incubation with anti-C3b(i) mAb.
• FIG. 6 Rosetting experiment using erythrocytes and opsonized LNCaP prostate cancer cells incubated in plasma in the presence of an anti-CRl X anti-C3b(i) bispecific monoclonal antibody complex (7G9 X 3E7).
• FIG. 8 The schematic illustrates the steps ofthe invention, all of which occur on the cell surface of tumor cells within the body ofthe cancer patient.
• human IgM either endogenous, or infused into the patient
• complement either endogenous, or infused into the patient as fresh plasma
• the resulting proteolytic fragment C3b(i) is deposited on the surface ofthe cancer cell.
• a mAb specific for the C3b(i) epitope is administered.
• the mAb can be associated with a toxic, enzymatic, genetic, differentiating, and/or imaging agent (therefore it is an "effector mAb”), which results in the destruction or imaging ofthe cancer cell.
• FIG. 10 Red cell binding experiment using erythrocytes and opsonized Rajii cells.
• Rajii cells treated with: washed whole blood reconstituted serum alone; washed whole blood reconstituted serum and Rituximab; washed whole blood reconstituted serum and anti-C3b(i) X anti-CRl bispecific monoclonal antibody complexes; and washed whole blood reconstituted serum, Rituximab, and anti-C3b(i) X anti-CRl bispecific monoclonal antibody complexes; washed whole blood reconstituted serum, Rituximab (15'), and anti- C3b(i) X anti-CRl bispecific monoclonal antibody complexes; and washed whole blood reconstituted serum, anti-C3b(i) X anti-CRl bispecific monoclonal antibody (15'), and Rituximab.
• the present invention provides methods for treating or preventing cancer, viral infection, microbial infection, or septic shock in an animal, said methods comprising administering to said animal IgG antibodies, IgM antibodies, and/or one or more complement components in combination with a therapeutically or prophylactically effective amount of one or more antibodies immunospecific for C3b(i) or C3b(i) linked to a second molecule.
• the present invention also provides methods for treating or preventing cancer, viral infection, or microbial infection in an animal, said methods comprising administering to said animal one or more antibodies immunospecific for one or more cancer antigens, viral antigens, or microbial antigens, respectively, in combination with one or more antibodies immunospecific for C3b(i) or C3b(i) linked to a second molecule.
• the present invention further provides methods for treating or preventing septic shock in an animal, said methods comprising administering to said animal one or more antibodies immunospecific for lipopolysaccharide (LPS), an endotoxin, or a constituent ofthe outer wall of a gram negative bacteria (e.g., the outer wall of E. coli) in combination with one or more antibodies immunospecific for C3b(i) or C3b(i) linked to a second molecule.
• LPS lipopolysaccharide
• endotoxin or a constituent ofthe outer wall of a gram negative bacteria (e.g., the outer
• monoclonal antibodies immunospecific for C3b(i) are conjugated to a therapeutic moiety such as an antibiotic, a chemotherapeutic cytotoxin, e.g., a cytostatic or cytocidal agent (e.g., paclitaxol, cytochalasin B or diphtheria toxin), a thrombotic or anti-angiogenic agent or a radioactive label.
• a chemotherapeutic cytotoxin e.g., a cytostatic or cytocidal agent (e.g., paclitaxol, cytochalasin B or diphtheria toxin), a thrombotic or anti-angiogenic agent or a radioactive label.
• monoclonal antibodies immunospecific for C3b(i) are conjugated to a detectable substrate such as, e.g., an enzyme, fluorescent marker, luminescent material, bioluminescent material, or radioactive material.
• bispecific antibodies which are immunospecific for C3b(i) and an effector cell receptor or antigen are administered to an animal, preferably a mammal and most preferably a human, to treat, or prevent cancer, viral infection, microbial infection, or septic shock.
• effector cell refers to a cell which is involved in a cell-mediated immune response. Examples of effector cells include, but are not limited to, monocytes, macrophages, dendritic cells, neutrophils, natural killer cells, lymphocytes and erythrocytes.
• anti-C3b(i) heteropolymer constructs bound ex vivo to an effector cell via a cell surface receptor are administered to an animal, preferably a mammal and most preferably a human, to treat or prevent cancer, viral infection or microbial infection.
• Cell surface receptors include, but are not limited to, CR1, CR2, CR3, CR4, human Fc ⁇ receptors CD 16, CD32 and CD64, and the Fc receptor for IgA, CD89.
• anti-C3b(i) heteropolymer constructs bound ex vivo to erythrocytes via CR1 are administered to an animal, preferably a mammal and most preferably a human, to treat, inhibit or prevent cancer, viral infection, microbial infection, or septic shock.
• bispecific diabodies which are antibody fragments immunospecific for C3b(i) and a complement component are administered to an animal, preferably a mammal and most preferably a human, to treat or prevent cancer, viral infection, microbial infection, or septic shock.
• the diabodies are capable of recruiting complement components.
• bispecific diabodies which are immunospecific for C3b(i) and Clq are administered to an animal, preferably a mammal and most preferably a human, to treat or prevent cancer, viral infection, microbial infection, or septic shock.
• the present invention encompasses the administration of IgG antibodies and/or IgM antibodies in combination with one or more anti-C3b(i) antibodies to animals to facilitate the opsonization of cancer cells, viruses, and microbes.
• IgG antibodies and/or IgM antibodies are administered to an animal prior to, subsequent to, or concomitantly with the administration of one or more antibodies immunospecific for C3b(i) to treat or prevent cancer, viral infection, microbial infection, or septic shock.
• IgG antibodies and/or IgM antibodies are administered to an animal prior to the administration of one or more antibodies immunospecific for C3b(i) to treat or prevent cancer, viral infection, microbial infection or septic shock.
• one or more antibodies immunospecific for one or more cancer cell antigens are administered to an animal prior to, subsequent to, or concomitantly with the administration of one or more antibodies immunospecific for C3b(i), in an amount effective for the treatment or prevention cancer.
• one or more antibodies immunospecific for one or more cancer cell antigens are administered to an animal prior to the administration of one or more antibodies immunospecific for C3b(i), in an amount effective for the treatment or prevention cancer.
• cancer cell antigens include, but are not limited to, improperly glycosylated cell-surface proteins and lipids.
• one or more antibodies used in cancer immunotherapy are administered to an animal prior to, subsequent to, or concomitantly with the administration of one or more anti-C3b(i) antibodies, in an amount effective for the treatment or prevention cancer.
• one or more antibodies used in cancer immunotherapy are administered to an animal prior to the administration of one or more anti-C3b(i) antibodies, in an amount effective for the treatment or prevention cancer.
• antibodies used in cancer immunotherapy include, but are not limited to: Herceptin® (Trastuzumab; Genetech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; Retuxan® (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non- Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA) which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG 2a antibody for the treatment of colorectal cancer; BEC2 (ImClone Systems Inc., NY) which is murine IgG antibody for the treatment of lung cancer; IMC-C225 (Imclone Systems Inc., NY) which is a chimeric IgG antibody for the treatment of head and neck cancer; Vitaxin (Medlmmune,
• antibodies used for the treatment or prevention of viral or microbial infection include, but are not limited to: Synagis® (Medlmmune, Inc., MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody for the treatment of patients with RSV infection; PRO542 (Progenies) which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir (Protein Design Labs, Inc., CA) which is a human antibody for the treatment of hepatitis B virus; Protovir (Protein Design Labs, Inc., CA) which is a humanized IgG ! antibody for the treatment of cytomegalo virus (CMV); WIN1 which immunospecifically binds to LPS (Novardis) and mouse monoclonal anti-Legionella Pneumonia LPS (Research Diagnostics, Inc. NJ).
• Synagis® Medlmmune, Inc., MD
• RSV humanized anti-respiratory syncytial virus
• the plasma or selectively enriched IgG and/or IgM antibodies may be obtained from normal animals or from animals with a particular type of cancer, a particular type viral infection, or a particular type of microbial infection.
• the plasma or selectively enriched IgG and/or IgM is obtained from an animal ofthe same species which receives the administration.
• the plasma may or may not be treated with EDTA, citrate or heparin to block the complement pathways.
• plasma or selectively enriched IgG and/or IgM antibodies from an animal, which contains antibodies immunospecific for cancer cell antigens (e.g., improperly glycosylated proteins or lipids) are administered to an animal prior to the administration of anti-C3b(i) antibodies.
• plasma or selectively enriched IgG and/or IgM antibodies from an animal which contains antibodies immunospecific for a viral antigen or a microbial antigen (e.g., anti-HIV gpl20 antibodies or anti-LPS antibodies) are administered to an animal prior to the administration of anti-C3b(i) antibodies.
• a viral antigen or a microbial antigen e.g., anti-HIV gpl20 antibodies or anti-LPS antibodies
• the present invention encompasses the administration of recombinant complement components or plasma as a source of complement components and one or more anti-C3b(i) antibodies for the treatment or prevention of cancer, viral infection, microbial infection, and septic shock
• one or more complement components or recombinant complement components are administered to an animal, preferably a mammal and most preferably a human, prior to, subsequent to, or concomitantly with the administration of antibodies immunospecific for C3b(i) in an amount effective for the treatment or prevention of cancer, viral infection, microbial infection, or septic shock.
• normal plasma as a source of complement components is administered to an animal prior to, subsequent to, or concomitantly with the administration of antibodies immunospecific for C3b(i) in an amount effective for the treatment or prevention of cancer, viral infection, microbial infection, or septic shock.
• a source of IgG and/or IgM antibodies and complement components is administered to an animal to insure efficient opsonization prior to the administration of antibodies specific for C3b(i).
• the administration of C3b(i) immunospecific antibodies in combination with IgG antibodies, IgM antibodies and/or complement components will initiate a chain reaction which results in increased complement activity and ultimately the killing of cancerous cells.
• the endogenous levels of IgG antibodies, IgM antibodies and/or complement components are analyzed to determine whether an animal, preferably a mammal and most preferably a human, requires the administration of IgG antibodies, IgM antibodies and/or complement components.
• Standard techniques known to those of skill in the art can be utilized to measure the endogenous levels of IgG antibodies, IgM antibodies and complement components in an animal's sera.
• the level of IgM antibodies or IgG antibodies in sera can be determined by titration ofthe sera against cell lines such as comparable cancer cell lines.
• the level of complement components and complement activity can be determined by, for example, in vitro tests for the ability to interact with complement proteins and the ability to lyse target cells opsonized with specific antibodies (Complement: A Practical Approach, Dodds and Sim, Oxford University Press 1997; Makrides et al., 1992, J. Biol. Chem. 264:24754-2476; and Weisman, H.
• the present invention also provides compositions comprising one or more anti-C3b(i) antibodies for use in the treatment, prevention, detection or diagnosis of cancer, viruses, and microbes.
• the present invention provides methods and kits for depleting cancerous cells in vitro utilizing C3b(i) specific antibodies.
• the invention also provides methods and kits for the detection, imaging, and diagnosis of cancer utilizing antibodies specific for C3b(i). Further, the invention provides pharmaceutical compositions comprising antibodies specific for C3b(i).
• IgG AND IgM ENRICHMENT the levels of IgG antibodies, IgM antibodies, and complement components in the sera or plasma of an animal are measured prior to the administration of anti-C3b(i) antibodies.
• animals determined to have low levels of IgG antibodies and/or IgM antibodies are administered normal plasma containing IgG antibodies and/or IgM antibodies (preferably, IgG antibodies or IgM antibodies that immunospecifically bind to one or more cancer cell epitopes (e.g., improperly glycosylated proteins or lipids expressed by cancer cells), viral epitopes, or microbial epitopes) prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of anti-C3b(i) antibodies for the treatment or prevention of cancer, viral infection, or microbial infection.
• cancer cell epitopes e.g., improperly glycosy
• animals determined to have low levels of IgG antibodies are administered plasma enriched for IgG antibodies prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of anti-C3b(i) antibodies for the treatment or prevention of cancer, viral infection, or microbial infection.
• IgM antibodies or IgG antibodies are selectively enriched utilizing standard techniques known to those of skill in the art. Such techniques include, but are not limited to, chromatography, centrifugation, and differential solubility.
• native or recombinant IgG antibodies or IgM antibodies known to immunospecifically bind to cancer cells, viruses or microbes are administered to an animal prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of anti-C3b(i) antibodies for the treatment or prevention of cancer.
• IgG antibodies or IgM antibodies that immunospecifically bind to one or more cancer cell antigens e.g. , improperly glycosylated proteins or lipids expressed by cancer cells
• viral antigens e.g., glycoprotein F of RSV and gpl20 of HIV
• microbial antigens e.g., LPS
• standard protein purification techniques include, but are not limited to, gel purification, chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, and differential solubility.
• Recombinant IgG antibodies and IgM antibodies can be produced utilizing standard techniques known to those of skill in the art.
• plasma as a source of IgG and IgM antibodies is administered to an animal the same day as the animal is administered antibodies immunospecific for C3b(i) or C3b(i) covalently linked to a second molecule for the treatment or prevention of cancer, viral infections or microbial infections.
• plasma as a source of IgG and IgM antibodies is administered to an animal a few minutes or hours (e.g., 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours) before the antibodies immunospecific for C3b(i) or C3b(i) covalently linked to a second molecule are administered to the animal for the treatment or prevention of cancer, viral infections or microbial infections.
• IgG antibodies and/or IgM antibodies are administered to an animal the same day as the animal is administered antibodies immunospecific for C3b(i) or C3b(i) covalently linked to a second molecule for the treatment or prevention of cancer, viral infections or microbial infections.
• IgG antibodies and/or IgM antibodies are administered to an animal a few minutes or hours (e.g. , 5 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours or 12 hours) before administering antibodies immunospecific for C3b(i) or C3b(i) covalently linked to a second molecule for the treatment or prevention of cancer, viral infections or microbial infections.
• animals determined to have low levels of complement, particularly C3 are infused with normal plasma prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of anti-C3b(i) antibodies for the treatment or prevention of cancer, viral infection, or microbial infection.
• the plasma is obtained from an animal ofthe same species that receives the plasma.
• animals determined to have low levels of complement are administered native or recombinant complement proteins (e.g., C3) prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of anti-C3b(i) antibodies for the treatment or prevention of cancer, viral infection, or microbial infection.
• complement proteins e.g., C3
• one or more complement components are administered to an animal the same day as the animal is administered anti-C3b(i) antibodies for the treatment or prevention of cancer, viral infection, or microbial infection.
• one or more complement components are administered to an animal a few hours (e.g., 1 hour, 2 hours, 4 hours, 6 hours, 8 hours or 12 hours) before administering antibodies anti-C3b(i) antibodies for the treatment or prevention of cancer, viral infection, or microbial infection.
• Complement components, in particular complement component C3 can be purchased from a company or purified utilizing standard protein purification techniques known to those of skill in the art.
• nucleic acid sequences encoding complement components can be obtained from available sequence databases, e.g., GenBank. Both cDNA and genomic sequences can be cloned and expressed. Further, in accordance with the invention, the native or recombinant complement components administered to an animal for the treatment or prevention of cancer, viral infection, or microbial infection retain the ability to function in the classical and/or alternative complement pathways.
• nucleotide sequence encoding complement components or a functionally active analogs or other derivatives thereof can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence.
• an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence.
• the nucleotide sequence encoding human C3 as disclosed in Genbank Accession Numbers NM_000064 and K02765 can be inserted into an appropriate expression vector.
• nucleotide sequence encoding human CI subcomponents, human C2 or human C2 as disclosed in Genbank Accession Numbers NM_000063, NMJ301734, J04080, and AF019413, respectively can be inserted into an appropriate expression vector.
• the necessary transcriptional and translational signals can be supplied by the native complement component genes or its flanking regions.
• host-vector systems may be utilized to express the protein-coding sequence. These include, but are not limited to, mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. , baculovirus); and microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
• the expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used.
• the human complement component genes or sequences encoding functionally active portions ofthe human complement components are expressed.
• any ofthe methods known to one of skill in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional and translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).
• the expression of a nucleic acid sequence encoding a complement component or fragments thereof may be regulated by a second nucleic acid sequence so that the complement component or fragments thereof are expressed in a host transformed with the recombinant DNA molecule.
• the expression of complement components (e.g. , C3) may be controlled by any promoter or enhancer element known to one of skill in the art.
• a complement component may regulated by any constitutive promoter, inducible promoter, tissue specific promoter, or native complement component promoter known to one of skill in the art.
• Promoters which may be used to regulate the expression of a complement component include, but are not limited to, viral promoters such as the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.
• prokaryotic promoters such as the ⁇ -lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci.
• promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp.
• mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel.
• the promoter regulating the expression ofthe complement component is a constitutive promoter. In certain other embodiments, the promoter regulating the expression ofthe complement component is an inducible or tissue-specific promoter.
• the vector used to express a complement component comprises a promoter operably linked to a complement component (e.g. , C3)-encoding nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene).
• a complement component e.g. , C3
• selectable markers e.g., an antibiotic resistance gene
• Expression vectors containing nucleotide sequences encoding one or more complement components can be identified by three general approaches: (a) nucleic acid hybridization; (b) presence or absence of "marker" gene functions; and (c) expression of inserted sequences.
• the presence of a nucleotide sequence encoding a complement component (e.g., C3) in an expression vector(s) can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to the inserted nucleotide sequence.
• the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion ofthe nucleotide sequence encoding the complement component in the vector(s). For example, if a nucleotide sequence encoding C3 is inserted within the marker gene sequence ofthe vector, recombinant vectors containing the nucleotide sequence can be identified by the absence ofthe marker gene function.
• certain "marker" gene functions e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
• recombinant expression vectors can be identified by assaying for RNA or polypeptides encoded by the nucleotide sequence for the complement component. Such assays can be based, for example, on the physical or functional properties ofthe complement component in in vitro assay systems, e.g., binding of C3 with anti-C3 antibody.
• a host cell can be transfected with a nucleotide sequence encoding a complement component or a vector comprising a nucleotide sequence encoding a complement component using techniques known to those of skill in the art such as, for example, microinjection, electroporation, lipofection, and calcium phosphate precipitation.
• Host cells may be stably or transiently transfected with a nucleotide sequence encoding a complement component or a vector comprising a nucleotide sequence encoding a complement component.
• cell lines which stably express one or more complement components may be engineered.
• host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
• expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
• a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11 :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al, 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
• antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al, 1981, J. Mol. Biol.
• telomeres can be chosen which modulates the expression ofthe inserted sequences in the specific fashion desired.
• Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing ofthe complement protein expressed.
• Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation and phosphorylation) of proteins.
• expression in a bacterial system can be used to produce an unglycosylated core protein product.
• Expression in yeast will produce a glycosylated product.
• Expression in mammalian cells can be used to ensure "native" glycosylation of a heterologous protein.
• mammalian host cells include, but are not limited to, 293 T, CHO, NERY, BHK, Hela, COS, MDCK, 293, 3T3, MT2, U937, WI38, BT483, Hs578T, HTB2, BT20, T47D, CRL7030, Hs578Bst, lymphocytes and fibroblasts.
• different vector/host expression systems may effect processing reactions to different extents.
• Antibodies ofthe invention include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized or chimeric antibodies, single chain antibodies, sFvs, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any ofthe above which immunospecifically bind to cancer cell antigens, viral antigens, microbial antigens, C3b(i) or fragments thereof, or C3b(i) linked to a second molecule.
• antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which immunospecifically binds a cancer cell antigen, a viral antigen, a microbial antigen, C3b(i), or C3b(i) linked (e.g., covalently linked) to a second molecule.
• the immunoglobulin molecules ofthe invention can be of any type (e.g., IgG, IgE, IgM, IgD and IgA ), class, or subclass of immunoglobulin molecule.
• Polyclonal antibodies which may be used in the methods ofthe invention are heterogeneous populations of antibody molecules derived from the sera of immunized animals.
• Various procedures well known in the art may be used for the production of polyclonal antibodies to an antigen-of-interest.
• various host animals can be immunized by injection with an antigen of interest or derivative thereof, including but not limited to rabbits, mice, rats, etc.
• adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
• BCG Bacille Calmette-Guerin
• corynebacterium parvum Such adjuvants are also well known in the art.
• Monoclonal antibodies which may be used in the methods ofthe invention are homogeneous populations of antibodies to a particular antigen (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, C3b(i) or C3b(i) covalently linked to a second molecule).
• a monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture.
• Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof.
• the hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.
• the monoclonal antibodies which may be used in the methods ofthe invention include, but are not limited to, human monoclonal antibodies or chimeric human-mouse (or other species) monoclonal antibodies.
• Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).
• the invention further provides for the use of bispecific antibodies.
• Methods for making bispecific antibodies are known in the art.
• Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Because ofthe random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification ofthe correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low.
• antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
• the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding, present in at least one ofthe fusions.
• CHI first heavy-chain constant region
• 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. This provides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
• the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in PCT Publication No. WO 94/04690 published March 3,1994, which is incorporated herein by reference in its entirety.
• bispecific antibodies For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology,1986, 121:210. Using such techniques, a bispecific molecule which combines anti-C3b(i) antibody and an antibody specific for an effector cell receptor or antigen can be prepared for use in the treatment or inhibition of disease as defined herein.
• the invention provides for the use of functionally active fragments, derivatives or analogs of antibodies which immunospecifically bind to cancer cell antigens, viral antigens, and microbial antigens.
• Functionally active means that the fragment, derivative or analog is able to elicit anti-anti-idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived recognized.
• the antigenicity ofthe idiotype ofthe immunoglobulin molecule may be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen.
• synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay)
• inventions include fragments ofthe antibodies ofthe invention such as, but not limited to, F(ab')2 fragments, which contain the variable region, the light chain constant region and the CHI domain ofthe heavy chain can be produced by pepsin digestion ofthe antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges ofthe F(ab')2 fragments.
• the invention also provides heavy chain and light chain dimers ofthe antibodies ofthe invention, or any minimal fragment thereof such as Fvs or single chain antibodies (SCAs) (e.g., as described in U.S. Patent 4,946,778; Bird, 1988, Science 242:423-42; Huston et al, 1988, Proc. Natl. Acad. Sci. USA
• recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope ofthe invention.
• a chimeric and humanized monoclonal antibodies comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope ofthe invention.
• antibody 10 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 monoclonal and a human immunoglobulin constant region.
• animal species such as those having a variable region derived from a murine monoclonal and a human immunoglobulin constant region.
• Humanized antibodies are antibody molecules from non-human species having
• CDRs complementarily determining regions
• Fully human antibodies are particularly desirable for therapeutic treatment of human patients.
• Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
• the transgenic mice are immunized 35 in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
• 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. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
• Completely human antibodies which recognize 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.
• the invention provides fusion proteins ofthe antibodies ofthe invention (or functionally active fragments thereof), for example in which the antibody is fused via a covalent bond (e.g. , a peptide bond), at either the N-terminus or the C-terminus to an amino acid sequence of another protein (or portion thereof, preferably at least 10, 20 or 50 amino acid portion ofthe protein) that is not the antibody.
• a covalent bond e.g. , a peptide bond
• the antibody or fragment thereof is covalently linked to the other protein at the N-terminus ofthe constant domain.
• the antibodies ofthe invention include analogs and derivatives that are either modified, t.e, by the covalent attachment of any type of molecule as long as such covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
• the derivatives and analogs ofthe antibodies include those that have been further modified, e.g. , by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
• the antibodies ofthe invention include antibodies with modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors.
• the antibodies ofthe invention include antibodies with modifications in amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g. , PCT Publication No. WO 97/34631 , which is incorporated herein by reference in its entirety).
• anti-C3b(i) antibodies are immunospecific for C3b(i) or a fragment thereof (e.g., at least 5, at least 10, at least 15, at least 25, or more contiguous amino acid residues of C3b(i)).
• anti-C3b(i) antibodies are immunospecific for C3b(i) covalently linked to a second molecule.
• anti-C3b(i) antibodies are monoclonal antibodies or Fab fragments.
• anti-C3b(i) antibodies are immunospecific for C3b(i) bound to IgG or IgM antibodies and said anti-C3b(i) antibodies have minimal cross-reactivity with C3b(i) in the media or milieu.
• anti-C3b(i) antibodies are immunospecific for C3b(i) bound to a cancer cell or C3b(i) bound to a virus or microbe; and said anti-C3b(i) antibodies have minimal cross-reactivity with C3b(i) in the media or milieu.
• anti-C3b(i) antibodies administered to humans are humanized or human monoclonal antibodies.
• Anti-C3b(i) antibodies can be obtained from any organization (e.g., a university scientist or a company such as Research Diagnostics Inc. in New Jersey) or produced by any method known to one of skill in the art.
• anti-C3b(i) antibodies can be produced by chemical synthesis or recombinant expression techniques.
• the nucleotide sequence encoding an anti-C3b(i) antibodies can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
• an anti-C3b(i) antibody is monoclonal antibody 7C12, 2H11, 8E11 or 3E7 (see, e.g., Ferguson et al., 1995, Arthritis Rheum. 38:190; Taylor et al., 1989, J. Immunology 143:3626; Tosic et al., 1989, J. Immunol. Methods 120:241; and Sokoloff et al., 2000, Cancer Immunol. Immunother. 49:551-562, each of which is incorporated herein by reference in their entirety).
• an anti-C3b(i) antibody is an antibody that competes with 7C12, 2H11, 8E11 or 3E7 for binding to C3b(i) or a fragment thereof, as determined by an immunoassay such as, e.g. , an ELISA.
• an anti-C3b(i) antibody is monoclonal antibody 3E7.
• an anti-C3b(i) antibody is an antibody that competes with monoclonal 3E7 for binding to C3b(i) or a fragment thereof, as determined by an immunoassay such as, e.g., an ELISA.
• an anti-C3b(i) antibody is a bispecific monoclonal antibody comprising monoclonal antibody 3E7 and monoclonal antibody 7G9 5 (see, e.g. , Sokoloff et al., 2000, Cancer Immunol. Immunother. 49:551 -562).
• antibodies immunospecific for a cancer cell antigen for ⁇ ⁇ use in accordance with methods ofthe invention are monoclonal antibodies or Fab fragments.
• antibodies immunospecific for a cancer cell antigen which are administered to humans are humanized or human monoclonal antibodies.
• cancer cell antigen refers to an antigen (e.g., a protein, glycoprotein, polypeptide, peptide, or glycolipid) that is preferentially or differentially expressed on cancer cells ⁇ relative to non-cancerous cells, preferably normal cells.
• cancer cell antigens include, but are not limited, to improperly glycosylated proteins and lipids, CD20, Her-2, and PSMA.
• Antibodies immunospecific for a cancer cell antigen can be obtained from any organization (e.g., a university scientist or a company such as Genentech) or produced by 0 any method known to one of skill in the art such as, e.g. , chemical synthesis or recombinant expression techniques.
• the nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
• antibodies for the treatment or prevention cancer are used in accordance with the methods ofthe invention.
• antibodies available for the treatment of cancer include, but are not limited to, Herceptin® (Trastuzumab; Genetech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; Retuxan® (rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibody for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA) which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murine IgG 2a antibody for the treatment of colorectal cancer; BEC2 (ImClone Systems Inc., NY) which is murine IgG antibody for the treatment of lung cancer; IMC-C225 (Imclone Systems Inc., NY) which is
• LymphoCide (Immunomedics, Inc., NJ) which is a humanized IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart I D10 (Protein Design Labs, Inc., CA) which is a humanized IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart I D10 (Protein Design Labs, Inc., CA) which is a humanized IgG antibody for the treatment of non-Hodgkin's lymphoma; Smart I D10 (Protein Design Labs, Inc., CA) which
• antibodies immunospecific for a viral or microbial antigen for use in accordance with methods ofthe invention are monoclonal antibodies.
• antibodies immunospecific for a viral antigen or microbial antigen which are administered to humans are humanized or human monoclonal antibodies.
• viral antigen includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV ⁇ c gpl20, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, he ⁇ es simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen) which is capable of eliciting an immune response.
• polypeptide protein e.g., HIV ⁇ c gpl20, HIV nef, RSV F glycoprotein, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, he ⁇ es simplex virus glycoprotein (e.g., gB, gC, gD, and gE) and hepatitis B surface antigen
• microbial antigen includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, 0 pathogenic protozoa, or yeast polypeptide including, e.g. , LPS and capsular polysaccharide 5/8) which is capable of eliciting an immune response.
• microbial antigen includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., a bacterial, fungi, 0 pathogenic protozoa, or yeast polypeptide including, e.g. , LPS and capsular polysaccharide 5/8) which is capable of eliciting an immune response.
• Antibodies immunospecific for a viral or microbial antigen can be obtained from any organization (e.g., a university scientist or a company such as Genentech) or produced by any method known to one of skill in the art such as, e.g. , chemical synthesis or 5 recombinant expression techniques.
• the nucleotide sequence encoding antibodies immunospecific for a viral or microbial antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
• antibodies for the treatment or prevention viral or 0 microbial infection are used in accordance with the methods ofthe invention.
• antibodies available for the treatment of viral infection or microbial infection include, but are not limited to, Synagis® (Medlmmune, ie, MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody for the treatment of patients with RSV infection; PRO 542 (Progenies) which is a CD4 fusion antibody for the treatment of HIV infection; Ostavir (Protein Design Labs, Inc., CA) which is a human antibody for the treatment of hepatitis B virus; Protovir (Protein Design Labs, Inc., CA) which is a humanized IgG j antibody for the treatment of cytomegalo virus (CMV); and anti-LPS antibodies.
• Synagis® Medlmmune, ie, MD
• RSV humanized anti-respiratory syncytial virus
• PRO 542 Progenies
• the antibodies ofthe invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.
• a nucleic acid encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions ofthe sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification ofthe ligated oligonucleotides by PCR.
• a nucleic acid molecule encoding an antibody may be generated from a suitable source. If a clone containing the nucleic acid encoding the particular antibody is not available, but the sequence ofthe antibody is known, a nucleic acid encoding the antibody may be obtained from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends ofthe sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.
• a suitable source e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin
• antibodies specific for a particular antigen may be generated by any method known in the art, for example, by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
• a clone encoding at least the Fab portion ofthe antibody may be obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al, 1997 Proc. Natl. Acad. Sci. USA 94:4937).
• nucleic acid sequence encoding at least the variable domain ofthe antibody may be introduced into a vector containing the nucleotide sequence encoding the constant regions ofthe antibody (see, e.g., PCT Publication No.WO 86/05807; PCT Publication No. WO 89/01036; and U.S. Patent No. 5,122,464).
• Vectors containing the nucleotide sequence encoding the constant regions ofthe antibody see, e.g., PCT Publication No.WO 86/05807; PCT Publication No. WO 89/01036; and U.S. Patent No. 5,122,46404.
• nucleic acid encoding the antibody can be used to introduce the nucleotide substitutions or deletion necessary to substitute (or delete) the one or more variable region cysteine residues participating in an intrachain disulfide bond with an amino acid residue that does not contain a sulfhydyl group. Such modifications can be carried out
• 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 monoclonal 0 antibody and a human immunoglobulin constant region, e.g. , humanized antibodies.
• Single chain antibodies are formed by linking the heavy 5 and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chain polypeptide.
• Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al, 1988, Science 242:1038-1041).
• Antibody fragments which recognize specific epitopes may be generated by known techniques.
• such fragments include but are not limited to: the F(ab')2
• the vector for the production ofthe antibody may be produced by recombinant 5 DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
• An expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the antibody ofthe invention.
• the expression ofthe antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.
• the host cells used to express the recombinant antibody ofthe invention may be either bacterial cells such as Escherichia coli, or, preferably, eukaryotic cells, especially for the expression of whole recombinant immunoglobulin molecule.
• mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalo virus is an effective expression system for immunoglobulins (Foecking et al., 198, Gene 45:101; Cockett et al, 1990, Bio/Technology 8:2).
• host-expression vector systems may be utilized to express the immunoglobulin molecules ofthe invention.
• Such host-expression systems represent vehicles by which the coding sequences ofthe antibody may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express the immunoglobulin molecule ofthe invention in situ.
• These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B.
• subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing immunoglobulin coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing immunoglobulin coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the immunoglobulin coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expres-sion vectors (e.g., Ti plasmid) containing immunoglobulin coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mamm
• a number of expression vectors may be advantageously selected depending upon the use intended for the antibody being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
• vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, 1983, ⁇ MBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
• pG ⁇ X vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
• GST glutathione S-transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free gluta-thione.
• the pG ⁇ X vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
• Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
• the virus grows in Spodopterafrugiperda cells.
• the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
• a number of viral-based expression systems may be utilized, h cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
• an adenovirus transcription/translation control complex e.g., the late promoter and tripartite leader sequence.
• This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination.
• Insertion in a non-essential region ofthe viral genome will result in a recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts, (e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359).
• Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame ofthe desired coding sequence to ensure translation ofthe entire insert.
• These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
• telomeres may be shortened by the telomeres.
• a host cell strain may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function ofthe protein.
• Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing ofthe foreign protein expressed.
• eukaryotic host cells which possess the cellular machineiy for proper processing ofthe primary transcript, glycosylation, and phosphorylation ofthe gene product may be used.
• mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.
• cell lines which stably express an antibody may be engineered.
• host cells can be transformed with DNA controlled by appropriate expression control elements (e.g. , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
• appropriate expression control elements e.g. , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
• This method may advantageously be used to engineer cell lines which express the antibody Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody.
• a number of selection systems may be used, including but not limited to the he ⁇ es simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc.
• the expression levels of an antibody can be increased by vector amplification (for a
• the host cell may be co-transfected with two expression vectors ofthe invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
• the two vectors may contain identical selectable markers
• heavy and light chain polypeptides 25 which enable equal expression of heavy and light chain polypeptides.
• a single vector may be used which encodes both heavy and light chain polypeptides.
• the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
• the coding sequences for the heavy and light chains may comprise
• the antibody may be purified by any method known in the art for purification of an antibody, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other 5 standard technique for the purification of proteins.
• chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• centrifugation e.g., centrifugation, differential solubility, or by any other 5 standard technique for the purification of proteins.
• chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• centrifugation e.g., centrifugation, differential solubility, or by any other 5 standard technique for the purification of proteins.
• differential solubility e.g., differential solubility
• one or more antibodies immunospecific for one or more cancer cell antigens, viral antigens or microbial antigens are conjugated to a therapeutic agent.
• anti-C3b(i) antibodies are conjugated to a diagnostic or therapeutic agent.
• the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g. , determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
• detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See generally U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
• 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, dichlorotriazinylamine 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 125 I, I31 I, In or 9 ⁇ Tc.
• an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion.
• a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
• Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
• Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC)), and anti-mitotic agents (e.g.,
• anti-C3b(i) antibodies are conjugated to cobra venom factor.
• C3b(i) specific antibodies conjugated to cobra venom factor are utilized in vitro to deplete cancerous cells from, e.g., bone marrow obtained from an animal, preferably a mammal and most preferably a human, with cancer.
• Methods of conjugating antibodies to cobra venom factor are taught in U.S. Patent No. 5,773,243, which is inco ⁇ orated herein by reference in its entirety.
• the conjugates ofthe invention can be used for modifying a given biological response; thus, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
• the drug moiety may be a protein or polypeptide possessing a desired biological activity.
• Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, IL-1, IL-2, IL-4, IL-5 ,IL-6, IL-7, 11-8, 11-9, 11-10, IL-12, IL- 15, granulocyte macrophase colony stimulating factor ("GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
• GM-CSF granulocyte macrophase colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• Monoclonal Antibodies '84 Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Tho ⁇ e et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982); each of which is inco ⁇ orated herein by reference.
• an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is inco ⁇ orated herein by reference.
• An antibody with or without a therapeutic moiety conjugated to it can be used as a therapeutic and administered alone or in combination with cytotoxic factor(s) and/or cytokine(s).
• the invention provides for methods of depleting cancerous cells from non-cancerous tissues and/or cells in vitro (or ex vivo). In particular, the invention provides for methods of depleting cancerous cells by killing them or by separating them from non-cancerous cells.
• anti-C3b(i) antibodies alone or in combination with plasma are combined in vitro with tissues and/or cells obtained from an animal, preferably a mammal and most preferably a human.
• anti-C3b(i) antibodies alone or in combination with IgG antibodies, IgM antibodies and/or one or more complement components are combined in vitro with tissues and/or cells obtained from an animal, preferably a mammal and most preferably a human.
• anti-C3b(i) antibodies alone or in combination with antibodies immunospecific for cancer cell antigens are combined in vitro with tissues and/or cells obtained from an animal, preferably a mammal and most preferably a human.
• tissue obtained from an animal for the in vitro depletion of cancerous cells from non-cancerous cells is bone marrow and /or peripheral blood.
• monoclonal antibodies immunospecific for C3b(i) are incubated in vitro with tissues and/or cells obtained from an animal, preferably a mammal and most preferably a human.
• the monoclonal antibodies are immunospecific for C3b(i) covalently linked to IgM or IgG antibody which is bound to the cancer cells. In another preferred embodiment, the monoclonal antibodies are immunospecific for C3b(i) covalently linked to a glycoprotein or or glycolipid on the cancer cells, CD20, Her2 or PSMA.
• bispecific antibodies which are immunospecific for C3b(i) and an effector cell receptor or antigen are incubated in vitro with tissues and/or cells obtained from an animal, preferably a mammal and most preferably a human.
• bispecific antibodies which are immunospecific for C3b(i) and a complement component are incubated in vitro with tissues and/or cells obtained from an animal, preferably a mammal and most preferably a human.
• bispecific diabodies which are antibodies fragments immunospecific for C3b(i) and a complement component (e.g., Clq) are incubated in vitro with tissues and/or cells obtained from an animal, preferably a mammal and most preferably a human.
• the bispecific diabodies facilitate complement mediated lysis ofthe cancer cells.
• the anti-C3b(i) antibodies used in the in vitro depletion of cancer cells from tissues can be conjugated to detectable substances (e.g., various enzymes, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials) or therapeutic agents (e.g., cytostatic and cytocidal agents), which are disclosed in section 5.5.
• detectable substances e.g., various enzymes, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials
• therapeutic agents e.g., cytostatic and cytocidal agents
• anti-C3b(i) antibodies may be conjugated to cobra venom factor in order to use enhanced complement activation to lyse the cancer cells.
• tissues and/or cells thus depleted of cancerous cells are administered to an animal, preferably a mammal and most preferably a human.
• the tissues and/or cells are obtained from an animal with cancer prior to treatment for cancer, and tissues and/or cells depleted of cancerous cells are administered to the animal after the treatment.
• the in vitro depletion of cancerous cells using anti-C3b(i) antibodies is done prior to administration of autologous bone marrow peripheral blood, or peripheral blood stem cells.
• Anti-C3b(i) antibodies conjugated to detectable substances can be utilized to sort cancerous cells from non-cancerous cells by methods known to those of skill in the art.
• cancerous cells are sorted using a fluorescence activated cell sorter (FACS). Fluorescence activated cell sorting (FACS) is a well-known method for separating particles, including cells, based on the fluorescent properties ofthe particles
• cells particularly bone marrow cells or peripheral blood stem cells, obtained from an animal, preferably a mammal and most preferably a human, are incubated with fluorescently labeled C3b(i) specific antibodies for a time sufficient to allow the labeled antibodies to bind to the cells (e.g., 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes or more), preferably between 10 to 60 minutes.
• fluorescently labeled C3b(i) specific antibodies for a time sufficient to allow the labeled antibodies to bind to the cells (e.g., 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes or more), preferably between 10 to 60 minutes.
• cells are incubated with C3b(i) specific antibodies, the cells are washed, and the cells are incubated with a second labeled antibody that recognizes the C3b(i) specific antibodies.
• the cells are washed and processed through the cell sorter, allowing separation of cells that bind both antibodies to be separated from hybrid cells that do not bind both antibodies.
• FACS sorted particles may be directly deposited into individual wells of 96-well or 384-well plates to facilitate separation.
• magnetic beads can be used to separate cancerous cells from non-cancerous cells. Cancerous cells may be sorted using a magnetic activated cell sorting
• MCS Magnetic 5
• a magnetic field is then applied, to physically manipulate the selected beads.
• the beads are then mixed with the cells to allow binding.
• the invention provides for treatment or prevention of cancer, including, but not
• neoplasms e.g., myeloblastic, promyelocytic, myelomonocytic, monocytic,
• lymphoma e.g., Hodgkin's disease and non-Hodgkin's disease
• fibrosarcoma myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, Ewing's tumor, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, j e adenocarcinoma, renal cell carcinoma, hepatoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retin
• the treatment and/or prevention of cancer includes, but is not limited to, alleviating one or more symptoms associated with cancer, the inhibition or reduction ofthe progression of cancer, the promotion o the regression of cancer, and/or the ⁇ - promotion ofthe immune response.
• commercially available or naturally occurring anti-C3b(i) antibodies, functionally active fragments or derivatives thereof are used in the present invention.
• a composition ofthe invention is administered to an animal to ameliorate one or more symptoms associated with cancer. In certain other embodiments, a composition ofthe invention is administered to an animal to inhibit or reduce the progression of cancer. In certain other embodiments, a composition ofthe invention is administered to an animal to promote the regression of cancer.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies is administered to an animal in order to ameliorate one or more symptoms associated with cancer.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies is administered to an animal in order to promote ofthe regression of cancer and/or the inhibit or reduce the progression of cancer in an animal.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies and one or more antibodies immunospecific for one or more cancer cell antigens is administered to an animal in order to ameliorate one or more symptoms associated with cancer.
• compositions comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies and one or more antibodies immunospecific for one or more cancer cell antigens is administered to an animal in order to promote ofthe regression of cancer and/or the inhibit or reduce the progression of cancer in an animal.
• Anti-C3b(i) antibodies may be administered alone or in combination with other types of cancer treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).
• anti-tumor agents include, but are not limited to, cisplatin, ifosfamide, paclitaxel, taxanes, topoisomerase I inhibitors (e.g., CPT-11 , topotecan, 9- AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5- fluorouracil (5-FU), leucovorin, vinorelbine, temodal, and taxol.
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, after surgical resection of cancer.
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, in conjugation with chemotherapy or radiotherapy.
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of cancer prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of plasma to the animal.
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of cancer prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of IgG antibodies, IgM antibodies and/or one or more complement components to the animal.
• an animal preferably a mammal and most preferably a human
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of antibodies immunospecific for one or more cancer cell antigens.
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of cancer prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of antibodies currently used for the treatment of cancer.
• an animal preferably a mammal and most preferably a human
• antibodies include, but are not limited to, Herceptin® , Retuxan®, OvaRex, Panorex, BEC2, IMC-C225, Vitaxin, Campath I/H, Smart MI95, LymphoCide, Smart I D 10, and Oncolym.
• men with prostate cancer are administered anti-C3b(i) antibodies in conjugation with androgen ablation therapy.
• non-Hodgkin's lymphoma patients are treated with Retuxan® prior to the administration of anti-C3b(i) antibodies.
• animals with increased risk of cancer are administered a composition ofthe invention. Examples of such animals include, but are not limited to, humans prone to breast cancer.
• the invention provides methods for the treatment or prevention of viral infections in an animal, preferably a mammal and most preferably a human, said methods comprising the administration of a therapeutically or prophylactically effective amount of anti-C3b(i) antibodies or nucleic acid molecules encoding said antibodies.
• viral infections which can be treated or prevented in accordance with this invention include, but are limited to, viral infections caused by retroviruses (e.g., human T-cell lymphotrophic virus (HTLV) types I and II and human immunodeficiency virus (HIV)), he ⁇ es viruses (e.g., he ⁇ es simplex virus (HSV) types I and II, Epstein-Barr virus and cytomegalovirus), arenaviruses (e.g.
• retroviruses e.g., human T-cell lymphotrophic virus (HTLV) types I and II and human immunodeficiency virus (HIV)
• he ⁇ es viruses e.g., he ⁇ es simplex virus (
• lassa fever virus paramyxoviruses (e.g. , morbillivirus virus, human respiratory syncytial virus, and pneumovirus), adeno viruses, bunyaviruses (e.g., hantavirus), cornaviruses, filoviruses (e.g., Ebola virus), flaviviruses (e.g., hepatitis C virus (HCV), yellow fever virus, and Japanese encephalitis virus), hepadnaviruses (e.g., hepatitis B viruses (HBV)), orthomyoviruses (e.g., Sendai virus and influenza viruses A, B and C), papovaviruses (e.g., papillomavirues), picomaviruses (e.g., rhino viruses, entero viruses and hepatitis A viruses), poxviruses, reo viruses (e.g., rotavirues), togaviruses (e.g., rot
• a composition ofthe invention is administered to an animal to ameliorate one or more symptoms associated with a viral infection or a disease or disorder resulting, directly or indirectly, from a viral infection.
• a composition ofthe invention is administered to a human to ameliorate one or more symptoms associated with AIDS.
• a composition ofthe invention is administered to reduce the titer of a virus in an animal.
• a composition ofthe invention is administered to an animal to enhance or promote the immune response.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies is administered to an animal in order to ameliorate one or more symptoms associated with a viral infection.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies is administered to an animal in order to reduce the titer of a virus in an animal.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies and one or more antibodies immunospecific for one or more viral antigens is administered to an animal in order to ameliorate one or more symptoms associated with a viral infection.
• compositions comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies and one or more antibodies immunospecific for one or more viral antigens is administered to an animal in order to reduce the titer of a virus in an animal.
• Anti-C3b(i) antibodies may be administered alone or in combination with other types of anti- viral agents.
• anti- viral agents include, but are not limited to: cytokines (e.g., IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ ); inhibitors of reverse transcriptase (e.g., AZT, 3TC, D4T, ddC, ddl, d4T, 3TC, adefovir, efavirenz, delavirdine, nevirapine, abacavir, and other dideoxynucleosides or dideoxyfluoronucleosides); inhibitors of viral mRNA capping, such as ribavirin; inhibitors of proteases such HIV protease inhibitors (e.g., amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir,); amphotericin B; castanospermine as an inhibitor of glycoprotein processing; inhibitors of neuraminidase such as influenza virus neuraminidase inhibitors (e
• Such anti- viral agents may be administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of a viral infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of anti-C3b(i) antibodies to the animal.
• a viral infection e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of a viral infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of plasma to the animal.
• a viral infection e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of a viral infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of IgG antibodies, IgM antibodies and/or one or more complement components to the animal.
• a viral infection e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after
• anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of a viral infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of antibodies immunospecific for one or more viral antigens.
• a viral infection e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after
• Example of antibodies immunospecific for viral antigens include, but are not limited to, Synagis®, PRO542, Ostavir, and Protovir.
• the invention provides methods for the treatment or prevention of microbial infections in an animal, preferably a mammal and most preferably a human, said methods comprising the administration of a therapeutically or prophylactically effective amount of anti-C3b(i) antibodies or nucleic acid molecules encoding said antibodies.
• microbial infections which can be treated or prevented in accordance with this invention include, but are not limited to, yeast infections, fungal infections, protozoan infections and bacterial infections.
• Bacteria which cause microbial infections include, but are not limited to, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitidis, Corynebacterium diphtheriae , Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersiniapestis,
• a composition ofthe invention is administered to an animal to ameliorate one or more symptoms associated with a microbial infection or a disease or disorder resulting, directly or indirectly, from a microbial infection. In certain other embodiments, a composition ofthe invention is administered to reduce the number or microbes in an animal.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies is administered to an animal in order to ameliorate one or more symptoms associated with a microbial infection.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies is administered to an animal in order to reduce the number of microbes in an animal.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies and one or more antibodies immunospecific for one or more microbial antigens is administered to an animal in order to ameliorate one or more symptoms associated with a microbial infection.
• composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies and one or more antibodies immunospecific for one or more microbial antigens is administered to an animal in order to reduce the number of microbes in an animal.
• Anti-C3b(i) antibodies may be administered alone or in combination with other types of anti-microbial agents.
• anti-microbial agents include, but are not limited to: antibiotics such as penicillin, amoxicillin, ampicillin, carbenicillin, ticarcillin, piperacillin, cepalospolin, vancomycin, tetracycline, erythromycin, amphotericin B, nystatin, metronidazole, ketoconazole, and pentamidine.
• Such anti-microbial agents may be administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of a microbial infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of anti-C3b(i) antibodies to the animal.
• anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of a microbial infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after) or concomitantly with the administration of plasma to the animal.
• a mammal e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of a microbial infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of IgG antibodies, IgM antibodies and/or one or more complement components to the animal.
• one or more anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, for the prevention or treatment of a microbial infection prior to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), subsequent to (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week after), or concomitantly with the administration of antibodies immunospecific for one or more microbial antigens.
• Example of antibodies immunospecific for microbial antigens include, but are not limited to, antibodies immunospecific for LPS and capsular polysaccharide 5/8.
• animals with increased risk of a viral or bacterial infection are administered a composition ofthe invention. Examples of such animals include, but are not limited to human burn patients, infants, immunocompromised or immunodeficient humans, and the elderly.
• the present invention provides methods for the treatment or prevention of septic shock caused by lipopolysaccharide (LPS), an endotoxin, or a constituent ofthe outer cell wall of a gram negative bacteria, which can be released into the circulation.
• LPS lipopolysaccharide
• anti-C3b(i) antibodies are administered to an animal, preferably a mammal and most preferably a human, to treat or prevent septic shock.
• animals with increased risk of septic shock are administered a composition ofthe invention. Examples of such animals include, but are not limited to human burn patients, infants, immunocompromised or immunodeficient humans, and the elderly.
• one or more anti-C3(i) antibodies are administered to an animal, preferably a mammal and most preferably in human, prior to (e.g., 15 minuted, 30 minutes, 45 minutes, 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours before), subsequent to (e.g., 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours after) or concomitantly with any known technique for the treatment or prevention of septic shock in said animal.
• an animal preferably a mammal and most preferably in human, prior to (e.g., 15 minuted, 30 minutes, 45 minutes, 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours before), subsequent to (e.g., 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours after) or concomitantly with any known technique for the treatment or prevention of septic shock in said animal.
• septic shock examples include, but are not limited to, antithrombin, intravenous immunoglobulins, cytokine antagonists (e.g., anti-tumor necrosis factor (TNF) antibodies, soluble TNF receptor, anti-interleukin-1 (IL-1) antibodies, and soluble IL-1 receptor), antibiotics, and anti-inflammatory agents.
• cytokine antagonists e.g., anti-tumor necrosis factor (TNF) antibodies, soluble TNF receptor, anti-interleukin-1 (IL-1) antibodies, and soluble IL-1 receptor
• antibiotics e.g., anti-tumor necrosis factor (TNF) antibodies, soluble TNF receptor, anti-interleukin-1 (IL-1) antibodies, and soluble IL-1 receptor
• anti-inflammatory agents e.g., anti-tumor necrosis factor (TNF) antibodies, soluble TNF receptor, anti-interleukin-1 (IL-1) antibodies, and soluble IL-1 receptor
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies is administered to an animal in order to ameliorate one or more symptoms associated with septic shock.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies and IgG and/or IgM enriched plasma is administered to an animal in order to ameliorate one or more symptoms associated with septic shock.
• a composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies and one or more complement components is administered to an animal in order to ameliorate one or more symptoms associated with septic shock.
• composition comprising a therapeutically effective amount of one or more anti-C3b(i) antibodies and one or more antibodies immunospecific for lipopolysaccharide is administered to an animal in order to ameliorate one or more symptoms associated with septic shock.
• human anti-C3b(i) antibodies, derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis.
• the present invention provides methods for the treatment or prevention of cancer, viral infection, and microbial infection comprising administering nucleic acid molecules encoding one or more anti-C3b(i) antibodies.
• nucleic acids comprising sequences encoding one or more anti-C3b(i) antibodies are administered to treat or prevent cancer, viral infection or microbial infection, by way of gene therapy.
• nucleic acids comprising sequences encoding one or more anti-C3b(i) antibodies and one or more antibodies immunospecific for one or more cancer antigens are administered to treat or prevent cancer, by way of gene therapy.
• nucleic acids comprising sequences encoding one or more anti-C3b(i) antibodies and one or more antibodies immunospecific for one or more viral or microbial antigens are administered to treat or prevent viral or microbial infection, by way of gene therapy.
• nucleic acids comprising sequences encoding one or more anti-C3b(i) antibodies and one or more complement components are administered to treat or prevent cancer, viral infection, or microbial infection, by way of gene therapy.
• Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
• the nucleic acids produce their encoded protein that mediates a prophylactic or therapeutic effect. Any ofthe methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
• a composition comprising nucleic acid sequences encoding one or more anti-C3b(i) antibodies, one or more antibodies immunospecific for cancer antigens, viral antigens or microbial antigens, and/or one or more component components in an expression vector are administered to suitable hosts.
• the expression of nucleic acid sequences encoding anti-C3b(i) antibodies, antibodies immunospecific for cancer antigens, viral antigens or microbial antigens, and/or component components may be regulated by any inducible, constitutive, or tissue-specific promoter known to those of skill in the art.
• the nucleic acid to be introduced for pu ⁇ oses of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression ofthe nucleic acid is controllable by controlling the presence or absence ofthe appropriate inducer of transcription.
• nucleic acid molecules encoding the coding regions of anti-C3b(i) antibodies, antibodies immunospecific for cancer antigens, viral antigens or microbial antigens, and/or component components and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of said coding regions (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).
• nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
• the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product.
• This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retro irals or other viral vectors (see U.S. Patent No.
• microparticle bombardment e.g., a gene gun; Biolistic, Dupont
• coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc.
• nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
• the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al.); WO 92/22635 dated December 23, 1992 (Wilson et al.);
• nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al, 1989, Nature 342:435-438).
• viral vectors are used to express nucleic acid sequences.
• a retroviral vector can be used (see Miller et al, 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been to delete retroviral sequences that are not necessary for packaging ofthe viral genome and integration into host cell DNA.
• the nucleic acid sequences encoding the antibodies and/or complement components to be used in gene therapy are cloned into one or more vectors, which facilitates delivery ofthe gene into a patient.
• retroviral vectors More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
• Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al, 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.
• Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development
• adenovirus vectors are used to deliver antibodies and/or complement components to an animal in accordance with methods ofthe invention.
• Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Patent No. 5,436,146).
• AAV vectors are used to delivery antibodies and/or complement components to an animal in accordance with methods ofthe invention.
• Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
• the method of transfer includes the transfer of a
• the cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
• the nucleic acid is introduced into a cell prior to administration in vivo ofthe resulting recombinant cell.
• introduction can be carried out by any combination of
• the resulting recombinant cells can be delivered to a patient by various methods known in the art.
• Recombinant blood cells e.g. , hematopoietic stem or progenitor cells
• the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
• Cells into which a nucleic acid can be introduced for pu ⁇ oses of gene therapy encompass any desired, available cell type, and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells (e.g., T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, and granulocytes) and various stem or progenitor cells, in particular 0 hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
• the cell used for gene therapy is autologous to the patient.
• nucleic acid 5 sequences encoding anti-C3b(i) antibodies, antibodies immunospecific for cancer antigens, viral antigens or microbial antigens, and/or component components are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
• stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and 0 maintained in vitro can potentially be used in accordance with this embodiment ofthe present invention (see e.g.
• compositions ofthe invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
• in vitro assays to demonstrate the therapeutic or prophylactic utility of a composition include, Q the effect of a composition on a cell line, particularly one characteristic of a specific type of cancer, or a patient tissue sample.
• the effect ofthe composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays.
• Test compositions can be tested for their ability to augment activated immune cells by contacting ⁇ - activated immune cells with a test composition or a control composition and determining the ability ofthe test composition to modulate the biological activity ofthe activated immune cells.
• the ability of a test composition to modulate the biological activity of activated immune cells can be assessed by detecting the expression of cytokines or antigens, detecting the proliferation of immune cells, detecting the activation of signaling molecules, detecting the effector function of immune cells, or detecting the differentiation of immune
• cellular proliferation can be assayed by 3 H-thymidine inco ⁇ oration assays and trypan blue cell counts.
• Cytokine and antigen expression can be assayed, for example, by immunoassays including, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots,
• immunohistochemistry radioimmunoassays 10 immunohistochemistry radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and FACS analysis.
• the activation of signaling molecules can be assayed, for example, by
• T-cells 15 kinase assays and electromobility shift assays (EMS As).
• EMS As electromobility shift assays
• the effector function of T-cells can be measured, for example, by a 51c r -release assay (see, e.g., Palladino et al., 1987, Cancer Res. 47:5074-5079 and Blachere et al, 1993, J. Immunotherapy 14:352-356).
• Test composition can be tested for their ability to reduce tumor formation in patients (i.e., animals) suffering from cancer. Test compositions can also be tested for their ability
• Test compositions can also be tested for their ability to alleviate of one or more symptoms associated with cancer or an infectious disease (e.g., a viral or microbial infection). Test compositions can also be tested for their ability to decrease the time course ofthe infectious
• test compositions can be tested for their ability to increase the survival period of patients suffering from cancer or an infectious disease (e.g., a viral or microbial infection). Techniques known to those of skill in the art can be used to analyze or test the to function ofthe test compositions in patients.
• infectious disease e.g., a viral or microbial infection.
• TM determine whether administration of a specific composition is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a composition, and the effect of such composition upon the tissue sample is observed.
• compositions for use in therapy can be tested in suitable animal model
• 35 systems prior to testing in humans including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
• any animal model system known in the art may be used.
• compositions ofthe invention provide methods of preventing and treating cancer, viral infection, microbial infection, and septic shock by administrating to an animal (e.g., cows, pigs, horses, chickens, cats, dogs, humans, etc.) an effective amount of a composition ofthe invention.
• an animal e.g., cows, pigs, horses, chickens, cats, dogs, humans, etc.
• compositions ofthe invention are administered to ⁇ Q human burn patients, infants, immunocompromised or immunodeficient humans, or the elderly.
• Compositions ofthe invention include any one ofthe following or a combination of any ofthe following: one or more anti-C3b(i) antibodies, one or more antibodies immunospecific for one or more cancer antigens, one or more antibodies immunospecific for one or more viral antigens, one or more antibodies immunospecific for one or more
• a composition ofthe invention is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
• compositions of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a Q retroviral or other vector, etc.
• Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
• compositions may be administered by any convenient route, for example by infusion or bolus injection, by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered 5 together with other biologically active agents. Administration can be systemic or local.
• Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
• compositions ofthe invention may be desirable to administer the compositions ofthe invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
• the composition can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
• the composition can be delivered in a controlled release or sustained release system.
• a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al, 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321 :574).
• polymeric materials can be used in a controlled release system (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190 ; During et al., 1989, Ann. Neurol. 25:351;
• a controlled release system can be placed in proximity ofthe therapeutic target (e.g., the brain, kidney, stomach, pancreas, and lung), thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
• the therapeutic target e.g., the brain, kidney, stomach, pancreas, and lung
• Other controlled release systems are discussed in the review by Langer (1990,
• the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g. , by use of a retroviral vector (see U.S. Patent No.
• a nucleic acid can be introduced mtracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination.
• compositions comprise a prophylactically or therapeutically effective amount of one or more anti-C3b(i) antibodies, one or more antibodies immunospecific for one or more cancer antigens, one or more antibodies immunospecific for one or more viral antigens, one or more antibodies immunospecific for one or more microbial antigens, one or more complement components, or a combination thereof, and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
• Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
• Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E.W. Martin.
• Such compositions will contain a therapeutically effective amount ofthe compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
• the formulation should suit the mode of administration.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• pharmaceutical compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the pharmaceutical composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site ofthe injection.
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• compositions ofthe invention can be formulated as neutral or salt forms.
• Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
• compositions ofthe invention which will be effective in the treatment or prevention of cancer, viral infection, or microbial infection can be determined by standard clinical techniques.
• in vitro assays may optionally be employed to help identify optimal dosage ranges.
• the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness ofthe disease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances.
• suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight.
• Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
• Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
• the preferred dosage is 0J 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 is often possible.
• Labeled antibodies, derivatives and analogs thereof, which specifically bind to C3b(i) can be used for diagnostic pu ⁇ oses to detect, diagnose, or monitor cancer.
• anti-C3b(i) antibodies which immunospecifically bind to C3b(i) covalently bound to IgG or IgM antibodies, or C3b(i) covalently bound to a cancer cell are used for diagnostic pu ⁇ oses to detect, diagnosis, or monitor cancer.
• cancer is detected in the patient.
• the patient is an animal, preferably a mammal and most preferably a human.
• diagnosis is carried out by: a) administering to an animal an effective amount of a labeled molecule which immunospecifically binds to C3b(i) or C3b(i) covalently linked to a second molecule; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at any cancerous site in the animal (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates the presence of cancer.
• diagnosis is carried out by: a) administering to an animal an effective amount of a labeled molecule which immunospecifically binds to C3b(i) or C3b(i) covalently linked to a second molecule prior to, subsequent to, or concomitantly with the administration of IgG antibody, IgM antibody, plasma, one or more complement components, and/or one or more antibodies which immunospecifically bind to a cancer cell antigen; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at any cancerous site in the animal (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates the presence of cancer.
• Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
• the invention provides methods for the diagnosis or detection of cancer in an animal, said methods comprising imaging said animal at a time interval after administering to said animal an effective amount of a labeled antibody which immunospecifically binds to C3b(i) or C3b(i) covalently linked to a second molecule, said time interval being sufficient to permit the labeled antibody to preferentially concentrate at any cancerous site in said animal, wherein detection ofthe labeled antibody localized at the site in the subject indicates the presence of cancer.
• the invention also provides methods for the diagnosis or detection of cancer in an animal, said methods comprising imaging said animal at a time interval after administering to said animal an effective amount of a labeled antibody which immunospecifically binds to C3b(i) or C3b(i) covalently linked to a second molecule prior to, subsequent to, or concomitantly with the administration of IgG antibody, IgM antibody, plasma, one or more complement components, and/or one or more antibodies which immunospecifically bind to a cancer cell antigen, said time interval being sufficient to permit the labeled antibody to preferentially concentrate at any cancerous site in said animal, wherein detection ofthe labeled antibody localized at the site in the subject indicates the presence of cancer.
• the time interval following the administering for permitting the labeled molecule to preferentially concentrate at any cancerous site in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
• monitoring ofthe cancer is carried out by repeating the method for diagnosing the cancer, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
• the density of a tumor facilitates the detection of said tumor using anti-C3b(i) antibodies in accordance with the methods of the invention.
• Presence ofthe labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods ofthe invention include but are not limited to: computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
• CT computed tomography
• PET position emission tomography
• MRI magnetic resonance imaging
• sonography sonography
• the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Patent 5,441,050).
• the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument.
• the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography.
• the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
• MRI magnetic resonance imaging
• kits that can be used in the above methods.
• a kit comprises one or more antibodies immunospecific for C3b(i) or C3b(i) linked (e.g., covalently linked) to a second molecule, in one or more containers.
• a kit comprises one or more antibodies immunospecific for C3b(i) or C3b(i) linked (e.g., covalently linked) to a second molecule and IgM antibody, in one or more containers.
• a kit comprises one or more antibodies immunospecific for C3b(i) or C3b(i) linked (e.g. , covalently linked) to a second molecule and IgG antibody, in one or more containers.
• kits comprises one or more antibodies immunospecific for C3b(i) or C3b(i)linked (e.g., covalently linked) to a second molecule and one or more complement components, in one or more containers.
• a kit comprises one or more antibodies immunospecific for C3b(i) or C3b(i) linked (e.g., covalently linked) to a second molecule and one or more antibodies immunospecific for one or more cancer cell antigens, viral antigens, or microbial antigens, in one or more containers.
• kits comprises one or more antibodies immunospecific for C3b(i) or C3b(i) linked (e.g., covalently linked) to a second molecule, IgM or IgG antibody, and one or more complement components in one or more containers.
• a kit comprises one or more antibodies immunospecific for C3b(i) or C3b(i) linked (e.g., covalently linked) to a second molecule, one or more antibodies immunospecific for one or more cancer cell antigens, viral antigens or microbial antigens, and one or more complement components, in one or more containers.
• kits ofthe present invention contain anti-C3b(i) antibodies which immunospecifically bind to C3b(i) covalently bound to IgG or IgM antibody, or C3b(i) covalently bound to a cancer cell, virus, or microbe.
• the kits ofthe invention contain instructions for the use of the antibodies for the treatment, prevention or diagnosis of cancer, viral infections, or microbial infections.
• C3b(i) can function as a tumor-specific antigen.
• Antibodies specific for C3b(i) can be utilized to target tumor cells for the delivery of therapeutic or diagnostic agents, including cytotoxic, chemotherapeutic, immune-enhancing drugs, radioactive compounds, genetic material and immune effector cells.
• LNCaP and lineage-derived C4-2 human prostate cancer cell lines were utilized in this example to demonstrate the use of C3b(i) as a target for immunotherapy.
• the LNCaP/C4-2 progression model recapitulates progression of human neoplastic prostate disease from an androgen-responsive and minimally metastatic (LNCaP cells) to an androgen-refractory (defined as being able to proliferate in castrate hosts) and highly aggressive phenotype (C4-2 subline) (Thalmann et al, 1994, Cane. Res. 54:2577-81; Chung et al, 1996, Urol. Oncol. 2:99-128; Hyytinen et al, 1997, Br. J. Cane. 75:190-5). It shares remarkable similarities with clinical human prostate cancer both in its genotypic and phenotypic changes.
• LNCaP/C4-2 progression model has been shown to be a powerful tool for evaluating anti-prostate cancer therapeutic approaches both in vitro and in vivo (Chung et al., 1997, Acta Urol. Jap. 43:815-20), especially with regard to hormone-refractory disease, for which few effective or durable treatment options currently exist (Scher et al., 1994, Sem Oncol 21:630-56).
• LNCaP American Type Culture Collection, Rockville, MD
• C4-2 Urocor, Oklahoma City, OK
• human prostate cancer cell lines were maintained in T-media with 5% heat inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY). Cultures were maintained at 37 °C in humidified 5% CO 2 , split and harvested at 80 to 90% confluence, and treated, if applicable, at 25% confluence. Cells were collected using either phosphate buffered saline (PBS) with 2.5 mM ethylenediitrilotetraacetic acid (EDTA; Sigma, St. Louis, MO) or trypsin (Gibco, Grand Island, NY) diluted 1:10 in phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• EDTA ethylenediitrilotetraacetic acid
• trypsin Gabco, Grand Island, NY
• Serum samples were obtained with written informed consent from normal male and female volunteers (University of Virginia, Charlottesville, VA) and from men being followed for prostate disease (University of Virginia and Eastern Virginia Medical School, Norfolk, VA). Prostate disease patients had pathologic documentation of either benign or neoplastic prostate disease. Blood was drawn into SST gel and clot activator Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ), held at room temperature for 30 min, and then centrifuged for 20 min at 700 X g to obtain serum which was stored at -80°C.
• Alternative opsonization procedures included addition of 10 mM EDTA to sera to block all complement activation (or use of EDTA-containing plasma), addition of 10 mM ethylene glycol tetraacetic acid (EGTA) and 5 mM Mg (Mg-EGTA) to allow only alternative pathway activation, use of purified IgM (1 mg/ml, Sigma, St. Louis, MO), or use of IgM-depleted serum.
• IgM was removed from normal human sera (NHS) by incubating 2.5 ml of serum with 1.65 ml (settled volume) anti-human IgM agarose (Sigma, St. Louis, MO) on ice for 1 hr with gentle shaking.
• the depleted serum was separated from the agarose by centrifugation at 1600 X g and then stored at -80 °C.
• ELISA determinations demonstrated that >90% ofthe human IgM was specifically removed from the serum by this procedure, but the level of human IgG was reduced by less than 10%.
• CR1 complement receptor 1
• Anti-C3b(i) mAb 3E7 (IgGl), which bound to a different epitope and was not blocked by the other anti-C3b(i) mAb, was prepared by our previously described methods (Tosic et al., 1989, J. Immunol. Methods 120:241). The specificity of mAb 3E7 for C3b(i) was confirmed by indirect flow cytometry as follows. Both mAb 3E7 and 7C12 bound to serum-opsonized pig erythrocytes, as revealed by fluorescein-isothiocyanate(FITC)-labeled anti-mouse IgG.
• FITC fluorescein-isothiocyanate
• a bispecific mAb complex (a heteropolymer) was prepared by cross-linking mAb 3E7 with mAb 7G9 (Segal et al, 1995, Curr Protocol Immunol 2:131; and Taylor et al., 1997, J. Immunol. 97:842).
• Alternative opsonization procedures included addition of 10 mM EDTA to sera or the use of EDTA-containing plasma to block all complement activation, addition of 10 mM [ethylenebis (oxonitrilo)]-N,N,N'N' -tetraacetic acid (EGTA) and 5 mM Mg +2 (MgEGTA) to allow only alternative pathway activation, the use of purified IgM (1 mg/ml, Sigma, St. Louis, MO), or the use of IgM-depleted serum. In the latter case, IgM was removed from NHS by incubating 2.5 ml serum with 1.65 ml (settled volume) anti-(human IgM) linked to agarose (Sigma, St.
• naive cancer cells were incubated with the 125 I-Iabeled mAb.
• the level of binding ofthe mAb to the cancer cells was then determined by centrifuging the sample through oil and measuring radioactive counts in the cell pellets (Ross et al, 1985, J. Immunol. 135:2005).
• 1 5 I-labeled mAb 3E7 or 7C12 was added directly to the incubation mixture containing cancer cells and NHS. After incubation at 37 °C, the cells were spun through oil to measure mAb binding. Rosette experiments
• Anti-C3b(i) mAb 2H1 1 (which recognizes the same epitope bound by mAb 7C12 (Tosic et al., 1989, J. Immunol. Methods 120:241) or mAb 3E7 was covalently coupled to amine-terminated BioMag particles (Polysciences Inc., Warrington, PA) following the manufacturer's instructions. Cancer cells were labeled with 51 Cr (Ferguson et al., 1995, Arthritis Rlieum 38: 190), opsonized with an equal volume of NHS, washed three times and then subjected to the magnetic purging protocols (see below).
• 51 Cr-labeled cancer cells For experiments with blood cells in serum, blood was collected into EDTA, the cells were washed three times and reconstituted into serum (or into plasma/EDTA as a control), and then 51 Cr-labeled cancer cells were added.
• 51 Cr-labeled cancer cells (approx. 50,000 cells) were first mixed with aliquots of whole blood anti-coagulated in EDTA (complement activation is prevented) or in citrate (partially permissive for complement activation). After opsonization the samples were washed three times. More than 80%) of the 51 Cr label was retained by the cancer cells in all opsonization procedures.
• CD14 positive cells were identified by dual gating on CD45 and CD14 (FITC, Caltag, Burlingame, Calif., MHCDI401, isotype control MG2a01). In these measurements FluorCount Fluorospheres (Coulter, Miami, Fla., PN 2547053) were used to normalize sample volumes.
• LNCaP and C4-2 prostate cancer cell lines were evaluated as follows. 1 X 10 6 cells of each prostate cancer cell line were opsonized with 25%) NHS (diluted in BSA-PBS) or maintained in BSA-PBS at 37°C for 30 min. After washing twice with PBS, either 2 ug or 200 ng of 131 I-labeled 7C12+8E11 or 131 I-labeled irrelevant mAb (diluted in BSA-PBS) was added to each set of cells and incubated at room temperature for 30 min. The cells were washed twice with PBS, and plated in triplicate in 24- well tissue culture plates (Fisher Scientific, Pittsburgh, PA) in T-media + 5% FBS at 3 XI 0 4 cells per well.
• the plates were then placed in a humidified environment at 37 °C with 5% CO 2 . A single media change was performed on day 3. On 5 (LNCaP) and 6 (C4-2) subsequent days, beginning 24 hr after mAb treatment, the triplicate wells were harvested to evaluate cell killing by comparing differences in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) (Sigma, St. Louis, MO) assay results.
• MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide
• IgM is more readily revealed on the cancer cells when the experiment is conducted under conditions (Mg-EGTA) that block the classical pathway of complement activation, as C3b deposition via the classical pathway seems to partially block epitopes on IgM (see Table 1, below).
• the flow cytometry results demonstrate that, after opsonization with serum from a prostate cancer patient, significantly less C3b(i) and IgM are deposited on the tumor cells (FIG. IC, D). It is noteworthy, however, that C3b(i) deposition via the alternative pathway (Mg-EGTA-treated serum) is comparable for both the normal and cancer patient serum, suggesting that the alternative pathway of complement activation remains intact in serum from prostate cancer patients.
• IgM binding promotes robust cancer cell opsonization with C3b(T)
• C3b fragments on human IgM in immune complexes has been demonstrated in several reports (Mehta et al., 1986, J. Immunol. 136:1765; Taylor et al, 1989, J. Immunol. 143:3626; and Thornton et al, 1996, Clin. Exp. Immunol. 104:531). Therefore, some C3b(i) is complexed to the IgM on the cancer cell, and it is likely that C3b(i) is also covalently attached to glycoproteins and glycolipids on the cancer cell.
• the representative data in FIG. 1 indicates that the serum from a man with prostate cancer was less effective than NHS in depositing C3b(i) on cancer cells.
• mAb specific for a cancer cell antigen is cross-linked with r a mAb specific for an effector site (e.g. , Fc receptors on monocytes/macrophages, granulocytes, or natural killer cells)
• an effector site e.g. , Fc receptors on monocytes/macrophages, granulocytes, or natural killer cells
• Segal DM Bakacs T, Jost CR, Kuruez I, Sconocchia G, Titus JA (1995) T-cell targeted cytotoxicity.
• Fanger MW ed Bispecific antibodies. Landis. Austin, Tex, p 27-42).
• immunocompetent cells can be delivered directly, and specifically, to a tumor ⁇ Q via the guidance ofthe anti tumor mAb.
• a prototype for this approach was examined by testing whether human erythrocytes could bind to C3b(i)-opsonized cancer cells through bispecific mAb complexes (heteropolymers) specific for C3b(i) and the primate erythrocyte complement receptor (CR1).
• bispecific mAb complexes heteropolymers
• CR1 primate erythrocyte complement receptor
• Complement-opsonized cancer cells can be purged by immunomagnetic methods
• Table 4 Flow-cytometric analyses ofthe effects of purging on populations of T lymphocytes, granulocytes and monocytes.
• Whole blood anti-coagulated with citrate or EDTA was used or washed blood cells were reconstituted to 50% hematocrit in serum.
• Radiolabeled anti-C3b(i) mAbs can kill prostate cancer cells in vitro
• cancer-specific mAbs involves the coupling of radioactive agents to the mAbs to allow for the imaging or destruction of tumors (Glennie MJ, French RR. Targeting drugs, toxins, and radionuclides with bispecific antibodies. In: Fanger MW, editor. Bispecific Antibodies, ed. Austin: RG Landis Co.; 1995, p. 107-20). The potential of this approach was examined by labeling anti-C3b(i) mAbs with 131 I, and then testing their
• CD59 (protectin) by cancer cells blocks the action ofthe membrane attack complex which might otherwise lyse the cancer cell.
• the results presented herein demonstrate that one approach to treating cancer is to infuse a patient with normal human plasma (to supply IgM and, if necessary, complement) and to then deliver systemically anti-neoplastic agents to the cancer cells by conjugating the agents to anti-C3b(i) mAbs, which would circulate through the body and home to sites of opsonized tumor cells.
• mAb-based immunotherapy for cancer requires a very high level of selective and high avidity binding ofthe mAb to the tumor.
• the results indicate that at least 50,000 C3b(i) epitopes are available on opsonized prostate cancer cells and, based on the in vitro killing studies, this level of cancer-associated antigen should be sufficient for specific targeting ofthe cancer cell, enabling the delivery of abundant therapeutic or diagnostic agents.
• C3b(i) Tumor tissue-specific delivery of therapeutic agents is crucial to avoid undesirable injury to healthy tissue.
• C3b(i) as a target, it is important that complement activation be limited to tumor cells. Except for a few relatively rare disease conditions (Rosse et al., 1995, Blood 86;3277-86; Morgan BP. Complement: clinical aspects and relevance to disease, ed. London: Harcourt Brace Jovanovich; 1990), the complement system is highly regulated and C3b(i) is not deposited on normal tissue.
• C3b(i) deposition has been shown to be confined to areas of malignancy in human prostate tissue specimens, and is absent in benign (FIG. 5A) and hype ⁇ lastic regions (not shown).
• C3b(i) covalently linked directly to IgM or to a carbohydrate on the cancer cell should, however, contain unique and specific antigenic determinants different from those expressed by C3b fragments in solution, and it should be possible to generate mAb specific for such cell-associated molecules.
• mAb 3E7 which was able to bind to the cancer cells during the opsonization step (Fig. 3), did not block purging with the 2H11 beads (Table 2). It is likely that other mAb can be developed that will demonstrate even greater binding specificity for C3b(i) covalently bound to the cancer cells.
• C3b(i) targeting may provide a powerful orthogonal approach. Use of these procedures in tandem with C3b(i)-based targeting may allow for highly efficacious purging of cancer cells from bone marrow or peripheral blood stem cells before autologous transplantation.
• anti-C3b(i) mAbs coupled with toxic agents or radioisotopes can be administered to individuals.
• FIG. 7 When LNCaP and C4-2 cells were treated with 13 T-labeled specific anti-C3b(i) mAbs, only those cells that had been opsonized with NHS prior to treatment with the 131 I-anti-C3b(i) mAbs were killed (FIG. 7).
• This approach can also be utilized for diagnostic imaging pu ⁇ oses, similar to the PROSTASCINTTM scan, when tumor cell deposits are effectively opsonized and then targeted with anti-C3b(i) mAb-conjugated compounds (Petronis et al., 1998, Clin. Nuc. Med. 23:672-7).
• 51 Cr-labeled Raji cells (model for B cell lymphoma) were dispersed in either serum or in serum containing 0.01 M EDTA (which blocks complement activation). They were incubated for 30 minutes at 37 °C in the presence or absence of 5
• 51 Cr-labeled Raji cells were added to washed whole blood which was reconstituted in fresh serum. The samples were incubated for 30 minutes at
• aeruginosa It is important to note that only a fraction ofthe bacteria bind to the erythrocytes, and with time binding actually decreases (see Table 5).
• One possible explanation for the decrease is that C3b bound to the bacteria is converted to further breakdown products, C3bi and C3dg, and the primate erythrocyte complement receptor does not bind these ligands with any measurable affinity.
• bispecific antibodies for CRl and C3b(i) enhance the ability of Pseudomonas aeruginosa to bind to erythrocytes in the presence of serum.
• Green fluorescent (GP) Pseudomonas aeruginosa was incubated for varying periods of time at 37°C with 7G9x2H4 (a bispecific monoclonal antibody for complement receptor CRl and a Pseudomonas aeruginosa antigen), 7G9x7C12 (a bispecific monoclonal antibody for complement receptor CRl and C3b(i)), 7G9x3E7 (a bispecific monoclonal antibody for complement receptor CRl and C3b(i)), or no heteropolymer (HP) and normal human serum containing human erythrocytes at a 30-50 %> hematocrit.
• 7G9x2H4 a bispecific monoclonal antibody for complement receptor CRl and a Pseudomonas aeruginosa antigen
• 7G9x7C12 a bispecific monoclonal antibody for complement receptor CRl and C3b(i)

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Immunology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Biochemistry (AREA)
• Oncology (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Cell Biology (AREA)
• Epidemiology (AREA)
• Reproductive Health (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Communicable Diseases (AREA)
• Physics & Mathematics (AREA)
• Optics & Photonics (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Gynecology & Obstetrics (AREA)
• Pregnancy & Childbirth (AREA)
• Hematology (AREA)
• Virology (AREA)
• Urology & Nephrology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (2)

Family

ID=24911153

Family Applications (1)

Country Status (6)

Families Citing this family (90)

Family Cites Families (11)

• 2000
  • 2000-11-28 US US09/724,620 patent/US6572856B1/en not_active Expired - Fee Related
• 2001
  • 2001-11-28 EP EP01986051A patent/EP1355563A4/en not_active Withdrawn
  • 2001-11-28 WO PCT/US2001/044813 patent/WO2002043570A2/en not_active Ceased
  • 2001-11-28 CA CA002429987A patent/CA2429987A1/en not_active Abandoned
  • 2001-11-28 JP JP2002545557A patent/JP2004520287A/ja active Pending
  • 2001-11-28 AU AU2002236519A patent/AU2002236519A1/en not_active Abandoned

Also Published As

Similar Documents

Legal Events