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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—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

Definitions

• the invention relates to a synergistic combination antibody therapy for treatment of B cell malignancies, especially B cell lymphomas and leukemias.
• This synergistic antibody combination comprises at least one antibody having substantial B cell depleting activity (e.g., an anti-CD19, CD20, CD22 or CD37 antibody) and an antibody that modulates or regulates the immune system, e.g., by modulating B cell/T cell interactions and/or B cell activity, differentiation or proliferation (e.g., anti-B7, anti-CD40, anti-CD23 or anti-CD40L ).
• the invention encompasses combination antibody therapies for CD40+ malignancies, which include using anti- CD40L antibodies to prevent CD40L from binding to CD40.
• antibodies or other agents which can inhibit CD40/CD40L interaction further can be combined with chemotherapeutics, radiation and/or other antibodies, preferably B cell depleting antibodies, e.g., anti-CD19, anti-CD20, anti-CD22 and/or anti-CD40 antibodies, or fragments thereof.
• the immune system of vertebrates (for example, primates, which include humans, apes, monkeys, etc.) consists of a number of organs and cell types which have evolved to: accurately and specifically recognize foreign microorganisms ("antigen") which invade the vertebrate-host; specifically bind to such foreign microorganisms; and, eliminate/destroy such foreign microorganisms.
• Lymphocytes as well as other types of cells, are critical to the immune system and to the elimination and destruction of foreign microorganisms. Lymphocytes are produced in the thymus, spleen and bone marrow (adult) and represent about 30% of the total white blood cells present in the circulatory system of humans (adult).
• T cells are responsible for cell mediated immunity, while B cells are responsible for antibody production (humoral immunity).
• B cells are responsible for antibody production (humoral immunity).
• T cells and B cells can be considered interdependent — in a typical immune response, T cells are activated when the T cell receptor binds to fragments of an antigen that are bound to major histocompatability complex ("MHC") glycoproteins on the surface of an antigen presenting cell; such activation causes release of biological mediators (“interleukins” or "cytokines”) which, in essence, stimulate B cells to differentiate and produce antibody (“immunoglobulins”) against the antigen.
• MHC major histocompatability complex
• Each B cell within the host expresses a different antibody on its surface— thus one B cell will express antibody specific for one antigen, while another B cell will express antibody specific for a different antigen. Accordingly, B cells are quite diverse, and this diversity is critical to the immune system. In humans, each B cell can produce an enormous number of antibody molecules (i.e., about 10 7 to 10 8 ). Such antibody production most typically ceases (or substantially decreases) when the foreign antigen has been neutralized. Occasionally, however, proliferation of a particular B cell will continue unabated; such proliferation can result in a cancer referred to as "B cell lymphoma.”
• Non-Hodgkin's lymphoma is one type of lymphoma that is characterized by the malignant growth of B lymphocytes. According to the American Cancer Society, an estimated 54,000 new cases will be diagnosed, 65% of which will be classified as intermediate- or high-grade lymphoma. Patients diagnosed with intermediate-grade lymphoma have an average survival rate of two to five years, and patients diagnosed with high-grade lymphoma survive an average of six months to two years after diagnosis.
• B cell malignancies e.g., B cell lymphomas and leukemias may be successfully treated using antibodies specific to B cell antigens that possess B cell depleting activity.
• B cell antibodies that have been reported to possess actual or potential application for the treatment of B cell malignancies include antibodies specific to CD20, CD19, CD22, CD37 and CD40.
• CD20 is a cell surface antigen expressed on more than 90% of B-cell lymphomas, which does not shed or modulate in the neoplastic cells (McLaughlin et al, J. Clin. Oncol 16: 2825-2833 (1998b)).
• the CD20 antigen is a non-glycosylated, 35 kDa B-cell membrane protein involved in intracellular signaling, B-cell differentiation and calcium channel mobilization (Clark et al, Adv. Cancer Res. 52: 81-149 (1989); Tedder et al, Immunology Today 15: 450-454 (1994)).
• the antigen appears as an early marker of the human B-cell lineage, and is ubiquitously expressed at various antigen densities on both normal and malignant B-cell populations.
• the antigen is absent on fully, mature B-cells (e.g., plasma cells), early B- cell populations and stem cells, making it a suitable target for antibody mediated therapy.
• Anti-CD20 antibodies have been prepared for use both in research and therapeutics.
• One anti-CD20 antibody is the monoclonal Bl antibody (U.S. Patent No. 5,843,398).
• Anti-CD20 antibodies have also been prepared in the form of radionuclides for treating B-cell lymphoma (e.g., I- labeled anti-CD20 antibody), as well as a 89 Sr-labeled form for the palliation of bone pain caused by prostate and breast cancer metastasises (Endo, Gan To Kagaku Ryoho 26: 744-748 (1999)).
• a murine monoclonal antibody, 1F5 was reportedly administered by continuous intravenous infusion to B-cell lymphoma patients.
• non-human monoclonal antibodies typically lack human effector functionality, e.g., they are unable to, inter alia, mediate complement dependent lysis or lyse human target cells through antibody-dependent cellular toxicity or Fc-receptor mediated phagocytosis.
• non-human monoclonal antibodies can be recognized by the human host as a foreign protein; therefore, repeated injections of such foreign antibodies can lead to the induction of immune responses leading to harmful hypersensitivity reactions.
• murine-based monoclonal antibodies this is often referred to as a Human Anti-Mouse Antibody response, or "HAMA” response.
• HAMA Human Anti-Mouse Antibody response
• these "foreign" antibodies can be attacked by the immune system of the host such that they are, in effect, neutralized before they reach their target site.
• RITUXAN® (also known as Rituximab, MabThera®, IDEC- C2B8 and C2B8) was the first FDA-approved monoclonal antibody and was developed at IDEC Pharmaceuticals (see U.S. Patent Nos. 5,843,439; 5,776,456 and 5,736,137) for treatment of human B-cell lymphoma (Reff et al, Blood 83: 435-445 (1994)).
• RITUXAN® is a chimeric, anti-CD20 monoclonal (MAb) which is growth inhibitory and reportedly sensitizes certain lymphoma cell lines for apoptosis by chemotherapeutic agents in vitro (Demidem et al, Cancer Biotherapy & Radiopharmaceuticals 12: 177- (1997)). RITUXAN® also demonstrates anti-tumor activity when tested in vivo using murine xenograft animal models. RITUXAN® efficiently binds human complement, has strong FcR binding, and can efficiently kill human lymphocytes in vitro via both complement dependent (CDC) and antibody- dependent (ADCC) mechanisms (Reff et al, Blood 83: 435-445 (1994)). In macaques, the antibody selectively depletes normal B-cells from blood and lymph nodes.
• MAb monoclonal
• RITUXAN® has been recommended for treatment of patients with low-grade or follicular B-cell non-Hodgkin's lymphoma (McLaughlin et al, Oncology (Huntingt) 12: 1763-1777 (1998a); Maloney et al, Oncology 12: 63-76 (1998); Leget et al, Curr. Opin. Oncol. 10: 548-551 (1998)).
• RITUXAN® has been approved for therapy of relapsed stage III/ ⁇ V follicular lymphoma (White et al, Pharm. Sci.
• FCC follicular center cell lymphoma
• MCL mantle cell lymphoma
• DLCL diffuse large cell lymphoma
• RITUXAN® cyclophosphamide, vincristine, prednisone and doxorubicin
• CHOP e.g., cyclophosphamide, vincristine, prednisone and doxorubicin
• RITUXAN® has exhibited minimal toxicity and significant therapeutic activity in low-grade non-Hodgkin's lymphomas (NHL) in phase I and II clinical studies (Berinstein et al., Ann. Oncol. 9: 995-1001 (1998)).
• RITUXAN® which was used alone to treat B-cell NHL at weekly doses of typically 375 mg/M 2 for four weeks with relapsed or refractory low-grade or follicular NHL, was well tolerated and had significant clinical activity (Piro et al, Ann. Oncol. 10: 655-61 (1999); Nguyen et al, (1999); and Coiffier et al, Blood 92: 1927-1932 (1998)). However, up to 500 mg/M 2 of four weekly doses have also been administered during trials using the antibody (Maloney et al, Blood 90: 2188-2195- (1997)).
• RITUXAN® also has been combined with chemotherapeutics, such as CHOP (e.g., cyclophosphamide, doxorubicin, vincristine and prednisone), to treat patients with low-grade or follicular B-cell non-Hodgkin's lymphoma (Czuczman et al, J. Clin. Oncol. 17: 268-76 (1999); and McLaughlin et al, (1998a)).
• CHOP e.g., cyclophosphamide, doxorubicin, vincristine and prednisone
• CD40 is expressed on the cell surface of mature B-cells, as well as on leukemic and lymphocytic B-cells, and on Hodgkin's and Reed-Sternberg (RS) cells of Hodgkin's Disease (HD) (Valle et al, Eur. J. Immunol. 19: 1463-1467 (1989); and Grass et al., Leuk. Lymphoma 24: 393-422 (1997)).
• RS Reed-Sternberg
• CD40 is a B-cell receptor leading to activation and survival of no ⁇ nal and malignant B-cells, such as non-Hodgkin's follicular lymphoma (Johnson et al, Blood 82: 1848-1857 (1993); and Metkar et al, Cancer Immunol Immunoiher. 47: 104 (1998)). Signaling through the CD40 receptor protects immature B-cells and B-cell lymphomas from IgM- or Fas-induced apoptosis (Wang et al, J. Immunology 155: 3722-3725 (1995)).
• CD40 stimulation may inhibit neoplastic B-cell growth both in vitro (Funakoshi et al, Blood 83: 2787-2794 (1994)) and in vivo (Murphy et al, Blood 86: 1946-1953 (1995)).
• Anti-CD40 antibodies (see U.S. Patent Nos. 5,874,082 and 5,667,165) administered to mice increased the survival of mice with human B-cell lymphomas (Funakoshi et al, (1994); and Tutt et al., J. Immunol. 161: 3176-3185 (1998)).
• Methods of treating neoplasms, including B-cell lymphomas and EBV-induced lymphomas using anti-CD40 antibodies mimicking the effect of CD40L and thereby delivering a death signal are described in U.S. Patent No. 5,674,492 (1997), which is herein incorporated by reference in its entirety.
• CD40 signaling has also been associated with a synergistic interaction with CD20 (Ledbetter et al.
• a CD40 ligand, gp39 (also called CD40 Ugand, CD40L or CD154), is expressed on activated, but not resting, CD4 + Th cells (Spriggs et al, J. Exp. Med. 176: 1543-1550 (1992); Lane et al, Eur. J. Immunol.
• CD40 and CD40L have been cloned and characterized (Stamenkovi et al, EMBO J. 8: 1403-1410 (1989); Armixage et al, Nature 357: 80-82 (1992); Lederman et al, J. Exp. Med. 175: 1091-1101 (1992); and Hollenbaugh et al, EMBO J. 11: 4313-4321 (1992)).
• Human CD40L is also described in U.S. Patent No. 5,945,513.
• CD40L may play an important role in the cell contact-dependent interaction of tumor B-cells (CD40 + ) within the neoplastic follicles or Reed-Sternberg cells (CD40 + ) in Hodgkin's Disease areas (Carbone et al, Am. J. Pathol 147: 912-922 (1995)).
• Anti-CD40L monoclonal antibodies reportedly have been effectively used to inhibit the induction of murine AIDS (MAIDS) in LP- BM5-infected mice (Green et al, Virology 241 : 260-268 (1998)).
• MAIDS murine AIDS
• LP- BM5-infected mice Green et al, Virology 241 : 260-268 (1998).
• CD40L-CD40 signaling leading to survival versus cell death responses of malignant B-cells is unclear.
• APO-2L a apoptosis inducing TRAIL molecule
• Anxi-CD40 antibodies have also been prepared to prevent or treat antibody- mediated diseases, such as allergies and autoimmune disorders as described in U.S. Patent No. 5,874,082 (1999).
• Anti-CD40 antibodies reportedly have been effectively combined with anti-CD20 antibodies yielding an additive effect in inliibiting growth of non-Hodgkin's B-cell lymphomas in cell culture (Benoit et al,
• Anti-CD40L antibodies include monoclonal antibodies 3E4, 2H5, 2H8, 4D9-8, 4D9- 9, 24-31, 24-43, 89-76 and 89-79, as described in U.S. Patent No. 5,7474,037 (1998), and anti-CD40L antibodies described in U.S. Patent No. 5,876,718 (1999) used to treat graft-versus-host-disease.
• CD22 is a B-cell-specific molecule involved in B-cell adhesion that may function in homotypic or heterotypic interactions (Stamenkovic et al, Nature AA:1 (1990); Wilson et al, J. Exp. Med. 173:137 (1991); Stamenkovic et al, Cell 66:1133 (1991)).
• the CD22 protein is expressed in the cytoplasm of progenitor B and pre-B-cells (Dorken et al, J. Immunol 136:4470 (1986); Dorken et al, "Expression of cytoplasmic CD22 in B-cell ontogeny.
• CD22 is expressed by follicular mantle and marginal zone B-cells but only weakly by germinal center B-cells (Dorken et al, J. Immunol 136:4470 (1986); Ling et al, "B-cell and plasma antigens: new and previously defined clusters" In Leukocyte Typing 111. White Cell Differentiation Antigens, McMichael et al, eds., Oxford University Press, Oxford, p. 302 (1987)).
• in situ hybridization reveals the strongest expression of CD22 mRNA within the germinal center and weaker expression within the mantle zone (Wilson et al, J. Exp. Med. 173:137 (1991)).
• CD22 is speculated to be involved in the regulation of B-cell activation since the binding of CD22 mAb to B-cells in vitro has been found to augment both the increase in intracellular free calcium and the proliferation induced after cross-linking of surface Ig (Pezzutto et al, J. Immunol. 138:98 (1987); Pezzutto et al, J Immunol. 140:1791 (1988)). Other studies have determined, however, that the augmentation of anti-Ig induced proliferation is modest (Dorken et al, J. Immunol. 136:4470 (1986)).
• CD22 is constitutively phosphorylated, but the level of phosphorylation is augmented after treatment of cells with PMA (Boue et al, J. Immunol. 140:192 (1988)). Furthermore, a soluble form of CD22 inhibits the CD3- mediated activation of human T-cells, suggesting CD22 may be important in T-cell - B-cell interactions (Stamenkovic et al, Cell 66:1133 (1991)).
• Ligands that specifically bind the CD22 receptor have been reported to have potential application in the treatment of various diseases, especially B-cell lymphomas and autoimmune diseases.
• the use of labeled and non- labeled anti-CD22 antibodies for treatment of such diseases has been reported.
• Tedder et al, U.S. patent 5,484,892 that purportedly bind CD22 with high affinity and block the interaction of CD22 with other ligands.
• autoimmune diseases such as glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosis, arthritis, thrombocytopenia purpura, agranulocytosis, autoimmune hemolytic anemias, and for inhibiting immune reactions against foreign antigens such as fetal antigens during pregnancy, myasthenia gravis, insulin-resistant diabetes, Graves' disease and allergic responses.
• autoimmune diseases such as glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosis, arthritis, thrombocytopenia purpura, agranulocytosis, autoimmune hemolytic anemias, and for inhibiting immune reactions against foreign antigens such as fetal antigens during pregnancy, myasthenia grav
• Patent 5,789,557 disclose chimeric and humanized anti-CD22 monoclonal antibodies produced by CDR grafting and the use thereof in conjugated and unconjugated form for therapy and diagnosis of B-cell lymphomas and leukemias.
• the reference discloses especially such antibodies conjugated to cytotoxic agents, such as chemotherapeutic drugs, toxins, heavy metals and radionuclides.
• cytotoxic agents such as chemotherapeutic drugs, toxins, heavy metals and radionuclides.
• PCT applications WO 98/42378, WO 00/20864, and WO 98/41641 describe monoclonal antibodies, conjugates and fragments specific to CD22 and therapeutic use thereof, especially for treating B-cell related diseases.
• an anti-CD22 antibody of fragment may be directly or indirectly conjugated to a desired effector moiety, e.g., a label that maybe detected, such as an enzyme, fluorophore, radionuclide, electron transfer agent during an in vitro immunoassay or in vivo imaging, or a therapeutic effector moiety, e.g., a toxin, drug or radioisotope.
• a desired effector moiety e.g., a label that maybe detected, such as an enzyme, fluorophore, radionuclide, electron transfer agent during an in vitro immunoassay or in vivo imaging, or a therapeutic effector moiety, e.g., a toxin, drug or radioisotope.
• an anti-human CD22 monoclonal antibody of the IgGl isotype is commercially available from Leinco Technologies, and reportedly is useful for treatment of B-cell lymphomas and leukemias, including hairy cell leukemia. (Campana, D. et al, J. Immunol. 134:1524 (1985)). Still further, Dorken et al, J. Immunol. 150:4719 (1993) and Engel et al, J. Immunol. 150:4519 (1993) both describe monoclonal antibodies specific to CD22. D. Anti-CD 19 Antibodies
• anti-CD 19 antibodies for classifying the status and prognosis of leukemias has been reported.
• at least one B cell depleting antibody preferably selected from an anti-CD20, anti- CD19, anti-CD22 or anti-CD37 antibody and at least one immunomodulatory antibody preferably selected from an anti-B7, anti-CD23, anti-CD40, anti-CD40L or anti-CD4 antibody.
• compositions and kits for treatment of B cell malignancies, in B cell lymphomas and leukemias that include at least one immunoregulatory or immunomodulatory antibody and at least one B cell depleting antibody.
• the immunoregulatory or immunomodulatory antibody will comprise an anti-CD40, anti-CD40L or anti-B7 antibody and the B cell depleting antibody will be specific to CD20, CD 19, CD22 or CD37.
• the composition will comprise an anti-CD40L or anti-B7 antibody and an anti-CD20 antibody.
• Another object of the invention is to provide a combination therapy for the treatment of a B-cell lymphoma or a B-cell leukemia comprising an anti-CD40L antibody or antibody fragment or CD40L antagonist and at least one of the following (a) a chemotherapeutic agent or a combination of chemotherapeutic agents, (b) radiotherapy, (c) an anti-CD20 antibody or fragment thereof, (d) an anti-CD40 antibody or fragment thereof, (e) an anti-CD 19 antibody or fragment thereof; (f) an anti-CD22 antibody or fragment thereof, (g) cytokines, and combinations thereof, where antibodies may be conjugated with a toxin or a radiolabel, or may be engineered with human constant regions as to elicit human antibody effector mechanisms, i.e. resulting in apoptosis or death of targeted cells.
• Fig. 1 Sensitivity of B-lymphoma cells to adriamycin after 4 hour exposure.
• Fig. 2. Anti CD40L (IDEC-131) overrides CD40L mediated resistance to killing by ADM of B-lymphoma cells.
• Panel B Effect of RITUXAN® on normal and sCD40L pre-treated DHL-4 cells.
• FIG. 3 (Panel A) Blocking of CD40L mediated cell survival of B-CLL by anti-CD40L antibody (IDEC-131). (Panel B) Blocking of CD40L mediated survival ofB-CLL by Rituxan®.
• Fig. 4 FACS analysis comprising HLA-DR expression in CD19 + CLL cells cultured with sCD40L and not cultured with sCD40L.
• the present invention provides a novel combination antibody regimen that involves the administration of at least one immunoregulatory or immunomodulatory antibody, e.g., an anti-B7 or anti-CD40 or anti-CD40L antibody and at least one B cell depleting antibody, e.g., an anti-CD20, anti-CD19, anti-CD22 or anti-CD37 antibody having substantial B cell depleting activity.
• at least one immunoregulatory or immunomodulatory antibody e.g., an anti-B7 or anti-CD40 or anti-CD40L antibody
• at least one B cell depleting antibody e.g., an anti-CD20, anti-CD19, anti-CD22 or anti-CD37 antibody having substantial B cell depleting activity.
• the complementary mechanisms of action will yield a more durable and potent clinical response as it is believed that the B cell depleting antibody will deplete activated B cells which may be resistant to the action of immunoregulatory or immunomodulatory antibodies such as anti-B7 or anti-CD40L antibodies.
• activated B cells can otherwise serve as effective antigen presenting cells for T cells as well as antibody producing cells.
• such activated B cells may include malignant cells which unless eradicated by give rise to new cancer cells and tumors.
• antibody as used herein is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide thereof.
• An antibody can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments.
• antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
• Antibody fragments comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof.
• antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
• Antibody fragments may be isolated using conventional techniques. For example, F(ab 1 ) 2 fragments can be generated by treating antibodies with pepsin. The resulting F(ab ! ) fragment can be treated to reduce disulfide bridges to produce Fab 1 fragments.
• “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
• VH variable domain
• Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
• variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs).
• the variable domains of native heavy and light chains each comprise four FRs, largely adopting a 13 -sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the B -sheet structure.
• the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).
• the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
• Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
• Fv is the minimum antibody fragment which contains a complete antigen- recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
• the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
• Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
• Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group.
• F(ab')Z antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
• the "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.
• antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgGI, IgG2, IgG3, IgG4, IgA, and IgA2.
• the heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma and mu, respectively.
• the heavy-chain constant domains will complete the gamma- 1, gamma-2, gamma-3 and gamma-4 constant region.
• these constant domains will also comprise modifications to enhance antibody stability such as the P and E modification disclosed in U.S. Patent No. 6,011,138 incorporated by reference in its entirety herein.
• P and E modification disclosed in U.S. Patent No. 6,011,138 incorporated by reference in its entirety herein.
• the , subunit structures and three dimensional configurations of different classes of immunoglobulins are well known.
• Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
• the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
• diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH - VL).
• VH heavy-chain variable domain
• VL light chain variable domain
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
• the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,XJ.S. Patent No. 4,816,567).
• the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol.
• humanized antibody is meant an antibody derived from a non-human antibody, typically a murine antibody, that retains or substantially retains the antigen- binding properties of the parent antibody, but which is less immunogenic in humans. This may be achieved by various methods, including (a) grafting the entire non- human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting only the non-human complementarity determining regions (CDRs) into human framework and constant regions with or without retention of critical framework residues; and (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues.
• CDRs complementarity determining regions
• human antibody is meant an antibody containing entirely human light and heavy chain as well as constant regions, produced by any of the known standard methods.
• primary antibody is meant a recombinant antibody which has been engineered to contain the variable heavy and light domains of a monkey (or other primate) antibody, in particular, a cynomolgus monkey antibody, and which contains human constant domain sequences, preferably the human immunoglobulin gamma 1 or gamma 4 constant domain (or PE variant).
• the preparation of such antibodies is described in Newman et al, Biotechnology, 10: 1458-1460 (1992); also in commonly assigned 08/379,072, 08/487,550, or 08/746,361, all of which are incorporated by reference in their entirety herein.
• These antibodies have been reported to exhibit a high degree of homology to human antibodies, i.e., 85-98%, display human effector functions, have reduced immunogenicity, and may exhibit high affinity to human antigens.
• antibody fragment an fragment of an antibody such as Fab, F(ab') 2 , Fab' and scFv.
• chimeric antibody an antibody containing sequences derived from two different antibodies, which typically are of different species. Most typically, chimeric antibodies comprise human and murine antibody fragments, and generally human constant and murine variable regions.
• B Cell Depleting Antibody therein is an antibody or fragment that upon administration, results in demonstrable B cell depletion.
• such antibody will bind to a B cell antigen or B cell marker expressed on the surface of a B cell.
• B cell depleting antibody after administration, typically within about several days or less, will result in a depletion of B cell number by about 50% or more.
• the B cell depleting antibody will be RITUXAN® (a chimeric anti- CD20 antibody) or one having substantially the same or at least 20-50% the cell depleting activity of RITUXAN®.
• a "B cell surface marker” or "B cell target” or “B cell antigen” herein is an antigen expressed on the surface of a B cell which can be targeted with an antagonist which binds thereto.
• Exemplary B cell surface markers include the CD 10, CD 19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD 86 leukocyte surface markers:
• the B cell surface marker of particular interest is preferentially expressed on B cells compared to other non-B cell tissues of a mammal and may be expressed on both precursor B cells and mature B cells.
• the marker is one, like CD20 or CD 19, which is found on B cells tliroughout differentiation of the lineage from the stem cell stage up to a point just prior to terminal differentiation into plasma cells.
• the preferred B cell surface markers herein are CD 19 and CD20.
• Immunoregulatory Antibody refers to an antibody that elicits an effect on the immune system by a mechanism different from B cell depletion, e.g., by CDL and/or ADCC activity.
• examples of such include antibodies that inhibit T cell immunity, B cell immunity, e.g. by inducing tolerance (anti-CD40L, anti-CD40) or other immunosuppressant antibodies, e.g., those that inhibit B7 cell signaling (anti- B7.1, anti-B7.2, anti-CD4, anti-CD23, etc.).
• the immunoregulatory antibody may possess the ability to potentiate apoptosis.
• an antibody that is normally a B cell depleting antibody can be engineered to become immunoregulatory by substantiating human constant regions as to take advantage of different effector mechanisms.
• a "B cell surface marker” herein is an antigen expressed on the surface of a B cell which can be targeted with an antagonist which binds thereto.
• Exemplary B cell surface markers include the CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85 and CD86 (B7.2) leukocyte surface markers.
• the B cell surface marker of particular interest is preferentially expressed on B cells compared to other non-B cell tissues of a mammal and may be expressed on both precursor B cells and mature B cells.
• the marker is one, like CD20 or CD 19, which is found on B cells throughout differentiation of the lineage from the stem cell stage up to a point just prior to terminal differentiation into plasma cells.
• the preferred B cell surface markers herein are CD 19, CD20, CD23, CD80 and CD86.
• the "CD20” antigen is a -35 kDa, non-glycosylated phosphoprotein found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and remains until plasma cell differentiation. CD20 is present on both normal B cells as well as malignant B cells. Other names for CD20 in the literature include "B-lymphocyte-restricted antigen” and "Bp35". The CD20 antigen is described in Clark et al PNAS (USA) 82:1766(1985).
• CD 19 antigen refers to a -90kDa antigen identified, for example, by the HD237-CD19 or B4 antibody (Kiesel et al. Leukemia Research II, 12: 1119 (1987)). Like CD20, CD 19 is found on cells throughout differentiation of the lineage from the stem cell stage up to a point just prior to terminal differentiation into plasma cells. Binding of an antagonist to CD 19 may cause internalization of the CD 19 antigen.
• the "CD22” antigen refers to an antigen expressed on B cells, also known as "BL-CAM” and "LybB” that is involved in B cell signaling and an adhesion. (See Nitschke et al, Curr. Biol. 7:133 (1997); Stamenkovic et al, Nature 345:74 (1990)).
• This antigen is a membrane immunoglobulin-associated antigen that is tyrosine phosphorylated when membrane Ig is ligated. (Engel et al, J. Etyp. Med. 181(4):1521 1586 (1995)). The gene encoding this antigen has been cloned, and its lg domains characterized.
• B7 antigen includes the B7.1 (CD80), B7.2 (CD81) and B7.3 antigen, which are transmembrane antigens expressed on B cells.
• Antibodies which specifically bind B7 antigens, including human B7.1 and B7.2 antigens are known in the art.
• Preferred B7 antibodies comprise the primatized® B7 antibodies disclosed by Anderson et al. in U.S. Patent No. 6,113,198, assigned to IDEC Pharmaceuticals Corporation, as well as human and humanized B7 antibodies.
• CD23 refers to the low affinity receptor for IgE expressed by B and other cells.
• CD23 will preferably be human CD23 antigen.
• CD23 antibodies are also known in the art. Most preferably, in the present invention, the CD23 antibody will be a human or chimeric anti-human CD23 antibody comprising human IgGI or IgG3 constant domains.
• a B cell "antagonist” is a molecule which, upon binding to a B cell surface marker, destroys or depletes B cells in a mammal and/or interferes with one or more B cell functions, e.g. by reducing or preventing a humoral response elicited by the B cell.
• the antagonist preferably is able to deplete B cells (i.e. reduce circulating B cell levels) in a mammal treated therewith. Such depletion may be achieved via various mechanisms such antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), inhibition of B cell proliferation and/or induction of B cell death (e.g. via apoptosis).
• Antagonists included within the scope of the present invention include antibodies, synthetic or native sequence peptides and small molecule antagonists which bind to the B cell marker, optionally conjugated with or fused to a cytotoxic agent.
• a CD40L antagonist is a molecule that specifically binds CD40L and preferably antagonizes the interaction of CD40L and CD40. Examples thereof include antibodies and antibody fragments that specifically bind CD40L, soluble CD40, soluble CD40 fusion proteins, and small molecules that bind CD40L.
• the preferred antagonist according to the invention comprises an antibody or antibody fragment specific to CD40.
• “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to a cell mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.
• FcRs Fc receptors
• NK Natural Killer
• NK cells express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII.
• FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
• an in vitro ADCC assay such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed.
• Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
• PBMC peripheral blood mononuclear cells
• NK Natural Killer
• ADCC activity of the molecule of interest maybe assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
• Human effector cells are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
• PBMC peripheral blood mononuclear cells
• NK natural killer cells
• monocytes cytotoxic T cells and neutrophils
• the effector cells may be isolated from a native source thereof, e.g. from blood or PBMCs as described herein.
• Fc receptor or “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
• the preferred FcR is a native sequence human FcR.
• a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRII subclasses, including allelic variants and alternatively spliced forms of these receptors.
• FcyRII receptors include FcyRIIA (an “activating receptor") and FcyRUB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
• Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain.
• Inhibiting receptor FcyRUB contains an immunoreceptor tyrosine-based inhibition motif (ITEM) in its cytoplasmic domain, (see review M. in Daeon, Annu. Rev. Immunol. 15:203-234 (1997)).
• FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995).
• FcR FcR
• FcRn neonatal receptor
• “Complement dependent cytotoxicity” or “CDC” refers to the ability of a molecule to lyse a target in the presence of complement.
• the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen.
• a CDC assay e.g. as described in Gazzano-Santoro et al., J Immunol. Methods 202: 163 (1996), may be performed.
• ⁇ "Growth inhibitory” antagonists are those which prevent or reduce proliferation of a cell expressing an antigen to which the antagonist binds.
• the antagonist may prevent or reduce proliferation of B cells in vitro and/or in vivo.
• Antagonists which "induce apoptosis" are those which induce programmed cell death, e.g. of a B cell, as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).
• the term "hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
• the hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g.
• "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.
• An antagonist "which binds" an antigen of interest e.g. a B cell surface marker, is one capable of binding that antigen with sufficient affinity such that the antagonist is useful as a therapeutic agent for targeting a cell, i.e. a B cell, expressing the antigen.
• an “anti-CD20 antibody” herein is an antibody that specifically binds CD20 antigen, preferably human CD20, having measurable B cell depleting activity, preferably having at least about 10% the B cell depleting activity of RITUXAN® (see U.S. Patent No. 5,736,137, incorporated by reference herein in its entirety).
• An “anti-CD22 antibody” herein is an antibody that specifically binds CD22 antigen, preferably human CD22, having measurable B cell depleting activity, preferably having at least about 10% the B cell depleting activity of RITUXAN® (see U.S. Patent No. 5,736,137, incorporated by reference herein in its entirety).
• an “anti-CD 19 antibody” herein is an antibody that specifically binds CD 19 antigen, preferably human CD 19, having measurable B cell depleting activity, preferably having at least about 10% the B cell depleting activity of RITUXAN® (see U.S. Patent No. 5,736,137, incorporated by reference herein in its entirety).
• An “anti-CD37 antibody” herein is an antibody that specifically binds CD37 antigen, preferably human CD37, having measurable B cell depleting activity, preferably having at least about 10% the B cell depleting activity of RITUXAN® (see U.S. Patent No. 5,736,137, incorporated by reference herein in its entirety).
• an "anti-B7 antibody” herein is an antibody that specifically binds B7.1 , B7.2 or B7.3, most preferably human B7.3, that inhibits B7/CD28 interactions and, which more does not substantially inhibit B7/CTLA-4 interactions, and even more preferably, the particular antibodies described in U.S. Patent 6,113,898, incorporated by reference in its entirety herein. It has recently been shown that these antibodies promote apoptosis. Therefore, they are well suited for anti-neoplastic applications.
• an "anti-CD40L antibody” is an antibody that specifically binds CD40L (also known as CD154, gp39, TBAM), preferably one having agonistic activity.
• a preferred anti-Cd40L antibody is one having the specificity of a humanized antibody disclosed in U.S. Patent No. 6,011,358 (assigned to IDEC Pharmaceuticals Corporation), incorporated by reference in its entirety herein.
• an "anti-CD4 antibody” is one that specifically binds CD4, preferably human CD4, more preferably a primatized or humanized anti-CD4 antibody.
• an "anti-CD40 antibody” is an antibody that specifically binds CD40, preferably human CD40, such as those disclosed in U.S. Patent 5,874,085, 5,874,082, 5,801,227, 5,674,442, snf 5,667,165, all of which are incorporated by reference herein.
• both the B cell depleting antibody and the immunoregulatory antibody will contain human constant domains.
• Suitable antibodies may include IgGl, IgG2, IgG3 and IgG4 isotypes.
• Specific examples of antibodies which bind the CD20 antigen include:
• antibodies which bind CD22 include LymphocideTM reported by Immunomedics, now in clinical trials for non-Hodgkin's lymphoma.
• antibodies that bind B7 antigen include the B7 antibody reported U.S. Patent 5,885,577, issued to Linsley et al, the anti-B7 antibody reported in U.S. Patent 5,869,050, issued in DeBoer et al, assigned to Chiron Corporation, and the primatized® anti-B7 antibodies disclosed in U.S. Patent 6,113,198 to Anderson et al., all of which are incorporated by reference in their entirety.
• antibodies that bind CD23 are well known and preferably include the primatized® antibodies specific to human CD23 reported by Reff et al, in U.S. Patent 6,011,138, issued on July 4, 1999, co-assigned to IDEC Pharmaceuticals Corp. and Seikakagu Corporation of Japan; those reported by Bonnefoy et al, No. 96 12741; Rector et al. J. Immunol. 55:481-488 (1985); Flores-Rumeo et al. Science 241:1038-1046 (1993); Sherr et al. J. Immunol, 142:481-489 (1989); and Pene et al, PNAS, USA 85:6820-6824 (1988).
• Such antibodies are reportedly useful for treatment of allergy, autoimmune diseases, and inflammatory diseases.
• rituximab or “RITUXAN®” herein refer to the genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen and designated “C2B8" in US Patent No. 5,736,B7, expressly incorporated herein by reference.
• the antibody is an IgGI kappa immunoglobulin containing murine light and heavy chain variable region sequences and human constant region sequences.
• Rituximab has a binding affinity for the CD20 antigen of approximately 8.0 nM.
• an "isolated" antagonist is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antagonist, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
• the antagonist will be purified (1) to greater than 95% by eight of antagonist as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
• Isolated antagonist includes the antagonist in situ within recombinant cells since at least one component of the antagonist's natural environment will not be present. Ordinarily, however, isolated antagonist will be prepared by at least one purification step.
• mammal for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
• Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease or disorder as well as those in which the disease or disorder is to be prevented. Hence, the mammal may have been diagnosed as having the disease or disorder or may be predisposed or susceptible to the disease.
• B Cell Malignancy
• this includes any B cell malignancy, e.g., B cell lymphomas and leukemias.
• B cell malignancy e.g., B cell lymphomas and leukemias.
• Preferred examples include Hodgkin's disease (all forms, e.g., relapsed Hodgkin's disease, resistant Hodgkin's disease) non-Hodgkin's lymphomas (low grade, intermediate grade, high grade, and other types).
• Examples include small lymphocytic/B cell chronic lymphocytic leukemia (SLL/B-CLL), lymhoplasmacytoid lymphoma (LPL), mantle cell lymphoma (MCL), follicular lymphoma (FL), diffuse large cell lymphoma (DLCL), Burkitt's lymphoma (BL), AIDS- related lymphomas, monocytic B cell lymphoma, angioimmunoblastic lymphoadenopathy, small lymphocytic, follicular, diffuse large cell, diffuse small cleaved cell, large cell immunoblastic lymphoblastoma, small, non-cleaved, Burkitt's and non-Burkitt's, follicular, predominantly large cell; follicular, predominantly small cleaved cell; and follicular, mixed small cleaved and large cell lymphomas.
• SLL/B-CLL small lymphocytic/B cell chronic lymphocytic
• lymphoma classifications include immunocytomal Waldenstrom's MALT-type/monocytoid B cell, mantle cell lymphoma B-CLL/SLL, diffuse large B-cell lymphoma, follicular lymphoma, and precursor B-LBL.
• B cell malignancies further include especially leukemias such as
• ALL-L3 (Burkitt's type leukemia), chronic lymphocytic leukemia (CLL), chronic leukocytic leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, lymphoblastic leukemia, lymphocytic leukemia, monocytic leukemia, myelogenous leukemia, and promyelocytic leukemia and monocytic cell leukemias.
• CLL chronic lymphocytic leukemia
• terapéuticaally effective amount refers to an amount of the antagonist which is effective for preventing, ameliorating or treating the B cell malignancy disease in question.
• immunosuppressive agent refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077, the disclosure of which is incorporated herein by reference), azathioprine; cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No.
• anti-idiotypic antibodies for MHC antigens and MHC fragments include cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone, methylprednisolone, and dexamethasone; cytokine or cytokine receptor antagonists including anti-interferon- ⁇ , ⁇ - or ⁇ -antibodies, anti-tumor necrosis factor- ⁇ antibodies, anti-tumor necrosis factor- ⁇ antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CDl la and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published 7/26/90), streptolanase; TGF- ⁇ ; streptodornas
• T-cell receptor fragments (Offner et al, Science, 251: 430-432 (1991); WO 90/11294; laneway, Nature, 341: 482 (1989); and WO 91/01133); and T cell receptor antibodies (EP 340,109) such as T10B9.
• cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
• the term is intended to include radioactive isotopes (e. g. At211 1131 1125 Y9o Re 186 Re lg8 sM153 Bi212 p32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
• chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
• examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime nitrogen mustards such as chiorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trof
• paclitaxel TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ
• doxetaxel Texotere, Rhone-Poulenc Rorer, Antony, France
• chiorambucil gemcitabine
• 6-thioguanine mercaptopurine
• methotrexate platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• anti-hormonal agents that act to regulate or inhibit hormone action on tumors
• anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
• cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones.
• cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor- ⁇ and - ⁇ ; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-1 ; platelet-growth factor; transforming growth factors (TGFs) such as TGF- ⁇ and TGF- ⁇ ; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferrin
• cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
• prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp.
• the prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, tl ⁇ ophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, 13 -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5fluorouridine prodrugs which can be converted into the more active cytotoxic free drug.
• cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
• a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the antagonists disclosed herein and, optionally, a chemotherapeutic agent) to a mammal.
• a drug such as the antagonists disclosed herein and, optionally, a chemotherapeutic agent
• the components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
• package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
• the methods and articles of manufacture of the present invention use, or incorporate, an antibody that has immunoregulatory activity, e.g. anti-B7, anti-CD23, anti-CD40L, anti-CD4 or anti-CD40 antibody, and an antibody that binds to a B cell surface marker having B depleting activity, e.g., anti-CD20, anti-CD22, anti-CD 19, or anti-CD37 antibody. Accordingly, methods for generating such antibodies will be described herein.
• the molecule to be used for production of, or screening for, antigen(s) may be, e.g., a soluble form of the antigen or a portion thereof, containing the desired epitope.
• cells expressing the antigen at their cell surface can be used to generate, or screen for, antagonist(s).
• Other forms of the B cell surface marker useful for generating antagonists will be apparent to those skilled in the art.
• Suitable antigen sources for CD40L, CD40, CD 19, CD20, CD22, CD23, CD37, CD4 and B7 antigen (e.g., B7.1, B7.2) antigen for producing antibodies according to the invention are well known.
• peptides can be synthetically prepared based upon the amino acid sequence. For example, with respect to CD40L, this is disclosed in Armitage et al. (1992).
• the CD40L antibody or anti-CD40L antibody will be the humanized anti-CD40L antibody disclosed in U.S. Patent 6,001,358, issued on June 14, 1999, and assigned to IDEC Pharmaceuticals Corporation.
• the antagonist may comprise soluble CD40, a CD40 fusion protein or a small molecule antagonist optionally fused to, or conjugated with, a cytotoxic agent (such as those described herein).
• Libraries of small molecules may be screened against the B cell surface marker of interest herein in order to identify a small molecule which binds to that antigen.
• the small molecule may further be screened for its antagonistic properties and/or conjugated with a cytotoxic agent.
• the antagonist may also be a peptide generated by rational design or by phage display (W098/35036 published 13 August 1998), for example.
• the molecule of choice may be a "CDR mimic" or antibody analogue designed based on the CDRs of an antibody, for example.
• the peptide may be antagonistic by itself, the peptide may optionally be fused to a cytotoxic agent or to an immunoglobulin Fc region (e.g., so as to confer ADCC and/or CDC activity on the peptide).
• a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thy
• Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g. 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
• the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
• Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
• the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
• Conjugates also can be made in recombinant cell culture as protein fusions.
• aggregating agents such as alum are suitably used to enhance the immune response.
• Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
• the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
• the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567).
• a mouse or other appropriate host animal such as a hamster
• lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
• lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).
• the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
• the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
• HAT medium hypoxanthine, aminopterin, and thymidine
• Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
• preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Virginia, USA.
• Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:300 1 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
• Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
• the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
• RIA radioimmunoassay
• ELISA enzyme-linked immunoabsorbent assay
• the binding affinity of the monoclonal antibody can, for example, be detennined by the 30 Scatchard analysis of Munson et al, Anal Biochem., 107:220 (1980).
• the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPML-1640 medium.
• the hybridoma cells may be grown in vivo as ascites tumors in an animal.
• the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
• DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oHgonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
• the hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E.
• Another method of generating specific antibodies, or antibody fragments, reactive against a CD40L, CD19, CD22, CD20, or CD40 protein or peptide is to screen expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with a CD40L, CD 19, CD20, or CD22 protein or peptide.
• a CD40L, CD 19, CD20, or CD22 protein or peptide For example, complete Fab fragments, V H regions and Fv regions can be expressed in bacteria using phage expression libraries.
• CD40L CD22, CD 19, or CD20.
• SCID-hu mouse available from Genpharm
• antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al, Nature, 348:552-554(1990). Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
• CD40L including human CD40L and mouse CD40L, and suitable monoclonal antibodies for use in the methods of the invention, are described in PCT Patent Application No. WO 95/06666 entitled "Anti-gp39 Antibodies and Uses Therefor;” the teachings of which are incorporated herein by reference in their entirety.
• Particularly preferred anti-human CD40L antibodies of the invention are MAbs 24-31 and 89-76, produced respectively by hybridomas 24-31 and 89-76.
• the 89-76 and 24- 31 hybridomas, producing the 89-76 and 24-31 antibodies, respectively, were deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, VA 20110-2209, on Sept. 2, 1994.
• the 89-76 hybridoma was assigned ATCC Accession Number HB 11713 and the 24-31 hybridoma was assigned ATCC Accession Number HB11712.
• the DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, et al, Proc. Natl Acad. ScL USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
• non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigencombining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
• a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature, 321:522-525 (1986); Reichmann et al, Nature,332:323-321 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
• humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• variable domains both light and heavy
• the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity.
• the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
• the human sequence which is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Suns et al, J. Immunol, 151:2296 (1993); Choxhia et al, J. Mol. Biol, 196:901 (1987)).
• Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains.
• the same framework may be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et ⁇ /., J. Immunol, 151:2623 (1993)).
• humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
• Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
• Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i. e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
• FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
• the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
• This technique modifies antibodies such that they are not antigenically rejected upon administration in humans.
• This technique relies on immunization of cynomolgus monkeys with human antigens or receptors. This technique was developed to create high affinity monoclonal antibodies directed to human cell surface antigens.
• Identification of macaque antibodies to human CD40L, CD20, CD22, CD40 or CD 19 by screening of phage display libraries or monkey heterohybridomas obtained using B lymphocytes from CD40L, CD20, CD22, CD40, or CD 19 immunized monkeys can be performed using the methods described in commonly assigned U.S. Application No. 08/487,550, filed June 7, 1995, incorporated by reference in its entirety herein.
• Antibodies generated using the methods described in these applications have previously been reported to display human effector function, have reduced immunogenicity, and long serum half-life.
• the technology relies on the fact that despite the fact that cynomolgus monkeys are phylogenetically similar to humans, they still recognize many human proteins as foreign and therefore mount an immune response.
• the antibodies generated in these monkeys have been discovered to have a high degree of amino acid homology to those produced in humans. Indeed, after sequencing macaque immunoglobulin light and heavy chain variable region genes, it was found that the sequence of each gene family was 85-98%) homologous to its human counterpart (Newman et al, 1992). The first antibody generated in this way, an anti-CD4 antibody, was 91-92% homologous to the consensus sequence of human immunoglobulin framework regions (Newman et al, 1992).
• the present invention relates, in part, to the use of monoclonal antibodies or primatized forms thereof which are specific to human CD40L antigen and which are capable of inl ibiting CD40 signaling or inhibiting
• CD40/CD40L interaction Blocking of the primary activation site between CD40 and CD40L with the identified antibodies (or therapeutically effective fragments thereof), while allowing the combined antagonistic effect on positive co-stimulation with an agnostic effect on negative signaling will be a useful therapeutic approach for intervening in relapsed forms of malignancy, especially B-cell lymphomas and leukemias.
• the functional activity of the identified antibodies is defined by blocking the signals of CD40 permitting it to survive and avoid IgM- or Fas-induced apoptosis.
• Manufacture of monoclonal antibodies which specifically bind human CD40L, as well as primatized antibodies derived therefrom can be performed using the methods described in U.S. Patent Nos.
• CD40 antibodies involves recombinant phage display technology. This technique is generally described supra.
• this will comprise synthesis of recombinant immunoglobulin libraries against the target, i.e., CD19, CD22, CD20, CD40, or CD40L antigen displayed on the surface of filamentous phage and selection of phage which secrete antibodies having high affinity to CD40L antigen.
• targets i.e., CD19, CD22, CD20, CD40, or CD40L antigen displayed on the surface of filamentous phage and selection of phage which secrete antibodies having high affinity to CD40L antigen.
• antibodies will be selected which bind to both human CD40L and CD40.
• the present inventors have created a unique library for monkey libraries which reduces the possibility of recombination and improves stability.
• this vector contains specific primers for PCR amplifying monkey immunoglobulin genes. These primers are based on macaque sequences obtained while developing the primatized technology and databases containing human sequences. Suitable primers are disclosed in commonly assigned 08/379,072, incorporated by reference herein.
• the second method involves the immunization of monkeys, i.e., macaques, against the desired antigen target, i.e., human CD 19, CD20, CD22, CD40 or CD40L.
• the desired antigen target i.e., human CD 19, CD20, CD22, CD40 or CD40L.
• the inherent advantage of macaques for generation of monoclonal antibodies is discussed supra.
• monkeys i.e., cynomolgus monkeys
• the resultant antibodies may be used to make primatized antibodies according to the methodology of Newman et al, (1992), and Newman et al, commonly assigned U.S. Serial No. 08/379,072, filed January 25, 1995, which are incorporated by reference in their entirety.
• antibodies obtained from cynomolgus monkeys are that these monkeys recognize many human proteins as foreign and thereby provide for the formation of antibodies, some with high affinity to desired human antigens, e.g., human surface proteins and cell receptors. Moreover, because they are phylogenetically close to humans, the resultant antibodies exhibit a high degree of amino acid homology to those produced in humans. As noted above, after sequencing macaque immunoglobulin light and heavy variable region genes, it was found that the sequence of each gene family was 85-88% homologous to its human counterpart (Newman et al, 1992).
• cynomolgus macaque monkeys are administered human, CD 19, CD20, CD22, CD40, or CD40L antigen, B cells are isolated therefrom, e.g., lymph node biopsies are taken from the animals, and B lymphocytes are then fused with KH6/B5 (mouse x human) heteromyeloma cells using polyethylene glycol (PEG). Heterohybridomas secreting antibodies which bind human CD40L antigen are then identified.
• PEG polyethylene glycol
• antibodies which bind to CD40L or CD40 it is desirable that they do so in a manner which interrupts or regulates CD40 signaling because such antibodies potentially may be used to inhibit the interaction of CD40L with CD40, with their counter-receptors. If antibodies can be developed against more than one epitope on CD40L or CD40, and the antibodies are utilized together, their combined activity may potentially provide synergistic effects.
• the disclosed invention involves the use of an animal which is primed to produce a particular antibody (e.g., primates, such as organgutan, baboons, macaque, and cynomolgus monkeys).
• a particular antibody e.g., primates, such as organgutan, baboons, macaque, and cynomolgus monkeys.
• Other animals which may be used to raise antibodies to human CD40L include, but are not limited to, the following: mice, rats, guinea pigs, hamsters, monkeys, pigs, goats and rabbits.
• Cell lines which express antibodies which specifically bind to human CD40L antigen are then used to clone variable domain sequences for the manufacture of primatized antibodies essentially as described in Newman et al, (1992) and Newman et al, U.S. Serial No. 379,072, filed January 25, 1995, both of which are incorporated by reference herein. Essentially, this entails extraction of RNA therefrom, conversion to cDNA, and amplification thereof by PCR using Ig specific primers. Suitable primers are described in Newman et al, 1992, and in U.S. Serial No. 379,072. Similar techniques will yield cell lines that express antibodies specific to CD40, CD 19, CD20, or CD22.
• Cell lines which express antibodies which specifically bind to human CD40L antigen are then used to clone variable domain sequences for the manufacture of primatized antibodies essentially as described in Newman et al, (1992) and Newman et al, U.S. Serial No. 379,072, filed January 25, 1995, both of which are incorporated by reference herein. Essentially, this entails extraction of RNA therefrom, conversion to cDNA, and amplification thereof by PCR using Ig specific primers. Suitable primers are described in Newman et al, 1992, and in U.S. Serial No. 379,072. Similar techniques will yield cell lines that express antibodies specific to CD40, CD 19, CD20, or CD22.
• Cell lines which express antibodies which specifically bind to human CD40L antigen are then used to clone variable domain sequences for the manufacture of primatized antibodies essentially as described in Newman et al, (1992) and Newman et al, U.S. Serial No. 379,072, filed January 25, 1995, both of which are incorporated by reference herein. Essentially, this entails extraction of RNA therefrom, conversion to cDNA, and amplification thereof by PCR using Ig specific primers. Suitable primers are described in Newman et al, 1992, and in U.S. Serial No. 379,072. Similar techniques will yield cell lines that express antibodies specific to CD40, CD 19, CD20, or CD22.
• the cloned monkey variable genes are then inserted into an expression vector which contains human heavy and light chain constant region genes.
• this is effected using a proprietary expression vector of IDEC, Inc., referred to as NEOSPLA.
• This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, human immunoglobulin kappa or lambda constant region, the dihydrofolate reductase gene, the human immunoglobulin gamma 1 or gamma 4 PE constant region and leader sequence.
• This vector has been found to result in very high level expression of primatized antibodies upon incorporation of monkey variable region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification.
• this expression system has been previously disclosed to result in primatized antibodies having high avidity (Kd ⁇ 10 "10 M) against CD4 and other human cell surface receptors. Moreover, the antibodies have been found to exhibit the same affinity, specificity and functional activity as the original monkey antibody.
• This vector system is substantially disclosed in commonly assigned U.S. Serial No. 379,072, incorporated by reference herein as well as U.S. Serial No. 08/149,099, filed on November 3, 1993, also incorporated by reference in its entirety herein. This system provides for high expression levels, i.e., > 30 pg/cell/day. Of course, the same methods can be used to produce cell lines that produce antibodies specific to CD 19, CD20, CD22, or CD40.
• human antibodies can be generated.
• transgenic animals e.g., mice
• transgenic animals e.g., mice
• the homozygous deletion of the antibody heavy-chain joining region PH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production.
• Transfer of the human germ-line immunoglobulin gene array in such germ line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Mad. Acad. Sci.
• phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
• V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
• the phage mimics some of the properties of the B cell.
• Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-57 1 (1993).
• V-gene segments can be used for phage display. Clackson et al, Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice.
• a repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self antigens) can be isolated essentially following the techniques described by Marks et al, J.Mol. Biol, 222:581-597 (1991), or Griffith et al, EMBO J. 12:725-734 (1993). See, also, US Patent Nos. 5,565,332 and 5,573,905.
• Human antibodies may also be generated by in vitro activated B cells (see US Patents 20 5,567,610 and 5,229,275). A preferred means of generating human antibodies using SCID mice is disclosed in commonly-owned, co-pending applications.
• antibody fragments Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al, Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al, Bio/Technology 10: 163-167 (1992)).
• F(ab')2 fragments can be isolated directly from recombinant host cell culture.
• the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; US Patent No. 5,571,894; and US Patent No. 5,587,458.
• the antibody fragment may also be a "linear antibody", e.g., as described in US Patent 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
• Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the B cell surface marker. Other such antibodies may bind a first B cell marker and further bind a second B cell surface marker. Alternatively, an anti-B cell marker binding arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus cellular defense mechanisms to the B cell.
• a triggering molecule such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to
• Bispecific antibodies may also be used to localize cytotoxic agents to the B cell. These antibodies possess a B cell marker-binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon- ⁇ , vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab)2 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 (Millstein et al, Nature, 305:537-539 (1983)).
• 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 of the 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 of the fusions.
• DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
• the 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 of the 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 WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 121:210 (1986).
• the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
• the preferred interface comprises at least apart of the CH3 domain of an antibody constant domain.
• one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
• Compensatory "cavities" of identical or similar size to the large side chains) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
• Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
• one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
• Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
• Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in US Patent No. 4,676,980, along with a number of cross-linking techniques.
• bispecific antibodies can be prepared using chemical linkage.
• Brennan et al, Science, 229:81(1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
• the Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
• One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
• the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
• bispecific antibodies have been produced using leucine zippers.
• the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
• the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
• the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (Vi by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
• V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
• sFv single-chain Fv
• Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Graber et al, J. Immunol, 152:5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).
• the antibodies used in the methods or included in the articles of manufacture herein are optionally conjugated to a cytotoxic agent.
• Conjugates of an antibody and one or more small molecule toxins such as a calicheamicin, a maytansine (US Patent No. 5,208,020), a trichothene, and CC 1065 are also contemplated herein.
• the antagonist is conjugated to one or more maytansine molecules (e.g. about 1 to about 10 maytansine molecules per antagonist molecule).
• Maytansine may, for example, be converted to May SS-Me which may be reduced to May-SH3 and reacted with modified antagonist (Charm et al. Cancer Research 52:127-131(1992)) to generate a maytansinoid-antagonist conj ugate.
• the antibody may be conjugated to one or more calicheamicin molecules.
• the calicheamicin family of antibiotics are capable of producing double stranded DNA breaks at sub-picomolar concentrations.
• Structural analogues of calicheamicin which may be used include, but are not limited to, ⁇ , ⁇ J , ⁇ J , N-acetyl- ⁇ PSAG and ⁇ (Hinman et al. Cancer Research 53:3336-3342 (1993) and Lode et al, Cancer Research 58: 2925-2928 (1998)).
• Enzymaticaliy active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published October 28, 1993.
• the present invention further contemplates antibody conjugated with a compound with nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
• a compound with nucleolytic activity e.g. a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase.
• a variety of radioactive isotopes are available for the production of radioconjugated antagonists. Examples include At 211 , 1 131 , 1 125 , Y 90 , Re 186 , RE 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu.
• Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyriyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-
• a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987).
• Carbon-14-labeled 1 isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic.acid is an exemplary chelating agent for conjugation of radionucleotide to the antagonist. See W094/11026.
• the linker may be a "cleavable linker" facilitating release of the cytotoxic drag in the cell.
• an acid-labile linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker (Charm et al. Cancer Research 52:127-131 (1992)) may be used.
• a fusion protein comprising the antibody and cytotoxic agent may be made, e.g. by recombinant techniques or peptide synthesis.
• the antibody may be conjugated to a "receptor” (such sxreptavidin) for utilization in tumor pretargeting wherein the antagonist- receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g. avidin) which is conjugated to a cytotoxic agent (e.g. a radionucleotide) .
• a "receptor” such as sxreptavidin
• a ligand e.g. avidin
• cytotoxic agent e.g. a radionucleotide
• the antibodies of the present invention may also be conjugated with a prodrug activating enzyme which converts a prodrag (e.g. a peptidyl chemotherapeutic agent, see W081/01145) to an active anti-cancer drug.
• a prodrug activating enzyme which converts a prodrag (e.g. a peptidyl chemotherapeutic agent, see W081/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Patent No. 4,975,278.
• the enzyme component of such conjugates includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
• Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drags; arylsulfatase useful for converting sulfate-containing prodrugs into free drags; cytosine deaminase useful for converting non-toxic5-fluorocytosine into the anti-cancer drag, fluorouracil; proteases, such as serratia protease, thermorysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drags; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydratecleaving enzymes such as 13-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drags;
• 13-lactamase useful for converting drags derivatized with 13-lactams into free drugs
• penicillin amidases such as penicillin V amidase or penicillin G amidase, useful for converting drags derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drags.
• antibodies with enzymatic activity also known in the art as "abzymes” can be used to convert the prodrugs of the invention into free active drags (see, e.g., Massey, Nature 328:457-458 (1987)).
• Antagonist-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
• the enzymes of this invention can be covalently bound to the antagonist by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above.
• fusion proteins comprising at least the antigen binding region of an antagonist of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al, Nature, 312:604-608 (1984)).
• the antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
• nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
• Liposomes containing the antagonist are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and W097/38731 published October 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
• Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG derivatized phosphatidylethanolamme (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.
• Fab' fragments of an antibody of the present invention can be conjugated to the liposomes as described in Martin et al J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction.
• a chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al. J.National Cancer Inst. 81(19)1484 (1989).
• Amino acid sequence modification(s) of protein or peptide antagonists described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
• Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody encoding nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the antagonist. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
• the amino acid changes also may alter post-translational processes of the antagonist, such as changing the number or position of glycosylation sites.
• a useful method for identification of certain residues or regions of the antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells Science, 244:1081-1085 (1989).
• a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen.
• Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution.
• Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polyp eptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
• terminal insertions include an antagonist with an N-terminal methionyl residue or the antagonist fused to a cytotoxic polypeptide.
• Other insertional variants of the antagonist molecule include the fusion to the N- or C-terminus of the antagonist of an enzyme, or a polypeptide which increases the serum half-life of the antagonist.
• variants are an amino acid substitution variant. These variants have at least one amino acid residue in the antagonist molecule replaced by different residue.
• the sites of greatest interest for substitutional mutagenesis of antibody antagonists include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened.
• Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
• Naturally occurring residues are divided into groups based on common side-chain properties:
• hydrophobic norleucine, met, ala, val, leu, ile
• neutral hydrophiuic cys, ser, thr
• cysteine residue not involved in maintaining the proper conformation of the antagonist also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
• cysteine bonds may be added to the antagonist to improve its stability (particularly where the antagonist is an antibody fragment such as an Fv fragment).
• a particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
• a parent antibody e.g. a humanized or human antibody
• the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
• a convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-1 sites) are mutated to generate all possible amino substitutions at each site.
• the antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed.
• alanine scanning mutagenesis can be performed to identified hypervariable region residues contributing significantly to antigen binding.
• Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antagonist. By altering is meant deleting one or more carbohydrate moieties found in the antagonist, and/or adding one or more glycosylation sites that are not present in the antagonist.
• N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
• the tripeptide sequences asparagine-X-serine and asparagine- X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
• O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly seine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
• glycosylation sites are conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
• the alteration may also be made by the addition of, or substitution by, one or more seine or threonine residues to the sequence of the original antagonist (for O-linked glycosylation sites).
• Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antagonist.
• ADCC antigen-dependent cell-mediated cyotoxicity
• CDC complement dependent cytotoxicity
• This may be achieved by introducing one or more amino acid substitutions in an Fc region of an antibody antagonist.
• cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
• the homodimeric antibody thus generated may have improved internalization capability and/or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J.
• Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1993).
• an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:2 19-230 (1989).
• a salvage receptor binding epitope into the antagonist (especially an antibody fragment) as described in US Patent 5,739,277, for example.
• the term "salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGI, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
• Therapeutic formulations comprising antagonists used in accordance with the present invention are prepared for storage by mixing an antagonist having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington 's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
• Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine,
• the immunomodulatory antibody and the B cell depleting antibody may be in the same formulation or may be administered in difficult formulations.
• the composition may further include other non-antibody antagonists, e.g., CD40L or B7 antagonists. Examples there of include soluble CD40, B7 and fusions thereof. Administration can be concurrent or sequential, and may be effective in either order. Exemplary anti-CD20 antibody formulations are described in W098/56418, expressly incorporated herein by reference.
• This publication describes a liquid multidose formulation comprising 40 mg/n L rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum shelf life of two years storage at 2-8°C.
• Another anti-CD20 formulation of interest comprises 1 Omg/mL rituximab in 9.0 mg/niL sodium chloride, 7.35 mg/niL sodium citrate dihydrate, 0.7mg/niL polysorbate 80, and Sterile Water for Injection, pH 6.5.
• Lyophilized formulations adapted for subcutaneous administration are described in W097/04801
• Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the mammal to be treated herein.
• the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
• it may be desirable to further provide a chemotherapeutic agent, cytokine or immunosuppressive agent e.g. one which acts on T cells, such as cyclosporin or an antibody that binds T cells, e.g. one which binds LFA-1).
• the effective amount of such other agents depends on the amount of antagonist present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
• the active ingredients may also be entrapped in microcapsules prepared, for example, by 30 coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drag delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
• sustained-release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
• B Cell depleting Antibody and Immunoregulatory Antibody A composition comprising B cell depleting antibody and/or an immunoregulatory antibody will be formulated, dosed, and administered in a fashion consistent with good medical practice.
• Factors for consideration in this context include the particular B cell malignancy or disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease or disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
• the therapeutically effective amount of the antagonist to be administered will be governed by such considerations.
• the B cell depleting antibody and the immunoregulatory antibody may be in the same or in different formulations. These antagonist formulations can be administered separately or concurrently, and in either order.
• the B cell depleting antibody specific to the B cell antigen target e.g., CD20, CD 19, CD22, CD37 or CD22
• the immunoregulatory antibody e.g., an anti-CD40L antibody, anti-CD40 antibody, or anti-B7 antibody.
• the CD40L antibody will be the humanized anti-CD40L antibody disclosed in U.S. Patent 6,001,358 and the anti-B7 antibody the primatized antibody disclosed in US Patent 6,113,898.
• this antibody has recently been show to possess apoptotic activity. Also the preferred CD40L antibody has been shown to have efficacy in treatment of both T and B cell autoimmune diseases. Also, unlike another humanized anti-CD40L antibody (5c8) reported by Biogen, this antibody is not known to cause any adverse toxicity.
• the therapeutically effective amount of an antibody administered parenterally per dose will typically be in the range of about 0.1 to 500 mg/kg of patient body weight per day, with the typical initial range of antagonist used being in the range of about 2 to 100 mg/kg.
• the preferred B cell depleting antibody is RITUXAN®.
• Suitable dosages for such antibody are, for example, in the range from about 20mg/m2 to about 1000mg/m2.
• the dosage of the antibody may be the same or different from that presently recommended for RITUXAN® for the treatment of non-Hodgkin's lymphoma.
• one may administer one or more initial doses) of the antibody followed by one or more subsequent dose(s), wherein the mg/m 2 dose of the antibody in the subsequent doses) exceeds the mg/m dose of the antibody in the initial dose(s).
• the initial dose may be in the range from about 20mg/m 2 to about 250mg/m 2 (e.g. from about 50mg/m 2 to about 200mg/m 2 ) and the subsequent dose may be in the range from about 250mg/m 2 to about lOOOmg/m 2 .
• the antagonist is administered as close to the first sign, diagnosis, appearance, or occurrence of the disease or disorder as possible or during remissions of the disease or disorder.
• the antibodies are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration.
• Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
• the antibody may suitably be administered by pulse infusion, e.g., with declining doses of the antibody.
• the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
• the combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
• administration of antibodies to the patient contemplates administration of antibodies by gene therapy.
• Such administration of nucleic acid encoding the antibodies is encompassed by the expression "administering a therapeutically effective amount of an antagonist". See, for example, W096/07321 published March 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.
• nucleic acid (optionally contained in a vector) into the patient's cells
• in vivo and ex vivo the nucleic acid is injected directly into the patient, usually at the site where the antagonist is required.
• ex vivo treatment the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e.g. U.S. Patent Nos. 4,892,538 and 5,283,187).
• techniques available for introducing nucleic acids into viable cells There are a variety of techniques available for introducing nucleic acids into viable cells.
• the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
• Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAF-dextran, the calcium phosphate precipitation method, etc.
• a commonly used vector for ex vivo delivery of the gene is a retroviras.
• the currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example).
• viral vectors such as adenovirus, Herpes simplex I virus, or adeno associated virus
• lipid-based systems useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example.
• an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
• proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g.
• capsid proteins or fragments thereof tropic for a particular cell type antibodies for proteins which undergo intemalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life.
• the technique of receptor-mediated endocytosis is described, for example, by Wu et al, .1 Biol Chem 262:4429-4432 (1987); and Wagner et al, Proc. Natl. Acad. Sci. USA 87:3410-3414(1990).
• Wu et al .1 Biol Chem 262:4429-4432 (1987); and Wagner et al, Proc. Natl. Acad. Sci. USA 87:3410-3414(1990).
• an article of manufacture containing materials useful for the treatment of the diseases or disorders described above is provided.
• the article of manufacture comprises a container and a label or package insert on or associated with the container.
• Suitable containers include, for example, bottles, vials, syringes, etc.
• the containers maybe formed from a variety of materials such as glass or plastic.
• the container holds or contains a composition which is effective for treating the disease or disorder of choice and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). As whole, there may be one or several compositions.
• At least one active agent in one of those compositions is an antibody having B cell depleting activity and at least one antibody is an immunoregulatory antibody such as an anti-CD40L, anti-CD40, anti-CD23, anti-CD4 or anti-B7 antibody.
• the label or package insert indicates that the composition is used for treating a patient having or predisposed to B cell malignancy, such as those listed hereinabove.
• the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
• BWFI bacteriostatic water for injection
• the antibodies of the invention may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce such effect to a therapeutic or prophylactic degree.
• Such antibodies of the invention can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.
• the routine of administration of the antibody (or fragment thereof) of the invention may be oral, parenteral, by inhalation or topical.
• parenteral as used herein includes intravenous, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred.
• the daily parenteral and oral dosage regimes for employing compounds of the invention to prophylactically or therapeutically induce immunosuppression, or to therapeutically treat carcinogenic tumors will generally be in the range of about 0.05 to 100, but preferably about 0.5 to 10, milligrams per kilogram body weight per day.
• the antibodies of the invention may also be administered by inhalation.
• inhalation is meant intranasal and oral inhalation administration.
• Appropriate dosage forms for such administration such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.
• the preferred dosage amount of a compound of the invention to be employed is generally within the range of about 10 to 100 milligrams.
• the antibodies of the invention may also be administered topically.
• topical admimstration non-systemic administration and includes the application of an antibody (or fragment thereof) compound of the invention externally to the epidermis, to the buccal cavity and instillation of such an antibody into the ear, eye and nose, and where it does not significantly enter the blood stream.
• systemic administration is meant oral, intravenous, intraperitoneal and intramuscular admimstration.
• the amount of an antibody required for therapeutic or prophylactic effect will, of course, vary with the antibody chosen, the nature and severity of the condition being treated and the animal.
• DHT-4 cells The concept that anti-CD40L antibody could block CD40L-CD40 mediated survival of malignant B-cells from chemotherapy induced toxicity/apoptosis was tested in vitro using IDEC-131, and the B-lymphoma cell line, DHL-4 (Roos et al, Leuk. Res. 10: 195-202 (1986)) exposed to adriamycin (ADM).
• IDEC-131 is a humanized version of the murine, monoclonal anti-human CD40L antibody, 24-31. initially, the minimum concentration of ADM cytotoxic to DHL-4 cells was determined by exposing DHL-4 cells for 4 hours to different concentrations of ADM.
• DHL-4 cells The cell cytotoxicity of DHL-4 cells after 5 days in culture was measured by Alamar Blue, a dye-reduction assay by live cells (see Gazzano-Santoro et al, J. Immunol. Meth. 202: 163-171 (1997)). Briefly, 1 x 10 5 DHL-4 cells in growth medium (RMPI-1640 plus 10% Fetal Calf Serum) were incubated with varying concentrations of ADM (1 x 10 "6 M to 1 x 10 "8 M) in cell culture tubes at 37 ° C. for 4 hours.
• ADM 1 x 10 "6 M to 1 x 10 "8 M
• the results are expressed as relative fluorescence units (RFU).
• the percentage of cytotoxicity was calculated as follows: [1- (average RFU of test sample ⁇ Average RFU of control cells)] x 100%. Titration curve of ADM cytotoxicity was established and minimal concentrations of the drag for cytotoxicity was selected for subsequent assays.
• the results, as displayed in Fig. 1, shows cell cytotoxicity of DHL-4 cells cultured for 5 days after being exposed to ADM (2 x 10 "7 M and 4 x 10 "8 M of ADM) for 4 hours prior to culture. Cells were washed once after exposure and cultured in growth medium for 5 days and cytotoxicity determined by Alamar Blue dye-uptake assay, as described above. Additionally, the DHL-4 cells were characterized for the membrane expression of selected CD molecules by flow cytometry. DHL-4 cells have been found to express CD 19, CD20, CD40 molecules, but no expression of CD40L was detected.
• Example 2 Anti -CD40L antibody overrides CD40L mediated resistance to killins by to killins, by adriamy in of -lymphoma cells
• Fig. 2A shows the effect of an anti-CD40L antibody on CD40L-CD40 mediated resistance of DHL-4 cells to cell death induced by ADM.
• DHL-4 cells 0.5 x 10 6 cells/ml
• soluble CD40L sCD40L, P. A. Brams, E. A. Padlan, K. Hariharan, K. Slater, J. Leonard, R. Noelle, and R. Newman, "A humanized anti-human CD 154 monoclonal antibody blocks CD 154-CD40 mediated human B cell activation," (manuscript submitted)) for 1 hour at 37°C.
• sCD40L (10 ⁇ g/ml) was added to cultures that have been continuously exposed to sCD40L during ADM treatment and to cultures that had no sCD40L during ADM exposure, h addition, IDEC-131 at 10 ⁇ g/ml was added to cultures to determine its effect on DHL-4 cells incubated with sCD40L and ADM. After 5 days, the cytotoxicity was measured by Alamar Blue dye-uptake assay, as described.
• IDEC-131 alone had no effect on DHL-4 cells treated with sCD40L, which indicates that the antibody, by itself, does not have any direct inhibitory or cytotoxic activities on DHL-4 cells (Fig. 2B).
• DHL-4 cells pre-incubated with and without sCD40L were cultured in the presence of different concentrations of IDEC-131, RITUXAN®, the anti-CD20 antibody CE9.1, and anti-CD4 antibodies (Anderson et al., Clin. Immunol. & Immunopathol 84: 73-84 (1997)). After 5 days, the cytotoxicity/proliferation of DHL-4 cells was determined by Alamar Blue assay, as described above. Fig.
• CD40L-CD40 sienalins prevents apoptosis of B-lymphoma cells by anti-CD20 antibody.
• DHL-4 cells 0.5 to 1 x 10 6 cells/ml
• sCD40L 10 ⁇ g/ml
• control antibody CE9.1; an anti-CD4 antibody
• Table I shows resistance of RITUXAN® induced apoptosis in DHL-4 lymphoma cells by exposure to sCD40L.
• activation of caspase-3 was used as the surrogate marker since our previous studies revealed good correlation between caspase-3 and Tunel assay.
• Cross-linking of RITUXAN® on the DHL-4 cell surface in the presence of sCD40L decreased levels of apoptosis, whereas cells not exposed to sCD40L apoptosed.
• cultures incubated in the presence of an antibody of the same isotype, control antibody (CE9.1) resulted in no apoptosis of the cells.
• CE9.1 control antibody
• Example 4 Effect of IDEC-131 on the survival of chronic lymphocytic leukemia (CLL) cells
• CLL chronic lymphocytic leukemia
• B-CLL cells were cultured with and without IDEC-131 in the presence of CD40L in vitro.
• Peripheral blood mononuclear cells (PBMC) were isolated from a CLL patient's blood using a Ficoll-Hypaque gradient centrifugation. Viability was determined by Trypan blue dye exclusion and was >98%. Flow cytometric analysis revealed that >70% of the lymphocytes were CD 19 + /CD20 + .
• CLL cells were cultured in CLL growth medium (e.g., RPMI-1640 medium supplemented with 5% FCS or 2% of autologous donor plasma, supplemented with 2 mM L-Glutamine and 100 U/ml Penicillin-Streptomycin).
• CLL growth medium e.g., RPMI-1640 medium supplemented with 5% FCS or 2% of autologous donor plasma, supplemented with 2 mM L-Glutamine and 100 U/ml Penicillin-Streptomycin.
• CD19 + B-cells were purified using CD19 + DynabeadsTM as per manufacture's instructions (Dynal, Cat. #111.03/111.04) and cultured as above.
• CLL or purified B-CLL cells cultured in growth medium mostly under went spontaneous apoptotic cell death. However, culturing these cells in the presence of sCD40L extended their viability in cultures.
• Table II indicates the cell viability of CD 19 + B-CLL cells grown in the presence or absence of sCD40L (5 ⁇ g/ml) at different time points and indicates the longer survival of CLL cells.
• B-CLL cells from Patient #1 cultured with sCD40L had > 60% viability for greater than 2 weeks, whereas cells grown in the absence of sCD40L had less than 10% viability.
• a equals the percent viability determined by Trypan blue dye exclusion.
• Fig. 3 A shows the effect of IDEC-131 on the growth and survival of B-CLL cells after 7 days in culture.
• Purified B-CLL cells from a CLL patient (2 x 10 6 cells/ml) were divided into two culture tubes. Cells in one tube were mixed with sCD40L (5 ⁇ g/ml) in equal volume of growth medium, whereas the other tube was incubated with equal volume of growth medium as control. After 1 hour of incubation at 37° C, cells were gently mixed and 100 ⁇ l of cell suspension media added to each well of a 96-well flat bottom plate in duplicate with and without varying concentrations of IDEC-131 (10 ⁇ g/ml to 0.3 ⁇ g/ml).
• CLL cells from CLL patients were cultured (5 x 10 5 cells/ml) with and without 5 ⁇ g/ml of CD40L at 37 ° C.
• the class II molecule, HLA-DR expression was determined on CD 19 + cells by flow cytometry using standard procedures. Briefly, cultured lymphocytes were harvested at different time points and analyzed for surface expression of molecules using antibodies coupled to either fluorescein (FITC) or phycoerythrin (PE) for single or double staining using a FACScan (Becton-Dickinson) flow cytometer.
• FITC fluorescein
• PE phycoerythrin
• anti-CD45-FITC to gate lymphocyte population on a scatter plot
• anti-CD 19-PE Pharmingen, Cat. # 30655
• anti-CD20-FITC Pharmingen; Cat. #33264
• anti-CD3-FITC antibodies Pharmingen; Cat. #30104
• anti-CD 19-RPE and anti-HLA-DR-FITC antibodies Pharmingen; Cat. #32384
• Fig. 4 shows the comparison of HLA-DR expression in CD 19 + CLL cells cultured with sCD40L and those cells not cultured with sCD40L. A higher level of HLA-DR expression was detected on B-CLL cells cultured in the presence of sCD40L (Table III).
• IDEC-131 For treatment of a CD40 + malignancy, IDEC-131 at about 10 to about 50 mg/ml in a formulation buffer 10 mM Na-citrate, 150 mM NaCl, 0.02% Polysorbate 80 at pH 6.5 is infused intravenously (iv) to a subject. IDEC-131 is administered before, after or in conjunction with RITUXAN®. The RITUXAN® dosage infused ranges from about 3 to about 10 mg/kg of subject weight.
• IDEC-131 is infused at a dosage ranging from about 3 to about 10 mg per kg of patient weight immediately prior to the initiation of the CHOP cycle. IDEC-131 administration will be repeated prior to each CHOP cycle for a total of 4 to 8 cycles.
• IDEC-131 is to be administered in combination with CHOP and RITUXAN®, IDEC-131 is admimstered as discussed above in Example 6, followed by the schedule specified for CHOP-IDEC-131 admimstration in Example 7. Alternatively, the same regimen is effected wherein IDEC-131 (anti-CD40L) is substantially within an anti-B7 antibody. All references discussed above are hereby incorporated by reference in their entirety.

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• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

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• 2001
  • 2001-05-16 JP JP2002508475A patent/JP2004502742A/ja not_active Abandoned
  • 2001-05-16 EP EP01939050A patent/EP1305045A1/en not_active Withdrawn
  • 2001-05-16 MX MXPA03000306A patent/MXPA03000306A/es not_active Application Discontinuation
  • 2001-05-16 AU AU2001264612A patent/AU2001264612C1/en not_active Ceased
  • 2001-05-16 KR KR1020087009742A patent/KR20080039547A/ko not_active Application Discontinuation
  • 2001-05-16 SG SG200500152-4A patent/SG136804A1/en unknown
  • 2001-05-16 KR KR10-2003-7000429A patent/KR20030031957A/ko not_active Application Discontinuation
  • 2001-05-16 WO PCT/US2001/015677 patent/WO2002004021A1/en active IP Right Grant
  • 2001-05-16 CA CA002415100A patent/CA2415100A1/en not_active Abandoned
  • 2001-05-16 IL IL15376401A patent/IL153764A0/xx unknown
  • 2001-05-16 AU AU6461201A patent/AU6461201A/xx active Pending
  • 2001-05-16 CN CN01814063A patent/CN1446104A/zh active Pending
• 2003
  • 2003-01-10 NO NO20030128A patent/NO20030128L/no not_active Application Discontinuation

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