Combination therapy for treatment of autoimmune diseases using b cell depleting/immunoregulatory ant

专利摘要:
The present invention provides for example a combination of an immunomodulatory antibody such as an anti-B7.1 or anti-B7.2 or anti-CD40L antibody and one or more B cell antibodies such as CD19, CD20, CD22, CD23, or CD37. For the treatment of autoimmune diseases, characterized in that such antibodies are used in isolation, or in combination, and in any order for an extended period of time.
公开号:KR20040023565A
申请号:KR10-2003-7003843
申请日:2001-09-18
公开日:2004-03-18
发明作者:한나네이빌
申请人:아이덱 파마슈티칼즈 코포레이션；
IPC主号:

专利说明:

조합 COMBINATION THERAPY FOR TREATMENT OF AUTOIMMUNE DISEASES USING B CELL DEPLETING / IMMUNOREGULATORY ANTIBODY COMBINATION}
[3] In recent years, the use of antibodies for the treatment of cancer, particularly non-Hodgkin's lymphomas, leukemias, viral mediated diseases, and autoimmune diseases, has been widely accepted. In particular, the use of anti-CD20 or anti-CD22 antibodies with cell depletion activity has been reported for the treatment of cancer, eg, non-Hodgkin's lymphoma and related B cell lymphoma. In addition, among others, the use of B cell depleting antibodies specific for CD19 and CD37 has been reported.
[4] In addition, the use of various immunomodulatory antibodies, ie antibodies that induce therapeutic benefit by modulating (ie enhancing or inhibiting specific immune pathways), has been reported. For example, such antibodies modulate the differentiation, proliferation, activation, and / or function of T or B cells, or other cells involved in the regulation of immunity. Such immunomodulatory antibodies bind ligands or receptors on immune cells, generally B or T cell antigens involved in the regulation of humoral or cellular immunity. Examples of such ligands are immune signaling molecules such as B7.1, B7.2, and T cell regulatory molecules such as CD40-L, CD40, and CD4. Discussion of the function of some of these antigens and the conventional use of antibodies specific to them for treatment are briefly discussed below.
[5] CD40L is a receptor expressed on the activating surface of helper cells and a ligand expressed on the surface of B-lymphocytes as well as counterreceptors for CD40, other antigen-presenting cells. The contact-dependent interaction of CD40L on activated T cells with CD40 expressed on B and other antigen-presenting cells, called “T cell helper function”, leads to activation and differentiation of B lymphocytes and the humoral immune response. It helps to adjust. Such regulation is involved in the regulation of the specificity, secretion and isotype-encoding function of antibody molecules. The process by which T cells help differentiation of B cells has been divided into two distinct phases: induction and effector phases (Vitetta et al., Adv. Immunol. 45: 1 (1989); Noelle et al., Immunol. Today 11: 361 (1990)).
[6] The basic principles of the molecule for help of T cells in humoral immunity through the interaction of CD40 and its ligands gp39 (also known as CD40L and CD154) are now well understood. In essence, activated T helper cells are known to express lymphokine genes and membrane proteins essential for the mutual activation of CD40L, cognate, antigen-presenting B cells. The interaction of CD40L with its receptor CD40 on B cells induces B cell invasion and induces B cell responses to the growth and differentiation effects of lymphokines.
[7] In addition, CD40L is known to play a broader and more universal role in the T cell immune process, ie its role in the regulation of T cell support and humoral immunity. This role of CD40L is also not well understood. For example, the pathology of T cell mediated autoimmune diseases including multiple sclerosis, type 1 diabetes, inflammatory bowel disease, ovarian inflammation, and thyroiditis presumably plays a role in disease pathology by ignoring the role of T effector cells. It has been hypothesized that it is involved in the presence of specific CD40 ligands expressing inhibitor cell numbers. In addition, CD40 and CD40L have been reported to contribute to autoimmune diseases by playing a peripheral or central role in resistance (Durie et al., Res. Immunol. Vol 145 (3): 200-205 (1994)).
[8] The use of antagonists of CD40L for the treatment of both B-cell mediated and T-cell mediated autoimmune diseases has been reported. For example, EP 555,880, US Pat. No. 5,474,771, and WO 93/09212 disclose the use of CD4OL antagonists for the treatment of humoral autoimmune diseases. The use of CD40L antagonists for treating T cell mediated autoimmune diseases is disclosed in US Pat. No. 5833987, and its PCT counterpart application PCT / US96 / 09B7.
[9] As discussed above, it has also been reported to use a molecule that specifically binds to a target antigen on B lymphocytes and depletes B cells. Perhaps the best accepted B cell target for therapy is the CD20 antigen, Rituxan, which is FDA approved. , Chimeric monoclonal antibody directed against CD20 antigen for the treatment of non-Hodgkin's lymphoma.
[10] The CD20 antigen (also known as human B-lymphocyte-limiting differentiation antigen, -p-33) is a hydrophobic intramembrane protein with a molecular weight of about 35 kD located in pre-B and mature B lymphocytes (Valentine et al. J. Biol. Chem. 264 (19): 11282-11287 (1989); and Einfeld et al. EMBO J. 7 (3): 711-717 (1988)). The antigen is also expressed in at least 90% of B cell non-Hodgkin's lymphoma HL (Anderson et al. Blood 63 (6): 1424-1433 (1984)), but hematopoietic stem cells, pro-B cells, It is not found in normal plasma cells or other normal tissues (Tedder et al. J. Immunol. 135 (2): 973-979 (1985)). CD20 controls the early stages in the activation process for cell cycle initiation and differentiation (Tedder et al., Supra) and possibly functions as calcium ion channels (Tedder et al. J. Cell. Biochem. 14D: 195 (1990) )).
[11] Because CD20 is expressed in B cell lymphomas, this antigen may serve as a candidate for the "targeting" of such lymphomas. In essence, this targeting can be generalized as follows: administering to the patient an antibody specific for the CD20 surface antigen of B cells; Such anti-CD20 antibodies specifically bind to CD20 antigens of both normal and malignant (surface) B cells; Antibodies bound to the CD20 surface antigen cause destruction and depletion of tumor B cells. In addition, chemical agents or radiolabels with the potential to destroy tumors may be conjugated to anti-CD20 antibodies such that the agent is specifically "carried" into tumor B cells. Regardless of the approach, the primary goal is to destroy the tumor; Specific approaches can be determined by the specific anti-CD20 antibody employed, and therefore the approaches available for targeting CD20 antigens can vary considerably.
[12] CD19 is another antigen expressed on the surface of B lineage cells. Like CD20, CD19 is found on cells throughout lineage differentiation from the stem cell stage to just before final differentiation into plasma cells (Nadler, L. Lymphocyte Typing II2: 3-37 and Appendix, Renling et al. Eds. (1986) by Springer Verlag. Unlike CD20, antibodies that bind to CD19 result in internalization of the CD19 antigen. The CD19 antigen is identified among others by the HD237-CD19 antibody (also called "B4" antibody) (Kiesel et al. Leukemia Research II, 12: 1119 (1987)). CD19 antigen is present in 4-8% of peripheral blood mononuclear cells and in at least 90% of B cells isolated from peripheral blood, spleen, lymph nodes or tonsils. CD19 is not detected in peripheral blood T cells, monocytes or granulocytes. Virtually all non T cell acute lymphocytic leukocytes (ALL), B cell chronic lymphocytic leukocytes (CLL), and B cell lymphoma express CD19 detectable by antibody B4 (Nadler et al., J. Immunol). 131: 244 (1983); and Nadler et al. In Progress in Hematology Vol. XII pp. 187-206. Brown, E. ed. (1981) by Grune & Stratton, Inc.).
[13] CD22 is another antigen expressed on the surface of B lineage cells. This antigen is also referred to by the names "BL-CAM" and "LyB8". This antigen is a membrane immunoglobulin-related protein having a molecular weight of about 140,000 that is tyrosine-phosphorylated when the membrane Ig is ligated thereto (Engel et al. J. R & PMed 181 (4): 1521-1526 91995; Campbell and Eur J.Immunol. 25: 1573). This antigen is a negative regulator of B-cell receptor signaling (Nitschke, et al., Curr. Biol. 7: 133 (1997)); It has been reported to promote monocyte erythrocyte artism (Stemenkoul et al. Nature 345: 74 (1990)). CD22-specific naked antibodies, called Lymophocide ^™ , are currently in clinical trials for the treatment of painless non-Hodgkin's lymphoma by Immunomedics, Inc. In addition, the use of yttrium 90 labeled forms of these same antibodies for the treatment of painless and aggressive non-Hodgkin's lymphoma is also in clinical trials.
[14] CD23 is another antigen expressed on B cells and is a low affinity receptor for IgE, also known as FcERII. The use of antibodies that bind CD23 for the treatment of inflammatory, autoimmune and allergic disorders is proposed in the patent and non-patent literature.
[15] B7.1 and B7.2 include other examples of B cell antigens that have been reported to have therapeutic utility in the use of ligands that specifically bind and act as immunomodulators. In particular, anti-B7, in particular B7.1 (CD80), B7.2 (CD86), or B7.3 which is expressed on the surface of B cells, binds to intra and external membrane glycoproteins as immunosuppressive agents and various diseases It has been reported to have potential applicability for the treatment of. For example, U. S. Patent No. 5,869, 040, registered to DeBoer et al. On Feb. 9, 1999, and assigned to Chiron Corporation, discloses another immunosuppressive agent to treat transplant rejection, graft-versus-host disease and rheumatoid arthritis. The use of anti-B7.1 antibodies together is disclosed. In addition, US Pat. No. 5,885,579, registered to Linsley et al. On March 23, 1999, discloses B7 positive cells by administering a ligand specific for the B7 antigen, eg, B7.1 (CD80) or B7.2 (CD86). Disclosed is the treatment of an immune disease involved in T cell interaction with.
[16] In addition, US Pat. No. 6,113,198 (Anderson et al.), In contrast to previous anti-B7 antibodies, does not inhibit B7.1 / CTLA-4 interactions and is specific for the B7-1 antigen useful for the treatment of diseases including autoimmune diseases. The use of an antibody is disclosed. However, the combined use of antibodies specific for CD40L with such antibodies is not disclosed, and the use of such antibodies with reported B cell depleting antibodies is not disclosed.
[17] In particular, rituximab (RITUXAN ) Antibody is a genetically engineered chimeric mouse / human monoclonal antibody directed against the CD20 antigen. RITUXAN Is indicated for the treatment of relapsed or refractory low grade or follicular, CD2O positive, B cell non-Hodgkin's lymphoma patients (US Pat. No. 5,736, B7, registered for Anderson et al. On April 7, 1998). The results of studies on the in vitro mechanism of action are RITUXAN It has been demonstrated that this human complement binds and degrades lymphoid B cell lines through the cytotoxicity (CDC) of complement-dependent cells (Reff et al. Blood 83 (2): 435-445 (1994)). It also has important activity in assays for antibody-dependent cytotoxicity (ADCC). More recently, RITUXAN Has been shown to have anti-proliferative effects and directly induce apoptosis in tritiated thymidine consolidation assays, whereas other anti-CD19 and CD20 antibodies did not (Maloney et al. Blood 88 (10): 637a). (1996)). Also RITUXAN And synergy between chemotherapy and toxins was observed experimentally. In particular, RITUXAN Sensitizes drug-resistant human B cell lymphoma cells lines against the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin and lysine (Demidem et al. Cancer Chemotherapy & Radiopharmaceuticals 12 (3): 177-186 (1997). )). RITUXAN in vivo Depletes B cells very effectively from peripheral blood, lymph nodes, and bone marrow of cynomolgus monkeys, probably through complement and cell-mediated methods (Reff et al. Blood 83 (2): 435-445 (1994). )).
[18] Perrotta and Abuel Blood: 92 Abstract # 3360 from the ASH 40th Annual Meeting (November 1998) is RITUXAN An anecdotal report of a 50 year old woman with idiopathic thrombocytopenic purpura (ITP) in response to
[1] This application is filed on September 18, 2000, entitled "Combination Therapy for the Treatment of Autoimmune Diseases Including CD40L Antagonist and Antibodies to B7, CD19, CD20, CD22 or CD23." 60 / 233,607 to Nabil Hanna; And US Provisional Patent Application No. 60 / 257,147 to Nabil Hanna, filed Dec. 22, 2000, entitled "Combination Therapy for the Treatment of Autoimmune Diseases Using a B Cell Depletion / Immunomodulatory Antibody Combination." Claims are made from this application.
[2] The present invention provides novel combination therapies for the treatment of autoimmune diseases. In particular, the present invention relates to the combined use of immunomodulatory antibodies, preferably antibodies modulating T and / or B cell differentiation, proliferation and / or function, and B cell depleting antibodies for the treatment of autoimmune diseases. Such antibodies may be administered separately or in combination, and in either order.
[19] The present invention utilizes a combination of one or more immunomodulatory antibodies and one or more B cell depleting antibodies, eg, antibodies targeting CD20, CD19, CD22, CD23, or CD37, to autoimmune diseases, preferably B cell mediated autologous. It is about treating an immune disease. Isolation or combined administration of this type of antibody leads to elevated benefits when used in the treatment of autoimmune diseases. This result occurs because B cell depleted antibodies act to deplete B cell numbers, reducing the amount of circulating IgE and other antibodies involved in the pathology of autoimmunity. However, B cell depleted antibodies such as RITUXAN Tends to preferentially deplete activated B cells. In contrast, immunomodulatory antibodies, such as anti-B7 and anti-CD40L antibodies, induce their immunomodulatory effects, ie immunosuppression against inactivated B cells (ie, inactivated antigen presenting B cells). Thus, it is hypothesized that the use of these two functionally distinct forms of antibodies leads to synergistic benefits in that both activated and inactivated B cells can be easily removed from circulation. This will significantly reduce the level of circulation of autoimmune antibodies because the levels of antibody-producing B cells are dramatically reduced. This will provide significant therapeutic benefit, particularly for autoimmune diseases in which B cells, and more particularly autoantibodies, are actively involved in disease pathology.
[20] As discussed below, preferred immunomodulatory antibodies include anti-B7.1 or anti-B7.2, anti-CD40, anti-CD40L, and anti-CD4 antibodies. Preferred examples of B cell depleting antibodies include antibodies specific for CD20, CD19, CD21, CD37 and CD22.
[21] In its broadest aspect, the present invention provides an antibody comprising (i) an immunomodulatory antibody, preferably an antibody that inhibits non-activated B cells; And (ii) B cell depleting antibodies; Such antibodies can be isolated or combined to provide combination therapies for treating autoimmune diseases such as rheumatoid arthritis, SLE, ITP by combined usage that can be administered in either order.
[22] In a more particular aspect, the invention provides an antibody comprising (i) an antibody against B7.1 or B7.2 and / or anti-CD40L, and (ii) a B cell selected from anti CD20, anti-CD19, anti-CD22 or anti-CD37. Treating autoimmune diseases by combined use of depleting antibodies.
[23] The present invention also includes a container and one or more compositions contained therein, for example, immunomodulatory antibodies and B cell depletion, such as anti-CD40L or anti-B7.1 or anti-B7.2 antibodies (immunoregulatory antibodies). An article of manufacture for the treatment of an autoimmune disease comprising an effective amount of an antibody or fragment thereof, anti-CD20, anti-CD19, anti-CD22 or anti-CD37 (B cell depleted antibody).
[24] I. Definition
[25] A “B cell depleted antibody” herein is an antibody or fragment that binds to a B cell label that causes apparent B cell depletion upon administration. Preferably, such antibodies will deplete at least about 50% of B cell numbers generally within approximately several days after administration. In a preferred embodiment, the B cell depleting antibody is RITUXAN (Chimeric anti CD20 antibody) or an antibody having substantially the same or greater cell depletion activity. This antibody has been demonstrated to provide substantially 90% B cell depletion within 24 hours of administering an effective amount.
[26] "Immunomodulatory antibody" means an antibody that has an effect on the immune system by activated B cell depletion and other mechanisms. Examples thereof include, for example, antibodies that inhibit T cell immunity, B cell immunity by inducing resistance (anti-CD40L, anti-CD40), or other immunosuppressive antibodies (anti-B7.1, anti-B7.2). , Or anti-CD4). In some cases, immunomodulatory antibodies may also have the ability to enhance apoptosis.
[27] A "B cell surface label" herein is an antigen expressed on the surface of a B cell that can be targeted using an antagonist that binds to it. Exemplary B cell surface labels include 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 labels. Of particular interest are B cell surface labels that are preferentially expressed on B cells as compared to other non-B cell tissues in mammals, and that can be expressed in both precursor B cells and mature B cells. In one embodiment, the label is something like CD20 or CD19 and is found on B cells throughout lineage differentiation from the stem cell stage to just before final differentiation into plasma cells. Desired B cell surface labels here are CD19, CD20, CD23, CD80 and CD86.
[28] The "CD20" antigen is a -35 kDa non-glycosylated phosphoprotein found on at least 90% surface of B cells from peripheral blood or lymphoid organs. CD20 is expressed during early pro-B cell development and remains until plasma cell differentiation. CD20 is present on both normal B and malignant B cells. Other names of CD20 in the literature include "B-lymphocyte-limiting antigen" and "Bp35". CD20 antigens are described in Clark et al. PNAS (USA) 82: 1766 (1985).
[29] "CD19" antigen means a -90kDa antigen, for example identified by HD237-CD19 or B4 antibody (Kiesel et al. Leukemia Research II, 12: 1119 (1987)). Similar to CD20, CD19 is found on cells throughout lineage differentiation from the stem cell stage to just before final differentiation into plasma cells. Binding of antagonists to CD19 can cause internalization of the CD19 antigen.
[30] By "CD22" antigen is meant an antigen expressed on B cells, also known as "BL-CAM" and "LybB", which are involved in B cell signaling and adhesion (Nitschke et al., Curr. Biol. 7: 133 ( 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)) was screen cloning a gene coding for the antigen, and characterized by its 1g domain.
[31] B7 antigens include B7.1 (CD80), B7.2 (CD86) and B7.3 antigens, which are intramembrane antigens expressed on B cells. Antibodies that specifically bind to B7 antigens, including human B7.1 and B7.2 antigens, are known in the art. Desired B7 antibodies are primatized as disclosed in US Pat. No. 6,113,198, registered for Anderson et al. And assigned to IDEC Pharmaceuticals. B7 antibodies and human and humanized B7 antibodies.
[32] CD23 refers to a low affinity receptor for IgE expressed by B and other cells. In the present invention, CD23 is preferably a human CD23 antigen. CD23 antibodies are also known in the art. In the present invention, it is most preferred that the CD23 antibody is a human or chimeric anti-human CD23 antibody comprising a human IgGI or IgG3 constant domain, and a depleted anti-CD23 antibody disclosed in US Pat. No. 6,011,138.
[33] An "autoimmune disease" is a non-malignant disease or disorder that arises from and is directed to an individual's native tissue. Non-malignant autoimmune diseases herein specifically exclude malignant or cancerous diseases or conditions, in particular B cell lymphoma, acute lymphocytic leukocytes (ALL), chronic lymphocytic leukocytes (CLL), hematopoietic leukemia and chronic myeloid Excludes constitutive leukemia. Examples of such diseases or disorders include inflammatory responses (eg, atopic dermatitis), such as inflammatory skin diseases including psoriasis and dermatitis; Systemic scleroderma and sclerosis; Reactions associated with inflammatory bowel disease (Crohn's disease and ulcerative colitis); Respiratory distress syndrome (adult respiratory distress syndrome; including ARDS); dermatitis; meningitis; encephalitis; Uveitis; colitis; Glomerulonephritis; Allergic conditions such as eczema and asthma and other conditions involved in T cell infiltration and chronic inflammatory responses; Atherosclerosis; Leukocyte adhesion defects; Rheumatoid arthritis; Systemic lupus erythematosus (SLE); Diabetes (eg, type 1 diabetes or insulin dependent diabetes); Multiple sclerosis; Rhinoid syndrome; Autoimmune thyroiditis; Allergic encephalomyelitis; Sjogren's syndrome; Juvenile diabetes; And immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes commonly found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; And pernicious anemia (Addison disease); Diseases involving leukocyte leakage; Central nervous system (CNS) inflammatory disorders; Multiple organ injury syndrome; Hemolytic anemia (including hanglobinemia); Myasthenia gravis; Antigen-antibody complex mediated disease; Anti-glomerular basement membrane disease; Antiphospholipid syndrome; Allergic neuritis; Graves' disease; Lambert-eaton duty syndrome; Bullous whey; pemphigus; Autoimmune polyendocrine disease; Lighter disease; Rigid human syndrome; Beset's disease; Giant cell arteritis; Immunocomplex nephritis; IgA nephropathy; IgM Multiple Neuropathy; Immune thrombocytopenic purpura (ITP), autoimmune thrombocytopenia and ovarian inflammation.
[34] B cell “antagonists” destroy or deplete mammalian B cells and / or one or more B cells, for example, by binding to B cell surface labels, for example by reducing or disrupting humoral responses induced by B cells. Molecules that interfere with function. In contrast, B cell depleted antibodies deplete B cells (ie, reduce circulating B cell levels) in mammals treated with them. This depletion can lead to a variety of mechanisms, such as antibody-dependent cell mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC), inhibition of B cell proliferation and / or induction of B cell death (eg, via apoptosis). It is possible through. Antagonists included within the scope of the present invention include antibodies, synthetic or natural sequence peptides and small molecule antagonists that are bound to B cell labels and optionally conjugated or fused to cytotoxic agents.
[35] CD40L antagonists are molecules that specifically bind to CD40L and preferably antagonize the interaction of CD40L and CD40. Examples thereof include CD40L, soluble CD40, soluble CD40 fusion proteins, and antibodies and antibody fragments that specifically bind to small molecules that bind to CD40L. Desired antagonists according to the present invention comprise antibodies or antibody fragments specific for CD40.
[36] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" mean that non-specific cytotoxic cells (ie, Natural Killer (NK) cells, neutrophils, and macrophages) that express Fc receptors (FcRs) are on target cells. It refers to a cell-mediated response to lysing target cells after recognition of the antibody bound to the ADCC, NK cell-mediated primary cells express only FcγRIII, and monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991), US Pat. No. 5,500,362 to assess ADCC activity of molecules of interest. In vitro ADCC assays can be performed as described in US Pat. No. 5,821,337. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Interested The ADCC activity in vivo, for example, the literature (Clynes et al PNAS (USA) 95: 652-656 (1998).) Can be evaluated in an animal model such as that described in.
[37] A "human effector cell" is a white blood cell that expresses one or more FcRs and acts as an effector. Preferably, these cells express at least FcγRIII and have ADCC effector action. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred. Effector cells may be isolated from their natural source, such as blood or PBMCs as described herein.
[38] The term "Fc receptor" or "FcR" is used to refer to a receptor that binds to the Fc region of an antibody. Desired FcRs are natural sequence human FcRs. In addition, desired FcRs are FcRs that bind IgG antibodies (γ receptors), including receptors of the FcγRI, FcγRII and RcγRIII subclasses, including allelic variants and optionally splicing forms of these receptors. FcγRII receptors include FcγRIIA (“activating receptor”) and FcγRUB (“inhibiting receptor”), which have essentially similar analogous amino acid sequences in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activating motif (ITAM) in the cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are described 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. . 126: is described in reference 330-41 (1995)). Other FcRs, including those to be identified in the future, are included in the term "FcR" herein. The term also includes neonatal receptor, FcRn, which allows mother IgGs to be delivered to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249) 1994).
[39] "Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to dissolve a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component (Clq) of the complement system to molecules (eg antibodies) complexed with syngeneic antigens. To assess complement activation, a CDC assay can be performed as described, for example, in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).
[40] An "growth inhibition" antagonist is an antagonist that prevents or reduces the proliferation of cells expressing the antigen to which the antagonist binds. For example, this antagonist may interfere with or reduce the proliferation of B cells in vivo and / or ex vivo.
[41] Antagonists that "induce apoptosis" include, for example, as identified by binding of Annexin V, fragmentation of DNA, cell contraction, endoplasmic reticulum expansion, cell fragmentation, and / or membrane vesicles (called apoptosis). For example, it is an antagonist that induces the planned cell death of B cells.
[42] The term "antibody" is used herein in its broadest sense and specifically refers to a fully monoclonal antibody, a polyclonal antibody, a multispecific antibody formed of two or more intact antibodies, as long as it exhibits the desired biological activity. Such as bispecific antibodies) and antibody fragments.
[43] An "antibody fragment" consists of a portion of an intact antibody, preferably comprising an antigen-binding or variable region thereof. Examples of 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.
[44] “Native antibodies” are generally about 150,000 Daltons heterotetramer glycoproteins composed of two identical light (L) chains (light chains) and two identical heavy (H) chains (heavy chains). Each light chain is bound to the heavy chain by one covalent disulfide bond, but the number of disulfide bonds varies in the heavy chain of another immunoglobulin isotype. Each heavy and light chain also has in-chain disulfide bridges at regular intervals. Each heavy chain has a variable domain (VH) at one end followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end; The constant domain of the light chain is in line with the first constant domain of the heavy chain, and the variable domain of the light chain is in line with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
[45] The term "variable" refers to the fact that any portion of the variable domain differs greatly in sequence between antibodies and is used in the binding and specificity of each specific antibody to its specific antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three parts called hypervariable regions of both the light and heavy chain variable domains. More conserved portions of the variable domains are called framework regions (FRs). The variable domains of the natural heavy and light chains each comprise four FRs, predominantly 13-sheet structures, linked to three hypervariable regions, which form loop links, and in some cases B-sheet structures To form. The hypervariable regions of each chain coexist with FRs and form the antigen-binding site of the antibody with the hypervariable regions from other chains (Kabat el al., Sequences of Proteins of Immunological Interest, 5th Ed. See Sevice, National Institutes of Health, Bethesda, MD. (1991). The constant domains are not directly related to the binding of the antibody to the antigen but exhibit various effector functions such as antibody involvement in antibody dependent cellular cytotoxicity (ADCC).
[46] Papain digestion of antibodies results in two identical antigen-binding fragments, called “Fab” fragments, each having a single antigen-binding site, and a residual “Fc” fragment whose name is readily capable of crystallizing. Pepsin treatment yields an F (ab ') 2 fragment having two antigen-binding sites and capable of crosslinking with the antigen.
[47] "Fv" is the minimum antibody fragment with a complete antigen-recognition and antigen-binding site. This region consists of a dimer with one heavy chain and one light chain variable domain that are tightly covalently bound. In this structure, three hypervariable regions of each variable domain interact to define an antigen-binding site on the VH-VL dimer surface. Collectively, six hypervariable regions confer antigen binding specificity to the antibody. However, even a single variable domain (or half of the Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigens, even if they have a lower affinity than the entire binding site.
[48] Fab fragments also comprise 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 several residues at the carboxy terminus of the heavy chain CHI domain comprising one or more cysteines from the antibody hinge region. Fab'-SH refers herein to Fab 'in which the cysteine residue (s) of the constant domains have one or more free thiol groups. F (ab ') Z antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.
[49] The “light chains” of antibodies of certain vertebrate species (immunoglobulins) can be assigned to one of two distinctly different forms called κ and λ, based on the amino acid sequences of their constant domains.
[50] Depending on the amino acid sequence of the constant domain of the heavy chains, antibodies can be assigned to different classes. There are five main classes of complete antibodies: IgA, IgD, IgE, IgG and IgM, some of which can be further subdivided into subclasses (isotypes) such as IgGI, IgG2, IgG3, IgG4, IgA and IgA2. have. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively. It is preferred that the heavy chain constant regions complete the γ-1, γ-2, γ-3 and γ-4 constant regions. These constant domains also preferably comprise variants for enhancing antibody stability, such as the P and E variants disclosed in US Pat. No. 6,011,138, which is incorporated herein by reference in its entirety. Subunit structures and three-dimensional structures of other immunoglobulin classes are well known.
[51] "Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, which domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide binder between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol . 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).
[52] The term "diebody" refers to a small antibody fragment having two antigen-binding sites, which fragments are linked to the light chain variable domain (VL) within the same polypeptide chain (VH-VL). VH). Using a binding agent that is too short to pair between two domains on the same chain allows the domain to pair with the complementary domains of the other chain to form two antigen binding sites. Diamond bodies are described, for example, in EP 404,097; WO 93/11161; And Hollinger et al., Proc. Nad. Acad . Sci. USA. 90: 6444-6448 (1993).
[53] As used herein, the term "monoclonal antibody" means an antibody that is substantially obtained from a homogeneous antibody population, ie the individual antibodies that make up this population are identical except for possible naturally occurring mutations that may be present in small amounts. Do. Monoclonal antibodies are highly specific for a single antigenic site. In addition, unlike conventional (polyclonal) antibody preparations that generally include other antibodies against other determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to specificity, monoclonal antibodies are advantageous in that they are synthesized in hybridoma cultures that are not contaminated by other immunoglobulins. The modifier “monoclonal” refers substantially to the characteristics of the antibodies obtained from antibodies of the homogeneous population and does not mean that the antibodies must be produced in any particular way. For example, the monoclonal antibodies used in accordance with the present invention may first be made by the hybridoma method described in Kohler et al. , Nature, 256: 495 (1975), or by using recombinant DNA methods. (See, eg, US Pat. No. 4,816,567). "Monoclonal antibodies" are also described, eg, in Clackson et al. , Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol ., 222: 581-597 (1991). The methods described in can be used to isolate from phage antibody libraries.
[54] Monoclonal antibodies herein are particularly identical or homologous to the corresponding sequences in which the portions of the heavy and / or light chains are derived from a particular species or belong to a specific antibody class or subclass, or Residues in a chain may refer to "chimeric" antibodies (immunoglobulins) that are identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies as long as they exhibit the desired biological activity. (US Pat. No. 4,816,567; Mossison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1980)). Chimeric antibodies of interest herein are “primatizations” comprising variable region antigen-binding sequences derived from non-human primates (eg, Old World Monkey, apes, etc.) and human constant region sequences. (primatized) "antibody.
[55] A “humanized” form of a non-human (eg mouse) antibody is a chimeric antibody comprising a minimal sequence derived from a non-human immunoglobulin. In most parts, a humanized antibody is a family of non-human species (donor antibodies) such as mice, rats, rabbits, or non-human primates that have the desired specificity, affinity, and capacity as residues from the hypervariable regions of the receptor. Human immunoglobulin (receptor antibody) that is replaced by residue from the variable region. In some cases, skeletal region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues not found in receptor antibodies or donor antibodies. This modification further improves antibody performance. In general, humanized antibodies comprise substantially one or more, and generally, all or substantially all hypervariable loops corresponding to domains of non-human immunoglobulins and all or substantially all FRs are domains of human immunoglobulin sequences Will consist of two variable domains. Humanized antibodies will also optionally comprise one or more portions of an immunoglobulin constant region (Fc), generally a region of human immunoglobulin. For more details see Jones et al., Nature 32 1: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2 : 593-596 (1992).
[56] As used herein, the term "hypervariable region" refers to an amino acid residue of an antibody that enables antigen-binding. The hypervariable regions are amino acid residues from the "complementarity determining regions" or "CDRs" (eg residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) and heavy chain variable of the light chain variable domain). 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the domains; Kabat et al., Sequence of Proteins of Immunological Interest, 5th Ed.Public Health Service., National Institutes of Health, Bethesda, MD. (1991)) and / or such residues from “overvariable loops” (eg residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) of the light chain variable domain) Domains 26-32 (H1), 53-55 (H2) and 96-101 (H3); Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.
[57] An antagonist that "binds" with an antigen of interest, such as, for example, a B cell surface label, is capable of binding to the antigen with sufficient affinity such that the antagonist is useful as a therapeutic agent that targets expressing cells (eg, B cells). It is an antagonist.
[58] As used herein, the term "anti-CD20 antibody" means RITUXAN. Measurable B cell depletion activity, preferably RITUXAN, when administered at the same amount and conditions as Is an antibody that specifically binds to a CD20 antigen, preferably human CD20, having at least about 10% of its B cell depleting activity (see US Pat. No. 5,736,137, which is incorporated herein by reference in its entirety).
[59] As used herein, the term "anti-CD22 antibody" refers to RITUXAN. Measurable B cell depletion activity, preferably RITUXAN, when administered at the same amount and conditions as Antibody that specifically binds to a CD22 antigen, preferably human CD22, having at least about 10% of its B cell depleting activity (see US Pat. No. 5,736,137, which is incorporated herein by reference in its entirety).
[60] As used herein, the term "anti-CD19 antibody" means RITUXAN. Measurable B cell depletion activity, preferably RITUXAN, when administered at the same amount and conditions as Antibody that specifically binds to a CD19 antigen, preferably human CD19, having at least about 10% of its B cell depleting activity (see US Pat. No. 5,736,137, which is incorporated herein by reference in its entirety).
[61] As used herein, the term "anti-CD23 antibody" means RITUXAN. When administered at the same amount and condition as, has measurable B cell depletion activity, preferably RITUXAN Antibody that specifically binds to a CD23 antigen, preferably human CD23, having at least about 10% of its B cell depleting activity (see US Pat. No. 5,736,137, which is incorporated herein by reference in its entirety).
[62] As used herein, the term "anti-CD37 antibody" means RITUXAN. Measurable B cell depletion activity, preferably RITUXAN, when administered at the same amount and conditions as Is an antibody that specifically binds to a CD37 antigen, preferably human CD37, having at least about 10% of its B cell depleting activity (see US Pat. No. 5,736,137, which is incorporated herein by reference in its entirety).
[63] An “anti-B7 antibody” herein is an antibody that specifically binds B7.1, B7.2 or B7.3, most preferably human B7.1. This antibody preferably inhibits B7 / CD28 interactions, more preferably B7.1 / CD28 interactions, and substantially no further inhibits B7 / CTLA-4 interactions. More preferably, the anti-B7.1 antibody is one of the specific antibodies described in US Pat. No. 6,113,898, which is incorporated herein by reference in its entirety. These antibodies have recently been found to promote apoptosis. Thus, they are very suitable for use in anti-tumor applications.
[64] An “anti-CD40L antibody” is one that specifically binds to CD40L (also known as CD154, gp39, TBAM), preferably having agonistic activity. Desired anti-Cd40L antibodies are antibodies having the specificity of a humanized antibody disclosed in US Pat. No. 6,011,358, assigned to IDEC Pharmaceuticals, which is incorporated herein by reference in its entirety.
[65] An "anti-CD4 antibody" is an antibody that specifically binds to CD4, preferably human CD4, more preferably primatized or humanized anti-CD4 antibodies, preferably human gamma 4 anti-human cd4 antibodies.
[66] “Anti-CD40 antibodies” specifically bind to CD40, preferably human CD40, as disclosed in US Pat. Nos. 5,874,085, 5,874,082, 5,801,227, 5,674,442 and 5,667,165, which are incorporated herein by reference in their entirety. It is an antibody.
[67] Both B cell depleting antibodies and immunoregulatory antibodies preferably comprise human constant domains. Suitable antibodies can include IgGl, IgG2, IgG3 and IgG4 isotypes.
[68] Examples of antibodies that bind CD20 antigens are: "Rituximab"("RITUXAN"). ") (US Pat. No. 5,736,137, incorporated herein by reference); Yttrium- [90] -labeled 2B8 murine antibody" Y2B8 "(US Pat. No. 5,736,37, incorporated herein by reference); optionally labeled with 131I Mouse IgG2a "B1" antibody (BEXXARTM) (US Pat. No. 5,595,721, incorporated herein by reference); Mouse monoclonal antibody "1F5" ( Press et al. Blood 69 (2); 584-591 (1987) “Chimeric 2H7” antibodies (US Pat. No. 5,677,180, incorporated herein by reference).
[69] Examples of antibodies that bind CD22 include LimposideTM, an immunomedical company currently in clinical trials for non-Hodgkin's lymphoma. Examples of antibodies that bind the B7 antigen include B7 antibodies reported in US Pat. No. 5,885,577 (registered in Linsley et al.), Anti-reported in US Pat. No. 5,869,050 (registered in DeBoer et al., Assigned to Chiron Corporation). B7 antibodies, and primateization disclosed in US Pat. No. 6,113,198 (Anderson et al.) Anti-B7.1 antibodies are included, all of which are incorporated herein by reference in their entirety.
[70] Preferred examples of antibodies that bind CD23 include human CD23, reported in US Pat. No. 6,011,138 (Reff et al.), Issued July 4, 1999 and jointly assigned to IDEC Pharmaceuticals Ltd. and Seigagaku, Japan. Specific primateization Antibodies are included. Other anti-CD23 antibodies and antibody fragments are described in 9696741 (Bonnefoy et al. ); 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 have been reported to be useful in the treatment of allergies, autoimmune diseases and inflammatory diseases.
[71] As used herein, "rituximab" or "RITUXAN". Means a genetically engineered chimeric mouse / human monoclonal antibody against the CD20 antigen named “C2B8” in US Pat. No. 5,736, B7 (incorporated herein by reference). IgGI kappa immunoglobulin comprising light and heavy chain variable region sequences and human constant region sequences Rituximab has a binding affinity for a CD20 antigen of about 8.OnM.
[72] An “isolated” antagonist is one that has been identified and separated and / or recovered from components of the natural environment. Contaminant components of the natural environment are substances that interfere with the diagnostic or therapeutic use of antagonists and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antagonist is (1) at least 95% by weight of the antagonist, most preferably at least 99% by weight, and (2) the N-terminus using a spinning cup sequencer as determined by the Lowry method. Or to a degree sufficient to obtain at least 15 residues of the internal amino acid sequence, (3) homogeneous by SDS-PAGE under reducing or non-reducing conditions using Coomassie Blue or preferably silver staining. Since at least one natural environmental component of the antagonist is not present, isolated antagonists include antagonists in situ within recombinant cells. Ordinarily, however, isolated antagonists will be prepared by one or more purification steps.
[73] By "mammal" for therapeutic purposes is meant any animal classified as a mammal, including humans, livestock and breeding animals, and zoos, sporting or pet animals (eg, dogs, horses, cats, cattle, etc.). Preferably the mammal is a human.
[74] "Treatment" means both therapeutic treatment and preventive or preventive measures. Cases in need of treatment include those already suffering from a disease or condition and cases in which the disease or condition should be prevented. Thus, a mammal may be diagnosed as having a disease or condition, or may be susceptible to or easily infected with a disease.
[75] The term "therapeutically effective amount" means an amount of antagonist effective to prevent, ameliorate or treat the autoimmune disease in question.
[76] As used herein, the term "immunosuppressant" refers to a substance that acts to inhibit or mask the immune system of the mammal to be treated. This includes substances that inhibit cytokine production, downregulate or inhibit self-antigen expression, or mask MHC antigens. Examples of such agents include, but are not limited to, 2-amino-6-aryl-5-substituted pyrimidines (US Pat. No. 4,665,077, incorporated herein by reference); azathioprine; Cyclophosphamide; Bromocriptine; Danazol; Dapson; Glutaraldehyde (which masks MHC antigens as described in US Pat. No. 4,120,649); Anti-iodotype antibodies of MHC antigens and MHC fragments; Cyclosporin A; Steroids such as glucocorticosteroids such as, for example, prednisone, methylprednisone, and dexamethasone; Cytokines, including anti-interferon-α, β- or δ-antibodies, anti-tumor necrosis factor-α antibodies, anti-tumor necrosis factor-β antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies Or cytokine receptor antagonists; Anti-LFA-1 antibodies, including anti-CD 11a and anti-CD18 antibodies; Anti-L3T4 antibodies; Heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4 / CD4a antibodies; Soluble peptides comprising LFA-3- binding domains (WO 90/08187, published 7/26/90); Streptoranase; TGF-β; Streptodonase; RNA or DNA from the host; FK506; RS-61443; Deoxyspergualin; Rapamycin; T-cell receptor (Cohen et al., US Pat. No. 5,114,721); T-cell receptor fragments (Offner et al., Science 251: 430-432 (1991); WO 90/11294; Ianeway, Nature, 341: 482 (1989); and WO 91/01133); T cell receptor antibodies such as T1OB9 (EP 340,109) are included.
[77] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or interferes with the function of a cell and / or destroys a cell. The term includes radioactive isotopes (e.g., radioactive isotopes of At211 I131 I135 Y90 Re186 Re188 Sm153 Bi212 P32 and Lu), chemotherapeutic agents and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or small molecule toxins. , Or fragments thereof.
[78] A "chemotherapeutic agent" is a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN ^™ ); Alkyl sulfonates such as busulfan, impprosulfan and pifosulfan; Aziridine, such as benzodopa, carboquone, meturedopa and uredopa; Ethyleneimine and methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethyllomelamine; Chlorambucil, Chlornaphazine, Colophosphamide, Estramustine, Iposamide, Mecroretamine, Mechlorethamine Oxide Hydrochloride, Melparan, Novembihin, Fennesterine, Prednisostin, Nitrogen mustards such as trophosphamide, uracil mustards; Nitrosureas such as carmustine, chlorozotocin, potemustine, lomustine, nimustine, rannimustine; Aklacinomycin, Actinomycin, Utremycin, Authramycin, Azaserine, Bleomycin, Coctinomycin, Calicheamicin, Carabicin, Carminomycin, Carcinophylline, Chromomycins , Dactinomycin, daunorubicin, detorrubicin, 6-diazo-5-oxo-L-lorleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin , Mycophenolic acid, nogalamycin, olibomycin, peplomycin, port pyromycin, puromycin, quelamycin, rhorubicin, streptonigine, streptozosin, tubercidine, ubenimex, ginostatin, Antibiotics such as zorubicin; Anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogs such as denophtherine, methotrexate, ptereptherin, trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmoper, cytarabine, dideoxyuridine, doxyfluridine, enositabine, floxuridine, 5-FU; Androgens such as calusosterone, dromostanolone propionate, epithiostanol, mepitiostane, testosterone; Anti-adrenal such as aminoglutetimide, mitotan, trilostane; Folic acid supplements such as proline acid; Aceglaton; Aldophosphamide glycosides; Aminolevulinic acid; Amsacrine; Vestravusyl; Bisantrene; Edatraxate; Depopamine; Demecolsin; Diajikuon; Elponnitine; Elliptinium acetate; Etogluside; Gallium nitrate; Hydroxyurea; Lentinane; Rodidamine; Mitoguazone; Mitoxantrone; Fur mall; Nitracrine; Pentostatin; Penammet; Pyrarubicin; Grape filinic acid; 2-ethylhydrazide; Procarbazine; PSK ; Lakamic acid; Sizopyran; Spirogermanium; Tenuazone acid; Triazcuone; 2,2 ', 2 "-trichlorotriethylamine;urethane;vindesine;dacarbazine;mannosemusin;mitobronitol;mitolactol;fibrobroman;azaitocin; arabinoside (" Ara-C ") Cyclophosphamide; thiotepa; taxoids such as paclitaxel (TAXOL); , Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere, Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin and carboplatin; Vinplastin; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantron; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT-11; Topoisomerase inhibitor RF2000; Difluoromethylornithine (DMFO); Retinolic acid; Esperamicin; Capecitabine; And pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. This definition includes, for example, tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onafristone, and toremiphene. Estrogens; And anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; And anti-hormonal agents that modulate or inhibit hormonal action on tumors, such as pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.
[79] The term "cytokine" is a generic term for proteins that are released by one cell population and act on another cell as intercellular mediators. Examples of such cytokines are lymphokine, monocaine, and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; Parathyroid hormone; Thyroxine; insulin; Proinsulin; Relaxine; Prorelaxin; Glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); Liver growth factor; Fibroblast growth factor; Prolactin; Placental lactogen; Tumor necrosis factor-α and -β; Mullerian-inhibiting substances; Mouse gonadotropin-associated peptide; Inhibin; Actibin; Vascular endothelial growth factor; Integrin; thrombopoietin (TPO); Nerve growth factors such as NGF-13; Platelet growth factor; Transforming growth factors (TGFs) such as TGF-α and TGF-β; Insulin-like growth factor-I and -II; Erythropoietin (EPO); Osteoinduction factors; Interferons such as interferon-α, -β and -γ; Colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); And granulocyte-CSF (G-CSF); IL-1, IL-1a, IL-2, IL-g, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL- Interleukin (ILs) such as 15; Tumor necrosis factor such as TNF-α or TNF-β; And other polypeptide factors including LIF and kit ligand (KL). The term cytokine, as used herein, includes biologically active equivalents with proteins and natural sequence cytokines from natural sources or from recombinant cell culture.
[80] The term "prodrug" of the present application is a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is convertible to a parent form that is activated or more active with an enzyme. It means a precursor or derivative form of. Wihnan, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al ., "Prodrugs: A Chemical Approach to Targeted Drug Delivery", Directed Drug Delivery, Borchardt et al. , (Ed.), Pp. 247-267, Humana Press (1985). Drug precursors of the invention include phosphate-containing drug precursors, thiophosphate-containing drug precursors, sulfate-containing drug precursors, peptide-containing drug precursors, D-amino acid-modified drug precursors, glycosylated drug precursors, 13-lactam- Containing drug precursor, optionally substituted phenoxyacetamide-containing drug precursor or optionally substituted phenylacetamide-containing drug precursor, 5-fluorocytosine and other convertible to more cytotoxic free drugs Fluorouridine drug precursors include, but are not limited to. Examples of cytotoxic drugs that can be derivatized in the form of drug precursors for use in the present invention include, but are not limited to, the chemotherapeutic agents described above.
[81] A "liposome" is a small vesicle composed of various forms of lipids, phospholipids and / or surfactants useful for delivering drugs (such as antagonists and optionally chemotherapeutic agents disclosed herein) to a mammal. The components of liposomes are generally arranged in a bilayer form similar to the lipid arrangement of the biofilm.
[82] The term "package insert" is used to refer to instructions normally included in a commercially available therapeutic product package, including instructions, uses, dosages, methods of administration, contraindications, and / or related to the use of such therapeutic product. Or information about the notice.
[83] II. Production of antibodies
[84] The production method and article of manufacture of the present invention use an antibody having an immunomodulatory activity such as, for example, an anti-B7, anti-CD40L, or anti-CD40 antibody, and an antibody that binds to a B cell surface label having B depletion activity Or include. Thus, the method of producing such antibodies is described below.
[85] The molecule used to produce or screen the antigen (s) may be, for example, in soluble form of the antigen or portion thereof comprising the desired epitope. Alternatively or additionally, cells expressing the antigen at their cell surface can be used to generate or screen antagonist (s). Other forms of B cell surface markers useful for producing antagonists will be well known to those skilled in the art. Suitable antigen sources for the CD40L, CD40, CD19, CD20, CD22, CD23, CD37 and B7 antigens (eg B7.1, B7.2) for producing the antibodies according to the invention are well known.
[86] The CD40L antibody or anti-CD40L antibody is preferably a humanized anti-CD40L antibody disclosed in US Pat. No. 6,001,358, registered June 14, 1999 and assigned to IDEC Pharmaceuticals.
[87] Desired antagonists are antibodies, but antagonists other than antibodies are also contemplated. For example, the antagonist may include small molecule antagonists or soluble CD40, CD40 fusion proteins optionally fused or conjugated with a cytotoxic agent (as described herein). Libraries of small molecules can be screened for B cell surface labels of interest to identify small molecules that bind to the antigen. Small molecules may be further screened for their antagonistic properties and / or conjugated with cytotoxic agents.
[88] The antagonist may also be a peptide produced by rational design or by phage display (see, for example, WO98 / 35036 published August 13, 1998). In one embodiment, the selected molecule can be a “CDR analogue” or antibody analogue designed based on the CDRs of the antibody. Such peptides may themselves be antagonists, but the peptides may optionally be fused to a cytotoxic or immunoglobulin Fc region (eg, to impart ADCC and / or CDC activity to the peptide).
[89] Exemplary techniques for the production of antibody antagonists for use in accordance with the present invention are described.
[90] (i) polyclonal antibodies
[91] Polyclonal antibodies are preferably generated in animals by subcutaneous (sc) or intraperitoneal (ip) multiple injections of the relevant antigen and adjuvant. Relevant antigens include, for example, keyhole limpet hemocyanin, serum albumin, bovine tyroglobulin, or for example maleimidobenzoyl sulfosuccinimide esters (linked via cysteine residues), N-hydroxysuccinimide (lysine Immunized, such as a soy trypsin inhibitor using a bifunctional or inducer such as binding through residues), glutaraldehyde, succinic anhydride, SOCl ₂ , or R ¹ N = C = NR (where R and R ¹ are other alkyl groups) It may be useful to conjugate to proteins that are immunogenic in the species.
[92] Animals were challenged for antigens, immunogenic conjugates, or derivatives by combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's adjuvant and injecting the solution into the endothelium at multiple sites. Immunize. One month later the peptide or conjugate in the Freund's complete adjuvant is further stimulated by subcutaneous injection of 1/5 to 1/10 of the original amount at multiple sites in the animal. After 7 to 14 days, animal blood is collected and serum is analyzed for antibody titer. The animal is further stimulated until the titer is stable. It is desirable to further stimulate the animal with a conjugate of the same antigen, via another crosslinker and / or conjugated to another protein. The conjugate may also be made during recombinant cell culture as protein fusion. It is also suitable to use flocculants such as alum to enhance the immune response.
[93] (ii) monoclonal antibodies
[94] Monoclonal antibodies are obtained from the same, substantially homogeneous population of antibodies, except for possible naturally occurring mutations in which the individual antibodies that make up the population may be present in small amounts. Thus, the modifier "monoclonal" refers to the characteristics of an antibody that are not a mixture of isolate antibodies.
[95] For example, monoclonal antibodies can be made using the hybridoma method first described in Kohler et al., Nature , 256: 495 (1975) or by recombinant DNA method (US Pat. No. 4,816,567). have.
[96] In hybridomas, other suitable host animals, such as mice or hamsters, are immunized as described above to identify lymphocytes capable of producing or producing antibodies that will specifically bind to the protein used for immunization. Optionally, lymphocytes can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal Antibodies: Principles and Prctice, pp. 59-103 (Academic Press, 1986)].
[97] Thus prepared hybridoma cells are grown by inoculation into a suitable culture medium which contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas is usually hypoxanthine, a substance that prevents the growth of HGPRT-deficient cells. , Aminopterin and thymidine (HAT medium).
[98] Desired myeloma cells are effectively fused to support stable high levels of antibody production by selected antibody-producing cells and are sensitive to media such as HAT media. Among these, desired myeloma cell lines are obtained from MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center, Sandiego, California USA (SICDC), and from American Type Culture Collection, Manassas, Virginia USA (ATCC). Is a myeloma strain of rats, such as those derived from capable SP-2 or X63-Ag8-653 cells. Human myeloma and mouse-human heteromyeloma cell lines have also been described for producing human monoclonal antibodies [Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)].
[99] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is confirmed by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
[100] The binding affinity of monoclonal antibodies can be found in, for example, 30 Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).
[101] After identifying hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity, clones were subcloned by the limiting dilution method and standardized by Goding, Monoclonal Antibodies: Principles and Prctice, pp. 59-103 (Academic Press, 1986)]. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in an animal.
[102] Monoclonal antibodies secreted by the subclones are cultured by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, It is suitably separated from ascites fluid or serum.
[103] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of a murine antibody). do. Hybridoma cells serve as the desired source of such DNA. Once isolated, the DNA is placed in an expression vector and then transfected into host cells, such as E. coli cells, apes COS cells, Chinese Hamster Ovart (CHO) cells, or myeloma cells that otherwise do not produce immunoglobulin proteins. And monoclonal antibodies are synthesized in recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Phickthun, Immunol. Revs., 130: 151-188 (1992).
[104] In other embodiments, the antibody or antibody fragment can be isolated from the antibody phage library 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. In subsequent publications combinatorial infection as a method for constructing a very large phage library as well as the production of high affinity (nM range) human antibodies by chain reorganization [Marks et al., Bio / Technology, 10: 779-783 (1992)]. And in vivo recombination [Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 (1993). Thus, these techniques can be used as an alternative to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.
[105] DNA also replaces the coding sequences for human heavy and light chain constant domains instead of homologous mouse sequences [US Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci . USA, 81: 6851 (1984)], or all or part of a coding sequence for a non-immunoglobulin polypeptide can be modified by covalently binding to an immunoglobulin coding sequence.
[106] Generally, such non-immunoglobulin polypeptides are substituted with constant domains of an antibody, or with variable domains at one antigen-binding site of an antibody, thereby having a specificity for one antigen-combination site and another antigen having specificity for one antigen. Chimeric divalent antibodies comprising antigen combining sites are produced.
[107] (iii) humanized antibodies
[108] Methods for humanizing nonhuman antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues introduced into it from a non-human source. These nonhuman amino acid residues are often referred to as "import" residues, which are generally obtained from "import" variable domains. Humanization is accomplished by the method of Winter and co-workers by replacing the hypervariable region sequences in place of the sequences of the corresponding human antibodies [Jones et al., Nature , 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988). Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially fewer complete human variable domains have been replaced with corresponding sequences of non-human species. Indeed, humanized antibodies are generally human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues at similar sites in rodent antibodies.
[109] Selecting human variable domains of both light and heavy chains, which are used to make humanized antibodies, are of great importance in reducing antigenicity. According to the so-called "best-fit" method, the sequences of the variable domains of rodent antibodies are screened against the entire library of known human variable domain sequences. The human sequence closest to that of a rodent is then referred to as the human framework region (FR) of the humanized antibody [Suns et al., J. Immunol, 151: 2296 (1993); Chothia et al., J. Mol. Biol, 196: 901 (1987). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same backbone can be used for several different humanized antibodies [Carter et al., Proc. Nad. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol, 151: 2623 (1993).
[110] It is also important to humanize the antibody to possess high affinity for the antigen and other desired biological properties. To achieve this goal, according to a desired method, humanized antibodies are prepared by an analytical process of various conceptual humanized products using parental sequences and three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly used and known to those skilled in the art. Computer programs are available that depict and display possible three-dimensional shape structures of selected candidate immunoglobulin sequences. Examining these displays can analyze the possible role of residues in the function of candidate immunoglobulin sequences. In particular, residues that affect the ability of a candidate immunoglobulin to bind its antigen can be analyzed. In this way, FR residues can be selected from the beneficiaries and combined, and sequences can be imported such that desired antibody properties such as increased affinity for the target antigen (s) are achieved. In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.
[111] (iv) human antibodies
[112] As an alternative to humanization, human antibodies can be produced. For example, immunization can now produce transgenic animals (eg, mice) capable of producing the entire repertoire of human antibodies without endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain binding region (PH) gene in chimeric and embryonic mutant mice completely inhibits endogenous antibody production. Transferring the human embryo immunoglobulin gene sequence to such embryonic mutant mice will produce human antibodies by antigenic immunoassay [eg, Jakobovits et al., Proc. Mad. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); And US Pat. Nos. 5,591,669, 5,589,369 and 5,545,807.
[113] Alternatively, phage display technology (McCafferty et al., Nature 348: 552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from non-immunized donors. have. According to this technique, antibody V domain genes are cloned into the backbone into one of the major or minor coat protein genes of a filamentous bacteriophage such as M13 or fd, and displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particles contain a single stranded DNA copy of the phage genome, a gene encoding an antibody exhibiting these properties is also selected when selected based on the functional properties of the antibody. Thus, phage resemble some of the properties of B cells. Phage display can be done in a variety of formats (see, eg, Johnson, Kevin S. and Chiswell, David J., Current Opinion in structural Biology 3: 564-571 (1993)). Various sources of V-gene fragments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated various arrays of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice. A repertoire of V genes from non-immunized human donors can be constructed and antibodies to antigens of various arrangements are described in Marks et al., J. Mol. Biol. 222: 581-597 (1991), or Griffith et al., EMBO J. 12: 725-734 (1993), can be isolated (see also US Pat. Nos. 5,565,332 and 5,573,905).
[114] Human antibodies may also be produced by B cells activated in vitro (see US Pat. Nos. 5,567,610 and 5,229,275).
[115] (v) antibody fragments
[116] Various techniques have been developed for producing antibody fragments. Traditionally, these fragments are obtained through proteolysis of complete antibodies [eg, 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, antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and chemically combined to form F (ab ') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another approach, F (ab ') 2 fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments will be well known to those skilled in the art. In other embodiments, the antibody of choice is a single stranded Fv fragment (scFv) [WO 93/16185; US Patent No. 5,571,894; And US Pat. No. 5,587,458. The antibody fragment may be a "linear antibody", eg, as described in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.
[117] (vi) bispecific antibodies
[118] Bispecific antibodies are antibodies that have binding specificities for two or more different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of B cell surface labels. Other such antibodies may bind to the first B cell surface label and further bind to the second B cell surface label. Optionally, the anti-B cell labeling arm is a trigger molecule on leukocytes, such as T-cell receptor molecules (eg CD2 or CD3), or FcγRI (CD64), in order to focus cell defense mechanisms against B cells. , FcγRII (CD32) and FcγRIII (CD16) can be combined with cancer that binds to Fc receptors for IgG (FcγR). Bispecific antibodies can also be used to localize cytotoxic agents to B cells. These antibodies have B cell label-binding cancers and cancers that bind to cytotoxic agents (eg, saporin, anti-interferon a, vinca alkaloids, lysine A chains, methotrexate or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F (ab) 2 bispecific antibodies).
[119] Methods of making bispecific antibodies are known in the art. Traditional methods of producing full length bispecific antibodies are 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)]. . Because immunoglobulin heavy and light chains are randomly classified, these hybridomas produce a potential mixture of 10 different antibody molecules, and only one of them has the correct bispecific structure. In general, the purification of the exact molecule by affinity chromatography is rather arduous and the yield is low. Similar procedures are disclosed in WO 93/08829, and Traunecker et al., EMBO J, 10: 3655-3659 (1991).
[120] According to another approach, antibody variable domains (antibody-antigen combining sites) with the desired binding specificities are fused to immunoglobulin constant domain sequences. Fusion is preferably using an immunoglobulin heavy chain constant domain, which comprises at least a portion of the hinge, CH2 and CH3 regions. It is preferred to have a first heavy chain constant region (CHI) comprising a site necessary for light chain binding, present in one or more fusions. DNA encoding the immunoglobulin heavy chain fusion, and if necessary the immunoglobulin light chain, is inserted into a separate expression vector and co-transfected into a suitable host organism. This provides great flexibility in controlling the mutual ratios of the three polypeptide fragments in the examples where unequal proportions of the three polypeptide chains used in the construction provide optimum yields. However, when two or more polypeptide chains of the same ratio are expressed in high yield or the ratio is not very important, coding sequences for two or all three polypeptide chains may be inserted in one expression vector.
[121] In a preferred embodiment of this approach, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity of one cancer, and a hybrid immunoglobulin heavy chain light chain pair (which confers second binding specificity) of the other cancer. This asymmetric structure has been found to facilitate the separation of desired bispecific compounds from unwanted immunoglobulin chain combinations, since only half of the bispecific molecules have an immunoglobulin light chain for ease of separation. This approach is disclosed in WO 94/04690. For more details on generating bispecific antibodies, see, eg, Suresh et al., Methods in Enzymology, 121: 210 (1986).
[122] According to another approach described in US Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimer recovered from recombinant cell culture. The desired interface comprises at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). Complementary "cavities" of the same or similar size as the large side chains are made at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller ones (eg alanine or threonine). This provides a mechanism to increase the yield of heterodimers for other unwanted end products, such as homodimers.
[123] Bispecific antibodies include crosslinked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate may bind to avidin and the other may bind to biotin. Such antibodies have been proposed, for example, to target immune system cells against unwanted cells (US Pat. No. 4,676,980), and to treat HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies can be made using any convenient crosslinking method. Suitable crosslinkers are known in the art and are disclosed in US Pat. No. 4,676,980 with a number of crosslinking techniques.
[124] Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical bonds. Brenann et al., Science, 229: 81 (1985) describe a method by which complete antibodies are proteolytically generated to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent 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 reduced to melcaptoethylamine and reconverted to Fab'-thiol and mixed with equimolar amounts of other Fab'-TNB derivatives to form a bispecific antibody. The bispecific antibodies produced can be used as medicaments for the selective immobilization of enzymes.
[125] Recent advances in technology have made it easier to recover Fab'-SH fragments directly from E. coli that can be chemically bound to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of fully humanized bispecific antibody F (ab ') 2 molecules. Each Fab 'fragment is secreted separately from E. coli and chemically bound in vitro to form a bispecific antibody. The bispecific antibodies thus formed not only induce lytic activity of stem stem cytotoxic lymphocytes against human breast tumor targets but also could bind to cells that overexpress ErbB2 receptors and normal human T cells.
[126] Various techniques have also been described for making and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies can be produced using leucine zippers (Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)). Leucine zipper peptides from Fos and Jun proteins were bound to the Fab 'portion of two different antibodies by gene fusion. Antibody homodimers are reduced in the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. Hollinger et al., Proc. Natl. Acad. Sci. USA. 90: 6444-6448 (1993) provided a "dibody" technique that provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (V _H ) linked to the light chain variable domain (V _L ) by a binder that is too short to form a pair between two domains on the same chain. Thus, the V _H and V _L domains of one fragment pair with the complementary V _L and V _H domains of the other fragment, forming two antigen-binding sites. Other methods of making bispecific antibody fragments using single chain Fv (sFv) dimers have also been reported (see Gruber et al., J. Immunol., 152: 5368 (1994)).
[127] Antibodies having two or more valences are also contemplated. For example, trispecific antibodies can also be prepared (Tutt et al. J. Immunol. 147: 60 (1991)).
[128] III. Conjugates and Other Modifications of Antagonists
[129] Antibodies used in the methods of manufacture herein or included in articles of manufacture are optionally conjugated with cytotoxic agents.
[130] Chemotherapeutic agents useful for the production of such antibody-cytotoxic agent conjugates have been described above.
[131] Conjugates of antibodies and one or more small molecular toxins, such as calicheamicin, maytansine (US Pat. No. 5,208,020), tricotene, and CC1065, are also contemplated herein. In one embodiment of the invention, the antagonist is conjugated to one or more maytansine molecules (eg, about 1 to about 10 maytansine molecules per antagonist molecule). For example, maytansine is converted to May SS-Me, which can be reduced to May-SH3, and reacted with a modified antagonist [Charm et al. Cancer Research 52: 127-131 (1992)] maytansinoid-antagonist conjugates.
[132] Optionally, the antibody may be conjugated to one or more calicheamicin molecules. Antibiotics of the calicheamicin family can produce double stranded DNA single strands at sub-picomol concentrations. Structural analogs of calicheamicin that may be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG and 0 ^I ₁ [Hinman et al. Cancer Research 53: 3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928 (1998).
[133] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chains, unbound active fragments of diphtheria toxins, exotoxin A chains (from Pseudomonas aeruginosa), lysine A chains, abrine A chains, and modeline A. Chain, α- sarsine , Aleurites fordii protein, diantine protein, Phytolacaamericana protein (PAPI, PAPII, and PAP-S), Momordica Charantia inhibitor, Cursin, Crotins, sapaonaria opininalis inhibitors, gelonin, mitogeline, restrictocin, phenomycin, enomycin and tricortesene. See, for example, WO 93/21232 published October 28, 1993.
[134] The present invention also contemplates antibodies conjugated with compounds having nuclease activity (eg, DNA endonucleases or ribonucleases such as deoxyribonuclease (DNase)).
[135] Various radioisotopes can be used to produce radioconjugated antagonists. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu.
[136] Conjugates of antagonists and cytotoxic agents include N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxyl Latex, iminothiolane (IT), difunctional derivatives of imidoesters (such as dimethyl adimimidate HCL), active esters (such as disuccinimidyl suverate), aldehydes (such as glutaraldehyde), (bis bis-azido compounds (such as (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), (tolyene 2,6-diisocyanate) Such as diisocyanates and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins are described in Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotides to antagonists (see WO 94/11026). The binder may be a "degradable binder" that facilitates the release of cytotoxic drugs in the cell. For example, acid-labile binders, peptidase-sensitive binders, dimethyl binders or disulfide-containing binders [Chari et al. Cancer Research 52: 127-131 (1992).
[137] Alternatively, fusion proteins comprising antibodies and cytotoxic agents can be made, for example, by recombinant techniques or peptide synthesis.
[138] In another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting, and the antagonist-receptor conjugate is circulated using a clearing agent and then unbound after administration to the patient. After removal of the conjugate, a "ligand" (eg, avidin) conjugated to a cytotoxic agent (eg, radionucleotide) is administered.
[139] Antibodies of the invention may also be conjugated with drug precursor-activating enzymes that convert drug precursors (eg, peptidyl chemotherapeutic agents, see WO 81/01145) into active anticancer drugs (eg, WO 88/07378). And US Pat. No. 4,975,278).
[140] Enzyme components of such conjugates include enzymes that can act on the drug precursors in a way that converts the drug precursors into more active, cytotoxic forms.
[141] Enzymes useful in the methods of the invention include alkaline phosphatase useful for converting phosphate-containing drug precursors into free drugs; Arylsulfatase useful for converting sulfate-containing drug precursors into free drugs; Cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anticancer drug fluorouracil; Proteases such as seratia proteases, thermolysine, subtilisin, carboxypeptidase, and cathepsin (such as cathepsin B and L), useful for converting peptide-containing drug precursors into free drugs; D-alanylcarboxypeptidase useful for converting drug precursors containing D-amino acid substituents; Carbohydrate enzymes such as 13-galactosidase and neuraminidase, which are useful for converting glycosylated drug precursors into free drugs; 13-lactamase useful for converting 13-lactam induced drugs into free drugs; And penicillin amidases such as penicillin V amidase or penicillin G amidase, which are useful for converting drugs derived from their amine nitrogen into free drugs with phenoxyacetyl or phenylacetyl groups, respectively. Alternatively, antibodies with enzymatic activity, also known as "abzymes" in the art, can be used to convert the drug precursors of the invention to free active drugs. Massey, Nature 328: 457-458 (1987 )]. Antagonist-Abzyme conjugates can be prepared as described herein to deliver Abzyme to tumor cell populations.
[142] The enzymes of the present invention may be covalently linked to the antagonist by techniques known in the art, such as by using such heterodifunctional crosslinkers. Alternatively, a fusion protein comprising at least the antigen binding region of the antagonist of the invention bound to at least the functionally active portion of the enzyme of the invention can be constructed using recombinant DNA techniques known in the art. See Neuberger et al., Nature, 312: 604-608 (1984).
[143] Other variations of the antibodies are also contemplated herein. For example, the antibody can be bound to one of various nonproteinaceous polymers, such as, for example, polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol.
[144] The antibodies disclosed herein may be formulated with liposomes. Liposomes containing antagonists are described by Epstein et al., Proc. Mad. Acad Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad Sci. USA, 77: 4030 (1980); US Patent Nos. 4,485,045 and 4,544,545; And WO 97/38731, published October 23, 1997, by methods known in the art. Liposomes with enhanced circulation time are disclosed in US Pat. No. 5,013,556.
[145] Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes with the desired diameter. Fab ′ fragments of the antibodies of the invention are described by Martin et al., J. Biol. Chem. 257: 286-288 (1982), may be conjugated to liposomes. Optionally, chemotherapeutic agents can be included in the liposomes [Gabizon et al. J. National Cancer Inst . 81 (19) 1484 (1989).
[146] Amino acid sequence modifications of the protein or peptide antagonists described herein are also 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 can be prepared by introducing a suitable nucleotide change into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antagonist. If the final structure has the desired properties, any combination of deletions, insertions and substitutions that appear in the final structure may be made. Can lose. Amino acid changes can also alter post-translational treatment of the antagonist, such as by changing the number or location of glycosylation sites.
[147] Methods useful for identifying specific residues or regions of antagonists that are desired locations for mutagenesis are called "alanine scanning mutagenesis" as described in Cunningham and Wells Science, 244: 1081-1085 (1989). Here, the residues or groups of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu), and neutral or negatively charged amino acids that affect the interaction of the amino acid with the antigen ( Most preferably alanine or polyalanine). These amino acid positions that exhibit functional sensitivity to substitutions are then purified by introducing additional or other modifications at or about the substitution sites. Therefore, the site for introducing the amino acid sequence modification is predetermined, but the characteristics of the mutation itself do not need to be predetermined. For example, to analyze the performance of mutations at a given site, scanning or random mutagenesis is performed at the target codon or region and the expressed antagonist variants are screened for the desired activity.
[148] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions having a length from one residue to a polypeptide comprising more than one hundred residues, as well as insertions in sequence of single or multiple amino acid residues. Examples of terminal insertions include antagonists with N-terminal methionyl residues or antagonists fused to a cytotoxic polypeptide. Other inserts of the antagonist molecule include fusion to the N- or C-terminus of the antagonist of the enzyme, or to a polypeptide that increases the serum half-life of the antagonist.
[149] Another form of variant is an amino acid substitution variant. These variants have one or more amino acid residues in the antagonist molecule replaced with other residues. Sites of greatest interest for substitutional mutagenesis of antibody antagonists include hypervariable regions, but FR modifications are also contemplated. Conservative substitutions are shown in Table 1 under the heading of “desired substitutions”. If such substitutions result in a change in biological activity, then more substantial changes, which may be referred to as "exemplary substitutions" in Table 1 or described further below with reference to the amino acid classification, may be introduced and screen the product. .
[150] Original residues Exemplary Substitution Desired substitution Ala (A) val; leu; ile val Arg (R) lys; gin; asn lys Asn (N) gln; his; asp, lys; arg gln Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu (E) asp; gin asp Gly (G) ala ala His (H) asn; gin; lys; arg arg Ile (I) leu; val; met, ala; phe; norleucine leu Lea (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr tyr Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser TrP (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu
[151] Substantial modifications in the biological properties of the antibody include (a) the structure of the polypeptide backbone at the substitution site, such as, for example, a sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. The effect on maintaining is achieved by choosing significantly different substitutions. Naturally occurring residues are divided into groups based on common side chain properties:
[152] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[153] (2) neutral hydrophilic: cys, ser, thr;
[154] (3) acidic: asp, glu;
[155] (4) basic: asn, gln, his, lys, arg;
[156] (5) residues affecting chain orientation: gly, pro; And
[157] (6) Directionality: trp, tyr, phe.
[158] Non-conservative substitutions will require exchanging elements of one of these classes for another class.
[159] Any cysteine residue that is not involved in maintaining the proper structure of the antagonist may be substituted with serine, generally to improve the oxidative stability of the molecule and to prevent abnormal crosslinking. Conversely, cysteine bonds may be added to the antagonist to improve its stability, especially when the antagonist is an antibody fragment such as an Fv fragment.
[160] Particularly desired forms of substitutional variants include substitution of one or more hypervariable region residues of a parent antibody (eg a humanized or human antibody). In general, the resulting variants selected for further development will have improved biological properties compared to the parent antibody from which they are produced. A convenient way to generate such substitutional variants is affinity maturation using phage display. In short, several hypervariable region sites (eg 6-7 sites) are mutated to produce all possible amino substitutions at each site. The resulting antibody variants are thus displayed in monovalent format from the filamentous phage particles as a fusion to the gene III product of M13 assembled within each particle. Phage-displayed variants are then screened for their biological activity (eg, binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be useful to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and antigen. Residues adjacent to such contact residues are candidates for substitution according to the techniques described in detail herein. Once such variants are created, a panel of variants can be screened as described herein and an antibody can be selected that has good properties for further development in one or more related assays.
[161] Another form of amino acid variant of the antagonist modifies the original glycosylation pattern of the antagonist. By modifying 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.
[162] Glycosylation of polypeptides is generally either N-linked or O-linked. N-linking refers to the attachment of carbohydrate residues to the side chains of asparagine residues. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine (X is any amino acid except proline) are recognition sequences for enzymatic attachment of carbohydrate residues to asparagine side chains. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation may also be used as 5-hydroxyproline or 5-hydroxylysine, but most commonly serine or threonine, one of the saccharides N-acetylgalactosamine, galactose, or xylose in hydroxyamino acids. Says to attach.
[163] Adding a glycosylation site to an antibody is readily accomplished by modifying the amino acid sequence to include one or more such tripeptide sequences (for N-linked glycosylation sites). Such modifications may be made by adding or replacing one or more serine or threonine residues in the sequence of the original antagonist (to the O-linked glycosylation site).
[164] Nucleic acid molecules encoding amino acid sequence variants of the antagonist are prepared by a variety of methods known in the art. These methods include isolation from natural sources (for naturally occurring amino acid sequence variants) or oligonucleotide-mediated (or site directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of already prepared variant or non-variant plates of antagonists. By, but not limited to.
[165] It may be desirable to modify the antagonists used in the present invention to improve effector action such as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) of the antagonist. This can be accomplished by introducing one or more amino acid substitutions into the Fc region of the antibody antagonist. Alternatively or additionally, cysteine residue (s) can be introduced into the Fc region, whereby interchain disulfide bonds can be formed in this region. The resulting homodimer antibodies may thus have improved internalization capacity and / or increased complement-mediated cell death and antibody-dependent cellular cytotoxicity (ADCC) [Caron et al., J. Exp Med. 176: 1191-1195 (1992) and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimer antibodies with enhanced antitumor activity are described by Wolff et al. Cancer Research 53: 2560-2565 (1993), which may also be prepared using heterobifunctional crosslinkers. Optionally, antibodies can be engineered to have a double Fc region, thereby enhancing complement solubility and ADCC ability [Stevenson et al. Anti-Cancer Drug Design 3: 219-230 (1989).
[166] To increase the serum half-life of the antagonist, a salvage receptor binding epitope can be inserted into the antagonist (especially antibody fragments), as described, for example, in US Pat. No. 5,739,277. As used herein, “structural receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (eg, IgG1, IgG2, IgG3 or IgG4) that increases the serum half-life of an IgG molecule in vivo.
[167] IV. Pharmaceutical composition
[168] Therapeutic compositions comprising an antagonist used according to the present invention can be prepared by mixing an antagonist with a desired degree of purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer [ Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. . (1980)] for storage in the form of a lyophilized composition or aqueous solution. 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; (Octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzetonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; Preservatives such as 3-pentanol and m-cresol; Low molecular weight polypeptides (up to about 10 residues); Proteins such as serum albumin, gelatin, or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counterions such as sodium; Metal complexes (eg, Zn-protein complexes); And / or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ or polyethylene glycol (PEG).
[169] The immunoregulatory antibody or antibody fragment and the B cell depleting antibody may be the same composition or may be administered in different compositions. Administration can be simultaneous or sequential and can be effective in any order. Such administration may be effective with repeated administration of both antibodies for a long period of time.
[170] Exemplary anti-CD20 antibody compositions are described in WO 98/56418, which is expressly incorporated herein by reference. This publication discloses a liquid multidose comprising 40 mg / ml rituximab, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20, with a minimum shelf life of 2 years at 2-8 ° C. The composition is described. Other anti-CD20 compositions of interest include 10 mg / ml rituximab in 9.0 mg / ml sodium chloride, 7.35 mg / ml sodium citrate dihydrate, 0.7 mg / ml polysorbate 80, and sterile water for injection at pH 6.5 do.
[171] Lyophilized compositions suitable for subcutaneous administration are described in WO 97/04801. Such lyophilized compositions can be reconstituted at high protein concentrations using suitable diluents and the reconstituted compositions can be administered subcutaneously to the mammals to be treated herein.
[172] The compositions herein may also comprise one or more active compounds which have complementary activities which are necessary for the particular indication to be treated, preferably which do not adversely affect each other. For example, it would be desirable to further provide chemotherapeutic agents, cytokines or immunosuppressive agents (eg, antibodies that act on T cells, such as cyclosporine, or antibodies that bind T cells, such as those that bind LFA-1). Can be. The effective amount of such other agents depends on the amount of antagonist present in the composition, the form of the disease or condition or treatment, and the other factors mentioned above. They are generally used at the same dose and as the route of administration as used so far or at about 1 to 99% of the dose used so far.
[173] The active ingredients are hydroxymethylcellulose or gelatin-microcapsules and poly (methyl) in colloidal drug delivery systems (e.g. liposomes, albumin, microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions, respectively. Methacrylate) microcapsules, for example, may be encapsulated in microcapsules prepared by 30 coacervation techniques or by interfacial polymerization. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[174] Sustained-release preparations may also 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 embodied articles such as membranes, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L- Degradable lactic acid-glycolic acid copolymers such as copolymers of glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT ^™ (injectable microspheres consisting of lactic glycolic acid copolymer and leuprolide acetate), Poly-D-(-)-3-hydroxybutyric acid. The composition used for in vivo administration must be sterile. It can be easily sterilized by filtration through sterile filtration membranes.
[175] V. Treatment with B cell depleted antibodies and immunomodulatory antibodies
[176] Compositions comprising B cell depleting antibodies and / or immunomodulatory antibodies are formulated and administered in a manner consistent with good medical practice of Hananigan. Factors to be considered here include the particular autoimmune disease or condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease or condition, the site of delivery of the medicament, the method of administration, the dosing schedule, and other known practices. Arguments are included. The therapeutically effective amount of the antagonist administered will be determined by such considerations.
[177] As noted above, the B cell depleting antibody and immunomodulatory antibody may be the same or different compositions. These antibody compositions may be administered individually or simultaneously and in any order. Preferably, the B cell depleting antibody specific for a B cell antigen target, such as CD20, CD19, CD22, CD23 or CD37, is an immunomodulatory antibody such as an anti-CD40L antibody, anti-CD40, or anti-B7. 1, administered separately from anti-B7.2 antibody. Preferably, the CD40L antibody is a humanized anti-CD40L antibody disclosed in US Pat. No. 6,001,358. It has been shown to be effective in the treatment of both T and B cell autoimmune diseases, eg, in the treatment of multiple sclerosis and ITP. In addition, another similar humanized anti-CD40L antibody ( 5c8 ) has been reported by Biogen, and it is not known that this antibody causes any adverse toxicity.
[178] As a general assertion, the therapeutically effective amount of the parentally administered antibody per dose will be about 0.1 to 500 mg / kg body weight of the patient per day and the general initial range of antagonists used will be about 2 to 100 mg / kg. .
[179] Desired B cell depleted antibody is RITUXAN to be. Suitable dosages of such antibodies are, for example, about 20 mg / m 2 to about 1000 mg / m 2. Antibody Doses RITUXAN to Treat Non-Hodgkin's Lymphoma May be the same as or different from what is currently recommended for. For example, substantially less than 375 mg / m 2 of antibody, such as when the dosage is in the range of about 20 mg / m 2 to about 250 mg / m 2, for example from about 50 mg / m 2 to about 200 mg / m 2. One or more doses may be administered to the patient.
[180] In addition, a person may administer the antibody at one or more initial doses, followed by one or more continued doses, where the mg / m 2 dose of the antibody at the initial dose is mg / m2 of the antibody at the initial dose. Exceed the m 2 dose. For example, an initial dose of about 20 mg / m 2 to about 250 mg / m 2 (eg, about 50 mg / m 2 to about 200 mg / m 2) and continued dose of about 250 mg / m 2 to about 1000 mg / m M 2.
[181] However, as noted above, this suggested amount of all immunomodulatory antibodies is at substantial therapeutic discretion. An important factor in selecting the appropriate dosage and schedule is the result obtained, as indicated above. For example, relatively high doses may be needed initially to treat ongoing and acute diseases. In order to obtain the most effective results, depending on the autoimmune disease or disorder, the antagonist is administered closely with the first symptom, diagnosis, appearance, or possible occurrence of the disease or disorder or alleviation of the disease or disorder.
[182] The antibody is administered by a suitable method including parenteral, subcutaneous, intraperitoneal, pulmonary, and intranasal, and if necessary for local immunosuppressive treatment, including intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, it may be desirable for the antibody to be administered by pulse infusion using, for example, a decreasing dose of the antibody. Preferably the dosing is by injection, most preferably intravenous or subcutaneous, depending in part on whether the administration is short term or chronic.
[183] Other compounds such as chemotherapeutic agents, immunosuppressants and / or cytokines may be further administered with the antibodies herein. Combination administration includes co-administration using individual compositions or single pharmaceutical compositions, and continuous administration in any order, preferably with time for both (or all) active agents to exert their biological activity simultaneously.
[184] In addition to administering antibodies to patients, the present application also contemplates antibody administration by gene therapy. Such administration of the nucleic acid encoding the antibody is included in the expression "administering a therapeutically effective amount of an antagonist." Regarding the use of gene therapy to generate intracellular antibodies, see, for example, WO 96/07321 published March 14, 1996.
[185] There are two main approaches for introducing nucleic acid (optionally included in a vector) into a patient's cell; In vivo and ex vivo. For in vivo delivery, nucleic acids are generally injected directly into a patient at the site where an antagonist is needed. For in vitro treatment, the cells of the patient are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or encapsulated in a porous membrane that is implanted, for example, into the patient [US Pat. No. 4,892,538]. And 5,283,187. There are a variety of techniques available for introducing nucleic acids into viable cells. Techniques vary depending on whether the nucleic acid is transferred into cells cultured ex vivo or in vivo into cells of the desired host. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. A vector commonly used for ex vivo delivery of genes is a retrovirus.
[186] Currently in vivo nucleic acid transfer techniques include viral vectors (such as adenoviruses, herpes simplex I viruses or adeno-associated viruses) and lipid systems (lipids useful for lipid mediated gene transfer are, for example, DOTMA, DOPE and DC-Chol). Used transfections are included. In some circumstances, it is desirable to provide a nucleic acid source with an agent that targets a target cell, such as a cell surface membrane protein or an antibody specific for the target cell, a ligand for a receptor on the target cell, and the like. When liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis include, for example, capsid proteins or fragments thereof that stimulate specific cell types, antibodies to proteins that are internalized in the circulation, and intracellular localization. It can be used to promote targeting and / or uptake of proteins that target and enhance intracellular half-life. Techniques for receptor-mediated endocytosis are described, for example, in Wu et al. J. Biol. Chem. 262: 4429-4432 (1987); And Wagner et al., Proc. Nad. Acad. Sci. USA 87: 3410-3414 (1990). For a review of currently known gene labeling and gene therapy protocols, see Anderson et al., Science 256: 808-813 (1992). See also WO 93/25673 and references cited therein.
[187] VI. Manufacture
[188] In another embodiment of the present invention, an article of manufacture comprising a substance useful for the treatment of said disease or disorder is provided.
[189] The article of manufacture comprises a container and a label or package insert associated with or on the container. Suitable containers include, for example, bottles, vials, syringes and the like. The container may be formed from various materials such as glass or plastic. The container may contain or contain a composition effective for the treatment of the selected disease or condition and may have a sterile entry port (eg, the container may be a vial with a stopper that can be punctured by an intravenous solution bag or a hypodermic needle). have). In general, one or several compositions are possible. At least one active agent in one of these compositions is an antibody with B cell depleting activity, wherein the at least one antibody is an immunomodulatory antibody such as an anti-CD40L, anti-CD40, anti-CD4, or anti-B7 antibody. The label or package insert indicates that the composition is used to treat a patient with or susceptible to autoimmune diseases such as those listed herein. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer such as BWFI for injection, phosphate-buffered saline, Ringer's solution and dextrose solution. It may further comprise other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
[190] Further details of the invention are illustrated by the following non-limiting examples. The disclosures of all citations in this specification are expressly incorporated by reference.
[191] Example 1
[192] Rituximab at first The antibody is used to treat a patient who has been clinically diagnosed with rheumatoid arthritis (RA). This patient may or may not have B cell depleting antibodies, ie malignancies. Optionally, salicylate; Indomethacin, phenylbutazone, phenylacetic acid derivatives (e.g. ibuprofen and phenopropene), naphthalene acetic acid (naproxen), pyrrole-alkanoic acid (tomethine), indoleacetic acid (sulindac), halogenated anthranilic acid (meth Nonsteroidal anti-inflammatory drugs, such as clofenamate sodium), pyroxicam, zomepilac and diflunisal; Antimalarial agents such as chloroquine; Gold salts; Penicylamine; Or any one or more agents used in the treatment of RA, such as immunosuppressive agents such as methotrexate or corticosteroids, in further known treatment or reduced dosages for such drugs, for further treatment of the patient. However, RITUXAN It is desirable to treat patients only.
[193] Dosage schedules as follows:
[194] (A) 50Mg / M2 Ⅳ 1 day
[195] 150mg / m2 IV 8, 15 & 22 days
[196] (B) 150Mg / M2 Ⅳ 1 day
[197] 375mg / m2 at Ⅳ 8, 15 & 22
[198] (C) 375 Mg / M2 Ⅳ 1, 8, 15 & 22 days
[199] According to any of RITUXAN Is administered intravenously (IV) to RA patients.
[200] And then treated with the humanized anti-CD40L antibody disclosed in US Pat. No. 6,001,358 administered intravenously (IV) according to the same dosing regimen.
[201] Paulus index (Palus et al. Athritis Rheum. 33: 477-484 (1990)), ie morning stiffness, and improvement in pain and inflammatory articular number, erythrocyte sedimentation (ESR), And primary response by a two-point or more improvement over a five-point rating of disease severity as assessed by the patient and physician. RITUXAN And administration of the anti-CD40L antibody will alleviate one or more symptoms of RA in the patient treated as described above.
[202] Example 2
[203] For example, a patient diagnosed with autoimmune hemolytic anemia (AIHA), such as Hanglobin anemia or Coombs positive anemia, is RITUXAN Treat with antibody. AIHA is acquired hemolytic anemia caused by auto-antibodies that react with the red blood cells of a patient. The treated patient may also optionally have a B cell malignancy. The patient is initially treated with a composition comprising a humanized anti-human CD40L antibody and is administered at a dose of 500 mg / m 2 given in IV. This dose is given twice a week for a total of four weeks.
[204] And then the dosing schedule:
[205] (A) 50mg / M2 Ⅳ 1 day
[206] 150mg / m2 IV 8, 15 & 22 days
[207] (B) 150Mg / M2 Ⅳ 1 day
[208] 375mg / m2 at Ⅳ 8, 15 & 22
[209] (C) 375 Mg / M2 Ⅳ 1, 8, 15 & 22 days
[210] According to any of RITUXAN Is administered intravenously (IV) to the patient.
[211] Another additional therapies (glucocorticoids, prednisone, azathioprine, cyclophosphamide, vinca-laden platelets and danazol) are anti-CD40L antibodies and RITUXAN May be combined with therapy. RITUXAN throughout the course of therapy And treating the patient with the same anti-CD40L antibody as in the above examples only with other agents.
[212] The overall response rate is determined based on improved blood cell counts, reduced demand for blood transfusions, improved hemoglobin levels, and / or reduced signs of hemolysis as determined by standard chemical parameters. Anti-CD40L Antibody and RITUXAN Administration of will improve one or more of any symptoms of hemolytic anemia in the patient treated as described above.
[213] Example 3
[214] Adult immune thrombocytopenic purpura (ITP) is a relatively rare hematologic disorder that constitutes the most common immune-mediated thrombocytopenia. The disease usually manifests with severe thrombocytopenia, which may be associated with acute bleeding in the presence of normal to increased megakaryocytes in the bone marrow. Most ITP patients have IgG antibodies directed against target antigens on the outer surface of the platelet membrane, causing platelet sequestration in the spleen and accelerating reticulocyte destruction of platelets (Bussell, JB Hematol. Oncol. Clin. North Am. ( 4): 179 (1990)). Numerous therapeutic interventions have been shown to be effective in the treatment of ITP. Steroids are generally considered to be first-line therapy, after which most patients are candidates for intravenous immunoglobulin (IVIG), splenectomy, or other medications including vincristine or immunosuppressive / cytotoxic agents. Less than 80% of ITP patients initially respond to the steroid process, but a much smaller number of patients are fully and persistently relieved. Splenectomy has been recommended as a standard second-line treatment for steroid deficiency and prolongs remission in nearly 60% of cases, but can reduce immunity to infection. Splenectomy is a major surgical method that can be associated with significant morbidity (15%) and mortality (2%). Although only a small percentage of adult ITP patients have been alleviated, IVIG has also been used as second-line medication.
[215] Without the associated morbidity that occurs with corticosteroids and / or splenectomy, therapeutic options that interfere with the production of autoantibodies by activated B cells will provide an important therapeutic approach for the proportion of ITP patients.
[216] Patients who have been clinically diagnosed with ITP (eg, having platelet counts less than 75,000 / μL) may optionally be treated with rituximab (RITUXAN) with steroid therapy. Treatment with antibodies).
[217] Dosage schedules as follows:
[218] (A) 50mg / M2 Ⅳ 1 day
[219] 150mg / m2 IV 8, 15 & 22 days
[220] (B) 150mg / M2 Ⅳ 1 day
[221] 375mg / m2 at Ⅳ 8, 15 & 22
[222] (C) 375 mg / M2 IV 1, 8, 15 & 22 days
[223] RITUXAN to ITP patients according to any of Re-administer intravenous (IV).
[224] RITUXAN Following administration, the patient is treated using one of the primatized anti-B7.1 antibodies disclosed in US Pat. No. 6,113,898, which is incorporated herein by reference in its entirety. The anti-B7.1 antibody is administered intravenously at a dose of 500 mg / m 2 for three weeks, twice weekly in separate compositions.
[225] RITUXAN TO PATIENT And prior to infusion of the anti-B7.1 antibody composition, in a single dose of 25-50 mg of diphenhydramine (by intravenous injection) and 650 mg (orally) of acetaminophen, respectively. RITUXAN, using sterile syringes and 21 gauge or larger needles And the required amount of anti-B7.1 antibody is transferred from the vial into a IV bag containing sterile, pyrogen-free 0.9% sodium chloride, USP (saline solution). RITUXAN And the final concentration of anti-B7.1 antibody is approximately 1 mg / ml. The initial dose infusion rate starts at 25 mg / hr for the first 30 minutes and increases at a rate of up to 200 mg / hr in 50 mg / hr increments at 30 minute intervals. RITUXAN And if the initial course of the B7.1 antibody is well accepted, the infusion rate of subsequent courses increases at a rate of up to 300 mg / hr up to 100 mg / hr in increments of 30 minutes starting at 50 mg / hr. Vital signs (blood pressure, pulse, breathing, temperature) are examined until stable or four times every 15 minutes, then every hour until the end of the infusion.
[226] Overall response rate is RITUXAN It is determined based on platelet counts measured twice consecutively after 4 weeks of treatment and 2 weeks after 3 weeks of administration of the B7 antibody composition. Anti-B7.1 Antibody and RITUXAN Patients treated with will exhibit improved platelet counts as compared to patients treated with placebo.
[227] While the invention has been described above by way of examples and preferred embodiments, various modifications of the invention as well as those shown in the description from the above detailed description are included within the scope of the invention. Such modifications are intended to be included within the scope of the following claims.

权利要求:
Claims (41)
[1" claim-type="Currently amended] A therapeutically effective amount of an immunomodulatory antibody selected from anti-CD40L, anti-B7.1 (CD80), anti-B7.2 (CD86), CD40 antibody and anti-CD4 antibody and a therapeutically effective amount of an antibody having B cell depleting activity A method of treating autoimmune diseases in a mammal, comprising administering a combination of to a mammal, wherein the immunomodulatory antibody and the B cell depleting antibody are isolated or combined, and administered in any order. How to.
[2" claim-type="Currently amended] The method of claim 1,
The B cell depleting antibodies are 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).
[3" claim-type="Currently amended] The method of claim 1,
The immunomodulatory antibody is an anti-CD40L antibody or an anti-B7 antibody.
[4" claim-type="Currently amended] The method of claim 3,
The combination comprises an antibody binding to CD40L and an antibody binding to CD20, CD22, CD19, CD23 or CD37.
[5" claim-type="Currently amended] The method of claim 3,
Wherein said combination comprises an antibody binding to B7.1 or B7.2 and an antibody binding to CD19, CD20, CD22, CD23, or CD37.
[6" claim-type="Currently amended] The method of claim 1,
Wherein the immunomodulatory antibody is administered before the B cell depleting antibody.
[7" claim-type="Currently amended] The method of claim 1,
A method for administering a B cell depleted antibody prior to an immunomodulatory antibody.
[8" claim-type="Currently amended] The method of claim 1,
A method comprising administering a combination of B cell depleted antibodies and immunoregulatory antibodies.
[9" claim-type="Currently amended] The method of claim 1,
The autoimmune disease is psoriasis; dermatitis; Systemic scleroderma and sclerosis; Reactions associated with inflammatory bowel disease; Crohn's disease; Ulcerative colitis; Respiratory distress syndrome; Adult respiratory distress syndrome (ARDS); dermatitis; meningitis; encephalitis; Uveitis; colitis; Glomerulonephritis; Allergic conditions; eczema; asthma; Conditions involved in the infiltration of T cells and chronic inflammatory responses; Atherosclerosis; Leukocyte adhesion defects; Rheumatoid arthritis; Systemic lupus erythematosus (SLE); diabetes; Multiple sclerosis; Rhinoid syndrome; Autoimmune thyroiditis; Allergic encephalomyelitis; Sjogren's syndrome; Juvenile diabetes; Immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes; Tuberculosis; Sarcoidosis; Polymyositis; Granulomatosis; Vasculitis; Pernicious anemia (Addison disease); Diseases involving leukocyte leakage; Central nervous system (CNS) inflammatory disorders; Multiple organ injury syndrome; Hemolytic anemia; Myasthenia gravis; Antigen-antibody complex mediated disease; Anti-glomerular basement membrane disease; Antiphospholipid syndrome; Allergic neuritis; Graves' disease; Lambert-eaton duty syndrome; Bullous whey; pemphigus; Autoimmune polyendocrine disease; Lighter disease; Rigid human syndrome; Beset's disease; Giant cell arteritis; Immunocomplex nephritis; IgA nephropathy; IgM Multiple Neuropathy; Immune thrombocytopenic purpura (ITP), autoimmune thrombocytopenia and ovarian inflammation.
[10" claim-type="Currently amended] The method of claim 1,
The mammal is a human.
[11" claim-type="Currently amended] The method of claim 3,
Wherein no antibody is conjugated with a cytotoxic agent.
[12" claim-type="Currently amended] The method of claim 4, wherein
Wherein said antibody combination comprises a humanized or human anti-human CD40L or B7.1 antibody and a chimeric, humanized or human anti-CD20 antibody.
[13" claim-type="Currently amended] The method of claim 1.
The B cell depleting antibody is conjugated with a cytotoxic agent.
[14" claim-type="Currently amended] The method of claim 12,
The cytotoxic agent is a radionuclide.
[15" claim-type="Currently amended] The method of claim 14,
The antibody comprises Y2B8 or 131I-B1 (BEXXAR ^™ ).
[16" claim-type="Currently amended] The method of claim 1,
Administering the antibody intravenously.
[17" claim-type="Currently amended] The method of claim 1,
Administering said antibody by infusion.
[18" claim-type="Currently amended] The method of claim 3,
A method comprising administering to a mammal substantially an amount of an antibody of substantially 375 mg / m ² or less.
[19" claim-type="Currently amended] The method of claim 18,
Wherein said dose is in the range of about 20 mg / m ² to about 250 mg / m ² .
[20" claim-type="Currently amended] The method of claim 19,
The dosage is in the range of about 50 mg / m ² to about 200 mg / m ² .
[21" claim-type="Currently amended] The method of claim 1,
The amount mg / m ² dose of the antibody in the subsequent dose comprises administering a subsequent dose followed by, the first dose of the antibody, characterized in that that exceeds the mg / m ² doses of the antibody in the initial dose of How it is done.
[22" claim-type="Currently amended] The method of claim 6,
And said autoimmune disease is immune thrombocytopenic purpura (ITP).
[23" claim-type="Currently amended] The method of claim 6,
Said autoimmune disease is rheumatoid arthritis.
[24" claim-type="Currently amended] The method of claim 6,
The autoimmune disease is hemolytic anemia.
[25" claim-type="Currently amended] The method of claim 21,
Wherein said hemolytic anemia is Hanglobinemia or Cum's positive anemia.
[26" claim-type="Currently amended] The method of claim 6,
And said autoimmune disease is vasculitis.
[27" claim-type="Currently amended] The method of claim 1,
Essentially consisting of administration of an anti-B7.1 antibody and a B cell depleted anti-CD20 antibody.
[28" claim-type="Currently amended] At least one composition comprises a B cell depleting antibody, and at least another composition comprises an anti-CD40L or anti-B7.1 or anti-B7.2 antibody and is susceptible to an autoimmune disease or prone to autoimmune disease An article of manufacture comprising a container and one or more compositions contained therein, further comprising a package insert directing a user of the composition for treatment.
[29" claim-type="Currently amended] The method of claim 25,
The autoimmune disease is psoriasis; dermatitis; Systemic scleroderma and sclerosis; Reactions associated with inflammatory bowel disease; Crohn's disease; Ulcerative colitis; Respiratory distress syndrome; Adult respiratory distress syndrome (ARDS); dermatitis; meningitis; encephalitis; Uveitis; colitis; Glomerulonephritis; Allergic conditions; eczema; asthma; Conditions involved in the infiltration of T cells and chronic inflammatory responses; Atherosclerosis; Leukocyte adhesion defects; Rheumatoid arthritis; Systemic lupus erythematosus (SLE); Diabetes; multiple sclerosis; Rhinoid syndrome; Autoimmune thyroiditis; Allergic encephalomyelitis; Sjogren's syndrome; Juvenile diabetes; Immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes; Tuberculosis; Sarcoidosis; Polymyositis; Granulomatosis; Vasculitis; Pernicious anemia (Addison disease); Diseases involving leukocyte leakage; Central nervous system (CNS) inflammatory disorders; Multiple organ injury syndrome; Hemolytic anemia; Myasthenia gravis; Antigen-antibody complex mediated disease; anti-glomerular basement membrane disease; Antiphospholipid syndrome; Allergic neuritis; Graves' disease; Lambert-eaton duty syndrome; Bullous whey; pemphigus; Autoimmune polyendocrine disease; Lighter disease; Rigid human syndrome; Beset's disease; Giant cell arteritis; Immunocomplex nephritis; IgA nephropathy; IgM Multiple Neuropathy; An article of manufacture selected from the group consisting of immune thrombocytopenic purpura (ITP), autoimmune thrombocytopenia and ovarian inflammation.
[30" claim-type="Currently amended] Administering a combination of an antibody against B7.1, B7.2 or CD40L and an anti-CD20 antibody having substantial B cell depletion activity, wherein said antibody is isolated or combined, and administered in either order A method for treating multiple sclerosis, characterized in that
[31" claim-type="Currently amended] Administering a combination of an antibody against B7.1, B7.2 or CD40L and an anti-CD20 antibody having substantial B cell depletion activity, wherein said antibody is isolated or combined, and administered in either order A method of treating ITP, characterized in that.
[32" claim-type="Currently amended] Administering a combination of an antibody against B7.1, B7.2 or CD40L and an anti-CD20 antibody having substantial B cell depletion activity, wherein said antibody is isolated or combined, and administered in either order Lupus treatment method characterized in that.
[33" claim-type="Currently amended] Administering a combination of an antibody against B7.1, B7.2 or CD40L and an anti-CD20 antibody having substantial B cell depletion activity, wherein said antibody is isolated or combined, and administered in either order A method of treating diabetes, characterized in that.
[34" claim-type="Currently amended] Administering a combination of the antibody to an antibody against B7.1, B7.2 or CD40L and an anti-CD20 antibody having substantial B cell depletion activity, wherein said antibody is isolated or combined, and in either order A method of treating rheumatoid arthritis, characterized in that the administration.
[35" claim-type="Currently amended] Administering a combination of the antibody to an antibody against B7.1, B7.2 or CD40L and an anti-CD20 antibody having substantial B cell depletion activity, wherein said antibody is isolated or combined, and in either order A method of treating psoriasis, characterized in that the administration.
[36" claim-type="Currently amended] Administering a combination of an antibody against B7.1, B7.2 or CD40L and an anti-CD20 antibody having substantial B cell depletion activity, wherein said antibody is isolated or combined, and administered in either order A method of treating thyroiditis, characterized in that
[37" claim-type="Currently amended] Administering a combination of an antibody against B7.1, B7.2 or CD40L and an anti-CD20 antibody having substantial B cell depletion activity, wherein said antibody is isolated or combined, and administered in either order A method of treating dermatitis, characterized by the above-mentioned.
[38" claim-type="Currently amended] Administering a combination of an antibody against B7.1, B7.2 or CD40L and an anti-CD20 antibody having substantial B cell depletion activity, wherein said antibody is isolated or combined, and administered in either order A method of treating IBD, characterized in that.
[39" claim-type="Currently amended] The method of claim 1,
A method further comprising the administration of a synthetic immunoadjuvant drug.
[40" claim-type="Currently amended] The method of claim 39,
The immunosuppressive agent is cyclosporin or FK506.
[41" claim-type="Currently amended] The method of claim 39,
And further comprising administering the antibody targeted to the autoantibody.

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NO20031218L|2003-05-19|

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WO2002022212A2|2002-03-21|

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MXPA03002262A|2003-10-15|

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NO20031218D0|2003-03-17|

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US20060275284A1|2006-12-07|

---

WO2002022212A3|2003-02-27|

---

CA2422076A1|2002-03-21|

---

US20020058029A1|2002-05-16|

---

WO2002022212A8|2002-08-15|

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CN1592645A|2005-03-09|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-09-18|Priority to US23360700P

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2000-09-18|Priority to US60/233,607

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2000-12-22|Priority to US25714700P

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2000-12-22|Priority to US60/257,147

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2001-09-18|Application filed by 아이덱 파마슈티칼즈 코포레이션

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2001-09-18|Priority to PCT/US2001/029026

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2004-03-18|Publication of KR20040023565A

优先权:

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申请号 | 申请日 | 专利标题

---

US23360700P| true| 2000-09-18|2000-09-18|

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US60/233,607|2000-09-18|

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US25714700P| true| 2000-12-22|2000-12-22|

---

US60/257,147|2000-12-22|

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PCT/US2001/029026|WO2002022212A2|2000-09-18|2001-09-18|Combination therapy for treatment of autoimmune diseases using b cell depleting/immunoregulatory antibody combination|