|Title of Invention||
A METHOD FOR EVALUATING THE EFFICACY OF AN ANTIBODY USEFUL FOR EVALUATING DRUG SAFETY AND EFFICACY
|Abstract||The present invention relates to a method for evaluating the efficacy of an antibody that binds CD20 comprising measuring the ability of a biological sample from a patient treated with the CD20 antibody to block a biological activity of the CD20 antibody. The invention further relates to a method of detecting neutralizing antibodies to a therapeutic antibody that binds CD20 comprising exposing cells that express an antigen to which the therapeutic antibody binds to complement in the presence of the therapeutic antibody and a biological sample from a patient treated therewith, wherein the patient has an autoimmune disease; and determining complement-dependent cytotoxicity (CDC) activity of the therapeutic antibody, wherein a reduction in the CDC activity indicates the presence of neutralizing antibodies in the biological sample. The invention further provides a method of immunotherapy comprising administering an antibody that binds CD20 to a patient; and measuring the ability of a biological sample from the patient to block a biological activity of the CD20 antibody.|
|Full Text||This is a non-provisional application claiming priority under 35 USC §119 to provisional application no. 60/490,678 filed July 29, 2003, the entire disclosure of which is hereby incorporated by reference.
Field of the Invention
The present invention concerns an assay for detecting neutralizing antibodies against an antibody or antagonist, and uses for that assay.
Background of the Invention
Lymphocytes are one of many types of white blood cells produced in the bone marrow during the process of hematopoiesis. There are two major populations of lymphocytes: B lymphocytes (B cells) and T lymphocytes (T cells). The lymphocytes of particular interest herein are B cells.
B cells mature within the bone marrow and leave the marrow expressing an antigen-binding antibody on their cell surface. When a naive B cell first encounters the antigen for which its membrane-bound antibody is specific, the cell begins to divide rapidly and its progeny differentiate into memory B cells and effector cells called "plasma cells". Memory B cells have a longer life span and continue to express membrane-bound antibody with the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibody but instead produce the antibody in a form that can be secreted. Secreted antibodies are the major effector molecule of humoral immunity.
The CD20 antigen (also called human B-lymphocyte-restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes (Valentine et al. J. Biol. Chem. 264(19):! 1282-11287 (1989); and Einfeld et al. EMBO J. 7(3):711-717 (1988)). The antigen is also expressed on greater than 90% of B cell non-Hodgkin's lymphomas (NHL) (Anderson et al. 5/ooc/63(6):1424-1433 (1984)), but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells or other normal tissues (Tedder et al. J. Immunol.
135(2):973-979 (1985)). CD20 regulates an early step(s) in the activation process for cell cycle initiation and differentiation (Tedder et al, supra) and possibly functions as a calcium ion channel (Tedder et al. J. Cell. Biochem. 14D:195 (1990)).
Given the expression of CD20 in B cell lymphomas, this antigen can serve as a candidate for "targeting" of such lymphomas. In essence, such targeting can be generalized as follows: antibodies specific to the CD20 surface antigen of B cells are administered to a patient. These anti-CD20 antibodies specifically bind to the CD20 antigen of (ostensibly) both normal and malignant B cells; the antibody bound to the CD20 surface antigen may lead to the destruction and depletion of neoplastic B cells. Additionally, chemical agents or radioactive labels having the potential to destroy the tumor can be conjugated to the anti-CD20 antibody such that the agent is specifically "delivered" to the neoplastic B cells. Irrespective of the approach, a primary goal is to destroy the tumor; the specific approach can be determined by the particular anti-CD20 antibody which is utilized and, thus, the available approaches to targeting the CD20 antigen can vary considerably.
CD 19 is another antigen that is expressed on the surface of cells of the B lineage. Like CD20, CD19 is found on cells throughout differentiation of the lineage from the stem cell stage up to a point just prior to terminal differentiation into plasma cells (Nadler, L. Lymphocyte Typing II2: 3-37 and Appendix, Renling et al. eds. (1986) by Springer Verlag). Unlike CD20 however, antibody binding to CD 19 causes internalization of the CD 19 antigen. CD 19 antigen is identified by the HD237-CD19 antibody (also called the "B4" antibody) (Kiesel et al. Leukemia Research II, 12: 1119 (1987)), among others. The CD 19 antigen is present on 4-8% of peripheral blood mononuclear cells and on greater than 90% of B cells isolated from peripheral blood, spleen, lymph node or tonsil. CD 19 is not detected on peripheral blood T cells, monocytes or granulocytes. Virtually all non-T cell acute lymphoblastic leukemias (ALL), B cell chronic lymphocytic leukemias (CLL) and B cell lymphomas express CD19 detectable by the 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).
Additional antibodies which recognize differentiation stage-specific antigens expressed by cells of the B cell lineage have been identified. Among these are the B2 antibody directed against the CD21 antigen; B3 antibody directed against the CD22 antigen; and the J5 antibody directed against the CD10 antigen (also called CALLA). See US Patent No. 5,595,721 issued January 21, 1997 (Kaminski et al.).
The rituximab (RITUXAN®) antibody is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen. Rituximab is the antibody called "C2B8" in US Patent No. 5,736,137 issued April 7, 1998 (Anderson et a/.). RITUXAN® is indicated for the treatment of patients with relapsed or refractory low-grade or follicular, CD20 positive, B cell non-Hodgkin's lymphoma. In vitro mechanism of action studies have demonstrated that RITUXAN® binds human complement and lyses lymphoid B cell lines through complement-dependent cytotoxicity (CDC) (Reff et al. Blood 83(2):435-445 (1994)). Additionally, it has significant activity in assays for antibody-dependent cell-mediated cytotoxicity (ADCC). More recently, RITUXAN® has been shown to have anti-proliferative effects in tritiated thymidine incorporation assays and to induce apoptosis directly, while other anti-CD 19 and CD20 antibodies do not (Maloney et al. Blood 88(10):637a (1996)). Synergy between RITUXAN® and chemotherapies and toxins has also been observed experimentally. In particular, RITUXAN® sensitizes drug-resistant human B cell lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP, VP-16, diphtheria toxin and ricin (Demidem et al. Cancer Chemotherapy & Radiopharmaceuticals 12(3): 177-186 (1997)). In vivo preclinical studies have shown that RITUXAN® depletes B cells from the peripheral blood, lymph nodes, and bone marrow of cynomolgus monkeys, presumably through complement and cell-mediated processes (Reff et al. 5/ooJ83(2):435-445 (1994)).
Patents and patent publications concerning CD20 antibodies include US Patent Nos. 5,776,456, 5,736,137, 6,399,061, and 5,843,439, as well as US patent appln nos. US 2002/0197255A1 and US 2003/0021781A1 (Anderson et al); US Patent No. 6,455,04381 and WOOO/09160 (Grillo-Lopez, A.); WOOO/27428 (Grillo-Lopez and White); WOOO/27433 (Grillo-Lopez and Leonard); WOOO/44788 (Braslawsky et al.); WO01/10462 (Rastetter, W.); WO01/10461 (Rastetter and White); WO01/10460 (White and Grillo-Lopez); US appln no. US2002/0006404 and WO02/04021 (Hanna and Hariharan); US appln no. US2002/0012665 Al and WOO 1/74388 (Hanna, N.); US appln no. US2002/0009444A1, and WO01/80884 (Grillo-Lopez, A.); WO01/97858 (White, C.); US appln no. US2002/0128488A1 and WO02/34790 (Reff, M.);WO02/060955 (Braslawsky et a/.);WO2/096948 (Braslawsky et al.);WO02/079255 (Reff and Davies); US Patent No. 6,171,58681, and WO98/56418 (Lam et al.); WO98/58964 (Raju, S.); WO99/22764 (Raju, S.);WO99/51642, US Patent No. 6,194,55181, US Patent No. 6,242,19581, US Patent No. 6,528,62481 and US Patent No. 6,538,124 (Idusogie et al.); WOOO/42072 (Presta, L.); WOOO/67796 (Curd et al.); WOO 1/03734 (Grillo-Lopez et al.); US appln no. US 2002/0004587A1 and WO01/77342 (Miller and Presta); US appln no. US2002/0197256 (Grewal, I.); US Patent Nos. 6,090,36581, 6,287,53781, 6,015,542, 5,843,398, and 5,595,721, (Kaminski et al); US Patent Nos. 5,500,362, 5,677,180, 5,721,108, and 6,120,767 (Robinson et al); US Pat No. 6,410,39181 (Raubitschek et al); US Patent No. 6,224,86681 and WOOO/20864 (Barbera-Guillem, E.); WOO 1/13945 (Barbera-Guillem, E.); WOOO/67795 (Goldenberg); WOOO/74718 (Goldenberg and Hansen); WOOO/76542 (Golay et al); WOO 1/72333 (Wolin and Rosenblatt); US Patent No. 6,368,59681 (Ghetie et al); US Appln no. US2002/0041847A1, (Goldenberg, D.); US Appln no. US2003/0026801A1 (Weiner and Hartmann); WO02/102312 (Engleman, E.), each of which is expressly incorporated herein by reference. See, also, US Patent No. 5,849,898 and EP appln no. 330,191 (Seed et al); US Patent
No. 4,861,579 and EP332.865A2 (Meyer and Weiss); and WO95/03770 (Bhat et al.).
Publications concerning therapy with Rituximab include: Perotta and Abuel "Response of chronic relapsing ITP of 10 years duration to Rituximab" Abstract # 3360 Blood 10(l)(part 1-2): p. 88B (1998); Stashi et al "Rituximab chimeric anti-CD20 monoclonal antibody treatment for adults with chronic idopathic thrombocytopenic purpura" 5/ooc?98(4):952-957 (2001); Matthews, R. "Medical Heretics" New Scientist (7 April, 2001); Leandro et al. "Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion" Ann Rheum Dis 61:833-888 (2002); Leandro et al. "Lymphocyte depletion in rheumatoid arthritis: early evidence for safety, efficacy and dose response. Arthritis and Rheumatism 44(9): S370 (2001); Leandro et al. "An open study of B lymphocyte depletion in systemic lupus erythematosus", Arthritis & Rheumatism 46(l):2673-2677 (2002); Edwards and Cambridge "Sustained improvement in rheumatoid arthritis following a protocol designed to deplete B lymphocytes" Rhematology 40:205-211 (2001); Edwards et al. "B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune disorders" Biochem. Soc. Trans. 30(4):824-828 (2002); Edwards et al. "Efficacy and safety of Rituximab, a B-cell targeted chimeric monoclonal antibody: A randomized, placebo controlled trial in patients with rheumatoid arthritis. Arthritis and Rheumatism 46(9): SI97 (2002); Levine and Pestronk "IgM antibody-related polyneuropathies: B-cell depletion chemotherapy using Rituximab" Neurology 52: 1701 -1704 (1999); DeVita et al. "Efficacy of selective B cell blockade in the treatment of rheumatoid arthritis" Arthritis & Rheum 46:2029-2033 (2002); Hidashida et al. "Treatment of DMARD-Refractory rheumatoid arthritis with rituximab." Presented at the Annual Scientific Meeting of the American College of Rheumatology; Oct 24-29; Ne Orleans, LA 2002; Tuscano, J. "Successful treatment of Infliximab-refractory rheumatoid arthritis with rituximab" Presented at the Annual Scientific Meeting of the American College of Rheumatology; Oct 24-29; New Orleans, LA 2002.
US Patent Application No. 2003/0068664 (Albitar et al.) describes an ELISA assay for determining human anti-chimeric antibody (HACA) directed against Rituximab.
Summary of the Invention
Example 1 herein describes the development of a complement-dependent cytotoxicity (CDC) assay for detecting neutralizing antibodies against an antibody that binds a B cell surface marker, namely the CD20 antigen. The CDC activity was measured by incubating CD20 positive cells with human complement in the absence or presence of different concentrations of the CD20 antibody. Cytotoxicity was then measured by quantifying live cells. Serum matrix effect on assay performance was tested. Serum could be tolerated up to 40% without a significant shift in EC50 values. CD20 antibody-treated systemic lupus erythrematosis (SLE) patient serum samples with an antibody response (HACA) were then tested. The CDC activity of the CD20 antibody could be either completely or partially blocked with HACA sera, indicating neutralizing activities in treated samples. In comparison, serum samples obtained prior to CD20 antibody treatment showed no neutralizing activity. This assay characterizes the nature of any anti-drug antibody response; therefore it will be valuable for evaluating drug safety and efficacy.
Accordingly, the present invention provides a method for evaluating the efficacy of an antibody that binds CD20 comprising measuring the ability of a biological sample from a patient treated with the CD20 antibody to block a biological activity of the CD20 antibody.
The invention further provides a method of immunotherapy comprising administering an antibody that binds CD20 to a patient; and measuring the ability of a biological sample from the patient to block a biological activity of the CD20 antibody.
In another aspect, the invention concerns a method of detecting neutralizing antibodies to a therapeutic antibody comprising exposing cells that
express an antigen to which the therapeutic antibody binds to complement in the presence of the therapeutic antibody and a biological sample from a patient treated therewith; and determining complement-dependent cytotoxicity (CDC) activity of the therapeutic antibody, wherein a reduction in the CDC activity indicates the presence of neutralizing antibodies in the biological sample.
Additionally, a method of evaluating the efficacy of an antagonist that binds a B cell surface marker is provided which comprises measuring the ability of a biological sample from a patient treated with the antagonist to block a biological activity of the antagonist.
In yet a further embodiment, the invention concerns a method of immunotherapy comprising
administering an antibody that binds a B cell surface marker to a patient; and measuring the ability of a biological sample from the patient to block a biological activity of the antibody.
Detailed Description of the Preferred Embodiments I. Definitions
Unless indicated otherwise, by "biological sample" herein is meant a sample obtained from a patient herein. The sample may comprise antibodies that bind to the antibody or drug with which the patient has been treated, such as human anti-murine antibody (HAMA), human anti-chimeric antibody (HACA) or human anti-human antibody (HAHA). The biological sample may for example be serum, antibodies recovered from the patient, plasma, cell lysate, milk, saliva, and other secretions, but preferably serum.
The expression "biological activity" refers to a measurable function of an antibody or antagonist herein. Various activities are contemplated and include, but are not limited to, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis, inhibiting growth of cells (e.g. tumor cells), etc.
The ability of a biological sample (or antibodies raised by a patient against the drug in question) to "block" a biological activity of an antagonist or antibody refers to both partial and complete blocking of that activity.
A "B cell surface marker" herein is an antigen expressed on the surface of a B cell which can be targeted with an antagonist or antibody which binds thereto. Exemplary B cell surface markers include the CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD40, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80, CD81, CD82, CD83, CDw84, CD85 and CD86 leukocyte surface markers. The B cell surface marker of particular interest is preferentially expressed on B cells compared to other non-B cell tissues of a mammal and may be expressed on both precursor B cells and mature B cells. In one embodiment, the marker is one, like CD20 or CD 19, which is found on B cells throughout differentiation of the lineage from the stem cell stage up to a point just prior to terminal differentiation into plasma cells. The preferred B cell surface marker herein is CD20.
The "CD20" antigen is a -35 kDa, non-glycosylated phosphoprotein found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and remains until plasma cell differentiation. CD20 is present on both normal B cells as well as malignant B cells. Other names for CD20 in the literature include "B-lymphocyte-restricted antigen" and "Bp35". The CD20 antigen is described in Clark et al. PNAS (USA) 82:1766 (1985), for example.
As used herein, "B cell depletion" refers to a reduction in B cell levels in an animal or human generally after drug or antibody treatment, as compared to the level before treatment. B cell depletion can be partial or complete. B cell levels are measurable using well known techniques such as those described in Reff et al., Blood 83: 435-445 (1994), or US Patent No. 5,736,137 (Anderson et al.). By way of example, a mammal (e.g. a normal primate) may be treated with various dosages of the antibody or antagonist, and peripheral B-cell concentrations may be determined, e.g. by a FACS method that counts B cells.
A "B cell malignancy" is a malignancy involving B cells. Examples include Hodgkin's disease, including lymphocyte predominant Hodgkin's disease (LPHD); non-Hodgkin's lymphoma (NHL); follicular center cell (FCC) lymphoma; acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL); hairy cell leukemia; plasmacytoid lymphocytic lymphoma; mantle cell lymphoma; AIDS or HIV-related lymphoma; multiple myeloma; central nervous system (CNS) lymphoma; post-transplant lymphoproliferative disorder (PTLD); Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma); mucosa-associated lymphoid tissue (MALT) lymphoma; and marginal zone lymphoma/leukemia.
Non-Hodgkin's lymphoma (NHL) includes, but is not limited to, low grade/follicular NHL, relapsed or refractory NHL, front line low grade NHL, Stage 1I1/IV NHL, chemotherapy resistant NHL, small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, diffuse large cell lymphoma, aggressive NHL (including aggressive front-line NHL and aggressive relapsed NHL), NHL relapsing after or refractory to autologous stem cell transplantation, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL, etc.
An "autoimmune disease" herein is a disease or disorder arising from and directed against an individual's own tissues. Examples of autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), juvenile
onset diabetes, multiple sclerosis, allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis, agranulocytosis, vasculitis (including ANCA), aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, central nervous system (CNS) inflammatory disorders, multiple organ injury syndrome, mysathenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman's syndrome, Goodpasture's syndrome, Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection, graft versus host disease (GVHD), pemphigoid bullous, pemphigus, autoimmune polyendocrinopathies, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), including fludarabine-associated IIP, thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Grave's disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), Type I diabetes also referred to as insulin-dependent diabetes mellitus (IDDM) and Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre' syndrome, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa), ankylosing spondylitis, Berger's disease (IgA
nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, Celiac sprue (gluten enteropathy), cryoglobulinemia, amyotrophic lateral sclerosis (ALS), coronary artery disease, cold agglutinin disease, acquired factor VIII inhibitors, lupus nephritis, etc.
An "antagonist" that binds a B cell surface marker herein is a molecule which, upon binding to a B cell surface marker, destroys or depletes B cells in a mammal and/or interferes with one or more B cell functions, e.g. by reducing or preventing a humoral response elicited by the B cell. The antagonist preferably is able to deplete B cells in a mammal treated therewith. Such depletion may be achieved via various mechanisms such antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), inhibition of B cell proliferation and/or induction of B cell death (e.g. via apoptosis). Antagonists included within the scope of the present invention include antibodies, synthetic or native sequence peptides, immunoadhesins, small molecule antagonists which bind to the B cell marker, optionally conjugated with or fused to a cytotoxic agent. The preferred antagonist comprises an antibody.
"Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells in summarized is Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
"Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and carry out ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred.
The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and Fey RHI subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see Dae'ron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). FcRs herein include polymorphisms such as the genetic dimorphism in the gene that encodes FcyRIIIa resulting in either a phenylalanine (F) or a valine (V) at amino acid position 158, located in the region of the receptor that binds to IgGl. The homozygous valine FcyRIIIa (FcyRIIIa-158V) has been shown to have a higher affinity for human IgGl and mediate increased ADCC in vitro relative to homozygous phenylalanine FcyRIIIa (FcyRIIIa-158F) or heterozygous (FcyRIIIa-158F/V) receptors.
"Complement dependent cytotoxicity" or "CDC" refer to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.
"Growth inhibitory" antagonists or antibodies are those which prevent or reduce proliferation of a cell expressing an antigen to which the antagonist binds. For example, the antagonist or antibody may prevent or reduce proliferation of B cells in vitro and/or in vivo.
Antagonists or antibodies which "induce apoptosis" are those which induce programmed cell death, e.g. of a B cell, as may be determined by standard apoptosis assays, such as binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).
The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
"Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. 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.
"Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has
regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a P-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the |3-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Rabat et ai, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).
Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called a, 8, e, y, and \i, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma
method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991), for example.
The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al, Proc. Natl Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences (US Pat No. 5,693,780).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones etal., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; 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.
An antagonist or antibody "which binds" an antigen of interest, e.g. a B cell surface marker or CD20, is one capable of binding that antigen with sufficient affinity and/or avidity such that the antagonist or antibody is useful as a therapeutic agent for targeting a cell expressing the antigen.
For the purposes herein, "immunotherapy" will refer to a method of treating a mammal (preferably a human patient) with an antibody, wherein the antibody may be an unconjugated or "naked" antibody, or the antibody may be conjugated or fused with heterologous molecule(s) or agent(s), such as one or more cytotoxic agent(s), thereby generating an "immunoconjugate".
As used herein, a "therapeutic antibody" is an antibody that is effective in treating a disease or disorder in a mammal with or predisposed to the disease or disorder. Exemplary therapeutic antibodies include anti-HER2 antibodies including rhuMAb 4D5 (HERCEPTIN J (Carter et al, Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992), U.S. Patent No. 5,725,856); anti-CD20 antibodies (see below); anti-IL-8 (St John et al., Chest, 103:932 (1993), and International Publication No. WO 95/23865); anti-VEGF antibodies including humanized and/or affinity matured anti-VEGF antibodies such as the humanized anti-VEGF antibody huA4.6.1 AVASTIN_ (Kim et al., Growth Factors, 7:53-64 (1992), International Publication No. WO 96/30046, and WO 98/45331, published October 15, 1998); anti-PSCA antibodies (WO01/40309); anti-CD40 antibodies, including S2C6 and humanized variants thereof (WOOO/75348); anti-GDI la antibodies including Raptiva™ (US Patent No. 5,622,700, WO 98/23761, Steppe et al., Transplant Intl. 4:3-7 (1991), and Hourmant et al, Transplantation 58:377-380 (1994)); anti-IgE antibodies (Presta et al, J. Immunol 151:2623-2632 (1993), and International Publication No. WO 95/19181 ;US Patent No. 5,714,338, issued February 3, 1998 or US Patent No. 5,091,313, issued February 25, 1992, WO 93/04173 published March 4, 1993, or International Application No. PCT/US98/13410 filed June 30, 1998, US Patent No. 5,714,338); anti-CD18 antibodies (US Patent No. 5,622,700, issued April 22, 1997, or as in WO 97/26912, published July 31, 1997); anti-Apo-2 receptor antibody antibodies (WO 98/51793 published November 19, 1998); anti-TNF-_ antibodies including cA2 (REMICADEJ, CDP571 and MAK-195 (See, US Patent No. 5,672,347 issued September 30, 1997, Lorenz et al. J. Immunol. 156(4): 1646-1653 (1996), and Dhainaut et al. Crit. Care Med. 23(9):1461-1469 (1995)); anti-Tissue Factor (TF) antibodies (European Patent No. 0 420 937 Bl granted November 9, 1994); anti-human _4-_7 integrin antibodies (WO 98/06248 published February 19, 1998); anti-EGFR antibodies (chimerized or humanized 225 antibody as in WO 96/40210 published December 19, 1996); anti-CD3 antibodies such as OKT3 (US Patent No. 4,515,893 issued May 7, 1985); anti-CD25 or anti-Tac antibodies such as
CHI-621 (SIMULECTJ and ZENAPAX_ (See US Patent No. 5,693,762 issued December 2, 1997); anti-CD4 antibodies such as the cM-7412 antibody (Choy et al. Arthritis Rheum 39(l):52-56 (1996)); anti-CD52 antibodies such as CAMPATH-1H (Riechmann et al. Nature 332:323-337 (1988); anti-Fc receptor antibodies such as the M22 antibody directed against Fc_RI as in Graziano et al. J. Immunol. 155(10):4996-5002 (1995); anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkey et al. Cancer Res. 55(23Suppl): 5935s-5945s (1995); antibodies directed against breast epithelial cells including huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al Cancer Res. 55(23): 5852s-5856s (1995); and Richman et al Cancer Res. 55(23 Supp): 5916s-5920s (1995)); antibodies that bind to colon carcinoma cells such as C242 (Litton et al. EurJ. Immunol. 26(1): 1-9 (1996)); anti-CD38 antibodies, e.g. AT 13/5 (Ellis et al J. Immunol. 155(2):925-937 (1995)); anti-CD33 antibodies such as Hu M195 (Jurcic etal Cancer Res 55(23 Suppl):5908s-5910s (1995) and CMA-676 or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide (Juweid et al Cancer Res 55(23 Suppl):5899s-5907s (1995); anti-EpCAM antibodies such as 17-1A (PANOREXJ; anti-GpIIb/IIIa antibodies such as abciximab or c7E3 Fab (REOPROJ; anti-RSV antibodies such as MEDI-493 (SYNAGISJ; anti-CMV antibodies such as PROTOVIRj anti-HIV antibodies such as PRO542; anti-hepatitis antibodies such as the anti-Hep B antibody OSTAVIR_; anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitope antibody BEC2; anti-_v_3 antibody VITAX1N_; anti-human renal cell carcinoma antibody such as ch-G250; ING-1; anti-human 17-1A antibody (3622W94); anti-human colorectal tumor antibody (A33); anti-human melanoma antibody R24 directed against GD3 ganglioside; anti-human squamous-cell carcinoma (SF-25); and anti-human leukocyte antigen (HLA) antibodies such as Smart ID 10 and the anti-HLA DR antibody Oncolym (Lym-1).
Examples of antibodies which bind the CD20 antigen include: "C2B8" which is now called "Rituximab" ("RITUXAN®") (US Patent No. 5,736,137, expressly incorporated herein by reference); the yttrium--labeled 2B8 murine
antibody designated "Y2B8" or "Ibritumomab Tiuxetan" ZEVALIN® (US Patent No. 5,736,137, expressly incorporated herein by reference); murine IgG2a "Bl," also called "Tositumomab," optionally labeled with 131I to generate the "131I-B1" antibody (iodine 1131 tositumomab, BEXXAR™) (US Patent No. 5,595,721, expressly incorporated herein by reference); murine monoclonal antibody "1F5" (Press et al. Blood 69(2):584-591 (1987)); murine 2H7 and chimeric 2H7 antibody (US Patent No. 5,677,180, expressly incorporated herein by reference); humanized 2H7, including "humanized 2H7 v!6" (see below); huMax-CD20 (Genmab, Denmark); AME-133 (Applied Molecular Evolution); and monoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing III (McMichael, Ed., p. 440, Oxford University Press (1987)).
Examples of antibodies which bind the CD19 antigen include the anti-CDantibodies in Hekman et al Cancer Immunol. Immunother. 32:364-372 (1991) and Vlasveld et al. Cancer Immunol. Immunother. 40:37-47 (1995); and the B4 antibody in Kiesel et al Leukemia Research II, 12: 1119 (1987).
The terms "rituximab" or "RITUXAN®" herein refer to the genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen and designated "C2B8" in US Patent No. 5,736,137, expressly incorporated herein by reference. The antibody is an IgGi kappa immunoglobulin containing murine light and heavy chain variable region sequences and human constant region sequences. Rituximab has a binding affinity for the CD20 antigen of approximately S.OnM.
Purely for the purposes herein, "humanized 2H7 v!6" refers to an antibody comprising the variable light and variable heavy sequences shown below . Variable light-chain domain of hu2H7 v!6:
DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPS NLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSFNPPTFGQGTK VE1KR(SEQIDNO:1) Variable heavy-chain domain of hu2H7 v!6:
VVYYSNSYWYFDVWGQGTLVTVSS (SEQ ID NO: 2).
Preferably humanized 2H7 v!6 comprises the light chain amino acid sequence:
YEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 3);
and heavy chain amino acid sequence:
SVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4).
An "isolated" antagonist or antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antagonist or antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antagonist or antibody will be purified (1) to greater than 95% by weight of antagonist or antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-
PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antagonist or antibody includes the antagonist or antibody in situ within recombinant cells since at least one component of the antagonist's or antibody's natural environment will not be present. Ordinarily, however, isolated antagonist or antibody will be prepared by at least one purification step.
"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease or disorder as well as those in which the disease or disorder is to be prevented. Hence, the mammal may have been diagnosed as having the disease or disorder or may be predisposed or susceptible to the disease. The expression "therapeutical ly effective amount" refers to an amount of the antagonist or antibody which is effective for preventing, ameliorating or treating the disease or condition in question.
The term "immunosuppressive agent" as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Pat. No. 4,665,077, the disclosure of which is incorporated herein by reference); nonsteroidal antiinflammatory drugs (NSAIDs); azathioprine; cyclophosphamide; bromocryptine; danazol; dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as glucocorticosteroids, e.g., prednisone, methylprednisolone, dexamethasone, and hydrocortisone; methotrexate (oral or subcutaneous); hydroxycloroquine; sulfasalazine; leflunomide; cytokine or
cytokine receptor antagonists including anti-interferon-y, -p, or -a antibodies, anti-tumor necrosis factor-a antibodies (infliximab or adalimumab), anti-TNFa immunoahesin (etanercept), anti-tumor necrosis factor-p antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CDl la and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published 7/26/90); streptokinase; TGF-P; streptodornase; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin; T-cell receptor (Cohen et al, U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offher et al, Science, 251: 430-432 (1991); WO 90/11294; laneway, Nature, 341: 482 (1989); and WO 91/01133); and T cell receptor antibodies (EP 340,109) such as T10B9.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At211,1131,1125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,
ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins;
capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-a and -p; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-P; platelet-growth factor; transforming growth factors (TGFs) such as TGF-a and TGF-P; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -P, and -y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis factor such as TNF-a or TNF-P; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
The term "prodrug" as used in this application refers to a precursor or
derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 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). The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, p-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
By "foreign antigen" is meant a molecule or molecules which is/are not endogenous or native to a mammal which is exposed to it. The foreign antigen may elicit an immune response, e.g. a humoral and/or T cell mediated response in the mammal. Generally, the foreign antigen will result in the production of antibodies thereagainst. Examples of foreign antigens contemplated herein include immunogenic therapeutic agents, e.g. proteins such as antibodies, particularly antibodies comprising non-human amino acid residues (e.g. rodent, chimeric/humanized, and primatized antibodies); toxins (optionally conjugated to a targeting molecule such as an antibody, wherein the targeting molecule may also be immunogenic); gene therapy viral vectors, such as retroviruses and adenoviruses; grafts; infectious agents (e.g. bacteria and virus); alloantigens (i.e. an antigen that occurs in some, but not in other members of the same species) such as differences in blood types, human lymphocyte antigens (HLA), platelet antigens, antigens expressed on transplanted organs, blood components,
pregnancy (Rh), and hemophilic factors (e.g. Factor VIII and Factor IX).
By "blocking an immune response" to a foreign antigen is meant reducing or preventing at least one immune-mediated response resulting from exposure to a foreign antigen. For example, one may dampen a humoral response to the foreign antigen, i.e., by preventing or reducing the production of antibodies directed against the antigen in the mammal. Alternatively, or additionally, one may suppress idiotype; "pacify" the removal of cells coated with alloantibody; and/or affect alloantigen presentation through depletion of antigen-presenting cells.
The term "graft" as used herein refers to biological material derived from a donor for transplantation into a recipient. Grafts include such diverse material as, for example, isolated cells such as islet cells; tissue such as the amniotic membrane of a newborn, bone marrow, hematopoietic precursor cells, and ocular tissue, such as corneal tissue; and organs such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, tubular organs (e.g., intestine, blood vessels, or esophagus), etc. The tubular organs can be used to replace damaged portions of esophagus, blood vessels, or bile duct. The skin grafts can be used not only for burns, but also as a dressing to damaged intestine or to close certain defects such as diaphragmatic hernia. The graft is derived from any mammalian source, including human, whether from cadavers or living donors. Preferably the graft is bone marrow or an organ such as heart and the donor of the graft and the host are matched for HLA class II antigens.
The term "mammalian host" as used herein refers to any compatible transplant recipient. By "compatible" is meant a mammalian host that will accept the donated graft. Preferably, the host is human. If both the donor of the graft and the host are human, they are preferably matched for HLA class II antigens so as to improve histocompatibility.
The term "donor" as used herein refers to the mammalian species, dead or alive, from which the graft is derived. Preferably, the donor is human. Human donors are preferably volunteer blood-related donors that are normal on physical examination and of the same major ABO blood group, because crossing major
blood group barriers possibly prejudices survival of the allograft. It is, however, possible to transplant, for example, a kidney of a type O donor into an A, B or AB recipient.
The term "transplant" and variations thereof refers to the insertion of a graft into a host, whether the transplantation is syngeneic (where the donor and recipient are genetically identical), allogeneic (where the donor and recipient are of different genetic origins but of the same species), or xenogeneic (where the donor and recipient are from different species). Thus, in a typical scenario, the host is human and the graft is an isograft, derived from a human of the same or different genetic origins. In another scenario, the graft is derived from a species different from that into which it is transplanted, such as a baboon heart transplanted into a human recipient host, and including animals from phylogenically widely separated species, for example, a pig heart valve, or animal beta islet cells or neuronal cells transplanted into a human host.
By "gene therapy" is meant the general approach of introducing nucleic acid into a mammal to be treated therewith. The nucleic acid may encode a polypeptide of interest or may be antisense nucleic acid. One or more components of a gene therapy vector or composition may be immunogenic in a mammal treated therewith. For example, viral vectors (such as adenovirus, Herpes simplex 1 virus or retrovirus); lipids; and/or targeting molecules in the composition may induce an immune response in a mammal treated therewith.
The expression "desensitizing a mammal awaiting transplantation" refers to reducing or abolishing allergic sensitivity or reactivity to a transplant, prior to administration of the transplant to the mammal. This may be achieved by any mechanism, such as a reduction in anti-donor antibodies in the desensitized mammal, e.g. where such anti-donor antibodies are directed against human lymphocyte antigen (HLA).
"Neutralizing antibodies" herein refer to antibodies that not only bind to an antigen (e.g. a therapeutic antibody such as a CD20 antibody) of interest, but further inhibit, to some extent, a biological activity of that antigen.
II. Neutralizing Antibody Assay
This invention concerns, at least in part, an assay for detecting neutralizing antibodies to a therapeutic antibody, or to an antagonist that binds to a B cell surface marker (e.g. to an antibody that binds CD20). The assay determines the ability of a biological sample from a patient treated with the antibody or antagonist to block a biological activity of the antibody or antagonist. Blocking activity may indicate reduced efficacy of the antibody or antagonist.
The sample is generally obtained from the patient prior to and/or after the patient has been treated with the antibody or antagonist. Usually biological samples are obtained from the patient at a series of time-points, e.g. from pretreatment throughout the treatment cycle(s). In order to avoid drug interfering with the performance of the assay, a biological sample will normally be taken when drug washout occurs. For instance, sample at baseline, and at 3, 6 and 9 months may be tested. If the patient is retreated at a later date, sample from baseline and at 3 or 6 months may be tested for neutralizing antibody.
The biological sample may comprise antibodies that bind to the antibody or antagonist with which the patient has been treated, such as human anti-murine antibody (HAMA), human anti-chimeric antibody (HACA) or human anti-human antibody (HAHA). HAHA may be against either a humanized or human therapeutic antibody. In one embodiment, the sample is one which has been determined to contain such antibodies. For instance, serum from the patient may be found to comprise antibodies to the drug in question through the ELISA assay in Example 1 below or in US Patent Application No. 2003/0068664 (Albitar et a/.).
The biological sample used in the assay may be serum, plasma, cell lysate, milk, saliva, or other secretions, as well as antibodies recovered from any one or more of such biological specimans. Preferably serum from the patient is subjected to the assay herein.
Neutralizing antibodies may decrease the expected pharmacologic level of the infused drug, thereby decreasing efficacy or making the likelihood of response
more variable. Neutralizing antibodies can be associated with serum sickness or immune complex disease on retreatment. By way of example, where a neutralizing antibody response is seen, the treatment may be halted or postponed, or the dosage may be increased, or the patient may be given further agents which improve the efficacy of the antibody or antagonist, and/or which reduce any immune response thereto. Various immunosuppressive agents that can be combined with the treatment to reduce an immune response, where a neutralizing antibody response is observed, are known and exemplary such drugs are specifically noted herein.
In addition to the usage of the assay results by clinicians in the treatment of patients, the neutralizing property of anti-drug antibodies, in conjunction with HAMA, HACA, or HAHA data, demonstrate immunogenicity, or tendency of immunogenicity, as well as the nature of immunogenicity of an antibody or antagonist. This information is useful in evaluating drug safety and predicting potential immune responses of patients to therapies.
The present assay represents an improvement over the ELISA assay in US 2003/0068664, Albitar et al.), in that it provides an assessment of whether or not any antibody response to the drug in question can actually neutralize (at least to some extent) a biological activity of the drug, whether that drug be an antibody or an antagonist to a B cell surface marker. Thus, the information can be used to determine the efficacy of the antibody or antagonist to treat the patient. In the context of a CD20 antibody, or other antagonist that binds a B cell surface marker, the assay is thought to be particularly useful where treatment therewith only leads to partial B cell depletion, where B cell hyperactivation is occurring (e.g. as in SLE), or where persistent disease symptoms exist for years and years (e.g as in SLEandRA).
Use of the assay with respect to patients who are being treated with the antibody or antagonist to treat an autoimmune disease is especially desirable. Various autoimmune diseases are described herein, but exemplary ones includes rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Wegener's
disease, inflammatory bowel disease, idiopathic or immune thrombocytopenic purpura (IIP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis (MS), psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjogren's syndrome glomerulonephritis, autoimmune hemolytic anemia etc.
Where the antagonist or antibody binds to a B cell surface marker, such as the CD20 antigen, the patient may have an autoimmune disease, B cell malignancy, or the antagonist or antibody may be used to block an immune response to a foreign antigen (e.g. where the foreign antigen is an immunogenic therapeutic agent, or a graft).
In the preferred embodiment of the invention, the biological activity assay comprises a cell-based functional assay, such as an assay which determines complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis, or inhibition of cell growth.
Preferably, the assay studies CDC activity. According to this embodiment of the invention, cells expressing the antigen (e.g. a B cell surface marker, such as CD20) to which the antibody or antagonist binds may be exposed to complement (preferably human complement) in the presence (or absence) of the antibody or antagonist as well as a biological sample from a patient treated with the antibody or antagonist. The present application contemplates exposing the four components (cells, complement, antibody or antagonist and biological sample) simultaneously or sequentially in any order; all of these possibilities are encompassed by the expression "exposing cells that express an antigen to which the therapeutic antibody binds to complement in the presence of the therapeutic antibody and a biological sample from a patient treated therewith." However, according to the preferred embodiment of the invention, the biological sample (e.g. serum) is combined with the antibody or antagonist so as to allow for neutralization of the antibody or antagonist activity, and then cells and complement are added to this mixture.
Following the exposure step, CDC activity is determined, preferably by assessing cell viability (i.e. by quantifying live cells). Various methods are available for determining cell viability including determining loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue (see Moore et al. Cy to technology 17:1-11 (1995)), annexin V, or 7AAD relative to untreated cells, the AlamarBlue™ assay herein etc. A reduction in the antibody's or antagonist's ability to mediate CDC may indicate that neutralizing antibodies are present in the biological sample.
For a cell-based assay, one will generally use a cell line which expresses the antigen to which the antibody or antagonist binds. In the case of the CD20 antigen, various cells are available including, WIL2-S cells (ATCC CRL 8885, American Type Culture Collection), or a CD20 expressing lymphoblastoid B-cell line. CDC assays using CD20 positive cells have been described in Idusogie et al.J. Immunol. 164:4178-4184(2000); Idusogie et al., J. Immunol 166:2571-2575 (2001); Reffetal. 5/oorf83(2):435-445 (1994); US Patent No. 6,194,551 Bl (Idusogie et al.); and US Patent No. 5,736,137 (Anderson et al.).
Where the assay evaluates ADCC, the antibody or antagonist may be assayed for its ability to mediate Natural-Killer cell (NK cell) and/or peripheral blood mononuclear cell (PBMC) lysis of cells expressing the antigen to which the therapeutic antibody binds. In the case of the CD20 antigen, WIL2-S cells may be used, and Shields et al., J. Biol. Chem. 276:6591-6604 (2001) and WOOO/42072 (Presta, L.) describe an exemplary ADCC assay using those cells. See, also, Clynes et al. Nature Medicine 6:443-6 (2000). US Patent No. 5,736,137 (Anderson et al.) also describes an ADCC assay using CD20 positive cells.
Apoptosis refers to programmed cell death, e.g. of a B cell, and may be determined by a variety of different assays, such as binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). Assays which determine the ability of an antibody (e.g. Rituximab) to induce apoptosis have been described in Shan et al. Cancer Immunol Immunther 48:673-
83 (2000); Pedersen et d. Blood 99:1314-9 (2002); Demidem et al Cancer Chemotherapy & Radiopharmaceuticals 12(3): 177-186 (1997), for example.
The ability of an antagonist or antibody to inhibit growth of a cell, e.g. a cancerous B cell expressing the antigen to which the antagonist or antibody binds, can be assessed by a variety of different assays. Taji et al. Jpn J. Cancer Res 89:748-56 (1998) describe how to determine growth inhibition of CD20-positive B lymphoma cell lines by a CD20 antibody.
Using the neutralizing antibody assay herein, one may determine the efficacy of an antibody or antagonist (e.g. one which binds CD20), by measuring the ability of a biological sample from a patient treated with the antibody or antagonist to block a biological activity of the antibody or antagonist, wherein a reduction in the biological activity relative to a control sample is indicative that the patient is raising antibodies against the antibody or antagonist in question and/or that such antibodies can neutralize, at least to some degree, the biological activity of the antibody or antagonist. A significant response may be one which results in a safety related problem from neutralizing antibody development and/or the requirement to alter dosing of the primary drug in response to altered clearance of the drug. For instance, in comparison to the same amount of pre-treatment counterpart (e.g., HAMA, HACA, and HAHA negative), a sample neutralizing about 20% or greater activity of the antibody or antagonist drug (e.g. in the range from about 20% to about 100%) at a given concentration, may be considered positive for neutralizing antibody directed against the antibody or antagonist.
III. Production of Antagonists or Antibodies
The methods of the present invention use, or incorporate, an antagonist which binds to a B cell surface marker or a therapeutic antibody. Accordingly, methods for generating such antagonists or antibodies will be described here.
The antigen to be used for production of, or screening for, antagonist or antibody may be, e.g., a soluble form of the antigen or a portion thereof, containing the desired epitope. Alternatively, or additionally, cells expressing the antigen at their cell surface can be used to generate, or screen for, antagonist or
antibody. Other forms of the antigen useful for generating antagonist or antibody will be apparent to those skilled in the art. Preferably, the antigen is a B cell surface marker, such as the CD20 antigen.
While the preferred antagonist is an antibody, antagonists other than antibodies are contemplated herein. For example, the antagonist may comprise a small molecule antagonist optionally fused to, or conjugated with, a cytotoxic agent (such as those described herein). Libraries of small molecules may be screened against the B cell surface marker of interest herein in order to identify a small molecule which binds to that antigen. The small molecule may further be screened for its antagonistic properties and/or conjugated with a cytotoxic agent.
The antagonist may also be a peptide generated by rational design or by phage display (see, e.g., WO98/35036 published 13 August 1998). In one embodiment, the molecule of choice may be a "CDR mimic" or antibody analogue designed based on the CDRs of an antibody. While such peptides may be antagonistic by themselves, the peptide may optionally be fused to a cytotoxic agent so as to add or enhance antagonistic properties of the peptide.
In another embodiment, the antagonist is an immunoadhesin comprising a binding domain, e.g. a peptide or protein that binds to a B cell surface marker such as CD20 fused to an immunoglobulin, e.g. an immunoglobulin Fc region.
A description follows as to exemplary techniques for the production of antibody antagonists used in accordance with the present invention.
(i) Polyclonal antibodies
Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC12, or R'NONR, where R and R1 are different alkyl groups.
Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 jag or 5 u,g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.
(ii) Monoclonal antibodies
Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.
For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made
by recombinant DNA methods (U.S. Patent No. 4,816,567).
In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Coding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).
The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of 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)).
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al, Anal. Biochem., 107:220 (1980).
After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Coding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.
The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the 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 PlUckthun, Immunol. Revs., 130:151-188 (1992).
In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al, Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-
628 (1991) and Marks et al, J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, et al., Proc. NatlAcad. Sci. USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
(in) Humanized antibodies
Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such
"humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antibody (Sims et al, J. Immunol, 151:2296 (1993); Chothia et al, J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Prestae/a/., J. Immunol., 151:2623 (1993)).
It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR
residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.
(iv) Human antibodies
As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (Jn) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et at, Nature, 362:255-258 (1993); Bruggermann et al, Year in Immuno., 7:33 (1993); and US Patent Nos. 5,591,669, 5,589,369 and 5,545,807.
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 unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-
gene segments can be used for phage display. Clackson et al, Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al, J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734(1993). See, also, US Patent Nos. 5,565,332 and 5,573,905.
Human antibodies may also be generated by in vitro activated B cells (see US Patents 5,567,610 and 5,229,275).
(v) Antibody fragments
Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al, Bio/Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; US Patent No. 5,571,894; and US Patent No. 5,587,458. The antibody fragment may also be a "linear antibody", e.g., as described in US Patent 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
(vi) Bispecific antibodies
Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the B cell surface marker. Other such antibodies may bind a first B cell marker and further bind a second B cell surface marker. Alternatively, an anti-B cell marker binding arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRJI (CD32) and FcyRIIl (CD16) so as to focus cellular defense mechanisms to the B cell. Bispecific antibodies may also be used to localize cytotoxic agents to the B cell. These antibodies possess a B cell marker-binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies).
Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al, EMBOJ., 10:3655-3659 (1991).
According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 121:210 (1986). According to another approach described in US Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-
products such as homodimers.
Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (US Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in US Patent No. 4,676,980, along with a number of cross-linking techniques.
Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al, Science, 229: 81 (1985); Shalabyera/., J. Exp. Med, 175: 217-225 (1992).
Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al,, J. Immunol., 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollingere/o/., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-
chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol., 152:5368(1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991). Antibodies with three or more antigen binding sites are described in WOO 1/77342 (Miller and Presta), expressly incorporated herein by reference.
IV. Conjugates and Other Modifications of the Antagonist or Antibody
The antagonist or antibody used in the methods or included in the articles of manufacture herein is optionally conjugated to a cytotoxic agent.
Chemotherapeutic agents useful in the generation of such antagonist or antibody-cytotoxic agent conjugates have been described above.
Conjugates of an antagonist or antibody and one or more small molecule toxins, such as a calicheamicin, a maytansine (US Patent No. 5,208,020), a trichothene, and CC1065 are also contemplated herein. In one embodiment of the invention, the antagonist or antibody is conjugated to one or more maytansine molecules (e.g. about 1 to about 10 maytansine molecules per antagonist or antibody molecule). Maytansine may, for example, be converted to May-SS-Me which may be reduced to May-SH3 and reacted with modified antagonist or antibody (Chari et al. Cancer Research 52: 127-131 (1992)) to generate a maytansinoid-antagonist or antibody conjugate.
Alternatively, the antagonist or antibody is conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Structural analogues of calicheamicin which may be used include, but are not limited to, yi', 0.2, 013', N-acetyl-yi', PSAG and 01] (Hinman et al. Cancer Research 53: 3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)).
Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americcma proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published October 28, 1993.
The present invention further contemplates antagonist or antibody conjugated with a compound with nucleolytic activity (e.g. a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
A variety of radioactive isotopes are available for the production of radioconjugated antagonists or antibodies. Examples include At211,1131,1125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu.
Conjugates of the antagonist or antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled 1 -isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antagonist or antibody. See WO94/11026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used.
Alternatively, a fusion protein comprising the antagonist or antibody and cytotoxic agent may be made, e.g. by recombinant techniques or peptide synthesis.
The antagonists or antibodies of the present invention may also be conjugated with a prodrug-activating enzyme which converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Patent No. 4,975,278.
The enzyme component of such conjugates includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.
Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as 0-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; p-lactamase useful for converting drugs derivatized with p-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antagonist or antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.
The enzymes of this invention can be covalently bound to the antagonist or antibody by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody linked to at
least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature, 312: 604-608 (1984)).
Other modifications of the antagonist or antibody are contemplated herein. For example, the antagonist or antibody may be linked to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.
The antagonists or antibodies disclosed herein may also be formulated as liposomes. Liposomes containing the antagonist or antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published October 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of an antibody of can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al. J. National Cancer Inst. 81(19) 1484 (1989).
Amino acid sequence modification(s) of protein or peptide antagonists or antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antagonist or antibody. Amino acid sequence variants of the antagonist or antibody are prepared by introducing appropriate nucleotide changes into the antagonist or antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antagonist or antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the antagonist or antibody, such as changing the number or position of glycosylation sites.
A useful method for identification of certain residues or regions of the antagonist or antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a residue or group of target residues are identified (e.g., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed antagonist or antibody variants are screened for the desired activity.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antagonist or antibody with an N-terminal methionyl residue or the antagonist fused to a cytotoxic polypeptide. Other insertional variants of the antagonist or antibody molecule include the fusion to the N- or C-terminus of the antagonist or antibody of an enzyme, or a polypeptide which increases the serum half-life of the antagonist or antibody.
Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antagonist or antibody
molecule replaced by different residue. The sites of greatest interest for substitutional mutagenesis of antibody antagonists include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened.
Substantial modifications in the biological properties of the antagonist or antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gin, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
Any cysteine residue not involved in maintaining the proper conformation of the antagonist or antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antagonist or antibody to improve its stability (particularly where the antagonist is an antibody fragment such as an Fv fragment).
A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or in additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as
described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.
Another type of amino acid variant of the antagonist or antibody alters the original glycosylation pattern of the antagonist or antibody. By altering is meant deleting one or more carbohydrate moieties found in the antagonist or antibody, and/or adding one or more glycosylation sites that are not present in the antagonist or antibody.
Glycosylation of polypeptides is typically either N-linked or O-linked. N-1 inked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
Addition of glycosylation sites to the antagonist or antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antagonist or antibody (for O-linked glycosylation sites).
Antibodies with altered Fc region glycosylation are described in WO02/079255 (Reff and Davies) and WO 03/035835 (Presta), expressly incorporated herein by reference.
Nucleic acid molecules encoding amino acid sequence variants of the antagonist or antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antagonist or antibody.
It may be desirable to modify the antagonist or antibody of the invention with respect to effector function, e.g. so as to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antagonist or antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of an antibody antagonist. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cell-mediated cytotoxicity (ADCC). See Caron et al., J. Exp Med 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989). Antibodies with altered (increased or diminished) Clq binding and or CDC activity are described in US Patent Nos. 6,194,55181 and 6,538,12481 (Idusogieef al.\ expressly incorporated herein by reference. Antibodies with altered (increased or diminished) FcR binding and/or ADCC activity are described in WOOO/42072 (Presta, L.), expressly incorporated herein by reference.
To increase the serum half life of the antagonist or antibody, one may incorporate a salvage receptor binding epitope into the antagonist or antibody (especially an antibody fragment) as described in US Patent 5,739,277, for example. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgGj, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
Alternatively, or additionally, one may increase, or decrease, serum half-life by altering the amino acid sequence of the Fc region of an antibody to generate variants with altered FcRn binding. Antibodies with altered FcRn binding and/or serum half life are described in WOOO/42072 (Presta, L.), expressly incorporated herein by reference.
V. Pharmaceutical Formulations
Therapeutic formulations of the antagonists or antibodies used in accordance with the present invention are prepared for storage by mixing an antagonist or antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, 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 counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
Exemplary CD20 antibody formulations are described in WO98/56418, expressly incorporated herein by reference. This publication describes a liquid multidose formulation comprising 40 mg/mL rituximab, 25 mM acetate, 150 mM
trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 at pH 5.0 that has a minimum shelf life of two years storage at 2-8°C. Another CD20 formulation of interest comprises lOmg/mL rituximab in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7mg/mL polysorbate 80, and Sterile Water for Injection, pH 6.5.
Lyophilized formulations adapted for subcutaneous administration are described in WO97/04801 and US Patent No. 6,267,958 (Andyaefo/.). Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the mammal to be treated herein.
The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a cytotoxic agent, chemotherapeutic agent, cytokine or immunosuppressive agent. The effective amount of such other agents depends on the amount of antagonist or antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antagonist or antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
VI. Treatment with the Antagonist or Antibody
The present invention contemplates therapy of various diseases and disorders with antibodies and antagonists. Where the antibody or antagonist binds to a B cell surface marker, such as CD20, conditions to be treated include B cell malignancies (see US Patent No. 6,455,04361, Grillo-Lopez, expressly incorporated herein by reference), and autoimmune diseases (see WOOO/67796, Curd et a!., expressly incorporated herein by reference). The antagonist or antibody which binds to a B cell surface marker may also be used to block an immune response to a foreign antigen, e.g. where the foreign antigen is an immunogenic drug or transplant (see WOO 1/03734, Grillo-Lopez et al, expressly incorporated herein by reference).
For the various indications disclosed herein, a composition comprising the antagonist or antibody will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular 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 agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The therapeutically effective amount of the antagonist or antibody to be administered will be governed by such considerations.
As a general proposition, the therapeutically effective amount of the
antagonist or antibody administered parenterally per dose will be in the range of about 0.1 to 20 mg/kg of patient body weight per day, with the typical initial range of antagonist or antibody used being in the range of about 2 to 10 mg/kg.
The preferred antagonist is an antibody, e.g. an antibody such as Rituximab or humanized 2H7, which is not conjugated to a cytotoxic agent. Suitable dosages for an unconjugated antibody are, for example, in the range from about 20 mg/m2 to about 1000 mg/m2. In one embodiment, the dosage of the antibody differs from that presently recommended for Rituximab. Exemplary dosage regimens for the CD20 antibody include 375 mg/m2 weekly x 4 or 8; or 1000 mg x 2 (e.g. on days 1 and 15).
As noted above, however, these suggested amounts of antagonist or antibody are subject to a great deal of therapeutic discretion. The key factor in selecting an appropriate dose and scheduling is the result obtained, as indicated above. For example, relatively higher doses may be needed initially for the treatment of ongoing and acute diseases. To obtain the most efficacious results, depending on the disease or disorder, the antagonist or antibody is administered as close to the first sign, diagnosis, appearance, or occurrence of the disease or disorder as possible or during remissions of the disease or disorder.
The antagonist or antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antagonist or antibody may suitably be administered by pulse infusion, e.g., with declining doses of the antagonist or antibody. Preferably the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is briefer chronic.
One may administer other compounds, such as cytotoxic agents, chemotherapeutic agents, immunosuppressive agents and/or cytokines with the antagonists or antibodies herein. The combined administration includes
coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
For RA, and other autoimmune diseases, the antagonist or antibody (e.g. a CD20 antibody) may be combined with any one or more of the immunosuppressive agents, chemotherapeutic agents and/or cytokines listed in the definitions section above; any one or more disease-modifying antirheumatic drugs (DMARDs), such as hydroxycloroquine, sulfasalazine, methotrexate, leflunomide, azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, Staphylococcal protein A immunoadsorption; intravenous immunoglobulin (1VIG); nonsteroidal antiinflammatory drugs (NSAIDs); glucocorticoid (e.g. via joint injection); corticosteroid (e.g. methylprednisolone and/or prednisone); folate; an anti-tumor necrosis factor (TNF) antibody, e.g. etanercept/ENBREL™, infliximab/REMICADE™, D2E7 (Knoll) or CDP-870 (Celltech); IL-1R antagonist (e.g. Kineret); 1L-10 antagonist (e.g. Ilodecakin); a blood clotting modulator (e.g. WinRho); an IL-6 antagonist/anti-TNF (CBP 1011); CD40 antagonist (e.g. IDEC 131); Ig-Fc receptor antagonist (MDX33); immunomodulator (e.g. thalidomide or ImmuDyn); anti-CD5 antibody (e.g. H5gl .1); macrophage inhibitor (e.g. MDX 33); costimulatory blocker (e.g. BMS 188667 or Tolerimab); complement inhibitor (e.g. H5G1.1, 3E10 or an anti-decay accelerating factor (DAF) antibody); or IL-2 antagonist (zxSMART).
For B cell malignancies, the antagonist or antibody (e.g. a CD20 antibody) may be combined with a chemotherapeutic agent; cytokine, e.g. a lymphokine such as IL-2, IL-12, or an interferon, such as interferon alpha-2a; other antibody, e.g., a radiolabeled antibody such as ibritumomab tiuxetan (ZEVALIN®), iodine I131 tositumomab (BEXXAR™), 1311 Lym-1 (ONCOLYM™), 90Y-LYMPHOCIDE™; anti-CD52 antibody, such as alemtuzumab (CAMPATH-1H™), anti-HLA-DR-p antibody, such as apolizumab, anti-CD80 antibody (e.g. I DEC-114), epratuzumab, HulDIO (SMART 1D10™), CD 19 antibody, CD40
antibody or CD22 antibody; an immunomodulator (e.g. thalidomide or ImmuDyn); an inhibitor of angiogenesis (e.g. an anti-vascular endothelial growth factor (VEGF) antibody such as AVASTIN™ or thalidomide); idiotype vaccine (EPOCH); ONCO-TCS™; HSPPC-96 (ONCOPHAGE™); liposomal therapy (e.g. daunorubicin citrate liposome), etc.
The preferred chemotherapy agents for combining with a CD20 antibody (or antagonist that binds to a B cell surface marker) are alkylator or anthracycline-based chemotherapeutic agents or fludarabine-based chemotherapeutic agents; cisplatin, fludarabine, vinblastine, doxorubicin, cyclophosphamide, and/or vincristine. With respect to CD20 antibodies or other antibodies that bind to a B cell surface marker, particularly desirable chemotherapies for combining with the antibody include, but are not limited to: cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) (Czuczman et al. JClin Oncol 17:268-76 (1999)); cyclophosphamide, vincristine, and prednisone (CVP); fludarabine (e.g. for treating CLL); fludarabine, cyclophosphamide, and mitoxantrone (FCM); or doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD).
The antagonist or antibody may also be used in myeloablative regimens. For instance, the antagonist or antibody may be used for in vivo purging prior to stem cell collection, or post-transplantation, for eradication of minimal residual disease.
Aside from administration of protein antagonists to the patient the present application contemplates administration of antagonists or antibodies by gene therapy. See, for example, WO96/07321 published March 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.
There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells; in vivo and ex vivo. For in vivo delivery the nucleic acid is injected directly into the patient, usually at the site where the antagonist or antibody is required. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example,
encapsulated within porous membranes which are implanted into the patient (see, e.g. U.S. Patent Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retrovirus.
The currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al, Proc. Natl. Acad. Sci. USA 87:3410-3414 (1990). For review of the currently known gene marking and gene therapy protocols see Anderson et al, Science 256:808-813 (1992). See also WO 93/25673 and the references cited therein.
Further details of the invention are illustrated by the following non-limiting Examples. The disclosures of all citations in the specification are expressly incorporated herein by reference.
A Complement-Dependent Cvtotoxicitv Assay for Detecting Neutralizing Antibodies Against Rituximab
Rituximab exerts its biological function by depleting CD20+ B cells through antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or both. In vitro, the CDC activity can be measured by incubating CD20+ WIL2-S lymphoma cells with human complement in the absence or presence of different concentrations of Rituximab. Cytotoxicity is then measured by quantifying live cells using ALAMAR BLUE® (Gazzano-Santoroe?a/., J. Immunol Methods 202 163-171 (1997)).
In this example, serum samples from patients treated with Rituximab which resulted in HACA were identified. HACA positive serum, which was confirmed by immunodepletion, was then subjected to the neutralizing antibody assay described below. The HACA assay is a bridging format with Rituximab as the capture reagent and biotinylated Rituximab as the detection reagent. The assay has a calibrated standard curve prepared with polyclonal goat antibodies to Rituximab. The minimum dilution of a sample in the assay is 1/5, with the lowest standard at 1 RU (relative unit)/mL. A sample response below 5RU/mL (value corrected for 1/5 dilution factor) is considered negative for HACA.
An assay for detecting neutralizing antibodies against Rituximab was developed. The neutralizing antibody assay was performed using RPMI 1640 culture medium supplemented with 0.1% bovine serum albumin (BSA), 20 mM HEPES (pH 7.2 -7.4), and 0.1 mM gentamicin. The assay was developed and calibrated using affinity purified polyclonal goat antibodies to Rituximab. When assay was performed in buffer matrix, typically 1-10 _L of goat anti-Rituximab was preincubated with 50 _L of various concentrations of Rituximab (0-10 _g/mL) in a flat-bottomed 96-well tissue culture plate. After preincubation at room temperature for 1-2 hours, 50 _L of a 1/3 human complement diluted in assay medium, 50 _L of WIL2-S lymphoblast cells of 106 cells/mL suspended in assay medium were added, and the mixture was incubated for 2 hours at 37°C and
5% CO2 to facilitate complement-mediated cell lysis. 50 _L of undiluted AlamarBlue_ was then added and the incubation continued for 15-26 hours. The plates were allowed to cool to room temperature for 10 minutes by shaking and the fluorescence was read using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm. Relative fluorescence units (RFU) were plotted against Rituximab concentration using a 4-parameter curve-fitting program (Softmax). By comparing the two curves with and without antibody preincubation, the neutralizing ability of anti-Rituximab antibodies can be determined. If the anti-Rituximab antibodies neutralized twenty percent or greater activity of Rituximab at a given concentration, the anti-Rituximab was defined as positive for neutralizing capability. This could be further quantified by determining the amount of anti-Rituximab to neutralize 1 _g of Rituximab. It was determined that the molar ratio for the goat anti-Rituximab polyclonal antibodies to neutralize Rituximab is approximately 3 to 1.
Since most patient samples for testing are serum samples, the serum matrix effect on assay performance was evaluated. Inclusion of 5% and 10% normal human serum in the assay medium had minimum effect on a 4-parameter fit curve. Signal suppression of upper asymptote was observed when serum concentration was above 20%. However, serum could be tolerated up to 50% without a significant shift in IC50 values. These data demonstrated the feasibility of using CDC assay for detecting anti-Rituximab antibodies without further manipulating patient's serum samples. When testing serum samples, up to 50 _L of serum was incubated with 50 _L of Rituximab dilutions before complement and cell suspension addition. The rest of the procedures were the same as described above. For data analysis, the neutralizing ability of Rituximab-treated serum was compared individually with pre-treatment serum to determine neutralizing activity. The sensitivity/limit of detection of the assay in serum matrix was determined by spiking affinity purified goat anti-Rituximab into normal human serum. Using the current assay format, the lowest neutralizing antibody amount in serum that can be detected is approximately 1 _g/mL.
Rituximab treated systemic lupus erythematosus (SLE) patient samples with an antibody response (HACA+) by the ELISA assay above were tested in the neutralizing antibody assay. Significant differences were observed between baseline serum and serum following Rituximab treatment. The CDC activity was either completely or partially blocked with HACA+ sera, indicating neutralizing activities in the treated samples. In comparison, serum samples obtained prior to Rituximab treatment showed no neutralizing activity.
In summary, the Example describes a cell-based functional assay, complement-dependent cytotoxicity (CDC) assay, for detecting neutralizing activity in the serum of Rituximab treated patients. This assay will help largely in characterizing the nature of an anti-drug antibody response; therefore it will be of great value when evaluating drug safety and efficacy.
Example 2 Therapy of Autoimmune Disease
According to one embodiment of the invention herein, the assay described herein may be used in relation to a treatment regimen for patients with an autoimmune disease. Exemplary autoimmune diseases include rheumatoid arthritis (RA), including juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), including lupus nephritis, Wegener's disease, inflammatory bowel disease, idiopathic or immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis (MS), psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjogren's syndrome, glomerulonephritis, autoimmune hemolytic anemia, etc.
An antibody that binds CD20 (e.g. Rituximab or humanized 2H7) is administered to the patient in an amount effective to treat the autoimmune disease in question. For instance, the antibody may be dosed at 375 mg/m2 every week for 4 or 8 weeks, or 1000 mg on Days 1 and 15. The antibody is optionally combined with one or more other drugs that treat the autoimmune disease, such as
immunosuppressive agents, chemotherapeutic agents and/or cytokines listed in the definitions section above; any one or more of disease-modifying antirheumatic drugs (DMARDs) such as hydroxycloroquine, sulfasalazine, methotrexate, leflunomide, azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, Staphylococcal protein A immunoadsorption; intravenous immunoglobulin (IVIG); nonsteroidal antiinflammatory drugs (NSAlDs); glucocorticoid (e.g. via joint injection); corticosteroid (e.g. methylprednisolone and/or prednisone); folate; an anti-tumor necrosis factor (TNF) antibody, e.g. etanercept/ENBREL™, infliximab/REMICADE™, D2E7 (Knoll) or CDP-870 (Celltech); IL-1R antagonist (e.g. Kineret); 1L-10 antagonist (e.g. Ilodecakin); a blood clotting modulator (e.g. WinRho); an IL-6 antagonist/anti-TNF (CBP 1011); CD40 antagonist (e.g. IDEC 131); Ig-Fc receptor antagonist (MDX33); immunomodulator (e.g. thalidomide or ImmuDyn); anti-CD5 antibody (e.g. HSgl.l); macrophage inhibitor (e.g. MDX 33); costimulatory blocker (e.g. BMS 188667 or Tolerimab); complement inhibitor (e.g. h5Gl .1, 3E10 or an anti-decay accelerating factor (DAF) antibody); or IL-2 antagonist (zxSMART).
A biological sample of serum, which may comprise HACA (directed against Rituximab) or HAHA (directed against humanized 2H7), is obtained from the patient at baseline, and 3, 6 and 9 months. The serum is subjected to an EL1SA to determine whether HACA or HAHA is present therein. The assay is described in Example 1 above.
Serum which is demonstrated to contain HACA or HAHA is then tested for neutralizing antibodies as in Example 1 above. In comparison to the same amount of pre-treatment counterpart (i.e., HACA and HAHA negative), a sample neutralizing about 20% or greater activity of Rituximab or humanized 2H7 at a given concentration, may be considered positive for neutralizing antibody directed against Rituximab or humanized 2H7. A positive result is indicative of reduced efficacy of the antibody in treating the autoimmune disease.
Example 3 Therapy of B cell Malignancy
A patient with a CD20 positive B cell malignancy, such as Hodgkin's disease including lymphocyte predominant Hodgkin's disease (LPHD), non-Hodgkin's lymphoma (NHL), follicular center cell (FCC) lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, plasmacytoid lymphocytic lymphoma, mantle cell lymphoma, AIDS or HIV-related lymphoma, multiple myeloma, central nervous system (CNS) lymphoma, post-transplant lymphoproliferative disorder (PTLD), Waldenstrom's macroglobulinemia (lymphoplasmacytic lymphoma), mucosa-associated lymphoid tissue (MALT) lymphoma, or marginal zone lymphoma/leukemia, is treated according to this example.
An antibody that binds CD20 (e.g. Rituximab or humanized 2H7) is administered to the patient in an amount effective to treat the B cell malignancy in question. For instance, the antibody may be dosed at 375 mg/m2 every week for 4 or 8 weeks.
Optionally, the CD20 antibody is combined with one or more chemotherapeutic agents. The preferred chemotherapy agents for combining with a CD20 antibody are alkylator or anthracycline-based chemotherapeutic agents or fludarabine-based chemotherapeutic agents; cisplatin, fludarabine, vinblastine, doxorubicin, cyclophosphamide, and/or vincristine. Particularly desirable chemotherapies for combining with the antibody include, but are not limited to: cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) (Czuczman et al. JClin Oncol 17:268-76 (1999)); cyclophosphamide, vincristine, and prednisone (CVP); fludarabine (e.g. for treating CLL); fludarabine, cyclophosphamide, and mitoxantrone (FCM); or doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) etc.
A biological sample of serum, which may contain HACA (directed against Rituximab) or HAHA (directed against humanized 2H7), is obtained from the patient at baseline, and 3, 6 and 9 months. The serum is subjected to an ELISA to
determine whether HACA or HAHA is present therein. The assay is described in Example 1 above.
Serum which is demonstrated to contain HACA or HAHA is then tested for neutralizing antibodies as in Example 1 above. In comparison to the same amount of pre-treatment counterpart (i.e., HACA and HAHA negative), a sample neutralizing about 20% or greater activity of Rituximab or humanized 2H7 at a given concentration, may be considered positive for neutralizing antibody directed against Rituximab or humanized 2H7. The presence of neutralizing antibodies indicates reduced effectiveness of the antibody in treating the B cell malignancy.
Example 4 Blocking an Immune Response to a Foreign Antigen
In the present example, an anti-CD20 antibody is used to block an immune response to a foreign antigen such as a therapeutic protein (e.g. a murine antibody or an immunotoxin), gene therapy viral vector, blood factor (e.g. Factor VIII), platelets, or transplant etc.
A suitable dosage of the CD20 antibody is 375mg/m2 by four or eight infusions given every week. Administration of the CD20 antibody will reduce or eliminate an immune response in the patients, and thereby facilitate successful therapy.
For blocking an immune response to a transplant, the CD20 antibody may be used as part of combination immunosuppressive regimens for prophylaxis of acute rejection. In this setting, a CD20 antibody, such as Rituximab or humanized 2H7, is administered in the peri-transplant period as part of a sequential combination regimen that includes T cell directed agents such as cyclosporine, corticosteroids, mycophenolate mofetil, with or without an anti-IL2 receptor antibody. Hence, the CD20 antibody would be considered part of an induction regimen, to be used in conjunction with chronic immunosuppressive therapies. The CD20 antibody may contribute to prevention of an allorejection response by inhibiting alloantibody production and/or affecting alloantigen presentation through depletion of antigen-presenting cells.
Dosages of the further immunosuppressive agents are as follows: cyclosporine (5mg/kg/day); corticosteroids (1 mg/kg, gradually tapered off); mycophenolate mofetil (1 gram given twice a day); and anti-IL2 receptor antibody (1 mg/kg, five infusions given weekly). The CD20 antibody may also be combined with other induction immunosuppressive drugs, such as polyclonal anti-lymphocyte antibodies or monoclonal anti-CD3 antibodies; maintenance immunosuppressive drugs, such as calcineurin inhibitors (e.g., tacrolimus) and antiproliferative agents (such as azathioprine, leflunomide or sirolimus); or combination regimens that include blockade of T cell costimulation, blockade of T cell adhesion molecules of blockade of T cell accessory molecules.
Aside from prophylaxis of acute rejection, CD20 antibodies may be used to treat acute rejection. Suitable dosages of the CD20 are as described above. The CD20 antibody is optionally combined with a CD3 monoclonal antibody and/or corticosteroids in the treatment of acute rejection.
CD20 antibodies may also be used (a) later in the post-transplant period alone, or in combination with other immunosuppressive agents and/or costimulatory blockade, for treatment or prophylaxis of "chronic" allograft rejection; (b) as part of a tolerance-inducing regimen; or (c) in the setting of xenotransplantation.
A biological sample of serum, which may contain HACA (directed against Rituximab) or HAHA (directed against humanized 2H7), is obtained from the patient at baseline, and 3, 6 and 9 months. The serum is subjected to an ELISA to determine whether HACA or HAHA is present therein. The assay is described in Example 1 above.
Serum which is demonstrated to contain HACA or HAHA is then tested for neutralizing antibodies as in Example 1 above. In comparison to the same amount of pre-treatment counterpart (i.e., HACA and HAHA negative), a sample neutralizing about 20% or greater activity of Rituximab or humanized 2H7 at a given concentration, may be considered positive for neutralizing antibody directed against Rituximab or humanized 2H7. Where a neutralizing antibody response is
detected, this indicates the antibody has reduced ability to block an immune response to the foreign antigen in question.
1. A method for evaluating the efficacy of an antibody that binds CD20
comprising the steps:
a. measuring the ability of a biological sample treated with the CD20
antibody to block a biological activity of the CD20 antibody;
b. exposing CD20 positive cells to complement in the presence of the
CD20 antibody for measuring the ability of the biological sample
to block the biological activity; and
c. determining viability of the said exposed cells obtained from step
(b) and/or determining the presence of neutralizing antibodies by
measuring complement-dependent cytotoxicity (CDC) activity of
the said antibodies obtained from step (b), wherein a reduction in
the CDC activity indicates the presence of neutralizing antibodies,
for evaluating the efficacy of an antibody that binds CD20,
wherein the said method is useful for evaluating drug safety and efficacy.
2. A method as claimed in claim 1, wherein the biological activity is selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis, and inhibition of cell growth.
3. A method as claimed in claim 1, wherein the biological activity is complement-dependent cytotoxicity (CDC).
4. A method as claimed in claim 1, wherein the CD20 antibody is rituximab
5. A method as claimed in claim 1, wherein the CD20 antibody is humanized 2H7.
6. A method as claimed in claim 1, wherein the biological sample comprises
antibodies from the patient that bind the CD20 antibody.
7. A method as claimed in claim 1, wherein the biological sample comprises
serum from the patient.
8. A method for evaluating the efficacy of an antibody substantially as
described and shown with reference to the description and examples.
|Indian Patent Application Number||167/DELNP/2006|
|PG Journal Number||26/2009|
|Date of Filing||10-Jan-2006|
|Name of Patentee||GENENTECH, INC.|
|Applicant Address||1 DNA WAY SOUTH SAN FRANCISCO CALIFORNIA 94080-4990 U.S.A.|
|PCT International Classification Number||G01N 33/564|
|PCT International Application Number||PCT/US2004/020069|
|PCT International Filing date||2004-06-24|